2016年02期目次

  • Identification of immunogenicity evaluation of recombinant BCG-BE1726D

    ZHANG Hai-feng;FANG Xi-jing;BI Lan;Wuhan Institute of Biological Products Co., Ltd.;

    Objective To identify the expression of specific recombinant BCG-BE1726 D protein(rBCG-BE1726D) and evaluate its immunogenicity in mice. Methods Freeze-dried BCG-SSI and rBCG-BE1726 D strains were subjected to expand cultivation of which the supernatant was collected by ultrafiltration concentration, while bacteria by ultrasoniction and centrifugation, and determined for specific proteins by Western blot. BALB / c mice were immunized s.c. with rBCGBE1726 D and BCG strains, of which splenic lymphocytes were isolated 6 weeks later, then determined for the secretion level of interferon γ(IFNγ) by ELISPOT and ELISA, and for percentages of CD4~+ amd CD8~+ T lymphocytes by flow cytometry. Results Ag85 B, ESAT-6 and RV2626c proteins were detected in rBCG-BE1726 D strain, while only Ag85 B in BCG-SSI. After stimulation with Ag85 B, the number of cell spots in BCG-SSI and rBCG-BE1726 D groups were significantly higher than those in PBST control group(P < 0. 05). However, after stimulation with PPD, the number of cell spots in rBCG-BE1726 D group was significantly higher than those in BCG-SSI and PBST control groups(P < 0. 000 1).No significant differences were observed in the numbers of cell spots produced by stimulation with ESAT-6, Rv2626 c,Rv1733c and RpfD in various groups(P > 0. 05). After stimulation with Ag85 B, the secretion of IFNγ in BCG-SSI and rBCG-BE1726 D groups were significantly higher than that in PBST control group(P < 0. 05). However, after stimulation with PPD, the secretion level of IFNγ in rBCG-BE1726 D group showed no significance with that in BCG-SSI group(P >0. 05). The secretion levels of IFNγ after stimulation with ESAT-6, Rv2626c, Rv1733c and RpfD in various groups showed no significant difference(P > 0. 05). The total percentage of CD4~+ and CD8~+ cells as well as ratio of CD4~+ to CD8~+ T lymphocytes in rBCG-BE1726 D group were significantly lower, while the percentage of CD8~+ T lymphocytes was significantly higher, than those in BCG-SSI and PBST control groups(P < 0. 05). Conclusion Recombinant BCGBE1726 D showed high immunogenicity, which enhanced the immune response level of BALB / c mice significantly.However, the immunogenicity of r BCG-BE1726 D showed no obvious advantages as compared with BCG-SSI, so the protection of this vaccine should be further studied.

    2016 02 v.29 [Abstract][OnlineView][Download 356K]

  • Effect of α-mannatide as an adjuvant on immunogenicity of M.RCAg-1,a multiepitope protein vaccine against Plasmodium falciparum

    LI Yue;DENG Wei-wei;YAO Mei-xue;ZHANG Jia-jia;WANG Bing;WANG Heng;Department of Microbiology and Parasitology, Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences;

    Objective To investigate the effect of α-mannatide as an adjuvant on immunogenicity of M. RCAg-1, a multiepitope protein vaccine against Plasmodium falciparum. Methods Mice were randomly divided into four groups, and immunized s.c. with M. RCAg-1, M. RCAg-1 + α-mannatide, α-mannatide and M. RCAg-1 + Freund's adjuvant respectively on days 0, 14 and 28, 0. 1 ml for each. The serum Ig G level, antibody subtype and recognition of single epitopes by serum antibody were determined by ELISA after the third immunization, while the recognition of natural P. falciparum protein with antibody by IFA, and the secretion level of cytokines by ELISPOT. Results After the third immunization, the GMT in M. RCAg-1 + α-mannatide group was significantly higher than that in M. RCAg-1 group(P < 0. 05), while was equivalent to that in M. RCAg-1 + Freund's adjuvant group(P > 0. 05). The high titer Ig Gs induced in M. RCAg-1 + α-mannatide group were mainly IgG1. The antibodies in M. RCAg-1 + α-mannatide group recognized single epitopes and natural P. falciparum significantly. The number of specific lymphocyte clones secreting IL-4 induced by various epitopes and proteins of immunized mice was larger than that secreting interferon γ(P < 0. 05). Conclusion As an adjuvant of M. RCAg-1,α-mannatide induced high humoral and cellular immune responses and showed a certain protective effect against plasmod-ium infection.

    2016 02 v.29 [Abstract][OnlineView][Download 243K]

  • Fusion expression and immunogenicity of Neisseria meningitidis surface proteins fHBP and PorA

    WU Gen-peng;YUAN Ping;WANG Yue-hong;WANG Xiao;BI Hui;HUANG Guo-ying;XU Fan-hong;ZHU Wei;Shanghai Institute of Biological Products Co.,Ltd.;

    Objective To construct the recombinant plasmid for fusion expression of Neisseria meningitidis(Nm)surface proteins fHBP and PorA,and determine the immunogenicity of f HBP antigen and fHBP-PorA fusion antigen. Methods The f HBP and Por A gene sequences were screened and optimized,and linked to the C-terminus of fHBP by the designed linker. The constructed recombinant plasmids pET-24b-fHBP and pET-24b-fHBP-PorA were transformed to competent E. coli BL21(DE3) for expression under induction of IPTG. The expressed protein was purified by nickel ion affinity chromatography and gel filtration chromatography,with which rabbits were immunized. The prepared antisera were evaluated for immunogenicity by indirect ELISA,flow cytometry and serum bactericidal assay(SBA). Results Recombinant plasmids pET-24b-fHBP and p ET-24b-fHBP-PorA were constructed correctly as proved by restriction analysis and sequencing. Recombinant fHBP was expressed in a soluble form,while fHBP-PorA in a form of inclusion body. All the titers of antisera were more than 1. 0 × 10~5. Flow cytometry showed specific binding of antisera with Nm. However,the antisera against f HBP-Por A showed a certain cross reaction with various Nm strains. Both the bactericidal titers of antisera against fHBP and fHBP-PorA to Nm29019 strain were 1 ∶ 128. Conclusion The fHBP showed good antigenicity and immunogenicity,which might be a good candidate for development of vaccines against B group Nm. However,the multiple antigen fusion strategy should be further optimized.

    2016 02 v.29 [Abstract][OnlineView][Download 271K]

  • Effect of overexpression of heat shock protein 70 in BRL cells on expression of caspase 3 induced by hydrogen peroxide

    JI Hong;ZHANG Wei-hong;LIAN Shuai;WANG Hui;MA Li;LI Yue;GUO Jing-ru;GUO wen-jin;LI Shi-ze;YANG Huan-min;College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University;

    Objective To investigate the effect of overexpression of heat shock protein(Hsp) 70 in Buffalo rat liver(BRL) cells on expression of cleaved caspase 3 induced by hydrogen peroxide(H_2O_2).Methods BRL cells were divided into six groups,i.e.blank control,Ad-CMV-Null,Ad-CMV-Hsp70,H_2O_2,Ad-CMV-Null + H_2O_2 and Ad-CMV-Hsp70 +H_2O_2 groups.The cells in Ad-CMV-Null,Ad-CMV-Hsp70,Ad-CMV-Null + H_2O_2 and Ad-CMV-Hsp70 + H_2O_2 groups were added with 1 × 10~7 PFU/ml Ad-CMV-Null or Ad-CMV-Hsp70 and incubated for 48 h,then those in H_2O_2,Ad-CMV-Null +H_2O_2 and Ad-CMV-Hsp70 + H_2O_2 groups were treated with 150 μmol / L H_2O_2 for 2 h,and those in blank control,AdCMV-Null and Ad-CMV-Hsp70 groups were added with media and incubated.The transcription level of Hsp70 mRNA in cells was determined by real-time fluorescent quantitative PCR,while the expression levels of Hsp70 and cleaved caspase3 by Western blot.Results Comparing with those in H_2O_2 group,both the mRNA transcription and protein expression levels of Hsp70 in Ad-CMV-Hsp70 + H_2O_2 group increased significantly(P < 0.01),while the expression level of cleaved caspase 3 decreased significantly(P < 0.01).Conclusion Hsp70 protect BRL cells from apoptosis by inhibiting caspase 3 activation.

    2016 02 v.29 [Abstract][OnlineView][Download 229K]

  • Optimization of culture condition and rejuvantion of UT-7/EPO cells

    SONG Wan-ying;YANG Shu;JIA Jun-ting;MA Yu-yuan;ZHAO Fu-guang;ZHANG Jin-gang;College of Life Science, Jilin Agriculture University;

    Objective To rejuvenate the dying UT-7 / EPO cells after resuscitation and optimize their growth density.Methods UT-7 / EPO cells were rejuvanted by changing the media(modified RPMI1640, DMEM and IMDM), adjusting the serum concentration(10%, 15% and 20%), adding various contents(1, 3 and 5 U / ml) of growth factors and coculture with feeder cells derived from mouse peritoneal cavity. The growth curve of cells recovered to normal status was plotted by CCK-8 method, based on which the culture densities of UT-7 / EPO cells in various vessels(48-, 24-, 12- and6-well plates as well as 25 cm2flask) were optimized by density gradient culture. The cells on the second day after subculture, in logarithmic growth phase, were stored in frozen, then resuscitated, subcultured for three passages and cultured for 9 consecutive days to evaluate the stability. The cells after rejuvantion were identified with STR reagent from human origin and mouse cell STR site respectively. Results The growth status of UT-7 / EPO cells was not improved significantly by changing the medium, serum concentration and growth factor content. Co-culture with feeder cells recovered the UT-7 / EPO cells to a normal growth status. The culture densities of UT-7 / EPO cells in various vessels were optimized. The UT-7 / EPO cells after resuscitation grew well, of which the status and vigor were similar to those before storage in frozen. The test result for STR site from human origin showed a single or two allele peaks on the STR profile of UT-7 / EPO cells after rejuvenation, while no contamination with other cells from human origin was observed.However, the test result for murine STR site showed no contamination with feeder cells. Conclusion UT-7 / EPO cells were effectively rejuvenated after co-culture with feeder cells. However, the optimal growth density maintained the cells in a stable and normal growth status.

    2016 02 v.29 [Abstract][OnlineView][Download 409K]

  • Effect of various cells on plaque reduction neutralization test on Japanese encephalitis virus

    PANG Xue;ZHANG Rong;YAO Yu;SUN Fei-fei;LI He;Department of Bioengineering, College of Life Science and Biopharmacy,Shenyang Pharmaceutical University;

    Objective To determine plaque forming unit(PFU) of Japanese encephalitis virus(JEV) and the titer of inactivated JE vaccine by using various cells and analyze the effect of the cells on determination result of vaccine titer.Methods JEV P3 strain was inoculated onto the monolayer of MDBK, Vero, LLC-MK2 and BHK-21 cells, and determined for PFU by plaque assay. Mice were immunized with inactivated JE vaccine(Vero cells), of which the serum samples were collected, inactivated at 56 ℃ for 30 min, mixed with JEV P3 strain, inoculated onto monolayer of Vero, BHK-21 and LLC-MK2 cells respectively, and derermined for vaccine titer by plaque reduction neutralization test. Results JEV caused cytopathic effect(CPE) of all the four kinds of cells, while formed no plaque on MDBK cells. The virus titers in harvests of Vero, LLC-MK2 and BHK-21 cells were 10. 02, 9. 42 and 9. 21 PFU / ml respectively. In the turn of T values of titers of inactivated JE vaccine, the cells were Vero, LLC-MK2 and BHK-21 cells. The plaque reduction rate and T value showed significant difference in Vero and LLC-MK2 cells(P < 0. 05), while showed no significant difference in LLC-MK2 and BHK-21 cells(P > 0. 05). Conclusion JEV caused CPE in all the cells, while formed no plaque on MDBK cells. The plaque reduction rates and the corresponding T values of various cells for determination of vaccine titer were different, of which the T value was the highest in Vero cells, and the lowest in BHK-21 cells.

    2016 02 v.29 [Abstract][OnlineView][Download 204K]

  • Cloning and sequence analysis of a xylanase gene from Simmental cattle rumen

    FAN Yong-liang;LI Rui-yao;QU Kai-xing;HUANG Bi-zhi;ZHANG Hui-min;YANG Zhang-ping;College of Animal Science and Technology, Yangzhou University;

    Objective To clone a xylanase gene, Rua11 A, from Simmental cattle rumen, and analyze its sequence.Methods Rua11 A gene was directly cloned from the metagenome of rumen microorganism of simmental cattle by nested PCR and unilateral PCR, of which the sequence and encoded amino acid sequence were predicted and analyzed by bioin-formatic software. Results The DNA sequence of encoding region of Rua11 A gene, at a length of 1 278 bp, including a762 bp xylanase encoding region, a 102 bp binding domain coding region and a 411 bp carbohydrate binding module(CBM) coding region, were obtained by splicing, which encoded 425 amino acid residues consisting of a 31-aa signal peptide and a 394-aa mature peptide and showed stable physiochemical structure. There was an aqueous amino acid-rich region at N-terminus, while no transmembrane domain. The predicted structure of Rua11 A was of typical characteristics of glycoside hydrolase(GH) family 11, which belonged to family 4 of CBM. Alignment analysis revealed that the Rua11 A sequence contained catalytic active center and residues that were strictly conserved among the GH family 11 members.Conclusion The Rua11 A gene of Simmental cattle rumen was successfully cloned, of which the relevant sequences were analyzed. It laid a foundation of highly heterologous expression and molecular modification of Rua11 A.

    2016 02 v.29 [Abstract][OnlineView][Download 835K]

  • In vivo analysis of treatment of prostate cancer with prostate surface membrane antigen aptemer-cationic liposome-double siRNA complex

    FU Jie;WANG Xiao-lin;LIU Yu-jie;ZHANG Jing;WANG Fang;XIANG Shen-si;GAO Xin;WANG Qing-qing;SONG Hai-feng;Institute of Radiation Medicine, Academy of Military Medical Sciences;

    Objective To prepared a novel prostate surface membrane antigen(PSMA)aptamer-cationic liposome-double si RNA complex, and evaluate its targeting to prostate cancer cells and the inhibitory effect on cell proliferation. Methods Reverse evaporation technique was used to prepare the cationic liposomes-double siRNAs [targeting Polo-like kinase 1(PLK1)and B7-homolog 3(B7H3)genes]complex, and the ratios of cationic liposomes and protamine to siRNAs were optimized. PSMA aptamer A10 was inserted into cationic liposomes-double si RNAs to prepare A10-liposome-siRNAs complex. Nanoparticle size and Zeta potential were evaluated. Silence efficiency on target gene in LNCap cells was evaluated by Western blot. Flow cytometry competing assay was performed to investigate the affinity of the A10-liposome siRNAs complexes to LNCap cells. CCK8 proliferation assay were used to evaluate the inhibition of tested delivery system to prostate cancer cells. Results The optimal ratios of cationic liposome and protamine to total si RNAs were 300 ∶ 1 and 1. 7 ∶ 1 respectively. The A10-liposome-siRNAs complex showed relatively high affinity to the PSMA on LNCap cells, and highly silenced the PLK1 and B7H3 genes in cells, of which the inhibitory effect on tumor cell proliferation was superior to that of silence of PLK1 or B7H3 gene alone. The delivery system showed synergism in inhibiting the growth of tumor cells. Conclusion The prepared PSMA aptamer-cationic liposome-double siRNA complex targeted to prostate cancer cells and showed a synergism in inhibiting the cells, indicating potential application of the double functional siRNA drug delivery system for gene therapy.

    2016 02 v.29 [Abstract][OnlineView][Download 319K]

  • Screening of neutralizing antibody titers against human cytomegalovirus in human immunoglobulin preparations

    GONG Yan-yan;MA Jie;CHEN Zhen;ZHU Meng-zhao;LI Man;Shandong Taibang Biological Products Co., Ltd.;

    Objective To screen the neutralizing antibody titers against human cytomegalovirus(HCMV)in domestic and imported human intravenous immunoglobulin(IVIG) and human intravenous cytomegalovirus immunoglobulin(CMVIVIG). Methods The neutralizing antibody titers against HCMV in IVIG and CMV-IVIG were determined by microcytopathic effect(micro-CPE), and the results were compared. Results The neutralizing antibody titers against HCMV in imported CMV-IVIG preparations Cytogam and Cytotect were 3 648 and 3 104 U / g IgG, which were 3. 5 and3. 2 times of those in IVIG preparations Privigen(1 048 U / g IgG)and Intratect(980 U / g IgG), respectively. The mean neutralizing antibody titer in domestic CMV-IVIG preparations(7 195 U / g IgG)was 3. 9 times of those in 19 batches of domestic IVIG manufactured by 13 manufacturers(1 836 U / g IgG). The mean titer of neutralizing antibody against HCMV in domestic IVIG preparations were 1. 8 and 1. 9 times of those in Privigen and Intratect, while those in domestic CMV-IVIG preparations were 2. 0 and 2. 3 times of those in Cytogam and Cytotect, respectively. Conclusion The neutralizing antibody titers against HCMV in both domestic and imported CMV-IVIG preparations were more than 3 times higher than those in the corresponding IVIG preparations, indicating the feasibility of application of micro-CPE to the screening of material plasma,which provided an experimental basis for production of preparations for passive immunization against CMV-associated diseases.

    2016 02 v.29 [Abstract][OnlineView][Download 119K]

  • Effect of soluble guanglate cyclase activator cinaciguat on oxidative stress of diabetic rats

    ZHAO Qi;ZHANG Mei;SUN Bing;BAN Bo;LI Ya-nan;LI Yan-ying;School of Medicine, Shandong University;

    Objective To analyze the effect of soluble guanglate cyclase on oxidative stress of diabetic rats. Methods Mouse model of type 2 diabetes mellitus was established by feeding with high lipid and high glucose diet and intrape-ritoneal injection with streptozotocin(STZ). The model rats were randomly divided into as well as low[3. 5 μg /(kg·d)],moderate[7 μg /(kg·d)]and high(14 μg /(kg·d)]dose cinaciguat groups. The rats in various groups were raised for12 weeks, of which venous blood samples were collected 14 d after treatment with cinaciguat, and determined for glucose and lipid contents by full-automatic biochemical analyzer, for superoxide dismutase(SOD) activity and malonaldehyde(MDA)content by colorimetry, and for endothelin(ET-1)content by ELISA. Results Compare with that in model group,the blood glucose levels of rats treated with cinaciguat at low, moderate and high doses showed no significant decrease(each P > 0. 05), while the triglyceride(TG)and cholesterol(TC)contents decreased significantly(each P < 0. 05), the SOD activity increased significantly, and the MDA and ET-1 contents decreased significantly. Conclusion Cinaciguat might regulate the blood lipid content, decrease the oxidative stress level and improved the function of vascular endothelial cells.

    2016 02 v.29 [Abstract][OnlineView][Download 145K]

  • Preparation and effectiveness evaluation on internal quality control for quantitative determination of hepatitis B virus DNA

    WEI Zhen-hong;GUO Rui;HUANG Huan-yuan;JU Jun;The First Clinical Medical College, Lanzhou University;

    Objective To prepare and preliminarily evaluate the internal quality control for quantitative determination of hepatitis B virus(HBV) DNA. Methods Multiple sequence alignment analysis was performed on HBV genotypes, and primers were designed based on the conserved region of HBV S gene for amplification by PCR. The PCR products were inserted into vector pEASY-T1, and the constructed recombinant plasmids were identified by sequencing, then diluted to concentrations of 10~7 and 10~4 copies / ml and used as high(HBV-H) and low(HBV-L) HBV DNA level quality controls respectively. The quality controls were stored at-80 ℃ in frozen and, after the initial target value was confirmed,evaluated for precision and stability, of which the internal quality control chart was plotted. Results The result of DNA sequencing of constructed recombinant plasmid was consistent with that of target fragment. Both the precision and stability of prepared internal quality control met the experimental requirements. Conclusion The prepared internal quality controls may be used for internal quality control of quantitative determination of HBV DNA.

    2016 02 v.29 [Abstract][OnlineView][Download 171K]

  • Epidemiological characteristics of mumps in Jilin Province,China during 2009～2013

    CHENG Tao;TIAN Xin;CAO Feng-rui;LIN Lin;FU Si-mei;WANG Shuang;SHAN Yuan-chun;CHEN Chao;Jilin Provincial Center for Disease Control and Prevention;

    Objective To analyze the incidence and epidemiological characteristics of mumps in Jilin Province, China during 2009 ~ 2013 and provide a basis for the development of measures for prevention and control of the disease.Methods The data on epidemiological characteristics of cases and outbreaks of mumps reported to Chinese disease pre-vention and control information management system in 2009 ~ 2013 were analyzed by descriptive epidemiological method.Results A total of 28 778 cases of mumps were reported in Jilin Province in 2009 ~ 2013, with an annual incidence rate of 20. 98 / 100 000. The ratio of male to female patients was 1. 64 ∶ 1. The maximum incidence rate appeared in April to July and November to the January of next year. Most of the cases occurred in children at ages of 3 ~ 14 years, accounting for 74. 65% of total cases. However, 62% of cases occurred in students. All the nine outbreaks occurred in educational in-stitutions, five in primary schools and four in middle schools. Conclusion The epidemic in Jilin Province were significantly seasonal, so the monitoring on focus groups should be strengthened in epidemic seasons, and morning inspection should be performed in educational institutions when outbreaks occur.

    2016 02 v.29 [Abstract][OnlineView][Download 159K]

  • Epidemiological characteristics of hepatitis B in children at ages of 0～14 years before and after inclusion of hepatitis B vaccine into immunization program in Tongchuan City, Shaanxi Province, China

    HU Gai-xia;YAN Yong-ping;LIU Xin-li;LIN Song-feng;Forth Military Medical University;

    Objective To analyze the epidemic characteristics of hepatitis B(HB) in children at ages of 0 ~ 14 years before and after inclusion of HB vaccine into immunization program in Tongchuan City, Shaanxi Province, China, and provide a basis for further study on prevention and therapy strategies. Methods The epidemic characteristics and trends of HB in children at ages of 0 ~ 14 years in Tongchuan City before and after inclusion of HB vaccine into immunization programs were analyzed by descriptive epidemiology. Results The annual mean incidence reported in all the population in Tongchuan City before inclusion of HB vaccine into immunization program was 41. 73 / 100 000, while that in children at ages of 0 ~ 14 years was 29. 03 / 100 000, accounting for 13. 21% of total cases reported in the population. The in-time and complete immunization rate of HB vaccine in children born before inclusion of HB vaccine into immunization program was only 20. 83%, while that after inclusion was 76. 44%. The incidence of HB in children at ages of 0 ~ 14 years showed an increasing trend before, and a decreasing trend after inclusion of HB vaccine into the immunization program.The incidence of HB in Tongchuan City showed no significant seasonality, which was higher in males than in females. The highest incidence occurred in Wangyi District of the city. Conclusion The implementation of HB immunization program in Tongchuan City controlled the incidence of the disease in children effectively. However, high incidence reported in adults should be paid more attention.

    2016 02 v.29 [Abstract][OnlineView][Download 173K]

  • Analysis of results of electronic bronchoscopy in 393 patients with lung cancer

    WANG Xiao-ping;CHEN Li-jun;WAN Yi-xin;TAO Hong-yan;HUANG Hui-rong;WU Fan-qi;ZHANG Li;NI Jing-man;The Second Hospital of Lanzhou University;

    Objective To ana lyze the results of electronic bronchoscopy in 393 patients with lung cancer and investi-gate the role of electronic bronchoscopy in diagnosis of lung cancer. Methods The data on 393 patients with lung cancer in 2012 ~ 2014 in the Second Hospital of Lanzhou University were analyzed retrospectively,and subjected to medical statistic analysis by using SAS 9. 3 software. Results The lung tumors were distributed more in right lung than in left lung,and more in lobi superior than in lobi inferior. Of the 393 cases of lung cancer, 284 cases were of neoplasm type,47 cases of invasive type,8 cases of outer suppression type,33 cases of inflammation type,and 18 cases of neoplasm with invasive type under bronchoscope,while no obvious abnormalities were observed in 3 cases. The positive rates in biopsy and brushing examination were 94.10% and 60. 71% respectively,which showed significant difference(P <0. 000 1). The positive rates of squamous cell carcinoma and small cell cancer in biopsy were 96. 84% and 97. 67%respectively,which were significantly higher than those of adenocarcinoma(P < 0. 000 1). However,the positive rate of small cell cancer in brushing examination was significantly higher than that of adenocarcinoma(P < 0. 05) while showed no significant difference with that of squamous cell carcinoma(P > 0. 05). The positive rates of squamous cell carcinoma and adenocarcinoma in brushing examination showed no significant difference(P > 0. 05). The positive rate of central type carcinomas in biopsy(95. 72%) was higher(P < 0. 000 1) than,while that in brushing examination showed no significant difference with,that of peripheral type carcinomas(P > 0. 05). Conclusion Electronic bronchoscopy showed high confirmation rate,less trauma and high safety,which was an important method for diagnosis of lung cancer.

    2016 02 v.29 [Abstract][OnlineView][Download 222K]

  • Preparation and effect evaluation of directional and sustainedreleased IgY microcapsules

    CHEN Feng-lian;LIU Wen-juan;XIE Jiang;ZHANG Jian;HUANG Hong-mei;LING Dan;WU Jian-min;Guangxi Key Laboratory of Animal Vaccines and New Technology, Guangxi Veterinary Research Institute;

    Objective To prepare directional and sustained-released IgY microcapsules and evaluate its effect. Methods IgY microcapsules against diarrhea in piglets were prepared by sparay drying technique using gelatin and sodium alginate as the wall materials. An L_9(3_4)orthogonal experiment was performed to optimize the technological condition for preparation of the microcapsules based on the effect of formula of materials on the performance of microcapsules, the influencing factors of stability of encysted emulsion and condition for spray drying, serving the activity, encapsulation efficiency, in vitro release property and stability of IgY as the evaluation indexes. The microcapsules were evaluated for IgY activity,encapsulation efficiency as well as sustained-releases and stabilities in simulated gastric fluid(SGF) and simulated intestinal fluid(SIF). Results The optimal technological condition for preparation of microcapsules was as follows:the concentrations of gelatin and sodium alginate were 4% and 0. 75% respectively. IgY was mixed with gelatin-sodium alginate at a mass ratio of 3 ∶ 4 and emulsified, then dried at an inlet air temperature of 170 ℃ and a feeding rate of10 ml / min for 10 s, then dried by spraying at an outlet temperature of 70 ℃ for 10 s. The encapsulation efficiency of microcapsules prepared under the optimized condition was 77. 4%, while the activity was maintained at more than 85%.The release rate 2 h after digestion in SGF was 8. 0%, while that 4 h after digestion in SIF was 81. 2%. The activity of microcapsules was more than 50% after being heated at 90 ℃ for 30 s, while was more than 90% after storage at 25 ~30 ℃ for 10 months. However, after storage at 4 ℃ for 10 months, the activity decreased by only 5%. Conclusion The IgY microcapsules prepared by this process showed high biological activity and stability as well as sustained releasing properties with targeting effects on specific IgY.

    2016 02 v.29 [Abstract][OnlineView][Download 263K]

  • Preliminary analysis on determination of national standard for protein content by amino acid assay

    BI Hua;SHI Xin-chang;LIU Lan;HAN Chun-mei;DING You-xue;PEI De-ning;RAO Chun-ming;National Institutes for Food and Drug Control;

    Objective To investigate the feasibility of determination of the protein content of national standard for protein content by amino acid assay. Methods The standards were processed by conventional acid hydrolysis method, and analyzed by HITACHI L8800 type amino acid analyzer. The time for hydrolysis was optimized at first, then the standards were hydrolyzed for optimal time and determined for content by analysis of amino acid and calculation of the total sum of amino acid contents. The method was verified for precision and accuracy. Results After seven determinations, the protein content of the current national standard[(2005) National Biological Products Standard 0009]was defined as(95. 02 ±3. 99) mg / 100 mg, with a relative standard deviation(RSD) of 4. 20%. The recovery rates of various amino acids in several tests were 93. 56% ~ 105. 12%. The albumin content calculated in the standards was 20. 52 mg, which was 103. 48% of the measurements by Kjeldahl nitrogen calibration. Conclusion The amino acid assay for determination of protein content was highly automatic and of good repeatability, which might be used for the analysis of recombinant protein content.

    2016 02 v.29 [Abstract][OnlineView][Download 276K]

  • Development and verification of rocket electrophoresis method for determination of polysaccharide content in group ACYW135 meningococcal polysaccharide vaccine

    CAO Fang;WANG Liang-chao;YANG Ling;PAN Ling-ling;YANG Qian-min;LI Qiang;LI Hong-shuang;HU Hong-qiang;HU Wen-long;Zhejiang Weixin Biological Pharmaceutical Co., Ltd.;

    Objective To develop and verify a rocket electrophoresis method for determination of polysaccharide content in group ACYW135 meningococcal polysaccharide vaccine. Methods Specific antisera were prepared by immunizing rabbits with groups A, C, Y and W135 meningococci respectively, and added onto agarose gel plate for rocket electrophoresis using the bulks of polysaccharides of the corresponding groups as antigen references. A standard curve was plotted with the know polysaccharide concentration against the height of rocket peak, based on which the polysaccharide contents of samples were calculated according to the heights of their rocket peaks. The developed method was verified for specificity, linear reproducibility, accuracy and precision, and used for determination of polysaccharide contents in three batches of group ACYW135 meningococcal polysaccharide vaccine. Results The standard curve of the developed method showed good linearity, with R2 values of not less than 0. 95. The detection range of polysaccharide concentration by the method was 3 ~ 7 μg / ml, while the recovery rate was 90% ~ 110%. The RSDs of test results of reproducibility as well as precision by different staff were less than 5%. No cross reactions were observed between the antigens of various groups,indicating high specificity of the method. All the polysaccharide contents in three batches of vaccine met the Requirements for Group ACYW135 Meningococcal Polysaccharide Vaccine as an internal standard. Conclusion The developed rocket electrophoresis method may be used for determination of polysaccharide content in group ACYW135 meningococcal polysaccharide vaccine.

    2016 02 v.29 [Abstract][OnlineView][Download 233K]

  • Development and comparison of three methods for determination of antibody titer against respiratory syncytial virus

    LI Shu-xiang;LIU Jing;WANG Xin-yi;XU Jing;National Vaccine and Serum Institute;

    Objective To develop and compare three methods for determination of antibody titer against respiratory syn-cytial virus(RSV). Methods The concentration of coating antigen and enzyme-labeled the secondary antibody in ELISA were optimized by chessboard titration, and the method was determined for linear range and reproducibility. Meanwhile,micro-neutralization test and plaque reduction neutralization test were developed and determined for reproducibility. Fifty-five healthy human serum samples were determined by the developed three methods and commercial ELISA kit for RSV antibody separately, and the correlations between the test results were compared. Results The optimal concentration of coating antigen and optimal dilution of HRP-labeled goat anti-human Ig G were 10 μg / ml and 1 ∶ 30 000 respectively.The antibody concentration at a range of 38. 00 ~ 4. 375 U / ml showed good linear relationship to A_(450/630) value, with a R~2 value of 0. 981 4. All the coefficients of variation(CVs) of determination results of samples at high, moderate and low concentrations were less than 10%. Obvious CPE appeared 7 d after micro-neutralization test, while the confluent cells fell off. The CV of neutralizing titer of polyclonal antibody against RSV in six consecutive tests was 4. 2%. Obvious plaques were observed in Vero cells 7 d after infection with RSV, of which the borders were clear after crystal violet staining. The CV of antiserum titer against RSV in six consecutive tests was 6. 81%. The correlation coefficient(R) of determined results by the developed ELISA and the commercial ELISA kit was 0. 787, while that by micro-neutralization test and plaque reduction neutralization test was 0. 937. Conclusion Three methods for determination of antibody titer against RSV were developed successfully, and good correlations were observed between the determination results by the developed ELISA method and commercial ELISA kit and between those by micro-neutralization test and plaque reduction neutralization test.

    2016 02 v.29 [Abstract][OnlineView][Download 223K]

  • Development of a gas chromatography method for determination of polysorbate 80 content in monoclonal antibody preparations

    HUANG Yun-zhong;YANG Bin;YE Chi-ming;ZHOU Dong-mei;XU Jun;Suen Wen-cheng;Sunshine Lake Pharma Co., Ltd;

    Objective To develop, optimize and validate a gas chromatography(GC) method for determination of polysorbate 80 content in monoclonal antibody(Mc Ab)preparations. Methods Under the optimized hydrolysis condition,the polysorbate 80 in test samples was hydrolyzed with 1 mol / L potassium hydroxide solution to obtain oleic acid which was subsequently methyl esterified and determined by GC, based on which the polysorbated 80 content was analyzed using internal standard. The developed method was validated for specificity, systemic suitability, linearity, precision, accuracy and stability, and used for determination of polysorbate 80 contents in three batches of Mc Ab against TNF α. Results When polysorbate 80 and alkali was mixed at a volume ratio of 1 ∶ 1 at 40 ℃ for 4 h, the hydrolysis reaction was complete. The method showed good system suitability and specificity for determination of polysorbate 80 content, and good linearity at a polysorbated 80 concentration of 0. 1 ~ 4. 0 mg / ml, with a R~2 value of 0. 999 9. The RSD of polysorbated 80 contents in twelve samples prepared by two staff was 3. 72%. The mean spike recoveries of polysorbate at low, medium and high concentrations were 105. 52%, 104. 67% and 102. 51% respectively, with a RSD of 2. 37%. The treated samples showed high stability within 27 h. The polysorbate 80 contents in three batches of samples were 1. 032, 1. 016 and 1. 022 mg / ml, which were 103. 2%, 101. 6% and 102. 2% of the labeled contents, respectively. Conclusion The developed GC method was simple, accurate, reliable and might be used for the determination of polysorbate 80 content in Mc Ab preparations.

    2016 02 v.29 [Abstract][OnlineView][Download 187K]

  • Progress in research on charge heterogeneity of recombinant monoclonal antibodies for human use

    ZHANG Kun-ming;PAN Yong-bing;ZHANG Ai-hua;Department of Antibody, Wuhan Institute of Biological Products;

    Therapeutic monoclonal antibodies(mAbs) are biological macromolecules with complex post-translational modifications. Because of their heterogeneity, mAbs are not a pure but a mixture of a variety of product-related substances.A comprehensive understanding of these product-related substances and degradation processes occurring in mAbs is particularly important. As almost all of the variants can cause charge-related heterogeneity, charge variable has become both the most sensitive approach to monitor the degradation of mAb and a reference standard for product quality and consistency. In this paper, the progress in research on causes and detection methods of several major charge variants and the impact on the safety and efficacy of product are reviewed.

    2016 02 v.29 [Abstract][OnlineView][Download 205K]

  • Progress in research on acquired immunodeficiency syndrome vaccine

    XU Ji-wei;SONG Xuan;LIANG Wei-zi;YANG Ying;School of Life Sciences, Tianjin University;

    Acquired immunodeficiency syndrome(AIDS)caused by human immunodeficiency virus(HIV)infection has become a worldwide health problem. Exploiting an effective HIV vaccine is an urgent task. Once human is infected by HIV-1, CD4~+ T cells will be attacked and the immune system will lose the defending functions. Meanwhile, the variability of HIV makes it difficult to design the corresponding vaccines. Induction of neutralizing antibodies and cellular immunity stimulation will be a promising way to protect body from HIV invasion. This paper reviews the structural characteristics of HIV, design of AIDS vaccine, cellular and humoral immune responses induced by vaccines, as well as the prospect of vaccine development.

    2016 02 v.29 [Abstract][OnlineView][Download 161K]

  • Progress in research on keratinocytes from patients with condyloma acuminatum

    ZHU Wei;CAO Ping;Kunming University of Science and Technology;

    Keratinocyte(KC)damage may cause a variety of skin diseases, including condyloma acuminatum(CA)with a great harm and an increasing infection rate in recent years. There are a variety of methods for culture of KCs from the parts with skin lesion caused by CA. The isolation of KCs from human skin for in vitro culture has a history of more than 30 years, however, the results varied widely due to different conditions and methods. Therefore, the establishment of a simple, economic and biologically active method for isolation and culture of human KCs is of an important significance in treatment of CA. This paper reviews the progress in research on KCs in the parts with skin lesion caused by CA and pro-vides a reference for study on therapy of the disease.

    2016 02 v.29 [Abstract][OnlineView][Download 126K]

  • Progress in study on application of γ-glutamic acid in biological products

    SONG Xiao-feng;YUAN Zeng-yan;XU Ping-hui;Sanquan School, Xinxiang Medical College;

    Poly γ-glutamic acid(γ-PGA)is a naturally occurring poly amino acids with characteristics of water solubility, biocompatibility, biodegradability and non-toxicity towards human and the environment. This paper reviews the application of γ-PGA as a raw material in prophylactic and therapeutic biological products and novel biomaterial products and as stabilizer, cryoprotecive agent and flocculating agent in biological products. Meanwhile, the prospect of γ-PGA applied to diagnostic biological products is investigated.

    2016 02 v.29 [Abstract][OnlineView][Download 149K]
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@2012 Editorial Department of "Chinese Journal of Biologicals"