2010年04期目次

  • Construction and Immune Effect of Recombinant Retrovirus Containing Glycoprotein Gene of Rabies Virus

    ZHAO Na, ZHANG Shou-feng, HU Yong-hao, et al(Gansu Agriculture University, Lanzhou 730070, China)

    Objective To construct a recombinant retrovirus containing the glycoprotein(GP)gene of rabies virus and determine its immune effect in mice. Methods Plasmid pVAX-G containing the GP gene of rabies virus SRV9 strain was digested with restriction endonuclease, and the obtained gene fragment was cloned into retrovirus vector. The constructed recombinant plasmid pLNCX-G was transfected to packaging cell line PA317 in mediation of liposome, and positive cell clones were screened for cloning and amplification. BHK cells were infected with virus particles, in which the integration and transcription of GP gene were determined by PCR and RT-PCR. The titer of retrovirus was determined with NIH3T3 cells. KM mice were immunized with the constructed recombinant retrovirus, and determined for serum neutralizing antibody titer against rabies virus by fluorescent antibody virus neutralization(FAVN)test. Results Restriction analysis proved that recombinant plasmid pLNCX-G was constructed correctly. The GP gene of rabies virus was integrated to the genome of BHK cells and transcribed normally. The virus titer reached 1. 2 × 104 CFU / ml at the most. The positive rates of neutralizing antibody against rabies virus induced in mice by intramuscular and intraperitoneal injections were 6. 7% and 86. 7%, while the effective protection rates were 0 and 76. 9% respectively. Conclusion The recombinant retrovirus containing the GP gene of rabies virus SRV9 strain was successfully constructed, and a certain neutralizing antibody titers were induced in mice, which laid a foundation of further study.

    2010 04 v.23 [Abstract][OnlineView][Download 186K]

  • Construction and Expression of Bicistronic Recombinant Adenovirus with P1 and 3CD Genes of Enterovirus 71

    GE Xiao-qin, LI Hong-jun, WANG Wen-ju, et al(Institute of Medical Biology, Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To construct a bicistronic recombinant adenovirus with P1 and 3CD genes of enterovirus 71(EV71) and determine the cleavage effect of 3CD protease as well as the antigenicity of expressed product. Methods P1 and 3CD genes were amplified from EV71 strain isolated from Fuyang City, Anhui Province, China for construction of shuttle plasmids pShuttle-CMVP1-3CD, pShuttle-CMV-P1 and pShuttle-CMV-3CD, based on which recombinant adenoviruses rAd-P1-3CD, rAd-P1 and rAd-3CD were obtained by homologous recombination and transfected to 293 cells respectively. The transcription of target gene was determined by RT-PCR, and the expression of target protein by IFA. Results Both PCR and restriction analysis proved that recombinant adenoviruses rAd-P1-3CD, rAd-P1 and rAd-3CD were constructed correctly. RT-PCR showed transcription of target mRNA in the 293 cells transfected with the 3 kinds of recombinant adenoviruses. Only specific expressed product of rAd-P1-3CD was observed by IFA, while no specific expressed product of rAd-P1 or rAd-3CD. Conclusion The bicistronic recombinant adenovirus with P1 and 3CD genes of EV71 was successfully constructed, and the expressed product showed EV71-specific antigenicity, indicating that the P1 protein showed antigenicity only after being cleaved with 3CD protease.

    2010 04 v.23 [Abstract][OnlineView][Download 337K]

  • Effect of Coexpressing Protein Disulfide Isomerase on Expression of IFN β-HSA Fusion Protein in Pichia pastoris

    QU Lin, LEI Jian-yong, ZHANG Qi, et al (Department of Cellular and Molecular Pharmacology, College of Medicine and Pharmacology, Jiangnan University, Wuxi 214122, Jiangsu Province, China)

    Objective To investigate the effect of coexpressing protein disulfide isomerase(PDI)on the expression of IFN β-HSA fusion protein in Pichia pastoris. Methods Primers were designed according to the PDI gene sequence of P. pastoris reported in GenBank, with which the target gene fragment was amplified from the genome of P. pastoris by PCR and inserted into expression vector pPICZαA. The constructed recombinant plasmid pPICZαA-PDI was transformed to recombinant P. pastoris KM71 / pPIC9K-IFNβ-HSA, and the positive transformants for coexpressing PDI were screened and induced by methanol. The expressed product was identified by SDS-PAGE, and the expression level of IFNβ-HSA was determined by ELISA. Results PCR proved that recombinant plasmid pPICZαA-PDI was transformed to recombinant P. pastoris KM71 / pPIC9K-IFNβ-HSA. SDS-PAGE showed that PDI was expressed in a large quantity, and IFNβ-HSA fusion protein was expressed significantly in both P. pastoris KM71 / pPIC9K-IFNβ-HSA and P. pastoris KM71 / pPIC9K-IFNβ-HSA / pPICZαA-PDI. However, the expression level of IFNβ-HSA fusion protein in P. pastoris KM71 / pPIC9K-IFNβ-HSA / pPICZαA-PDI was 60% higher than that in P. pastoris KM71 / pPIC9K-IFNβ-HSA, which reached (22. 49 ± 3. 52)mg / L. Conclusion Coexpressing PDI promoted the secretory expression of foreign protein, which laid a foundation of further study on effect of over-expression of chaperones in P. pastoris on secretion of foreign protein.

    2010 04 v.23 [Abstract][OnlineView][Download 286K]

  • Inhibitory Effect of Recombinant Adenovirus Expressing Hemagglutinin-Neuramidinase Gene of Newcastle Disease Virus on Hepatoma HepG-2 Cells

    WEN Zhong-mei, LI Xiao, LIU Yan, et al(Department of Respiratory Medicine, First Hospital of Jilin University, Changchun 130021, China)

    Objective To investigate the inhibitory effect of recombinant adenovirus Ad-HN expressing hemagglutinin(HA)neuramidinase(NA)gene of Newcastle disease virus (NDV)on human hepatoma HepG-2 cells. Methods HepG-2 cells were infected with Ad-HN, and the inhibitory effect of Ad-HN on proliferation of HepG-2 cells was determined by MTT method. The apoptosis of HepG-2 cells was determined by Annexin V staining combined with flow cytometry. The morphologies of HepG-2 cells infected with Ad-HN and their nuclei were observed by AO / EB staining and DAPI staining. The activities of Caspase 1, 3, 6 and 8 were determined by substrate coloration. Results Ad-HN inhibited the proliferation of HepG-2 cells effectively. After infection with Ad-HN at a MOI of 100 for 48 h, the inhibition rate of HepG-2 cells reached a peak value of 17. 20% ～ 35. 04%. Annexin V staining showed that the apoptosis rate of HepG-2 cells 48 h after infection with Ad-HN was 29. 39%. The infection with Ad-HN induced the apoptotic character of HepG-2 cells, such as cell condensation as well as nuclear shrinkage and breakage. Meanwhile, the infection with Ad-HN significantly up-regulated the Caspase activity of HepG-2 cells. Conclusion Ad-HN induced the apoptosis of and showed inhibitory effect on HepG-2 cells.

    2010 04 v.23 [Abstract][OnlineView][Download 247K]

  • Prokaryotic Expression and Purification of OD Domain of Chronic Myeloid Leukemia Bcr / Abl Fusion Gene

    JI Mao-sheng, HUANG Zheng-lan, HUANG Shi-feng, et al(Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics of Education Ministry, Chongqing Medical University, Chongqing 400016, China)

    Objective To express the OD domain of chronic myeloid leukemia(CML)Bcr / Ab1 fusion gene in prokaryotic cells and purify the expressed product. Methods TAT, OD and HA gene fragments were cloned into prokaryotic expression vector pET32a(+)in turn, and the constructed recombinant plasmid pTAT-OD-HA was identified by PCR, restriction analysis and sequencing, then transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot, then purified by nickel ion affinity chromatography. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pTAT-OD-HA was constructed correctly. The relative molecular mass of expressed protein was about 30000. The expression level of recombinant protein reached a peak value 6 h after induction, which accounted for about 10% of total somatic protein. The expressed protein showed specific reaction with mouse anti-HA monoclonal antibody, and reached a purity of about 95% after purification. Conclusion TAT-OD-HA fusion protein was successfully expressed in prokaryotic cells and purified, which laid a foundation of further study on its role in CML.

    2010 04 v.23 [Abstract][OnlineView][Download 227K]

  • Effect of Reduced Glutachione on Function of Cardiac Mitochondria of Rats with Diabetic Cardiomyopathy

    ZHANG Ying-yu, FANG Chun-qian, CHEN Yun, et al(Affiliated People's Hospital of Jiangsu University, Zhenjiang 212002, Jiangsu Province, China)

    Objective To observe the effect of reduced glutachione(GSH)on the function of cardiac mitochondria of rats with diabetic cardiomyopathy(DCM)induced by streptozotocin(STZ). Methods Rat model of DCM was established by injecting i.p. SD rats with STZ. The model rats were divided into DCM and DCM + GSH groups randomly. The rats in DCM + GSH group were injected i.p. with GSH, once a day for 8 weeks, and those in DCM group with physiological saline. Normal SD rats injected i.p. with physiological saline once a day for 8 weeks were served as control(C group). The rats in various groups were determined by anti-oxidation indexes in sera and cardiac tissue homogenate, calcium ion-induced mitochondria swelling and membrane potential of cardiac mitochondria. Results The SOD activity in cardiac tissue homogenate of rats in DCM + GSH group was significantly higher than that in DCM group. Though the SOD activity in sera of rats in DCM + GSH group showed an increasing tendency, it showed no significant difference with that in DCM group. The MDA content in sera of rats in DCM + GSH group decreased significantly as compared with that in DCM group. The MDA content in cardiac tissue homogenate of rats in DCM + GSH group showed also a decreasing tendency, while showed no significant difference with that in DCM group. Though the sensitivity of cardiac mitochondria of rats to the swelling induced by high calcium ion concentration in DCM + GSH group increased as compared with that in DCM group, it had not recovered to the normal level. The membrane potential of cardiac mitochondria of rats in DCM + GSH group was significantly higher than that in DCM group. Conclusion GSH showed a certain protective effect on the hearts of rats with DCM induced by STZ, by a potential mechanism of enhancing the anti-oxidation ability of cardiac muscle and improving the function of mitochondria in cardiac tissue.

    2010 04 v.23 [Abstract][OnlineView][Download 173K]

  • Effect of MMP-2 Antisense Oligodeoxynucleotide on Proliferation and Invasion of Human Gastric Cancer SGC7901 Cells

    LIU Qian, WU Xiao-ling, LUO Hong-chun, et al(Department of Gastrointestinalogy, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China)

    Objective To investigate the effect of matrix metalloproteinase-2(MMP-2)antisense oligodeoxynucleotide (ASODN)on the proliferation and invasion of human gastric cancer SGC7901 cells. Methods Various concentrations of thiophsphorylate-modified ASODN of MMP-2 were transfected to moderately and lowly differential SGC7901 cells. The transcription level of MMP-2 mRNA was determined by RT-PCR, the abilities of in vitro migration and invasion of cells by Transwell test, and the proliferative activity of cells by MTT method. Results The transcription level of MMP-2 mRNA in SGC7901 cells after transfection with ASODN decreased significantly. Both in vitro migration and invasion abilities of cells were inhibited, and the inhibitory effect increased with the increasing ASODN concentration. However, the proliferative activity of cells showed no significant change. Conclusion MMP-2 ASODN inhibited the transcription level of MMP-2 mRNA as well as in vitro migration and invasion, while showed no significant correlation to the proliferative activity of SGC7901 cells.

    2010 04 v.23 [Abstract][OnlineView][Download 340K]

  • Prokaryotic Expression and Purification of Mouse IL-13 Receptor α2 Extracellular Domain

    WANG Li-li, WEI Ya-qiang, CAI Lei, et al (Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi'an 710032, China)

    Objective To express mouse IL-13 receptor α2 extracellular domain(sIL-13Rα2)in prokaryotic cells and purify the expressed product. Methods The sIL-13Rα2 gene was cloned into prokaryotic expression vector pROEX HTa, and the constructed recombinant plasmid pROEX HTa / sIL-13Rα2 was identified by restriction analysis, then transformed to E. coli BL21 (DE3)for expression under induction of IPTG. The expressed product was determined for expression level by thin layer scanning, analyzed for form, and identified by Western blot, then purified by nickel ion affinity chromatography. Results Restriction analysis proved that recombinant plasmid pROEX HTa / sIL-13Rα2 was constructed correctly. The expressed sIL-13Rα2 protein, with a relative molecular mass of about 36 200, reached a maximum expression level after induction for 6 h, which contained about 45% of total somatic protein. The recombinant sIL-13Rα2 protein, existing in a form of inclusion body, showed specific reaction with monoclonal antibody against mouse sIL-13Rα2, and reached a purity of more than 95% after purification. Conclusion Mouse sIL-13Rα2 was successfully expressed and purified, which laid a foundation of further study on its application to treatment of bronchial asthma.

    2010 04 v.23 [Abstract][OnlineView][Download 226K]

  • Cloning and Eukaryotic Expression of Porcine Interferon α Gene

    LI Peng-fei, LI Ming, CHENG Jian, et al (Liaoning Medical University, Jinzhou 121000, Liaoning Province, China)

    Objective To clone porcine interferon α(IFNα)gene and express in Pichia pastoris. Methods Lymphocytes were isolated from porcine peripheral blood and induced by phytohemagglutinin(PHA), from which total RNA was extracted with Trizol reagent for amplification of porcine IFNα gene by RT-PCR. The amplified gene was cloned into vector pPIC9K, and the constructed recombinant plasmid pPIC9K-IFNα was identified by PCR, restriction analysis and sequencing, then transformed to P. pastoris GS115 by electrotransformation. The recombinants were screened for expression under induction of methanol. The expressed product was identified by SDS-PAGE and Western blot. Results The porcine IFNα gene at a length of 521 bp was amplified by RT-PCR. PCR, restriction analysis and sequencing proved that recombinant plasmid pPIC9K-IFNα was constructed correctly. The expressed protein showed a relative molecular mass of about 19 000, and that in soluble form contained about 13% of total somatic protein. Specific immune reaction of the expressed protein with the corresponding antibody was observed. Conclusion Porcine IFNα gene was successfully cloned and expressed in P. pastoris, which laid a foundation of further study on biological characters of porcine IFNα.

    2010 04 v.23 [Abstract][OnlineView][Download 217K]

  • Preparation of Phage-displayed Diabetes Type 1 Vaccine by Lyophilization

    REN Wan-jun, HU Yun-zhang, HU Ning-zhu (Institute of Medical Biology, Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To prepare phage-displayed diabetes type 1 vaccine by lyophilization. Methods Various stabilizers were screened by orthogonal test and mixed with phage-displayed diabetes type 1 vaccine respectively, based on which the lyophilization curve was optimized. The vaccines before and after lyophilization were determined for titers and evaluated by various indexes. Results The formula of stabilizer was optimized as 10. 85% trehalose + 17. 37% glycine(w / v). The lyophilization curve was optimized as follows: S1:-40℃ 4 h, 1. 5℃ / min; S2:-15℃ 5 h, 1. 5℃ / min; S3: 25℃ 4 h, 1. 5℃ / min; vacuum: 0. 02 mbar. The decrease of vaccine titer after lyophilization was not more than 0. 5 pfu / ml. The freeze-dried vaccine showed both good appearance and good morphology under microscope, and its temperature for glass state transformation reached more than 220℃, while its residual moisture content was less than 3%. Conclusion The phage-displayed diabetes type 1 vaccine prepared by lyophilization using a stabilizer consisting of trehalose and glycine were of simple components, high thermal stability and low residual moisture content, and its lyophilization curve was simplified, while the time for storage was prolonged.

    2010 04 v.23 [Abstract][OnlineView][Download 290K]

  • Analysis of CD3+CD4+ and CD4+CD25+ Lymphocyte Subsets of Pigs with Dumbing Immune Response to Classical Swine Fever Vaccine

    WANG Wei, YANG Zhi, FU Ming-shan, et al (College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To analyze the distribution of CD3+CD4+ and CD4+CD25+ lymphocyte subsets in the pigs with dumbing immune response to classical swine fever(CSF)vaccine. Methods Fifteen CSF virus(CSFV)antibody-negative pigs were screened by ELISA and serum neutralizing antibody test from the female breeding pigs immunized with CSF vaccine, using 15 antibody-positive pigs as control. The anticoagulant blood of pigs was collected for isolation of peripheral blood lymphocytes(PBLs)in which the distribution of CD3+CD4+ and CD4+CD25+ lymphocyte subsets was determined by flow cytometry. Results The percentage of CD3+CD4+ lymphocyte subsets was significantly lower in PBLs of CSFV antibody-negative pigs(24. 09% ± 1. 29%)than in CSFV antibody-positive ones(37. 49% ± 1. 60%), while that of CD4+CD25+ lymphocyte subsets was significantly higher in the former(2. 34% ± 0. 20%) than in the latter (1. 64% ± 0. 13%). Conclusion The decrease of CD3+CD4+ and increase of CD4+CD25+ lymphocyte subsets in PBLs might be the important causes of dumbing immune response to CSF vaccine.

    2010 04 v.23 [Abstract][OnlineView][Download 213K]

  • Preparation and Immune Effect of Inactivated Porcine Reproductive and Respiratory Syndrome Vaccine

    WANG Xing-chen, LI Yu, WANG Gui-jun, et al (College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China)

    Objective To prepare an inactivated oil emulsion vaccine with a porcine reproductive and respiratory syndrome virus (PRRSV)Anhui strain. Methods PRRSV was subcultured and proliferated in Marc-145 cells, and prepared into inactivated oil emulsion vaccine which was subjected to examination on physical property as well as sterility and safety tests. The qualified vaccine was used for immunization of healthy piglets by intramuscular injection, and the serum antibody level and protection were determined. The immune effects of vaccine by various schedules, as well as those of the prepared vaccine and commercial inactivated vaccines, were compared. Results The optimal dosage of prepared vaccine for each piglet was 2 ml. The serum antibody level of immunized piglets reached a peak value 14 d after immunization. The protective rate of the prepared vaccine against challenge with the same virus strain was 80%(4 / 5). Both immunizations with two doses of inactivated vaccine and with one dose of inactivated vaccine and one dose of live attenuated vaccine showed good immune effects. The immune effect of prepared inactivated vaccine showed no significant difference with that of commercial inactivated vaccine No. 1, while superior to that of No. 2. Conclusion Inactivated PRRSV oil emulsion vaccine was successfully prepared, which was safe, effective, showed good immune effect and was worthy of popularization.

    2010 04 v.23 [Abstract][OnlineView][Download 159K]

  • Development of A Method for Determination of Immunogenicity of HIV Vaccine in Mice

    HUANG Wei-jin, ZHANG Chun-tao, ZHAO Chen-yan, et al (National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To preliminarily develop a method for determination of immunogenicity of HIV vaccine in mice and evaluate the immunogenicity. Methods BALB / c mice were immunized with one kind of HIV DNA vaccine(D)and two kinds of recombinant vaccinia virus-based HIV vaccines(M and R)respectively, and their sera and splenic lymphocytes were separated 6 weeks later. The secretions of cytokines in splenic lymphocytes were determined by ELISOPT and intracellular cytokine staining, and the proliferation of lymphocytes by MTS method. The anit-HIV IgG and IgA levels in sera were determined by ELISA. Results All the vaccines induced a broad of immune responses in mice. ELISPOT proved the secretions of IFNγ, IL-2, IL-4 and IL-6, and intracellular cytokine staining proved the secretions of IFNγ, IL-2 and IL-4 in splenic lymphocytes. The numbers of splenic lymphocytes positive in proliferation test of mice immunized with vaccines D and M were larger than that with vaccine R. Anti-HIV IgG were detected in 0 / 5, 1 / 5 and 5 / 5 of mice immunized with vaccines D, M and R respectively, while no anti-HIV IgA were detected. Conclusion A method for determination of immunogenicity of HIV vaccine in mice was preliminarily developed.

    2010 04 v.23 [Abstract][OnlineView][Download 228K]

  • Effect of Mycobacterium vaccae for Injection on Cellular Immune Function of Mice Infected with Mycobacterium tuberculosis

    TAO Li-feng, PU Jiang, WAN Rong-sheng, et al (Anhui Longcome Biological Pharmacy Co. Ltd, Hefei 230088, China)

    Objective To investigate the effect of Mycobacterium vaccae for injection on cellular immune function of mice infected with Mycobacterium tuberculosis as well as mechanism of the effect. Methods Mouse model of Mycobacterium tuberculosis infection was established by injecting i.v. C57 mice with Mycobacterium tuberculosis H37RV strain. The model mice were divided into model control, M. vaccae high, moderate and low dosage and isoniazid (INH)groups randomly, using healthy mice as normal control. The mice were killed 8 weeks after infection, and their lungs, spleens and livers were taken out for calculation of weight indexes and total pathological change indexes. The live bacteria in lung were counted, and the pathological changes of main organs were observed. The ratios of CD3+ T, CD4+ T, NK1.1+ T, NK and γδT cells in spleen as well as expressions of IFNγ and IL-4 in CD4+ T cells were determined by flow cytometry. Results The M. vaccae at various dosages decreased the pathological change of mice significantly. The total pathological change indexes of various organs, weight indexes of spleens and lungs as well as live bacterial counts of mice in M. vaccae moderate dosage group were significantly lower than those in model control group. The pathological change in lungs of mice in M. vaccae high, moderate and low dosage groups were mainly expressed as proliferative node, while those in model control group as necrotizing and proliferative nodes. The ratios of CD3+ and CD4+ T cells of mice in M. vaccae high, moderate and low dosage groups increased significantly, while the ratios of CD4 + IFNγ+ helper T cells showed no significant difference with those in model control group. The ratios of CD4+IL-4+ helper T cells of mice in M. vaccae moderate and low dosage groups decreased significantly, while those of γδT cells and NK cells increased significantly. Conclusion M. vaccae increased both natural and acquired cellular immunities of mice against Mycobacterium tuberculosis and showed good immunological intervention.

    2010 04 v.23 [Abstract][OnlineView][Download 353K]

  • Development of Purification Procedure of Inactivated Poliomyelitis Vaccine Prepared with Sabin Strain

    HENG Xie, GAO Na, WU Yin-jie, et al(Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To develop a purification procedure of inactivated poliomyelitis vaccine(IPV)prepared with Sabin strain. Methods Poliomyelitis virus was purified by Sepharose CL-6B gel filtration and DEAE-Sepharose Fast Flow ion exchange chromatography, subjected to control tests then sterilized by filtration and inactivated. Results All the removal rates of protein in poliomyelitis virus typesⅠ, Ⅱ and Ⅲ were not less than 98.81%, and the residual DNA contents were less than 100 pg / ml, while the virus titers were 8. 75, 8. 50 and 8. 63 lg CCID50 / ml respectively. The D antigen content in the virus after sterilization by filtration and inactivation decreased slightly. Conclusion A purification procedure of IPV prepared with Sabin strain was developed, and IPV with high purity was prepared.

    2010 04 v.23 [Abstract][OnlineView][Download 142K]

  • Development of A RT-PCR Method for Determination of Chinese Sacbrood Virus

    MA Ming-xiao, LI Ming, YUAN Chun-ying, et al(College of Animal Science and Veterinary Medicine, Liaoning Medical University, Jinzhou 121001, Liaoning Province, China)

    Objective To develop a RT-PCR method for determination of Chinese sacbrood virus(CSBV). Methods A pair of primers specific to structural protein gene of CSBV were designed according to the published CSBV gene sequence, with which the RNA of CSBV was extracted and reversely transcribed to cDNA as a template for PCR amplification. The developed RT-PCR method was optimized, verified for specificity and sensitivity, and used for determination of 31 clinical samples. Results The developed RTPCR method for CSBV showed high specificity and sensitivity, and its minimum detection limit using the recombinant plasmid containing CSBV structural protein gene as a template was 10 pg DNA. Twenty-two samples diagnosed as negative by clinical symptoms and electron microscopy were confirmed by the developed RT-PCR method, from 2 of which the target genes were amplified. Conclusion The developed RT-PCR method laid a foundation of determination, epidemiological investigation and establishment of animal model of CSBV infection.

    2010 04 v.23 [Abstract][OnlineView][Download 157K]

  • Production of Recombinant Human Tissue Type Plasminogen Activator by Culture of Recombinant CHO Cells in 30 L Bioreactor

    MEI Jian-guo, SHEN Zhi-qiang, ZHUANG Jin-qiu(Shandong Binzhou Animal Science & Veterinary Medicine Institute, Binzhou 256600, Shandong Province, China)

    Objective To produce recombinant human tissue type plasminogen activator(rht-PA)by culture of recombinant CHO cells in 30 L packed bed bioreactor. Methods The CHO cell strain for expression of rht-PA was resuscitated with IMDM containing 10% fetal bovine serum(FBS), then amplified and inoculated to a 30 L bioreactor under real-time monitoring by Biocommand Plus software. Serum-containing medium was used for culture of CHO cells at first then was changed into serum-free one. Perfusion culture mode was adopted in the whole course of culture, during which the glucose concentration in culture supernatant was determined every day, and expression level and bioactivity of rht-PA every other day. The expressed rht-PA was purified by LysineSepharose 4B and Zn2+-Sepharose 4B affinity chromatography and determined for specific activity, yield and purity. Results The whole culture course lasted for 51 d, consisting of serum-containing culture for 6 d for cell growth and serum-free culture for 45 d for rht-PA expression. The perfusion rate was 46. 7 L / d in average and 60 L / d at the most. About 2 100 L of culture supernatant was harvested. The expression level of rht-PA was 15. 15 mg / L in average and 19. 25 mg / L at the most. The mean bioactivity of rht-PA was about 8 000 IU / ml. The glucose consumed reached a peak value of 15. 97 g / L·d on day 13 after serum-free culture for expression. The specific activity, yield and purity of purified rht-PA were 6 × 105 IU / mg, 63% and more than 99% respectively. Conclusion A 30 L packed bed bioreactor was suitable for long-term continuous culture of recombinant CHO cells and high expression of rht-PA.

    2010 04 v.23 [Abstract][OnlineView][Download 169K]

  • Development of A Bioinformatic Method for Determination of RNA Viral Pathogen of Infectious Diseases

    YU Ting, BAO Hong, LIU Ai-zhong, et al (Department of Chinical Laboratory, The Second Hospital of Jilin University, Changchun 130041, China)

    Objective To develop a bioinformatic method for determination of RNA viral pathogen of infectious diseases using HIV as subject. Methods The sera of patients with AIDS were filtered then treated with DNase to remove endogenous DNA by DNase sequence-independent single primer amplification. Viral RNA was extracted from the sera and reversely transcribed to doublestranded cDNA which was digested with Taq Ⅰ and added with adapter molecule as a primer for non-specific amplification of pathogenic gene by PCR. The PCR products were cloned and identified by restriction analysis, and their sequences were compared with those reported in GenBank by using BLAST software. Results Several DNA fragments were obtained from sera of patients with AIDS by DNase sequence-independent single primer amplification. The inserted gene fragment was observed in constructed recombinant cloning vector after digestion with EcoRⅠ and Hind Ⅲ. All the sequences of PCR products were consistent with those of known pathogenic genes. Conclusion RNA viral pathogen may be detected by DNase treatment and DNase sequence-independent single primer amplification.

    2010 04 v.23 [Abstract][OnlineView][Download 244K]

  • Development of An ELISA Method for Rapid Quantitative Determination of Human Neutrophil Lipocalin

    WANG Hong-mei, JIA Fu-rong, SHENG Yan, et al (Changchun Brother Biotech Co. Ltd, Changchun 130012, China)

    Objective To develop, verify and preliminarily apply an ELISA method for rapid quantitative determination of human neutrophil lipocalin(HNL). Methods A double-antibody sandwich ELISA method for rapid and one-step quantitative determination of HNL was developed using rabbit anti-human HNL polyclonal antibody as coating antibody and HRP-labeled HNL monoclonal antibody as detection antibody, then verified and applied preliminarily. Results The optimal working concentration of coating antibody was 1 μg / ml, while the optimal dilution of detection antibody was 1 ∶ 1 000. The detection result showed a good linear relationship to the standard concentration at a range of 0. 1 ～ 10 μg / L, with a correlation coefficient (r)of more than 0. 98. The sensitivity of the developed ELISA method was 0. 103 μg / L. The intra-coefficients of variation of determination results of HNL standard at concentrations of 7. 2, 3. 6 and 0. 9 μg / L were 5. 6%, 6. 8% and 9. 8%, while the inter-coefficients of variation were 5. 7%, 6. 9% and 10. 5%, respectively. The mean recovery rate of standards at various concentrations was 102. 04%. Inhibition test showed high specificity of the developed method. The analysis of perfection showed no influence of dilution of test samples on detection result. The determination results in 8 consecutive working days showed high stability of the developed method. The HNL contents in 30 serum specimens from healthy adults determined by the developed ELISA method were (90. 73 ± 30. 12)μg / L. Conclusion An ELISA method for rapid quantitative determination of HNL was developed, which was simple, stable and suitable for routine detection in clinic.

    2010 04 v.23 [Abstract][OnlineView][Download 184K]

  • Pharmacokinetics of Polyethylene Glycol-modified Erythropoietin in Cercopithecoids

    ZHENG Jue-cun, SHEN Fu-bing, DENG Nian-hua, et al(Department of Clinical Laboratory Medicine, Chengdu Medical College, Chengdu 610083, China)

    Objective To analyze the pharmacokinetics of polyethylene glycol-modified erythropoietin (PEG-EPO)with a relative molecular mass of 40 000 in cercopithecoids. Methods A double antibody sandwich ELISA method for determination of PEG-EPO was developed and verified. Cercopithocoids were injected i.v. with PEG-EPO at dosages of 1, 3 and 9 mg / kg respectively, and the PEG-EPO concentrations in sera at various time points were determined. The pharmacokinetic parameters in various dosage groups were obtained by DAS software. Results The metabolism of PEG-EPO in cercopithecoids was fitted to two-department open model. The t1 / 2β of PEG-EPO at dosages of 1, 3 and 9 mg / kg were (51. 893 00 ± 5. 395 58), (42. 485 50 ± 3. 016 06)and (56. 461 30 ± 6. 819 32)h, while the AUC0～144 were(109. 32 ± 9. 82),(236. 23 ± 36. 16)and(722. 55 ± 84. 31)mg / L·h, respectively. Conclusion The modification with PEG at a relative molecular mass of 40 000 prolonged the half-life of EPO in cercopithecoids.

    2010 04 v.23 [Abstract][OnlineView][Download 154K]

  • Development of Method and Standard for Quality Control of Freeze-dried Functional Fragment GP38 of Pseudomonas Exotoxin A

    SHI Xin-chang, HAN Chun-mei, LI Xiang, et al(National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To develop a method and a standard for quality control of freeze-dried functional fragment GP38 of Pseudomonas exotoxin A(PEA), an analog of human gonad hormone releasing hormone(GHRH). Methods HeLa cells / crystal violet colorimetric method was used for determination of biological activity of GP38, and verified for precision and recovery rate. The purity of GP38 was determined by RP-HPLC, and the peptide maps of test sample and reference were compared by the CPM software developed by the authors. Other control tests were carried out according to the requirements in Chinese Pharmacopoeia (Volume Ⅲ, 2005). Results The intra-and inter-coefficients of variations (CVs)of determination results of bulk of GP38 by HeLa cells / crystal violet colorimetric method were 10% and 20%, while those of final product were 12% and 21%, respectively. The biological activities of bulk and final product of GP38 determined by the developed method were (3 736 ± 483)and (1 777 ± 260)U / ml respectively. The RP-HPLC purity of bulk was(97. 1 ± 0. 2)%, which met the quality standard(more than 95%). The similarity of peptide maps of test sample and reference repeatedly tested under the same digestion conditions was more than 0.99, while that digested for different times and repeatedly tested under the same conditions was more than 0. 90. All the other quality indexes met the requirements in Chinese Pharmacopoeia (Volume Ⅲ, 2005). Conclusion The developed method and standard may be used for the routine quality control of GP38.

    2010 04 v.23 [Abstract][OnlineView][Download 199K]

  • Influence of Cyclical Administration with Low-dose Cyclophosphamide on Regulatory T Cells of Melanoma-bearing Mice and Its Antitumor Effect

    LI Wei-bo, YIN Yu, CAI Jian-hui, et al (Second Hospital of Hebei Medical University, Shijiazhuang 050051, China)

    Objective To observe the influence of cyclical administration with low-dose cyclophosphamide(CTX)on regulatory T cells(Treg)of melanoma-bearing mice and its antitumor effect. Methods Melanoma-bearing mouse model was established by inoculating s.c. mice with melanoma B16 cells. The model mice were injected i.p. with CTX once or every 7 d for 3 times, and determined for CD4+CD25+Foxp3+ Treg content in spleens by flow cytometry. The onsets of autoimmune diseases of model mice in various groups were observed. Mouse bone marrow-derived dendritic cells (DCs)were cultured, then co-cultured with the splenic T lymphocytes of model mice and determined for the secretion level of IFNγ by ELISA. The size of tumor was measured, based on which a tumor growth curve was plotted. Results The optimal dosage of CTX was 100 mg / kg. The CD4+CD25+Foxp3+ / CD4+ ratio in spleens of model mice showed an increasing tendency with the increasing time for melanoma-bearing. The inhibitory effect of a single administration with CTX on Treg lasted for a short time. However, the cyclical administration with CTX prolonged the inhibitory effect, maintaining Treg at a relatively low level. Meanwhile, the cyclical administration with CTX increased the secretion level of IFNγ in splenic T lymphocytes of melanoma-bearing mice. Neither a single administration nor cyclical administration with CTX delayed the growth of tumor. No immune white spots or obvious chemotherapy-associated adverse effects were observed in mice treated with CTX by a single administration and cyclical administration. Conclusion The cyclical administration with CTX was more effective for regulating Treg, thus promoted the antigen specific activation of T lymphocytes by DCs and enhanced the anti-tumor effect of DC vaccine.

    2010 04 v.23 [Abstract][OnlineView][Download 254K]

  • Inhibitory Effect of Amlodipine on Murine Hepatoma H22 Cells and Its Mechanism

    LI Wei-ping, HUANG Wen-jing, SUN Yang, et al(Key Laboratory of Biochemistry and Molecular Pharmacology of Chongqing, Department of Pharmacology, Pharmaceutical College, Chongqing Medical University, Chongqing 400016, China)

    Objective To investigate the inhibitory effect of amlodipine on murine hepatoma H22cells and its potential mechanism. Methods The effect of amlodipine on the proliferation of H22 cells was determined by MTT method. H22 cells-bearing mouse model was established, based on which the inhibiting rate of amlodipine to tumor was observed. The pathological change of tumor tissue was observed by HE staining. The expression levels of bcl-2 and bax proteins in tumor tissue were determined by immunohistochemical method. Results The treatment with amlodipine for 48 h inhibited the proliferation of H22 cells significantly, and the inhibitory effect was dose-dependent. The median inhibiting concentration of amlodipine was 5. 6 × 10-3 mg / ml. In vivo test showed that intragastric administration with amlodipine at both dosages of 3 and 10 mg / kg·d for 10 d significantly inhibited the growths of tumors in H22 cells-bearing mice. HE staining showed that the nuclei of tumor cells of mice treated with amlodipine were stained lightly and arranged tightly. Immunohistochemical assay proved that the expression of bcl-2 protein in tumor tissues of mice treated with amlodipine was down-regulated, while that of bax protein was up-regulated. Conclusion Amlodipine showed significantly inhibitory effect on hepatoma, of which the mechanism might be associated with induction of tumor cell apoptosis.

    2010 04 v.23 [Abstract][OnlineView][Download 570K]

  • Extraction of Membrane Protein of Brucella melitensis and Development of Indirect ELISA for Serum Antibody

    YANG Yan-ling, TANG Jie, BAI Guang-yan, et al (College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To extract the 16 m membrane protein of standard virulent strain of Brucella melitensis, analyze its reactogenicity and develop an indirect ELISA for serum antibody. Methods The 16 m membrane protein of standard virulent strain of Brucella melitensis was extracted with sodium carbonate and determined for relative molecular mass by 12% SDS-PAGE. The immune reaction bands in membrane protein component were preliminarily determined by Western blot with sera of goats infected naturally with B. melitensis. The dilutions of antigen and antibody were optimized by block titration, based on which an indirect ELISA method was developed and verified for specificity and precision, then used for determination of serum antibody titer of immunized guinea pigs. The goat serum samples collected in clinic were determined by the developed ELISA, and the results were compared with those by Rhose bengal sodium salt plate agglutination test. Results The concentration of extracted 16 m membrane protein of B. melitensis was 9. 4 μg / μl. The relative molecular mass of membrane protein ranged from 17 000 to 80 000. Western blot showed several specific reaction bands. The optimal dilutions of membrane protein antigen and antibody were 1 ∶ 1 280 and 1 ∶ 10 respectively. The developed ELISA showed good specificity and precision. The serum antibody titer of guinea pigs determined by the developed ELISA was 1 ∶ 64 000. The antibody positive rate of 510 goat serum samples collected in clinic determined by the developed ELISA was 86. 3%, which was significantly higher than that by Rhose bengal sodium salt plate agglutination test (41. 2%). Conclusion The membrane protein of B. melitensis was successfully extracted, which showed good reactogenicity, and an indirect ELISA for serum antibody was developed.

    2010 04 v.23 [Abstract][OnlineView][Download 206K]

  • Prokaryotic Expression of Porcine Interferon γ and Preparation of Antisera

    TIAN Yuan, ZHU Ling-wei, ZHANG Guo-li, et al (College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To express porcine interferon γ(poIFNγ)in prokaryotic cells and prepare the antisera. Methods The gene fragment encoding mature polypeptide of poIFNγ was amplified by PCR and cloned into prokaryotic expression vector pET28a(+). The constructed recombinant plasmid pET-28a-poIFNγ was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed protein was purified and inoculated to mice, and the antisera were prepared and determined for titer by ELISA. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-poIFNγ was constructed correctly. The expressed product, mainly existing in a form of inclusion body, contained 40% of total somatic protein and reached a purity of 90% after purification. The titer of prepared antisera was 10-5. Conclusion Porcine interferon γ was successfully expressed in E. coli, and high titer antisera was prepared.

    2010 04 v.23 [Abstract][OnlineView][Download 191K]

  • Clinical Trial of Effect and Adverse Reaction of Domestic Therapeutic BCG for Prevention of Recurrence of Superficial Bladder Cancer

    HUANG Jian, WANG Guo-zhi, XU Chun-lan, et al (National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To observe the effect and adverse reaction of domestic therapeutic BCG, i.e. Chinese therapeutic BCG (CT-BCG)for prevention of recurrence of superficial bladder cancer. Methods A total of 117 patients with superficial bladder cancer after surgery were treated with 120 mg of CT-BCG, using 53 patients treated with 40 mg of milomycin-c (MI-C)as control. The CT-BCG and MI-C were infused into bladder through a vessel and excreted by the patients themselves 2 h later. The drugs were administered 2 or 3 weeks after surgery, at various intervals for one year. The recurrences and adverse reactions of patients were observed. Results The recurrence rates of patients treated with CT-BCG and MI-C were 18. 8% and 13. 2% respectively, which showed no significant difference. Most of adverse reactions were symptoms of cystitis, such as dysuria, frequent micturition and hemuresis, as well as mild fever. No mild fever were observed in the patients treated with MI-C, while the symptoms of cystitis were mild as compared with those treated with CT-BCG. Conclusion CT-BCG showed satisfactory preventive effect on recurrence of superficial bladder cancer after surgery, and caused only mild adverse reactions.

    2010 04 v.23 [Abstract][OnlineView][Download 123K]

  • Relationship of Angiopioetin-like Protein 3 Content in Human Sera to Hyperlipemia and Hyperglycemia

    XIAO Ya-xiong , WANG Ji-hong, KONG Yu-han (Biochemistry and Molecular Biology Lab of Experiment Teaching Center, Chongqing Medical University, Chongqing 400016, China)

    Objective To analyze the relationship of ANGPTL3 content in sera of patients with hyperlipemia(HL), hyperglycemia(HG)and HG complicated with HL(HG-HL)to glycometabolism and lipid metabolism disturbances in humans. Methods The subjects were divided into healthy control (C), HL, HG and HG-HL groups according to the results of physical examination, and determined for serum ANGPTL3 content by double antibody sandwich ELISA. Results The serum ANGPTL3 contents of subjects in HL, HG and HG-HL groups were significantly higher than those in C group. However, only the serum ANGPTL3 content in HG group was significantly higher in female subjects than in male subjects, while those in other groups showed no significant difference. Conclusion The ANGPTL3 levels in sera of patients with HL, HG and HG-HL were significantly higher than those in healthy persons, indicating that ANGPTL3 might play an important role in the onset of glycometabolism and lipid metabolism disturbances in humans.

    2010 04 v.23 [Abstract][OnlineView][Download 131K]

  • Progress in Research on Methods for Determination of Active Antigen Content of Rabies Vaccine

    LIU Jin-hua, DONG Guan-mu (National Institute for the Control of Pharmaceutical and Biological Products, Bei-jing 100050, China)

    Since antigen content is one of the major quality indexes of rabies vaccine, the development of a rapid, accurate and effective method for determination of antigen content is of importance significance. In this paper, the progress in research on methods for determination of active antigen content of rabies vaccine is reviewed, and various methods are evaluated comprehensively.

    2010 04 v.23 [Abstract][OnlineView][Download 133K]

  • Progress in Research on Role of Exosomes in Tumor Immunity

    ZHU Lei, LI Xiang, LIU Peng-fei, et al(Bioengineering Experimental Center, College of Pharmacology, Jilin University, Changchun 130021, China)

    Exosomes are small membrane vesicles formed by multivesicular body of many cells. Their function was considered as only degrading endocytic substances at first. However, the study during the past several years has proved that the specific function of exosomes are closely associated with the cells from which they are derived. Exosomes carry several unique proteins, such as adhesion protein and heat-shock protein, and play important roles in signal transduction. Since exosomes can deliver antigen to T cells, they are also important to immune system. As a novel tool for immune therapy, exosomes may be used for the treatment of tumors and immune tolerance. The mechanisms of exosomes in induction of anti-tumor immune response and immune suppression are reviewed in this paper.

    2010 04 v.23 [Abstract][OnlineView][Download 140K]

  • Advance in Dendritic Cell Vaccine and Cellular Immune Therapy of Cervical Cancer

    JIAO Qing-fang, SONG Fang-zhou, YI Fa-ping (Department of Immunology, Chongqing Medical University, Chongqing 400016, China)

    Cervical cancer is one of the most common tumors in gynecology, which ranks second on the list of morbidity of malignant tumors in women. Since dendritic cells are the initiator of immune response, DC-based immune therapy of tumor has become a focus of study. This paper reviews the anti-tumor mechanism of DCs as well as the application of DCs in immune therapy of cervical cancer.

    2010 04 v.23 [Abstract][OnlineView][Download 144K]
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