2008年08期目次

  • Mock Screening and Activity of Human Interleukin-18 Mutant

    YANG Jing-sheng, YU Gang, HU Yong, et al (Wuhan Institute of Biological Products, Wuhan 430030, China)

    Objective To screen appropriate human interleukin-18(hIL-18) mutant and determine its activity. Methods Model the molecular structures of human IL-18 receptor(IL-18R) and IL-18 / IL-18R complex by using protein homology modeling software. Mock construct the three-dimensional structure of IL-18 mutant in which the cysteine at 4 sites were substituted with the other 19 kinds of amino acids by modeler 8.2 software using wild IL-18 as template. Calculate the intermolecular energy of the complex to screen the program for mutation. The mock screened mutants were constructed by molecular biological technique, then expressed, purified and determined for activity. Results A total of 12 mutants were screened according to the theoretical prediction. Both restriction analysis and sequencing proved that the recombinant plasmids containing the mutant genes were constructed correctly. The mutant proteins in forms of inclusion bodies contained about 30% of total somatic protein and reached a purity of more than 90% after purification. The activities of several purified mutants such as C38Glu and C127Ser, at low concentrations, increased slightly, which were basically consistent with those predicted theoretically. Conclusion Several human IL-18 mutants were successfully constructed, which proved that pre-screening by bioinformatic method was helpful to promoting the study on IL-18 mutation.

    2008 08 [Abstract][OnlineView][Download 779K]

  • Establishment of A Large Capacity Human Naive Phage Antibody Library

    WU Yong-qiang, DONG Guan-mu, QIN Si-yuan, et al (Wuhan Institute of Biological Products, Wuhan 430060, China)

    Objective To establish a large capacity human naive phage antibody library. Methods The mRNA was extracted from human peripheral blood lymphocytes and transcribed to the first chain of cDNA, which was further used as a template for amplification of VH and VL gene fragments by PCR. Insert the PCR product of VL gene into the pDF vector containing Loxp and Loxp511 sequences, and transform the constructed recombinant plasmid to E. coli XL1-Blue to establish light chain library. Insert the PCR product of VH gene into the vector for light chain library, and transform the constructed recombinant plasmid to E.coli XL1-Blue to establish primary phage antibody library. Super-infect E. coli BS1365 with the primary phage antibody library to establish recombinant phage antibody library. Infect E. coli XL1-Blue with the recombinant phage antibody library to establish working phage antibody library. Results All the lengths of amplified VH and VL subclass genes were consistent with the theoretical values. Both the cloning efficacies of VH and VL genes were 100%. The capacity and titer of primary phage antibody library were 8 × 108 and 6 × 1013, and those of working phage antibody library were 8 × 1010 and not less than 1 × 1013 respectively. Conclusion A human naive phage antibody library with a capacity of 1010 was established.

    2008 08 [Abstract][OnlineView][Download 147K]

  • Identification of Newcastle Disease Virus Receptor on Natural Host Cell Membrane

    LIU Wei△, DING Zhuang, XUAN Hua, et al (△Department of Major Pathogen and Disease in Animals, National Key Laboratory of Preventive Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To identify the receptor of paramyxovirus F48E9 strain from chick origin on natural host cell membrane. Methods Extract the membrane protein of chick embryo fibroblast (CEF) by using membrane extraction kit. Determine the binding activity of isolated paramyxovirus F48E9 strain to cell membrane by a modified indirect ELISA. Identify the Newcastle disease virus (NDV) receptor on host cell membrane by virus overlay protein blot assay (VOPBA). Results Several suspected receptor bands with relative molecular masses of 35 000 to 60 000 were observed on the PVDF membrane blotted with CEF, which showed specific binding to NDV. Conclusion The receptor of NDV on natural host cell membrane was preliminarily identified. The properties of the suspected receptors as well as their roles in pathology of NDV were to be further studied.

    2008 08 [Abstract][OnlineView][Download 88K]

  • Stable Expression of Varicella-zoster Virus Glycoprotein E in CHO Cells

    CHEN Zhe-wen, JIN Yu-lan, HUANG Hai-wu, et al (Shanghai Institute of Biological Products, Shanghai 200052, China)

    Objective To stably express the glycoprotein E(gE) of varicella-zoster virus (VZV) in CHO cells. Methods Amplify the full-length gene encoding extracellular domain of gE of VZV by PCR and insert into vector PSGHVO. Co-transfect CHO cells with the constructed recombinant plasmid PSGHVO-gE and DHFR gene as a selection marker and screen the cell strains stably secreting human growth hormone(hGH)-gE fusion protein. Determine the expressed fusion protein in cell culture supernatant by ELISA, identify by Western blot and purify by Ni2+-NTA column chromatography. Results The target gene fragment at a length of 1 500 bp was observed on both the agarose gel electrophoretic profiles of PCR products of amplified gE gene and constructed recombinant plasmid PSGHVO-gE. Both ELISA and Western blot proved the expression of recombinant hGH-gE fusion protein in culture supernatant of CHO cells co-transfected with recombinant plasmid PSGHVO-gE and DHFR gene. The gE reached an expression level of about 20 mg / L and a purity of 90% after purification. Conclusion The gE of VZV was stably expressed in CHO cells, which laid a foundation of preparation of VZV surface antigen.

    2008 08 [Abstract][OnlineView][Download 221K]

  • Construction of Recombinant Fowlpox Virus for Co-expressing P1-2A-IL-18 Gene of Foot-and-mouth Disease Virus FMDV Type O and P1-2A-3C Gene of FMDV Type Asia I

    GU Chang-wei△, JIN Ning-yi, HUO Xiao-wei, et al (△Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun 130062, China)

    Objective To construct the recombinant fowlpox virus (rFPV) for co-expressing the P1-2A-IL-18 gene of foot-and-mouth disease virus (FMDV) type O and P1-2A-3C gene of FMDV type Asia I. Methods Digest recombinant plasmids T-P1-2A-IL-18 and T-P1-2A-3C with restriction endonuclease, and clone the obtained P1-2A-IL-18 and P1-2A-3C genes into expression vector pUTAL. Chick embryo fibroblast(CEF) was co-transfected with the constructed recombinant plasmid pUTAL-P1-2A-3C-P1-2A-IL-18 and FPV, then monoclonal recombinant virus strains were selected by 3 cycles of BrdU pressure screening and identified by RT-PCR and indirect IFA. Results Restriction analysis proved that recombinant plasmid pUTAL-P1-2A-3C-P1-2A-IL-18 was constructed correctly. RT-PCR and indirect IFA showed that P1-2A-3C-P1-2A-IL-18 gene cassette was successfully expressed in CEF transfected with rFPV. Conclusion The rFPV for co-expressing the P1-2A-IL-18 gene of FMDV type O and P1-2A-3C gene of FMDV type Asia I was successfully constructed.

    2008 08 [Abstract][OnlineView][Download 138K]

  • Cloning and Prokaryotic Expression of Genes Encoding Nonapeptide Inhibiting Newcastle Disease Virus Proliferation and Its Mutant

    XIE Jun-qiu, ZHANG Qing-hua, ZHANG Xue-yan, et al (Institute of Medical Biochemistry and Molecular Biology, College of Basic Medicine, Lanzhou University, Lanzhou 730000, China)

    Objective To clone and express the genes encoding nonapeptide and its mutant, both inhibiting the proliferation of Newcastle disease virus (NDV). Methods Design and synthesize the two-copy concatemer of genes encoding nonapeptide and its mutant and clone into vector pUC18. Digest the constructed recombinant plasmids pUC-Nonapeptide2-1 and pUC-Nonapeptide2-2 with restriction endonuclease, and insert the obtained target genes into expression vector pGEX-4T-1. Transform the constructed recombinant plasmids pGEX-Nonapeptide2-1 and pGEX-Nonapeptide2-2 to E.coli BL21 (DE3) for expression under induction of IPTG. Identify the expressed products by SDS-PAGE and Western blot. Results Recombinant plasmids pGEX-Nonapeptide2-1 and pGEX-Nonapeptide2-2 were constructed correctly as proved by restriction analysis, PCR and sequencing. The expressed Nonapeptide2 protein and its mutant contained 41% and 37% of total somatic protein respectively. Each of the expressed protein showed a single band with relative molecular mass of about 29 000 on SDS-PAGE profile, which was consistent with the theoretical value, and showed good reactogenicity as proved by Western blot. Conclusion The genes encoding nonapeptide inhibiting NDV proliferation and its mutant were successfully cloned and expressed.

    2008 08 [Abstract][OnlineView][Download 171K]

  • In Vitro Induction of Colon Cancer-specific CTLs with Prostate-specific Antigen

    WANG Yi△, SU Xiao-yun, SHI Yi, et al (△School of Pharmacy, Jilin University, Changchun 130021, China)

    Objective To determine the expression level of prostate-specific antigen (PSA) in colon cancer and explore the possibility of PSA as a new target for specific immunotherapy of colon cancer. Methods Determine the expression level of PSA mRNA in colon cancer cell line by RT-PCR, and that of PSA protein by immunohistochemical method. Induce the PBMCs of patients with colon cancer by PAP epitope peptide and determine the secretion level of PSA specific IFN-γ by ELISA. Determine the cytotoxicity of PSA peptide-specific CTLs against colon cancer cells by 51Cr release method. Results The expressions of PSA mRNA and PSA protein were observed in 4 kinds of colon cancer cells, i.e. colo201, colo205, SW480 and SW620. The induced CTLs showed specific killing effect on HLA-A24+ / PSA+ colon cancer cells. The cytotoxicity of CTLs was CD8+ T lymphocyte-dependent. Conclusion The results indicated the presence of PSA-specific CTLs precursors in PBMCs of patients with colon cancer. PSA might be used as a target for specific immunotherapy of colon cancer.

    2008 08 [Abstract][OnlineView][Download 165K]

  • Activities of Matrix Metalloproteinase-2 and -9 in Odontoblast of Healthy and Carious Teeth

    WANG Xiao-chun△, LIANG Jing-ping, GUO Bing-shi, et al (△Department of Oral Medicine, The Forth Hospital of Harbin Medical University, Harbin 150001, China)

    Objective To determine the activities of matrix metalloproteinase(MMP)-2 and -9 in odontoblast of healthy and carious teeth and explore the roles of MMP-2 and MMP-9 in pathogenic mechanism of dental caries. Methods Culture the odontoblast of healthy and carious teeth, and determine the activities of MMP-2 and MMP-9 in culture supernatant by zymogram. Results Both MMP-2 and MMP-9 activities in odontoblast of carious teeth were significantly higher than those of healthy teeth. Conclusion MMPs might play important roles in the progression of dental caries.

    2008 08 [Abstract][OnlineView][Download 142K]

  • Effect of RhoC Gene Silencing on Apoptosis and Migration of Hepatocellular Carcinoma Bel7402 Cells

    XIE Shu-li, SU Xue-jin, SUN Mei, et al (Department of Biopathology, Bethune Medical College of Jilin University, Key Laboratory of Education Ministry, Changchun 130021, China)

    Objective To study the effect of RhoC gene silencing on apoptosis and migration of hepatocellular carcinoma Bel7402 cells. Methods Silence the expression of RhoC gene in Bel7402 cells by RNAi. Determine the levels of apoptosis gene and apoptosis-related gene by FACS and RT-PCR, and the migration and growth of cells by scarification healing test and soft agar clone formation test. Results The apoptosis rate of cells transfected with recombinant plasmid containing RNAi was significantly higher than that with control plasmid, and the expression level of Bcl-2 gene in the former was significantly lower than that in the latter. The expression of Bax gene was observed in the cells transfected with recombinant plasmid containing RNAi. The scratch of cells transfected with control plasmid was healed within 48 h, but was not in those with recombinant plasmid containing RNAi. The formation rate of soft agar clones of cells transfected with recombinant plasmid containing RNAi was significantly lower than that with control plasmid. Conclusion RhoC gene silencing promotes the apoptosis and inhibits the migration and anchor independent growth of hepatocellular carcinoma Bel7402 cells.

    2008 08 [Abstract][OnlineView][Download 101K]

  • Construction of Eukaryotic Expression Vector for Pathogenic Gene MYOC of Primary Open Angle Glaucoma and Its Expression in COS-7 Cells

    WANG Jian-wen, LIU Jian-ju, ZHOU Wen-yan, et al (Department of Ophthalmology, Affiliated First Hospital of Harbin Medical University, Harbin 150001, China)

    Objective To construct the eukaryotic expression vector for pathogenic gene MYOC of primary open angle glaucoma(POAG) and express in COS-7 cells. Methods MYOC cDNA was amplified from human eye tissue(cornea limbus) by RT-PCR, purified and cloned into vector pGEM-T, then subcloned to eukaryotic expression vector pEGFP-N3. Transfect COS-7 cells with the constructed recombinant plasmid pEGFP-N3-MYOC, observe the expression of MYOC protein under fluorescent microscope and identify the expressed product by Western blot. Results Both restriction analysis and DNA sequencing proved that recombinant plasmid pEGFP-N3-MYOC was constructed correctly. Fluorescent microscopy showed that MYOC protein was expressed and located in cytoplasm, while green fluorescent protein(GFP) was distributed in the whole cells. Western blot revealed that expressed MYOC protein could be secreted to the extracellular region. Conclusion The eukaryotic expression vector for MYOC gene was successfully constructed and expressed in COS-7 cells, which laid a foundation of further study on pathogenic mechanism of POAG.

    2008 08 [Abstract][OnlineView][Download 152K]

  • Prokaryotic Expression and Identification of Murine Lipocalin 2

    WANG Shuang, ZHU Dao-yin, TIE Ru-xiu, et al (Department of Immunology, Chongqing Medical University, Chongqing 400016, China)

    Objective To express murine lipocalin 2 (lcn2) and study its biological activity. Methods Extract total RNA from murine macrophage RAW264.7 strain for amplification of lcn2 gene by RT-PCR. Insert the amplified lcn2 gene into prokaryotic expression vector pET-32a (+), and transform the constructed recombinant plasmid pET-32a (+)-lcn2 to E. coli BL21 (DE3) plysS for expression under induction of IPTG. The expressed product was identified by His-tag in-gel staining, purified by Ni2+-NTA affinity chromatography and determined for biological activity. Results Both nucleotide sequencing and restriction analysis proved that recombinant plasmid pET-32a(+)-lcn2 was constructed correctly. SDS-PAGE showed that expressed recombinant lcn2 protein, with a relative molecular mass of about 21 000, mainly existed in a form of inclusion body and contained 35% of total somatic protein. The expressed product reached a protein concentration of 1. 0 g / L after purification and showed a certain inhibitory effect on both E.coli and β-streptococcus. Conclusion Recombinant lcn2 protein was successfully expressed in prokaryotic cells and showed a certain bacleriostatic effect.

    2008 08 [Abstract][OnlineView][Download 287K]

  • Effect of Maternal Ethanol Heavy Consumption During Lactation on Sensitivity to Insulin of Rat Offspring in Adulthood

    DU Hong-wei, ZHANG Ming, GAO Hang, et al (Department of Pediatrics, First Hospital of Jilin University, Changchun 130021, China)

    Objective To observe the effect of maternal ethanol heavy consumption during lactation on the sensitivity to insulin of rat offspring in adulthood. Methods Female Wistar were given ethanol 4g / kg·d by gavage throughout the lactation period, using distilled water as control. The male offspring after birth were measured for bodyweight once a week until the age of 16 weeks, during which the diet taken after weaning was observed from the 4th week. Perform intravenous glucose tolerance test on the offspring at age of 16 weeks. Determine the skeletal muscle cell membrane glucose transporter 4(GLUT4) by Western blot, and the triglyceride (TG) contents in skeletal muscle and plasma by enzymological method. Results The maternal ethanol heavy consumption during lactation showed no significant effect on growth of offspring. Compared with those of control, the glucose tolerance level, insulin sensitivity index(S1), disposition index(DI) and glucose tolerance index (KG) of 16-week-old offspring of rats with ethanol heavy consumption during lactation decreased significantly, while the area under blood glucose curve increased significantly. However, the glucose effectiveness (SG), acute insulin response level to glucose, skeletal muscle cell membrane glucose transporter 4 (GLUT4) as well as triglyceride (TG) contents in skeletal muscle and plasma showed no significant difference from those of control. Conclusion Maternal ethanol heavy consumption during lactation induced insulin resistance in rat offspring, which was unassociated with GLTU4 and TG level in muscle tissue.

    2008 08 [Abstract][OnlineView][Download 157K]

  • Expression of Nontoxic Diphtheria Toxin Mutant CRM197 as A Carrier Protein

    WANG Chun-e, YE Qiang, LI Feng-xiang (National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To express the nontoxic mutant CRM197 of diphtheria toxin and study its effect as a carrier protein. Methods Express CRM197 in E. coli BL21(DE3) by gene engineering technique and purify the expressed product by nickel ion affinity chromatography. Link the purified CRM197 as a carrier protein to activated group A meningococcal polysaccharide (GAMP), using EDAC as linker. Immunize BALB / c mice with the prepared GAMP- CRM197 conjugate and determine the specific IgG against GAMP in sera by indirect ELISA. Results Recombinant CRM197 was expressed in E. coli mainly in a form of inclusion body. The expressed product contained about 25% of total somatic protein and showed good reactogenicity as proved by Western blot. The IgG level against GAMP induced by GAMP- CRM197 conjugate was significantly higher than those by GAMP and by the mixture of GAMP and rCRM197. Repeated immunization with the conjugate induced immunopotentiation, indicating the effect of CRM197 as a carrier protein. Conclusion Recombinant CRM197 was successfully expressed in E. coli, and the GAMP-rCRM197 conjugate prepared with the purified expressed product showed good immunogenicity. It laid an experimental foundation of preparation of other conjugate vaccine using CRM197 as a carrier protein.

    2008 08 [Abstract][OnlineView][Download 298K]

  • Cost-Benefit Analysis of Influenza Vaccination in Rizhao City, Shangdong Province, China

    XING Liang-hong, GE Chang-min, XING Liang-ju, et al (Sanitation and Anti-epidemic Station of Donggang District, Rizhao City, Rizhao 276800, Shandong Province, China)

    Objective To perform a cost-benefit analysis on influenza vaccination in Rizhao City, Shandong Province, China, and provide scientific basis for the vaccination. Methods After the epidemic season of influenza in 2007, 3 830 subjects from the kindergartens, colleges, enterprises and rest homes in Rizhao City, Shangdong Province, China were divided into child(at ages of not more than 7 years), adult (at ages of 20 ～ 59 years) and aged (at ages of not less than 60 years) groups and investigated for influenza vaccination rate, the percentages of outpatients and inpatients with all kinds of diseases and with respiratory diseases, as well as vaccination and medical costs, based on which a cost-benefit analysis was performed on influenza vaccination. Results Of the 3 830 subjects, 2 692(70. 29%) were inoculated with influenza vaccine. Compared with those unvaccinated, the percentages of outpatients and inpatients with all kinds of diseases and with respiratory diseases in the subjects vaccinated were significantly low, especially in child and aged groups, and the medical costs decreased at different extents. The total benefit-cost rates(BCRs) of influenza vaccination in decreasing all kinds of diseases and respiratory diseases were 1. 28 and 0. 77 respectively. Conclusion Influenza vaccination decreased the percentages of outpatients and inpatients with all kinds of diseases and with respiratory diseases as well as medical cost, especially in children and aged.

    2008 08 [Abstract][OnlineView][Download 22K]

  • Isolation, Purification and Activity of A Novel Antimicrobial Peptide from Skin of Rana catesbeiana

    ZHAO Rui-li△, HAN Wen-yu, HAN Jun-you, et al (△College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To isolate, purify and determine the activity of a novel antimicrobial peptide from the skin of Rana catesbeiana. Methods The crude extracts of antimicrobial peptide from skin of Rana catesbeiana were obtained by stimulation and lixiviation stimulation of skin respectively and determined for bacteriostatic activity and minimum bacteriostatic concentration, then purified by Sephadex G-50 gel filtration and RP-HPLC. The purified Rana catesbeiana antimicrobial peptide (RCABP) was analyzed for relative molecular mass, amino acid sequence and activity. Results Both the RCABP extracted by lixiviation and stimulation showed strong bacteriostatic activity. The purified RCABP, with a relative molecular mass of about 4 370 and consisted of 22 amino acids at N-terminus, and showed low homology to the antimicrobial peptides from other amphibian origins. It showed anticoagulant and thrombolytic activities, mild hemolytic activity and a certain activity of trypsin inhibitor, but no local hemorrgagic activity or the activity in hydrolysis of trypsin. Conclusion A novel antimicrobial peptide from skin of Rana catesbeiana was successfully isolated and purified.

    2008 08 [Abstract][OnlineView][Download 122K]

  • Synthesis and In Vitro Antitumor Antactivity of Integrin αvβ3 Antagonist-Chlorambucil Conjugate Toxin

    LI Jian, QIU Ai-dong, WANG Yu-long(Yangtze Delta Region Institute of Tsinghua University Zhejiang, Jiaxing 314050, Zhejiang province, China)

    Objective To synthesize integrin αvβ3 antagonist-chlorambucil conjugate toxin and determine its in vitro antitumor activity. Methods Synthesize integrin αvβ3 antagonist chemically and conjugate to chlorambucil through a amido bond. Determine the inhibitory effect of the conjugate toxin on human umbilical vein endothelial cells ECV304 and liver cancer HepG2 cells by MTT method. Results Nuclear magnetic resonance and mass spectrography showed correct structure of the synthetic conjugate toxin. The conjugate toxin reached a purity of more than 90%, and its inhibitory effect on HepG2 cells was weaker than that of chlorambucil. However, it showed highly specific inhibitory effect on ECV304 cells. Conclusion The synthetic integrin αvβ3 antagonist-chlorambucil conjugate toxin inhibited the proliferation of HepG2 and EVC304 cells, which provided a novel route for therapy of tumors.

    2008 08 [Abstract][OnlineView][Download 55K]

  • Preparation and Biological Activity of Recombinant Mini-plasmin

    ZHANG Qing-ni, XU Ji, LI Fei, et al (Shanghai Fuchunzhongnan Biotech Co. Ltd, Shanghai 201203, China)

    Objective To prepare recombinant mini-plasmin and determine its biological activity. Methods Transform recombinant plasmid pET11a-miniPlg to E. coli BL21(DE3) for expression under induction of IPTG. Inclusion body was extracted from the harvested bacteria, then re-naturalized, purified by chromatography, and activated by urokinase. The prepared mini-plasmin was preliminarily identified and determined for in vitro activity by chromophoric substrate method. Perform in vivo thrombolysis test on mini-plasmin in animal models, using recombinant tissue plasminogen activator(rt-PA) as control. Results The expressed mini-plasmin contained 30% ～ 35% of total somatic protein, and its recovery rate after purification, yield, relative molecular mass and specific activity were 15%, 4.93 mg / g wet bacteria, 38 527 and 115 IU / mg respectively. The amino acid sequences at N- and C- terminuses of the prepared mini-plasmin were consistent with the theoretical value. The mini-plasmin showed good thrombolytic effect in animal test but no significant influence on fibrinolysis and blood coagulation systems. Conclusion The prepared mini-plasmin showed good biological activity, and was superior to rt-PA in both thrombolytic effect and safety.

    2008 08 [Abstract][OnlineView][Download 176K]

  • Preparation and Preliminary Application of McAbs against Recombinant Human IL-12

    LI Li, MA Ying-zhe, ZHANG Jia-ying, et al (Experiment Center of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China)

    Objective To prepare the McAbs against recombinant human IL-12 (rhIL-12) and develop a double antibody sandwich ELISA method for determination of IL-12. Methods Immunize BALB / c mice with purified rhIL-12(p70) to prepare McAbs. Determine the serum IL-12 levels of patients with gastric carcinoma before and after surgery by the double antibody sandwich ELISA kit prepared with the McAbs. Results Three hybridoma cell strains stably secreting McAb against rhIL-12 were screened, of which 2 secreted IgG1 and the other one secreted IgG2a, each with a κ chain. The secreted McAbs reached a purity of more than 95% after purification. In the order of relative affinity, the secreted McAbs were EM5, EM6 and EV9. Both Western blot and ELISA showed good specificity of secreted McAbs. The minimum detection limit and linear range of rhIL-12 determined by the prepared double antibody sandwich ELISA kit were 20 and 25 ~ 3200 pg / ml respectively. The serum IL-12 level of patients before surgery determined by the kit were significantly lower than those of normal control, but increased significantly 10 d after surgery. The IL-12 levels of patients treated with immunopotent NE were significantly higher than those untreated. Conclusion The McAbs prepared using rhIL-12 as antigen may be used for the in vitro and in vivo immunological determination of IL-12, which lays a foundation of study, detection and clinical application of IL-12.

    2008 08 [Abstract][OnlineView][Download 167K]

  • Development of A Modified Double Antibody Sandwich ELISA for Determination of Recombinant Human Thrombopoietin Content

    HOU Xu-feng, ZHAO Hui-lin (Institute for Shenyang Sunshine Pharmaceutical Co. LTD, Shenyang 110027, China)

    Objective To develop a modified double antibody sandwich ELISA for determination of recombinant human thrombopoietin (rhTPO) content. Methods Select the optimal working concentrations of McAb for coating, first antibody and the enzyme-labeled second antibody by chessboard titration. Plot the standard curve to obtain the linear range, detection limit and quantification limit for determination of rhTPO. Evaluate the accuracy (recovery rate), precision and specificity of the developed method and compare with those of Lowry method. Results The optimal working concentrations of McAb for coating, first antibody and the enzyme-labeled second antibody were 1.0 μg / ml, 1 ∶ 5 000 and 1 ∶ 10 000 respectively. The linear range, detection limit and quantification limit for determination of rhTPO were 125 ～ 4 000, 75 and 189 pg / ml respectively. The recovery rates of rhTPO at various concentrations ranged from(97. 0 ± 2. 1)% to(105. 0 ± 8. 5)%, the variation coefficients of inter- and intra-assays of 6 batches of samples were (3. 2 ～ 8. 1)% and (5. 7 ～ 10. 4)% respectively. The developed method showed good specificity, and its determination result showed good relationship to that by Lowry method. Conclusion A modified double antibody sandwich ELISA for determination of rhTPO content was developed, which showed good accuracy, precision and specificity and might be used for the quantitative analysis of rhTPO during research and production.

    2008 08 [Abstract][OnlineView][Download 59K]

  • Curative Effect of Recombinant Human Interferon α2b Gel Combined with Aciclovir on Herpes Zoster

    PAN Bao-chang, HOU Xiu-wei, LI Xue-ping (Second Hospital of Jilin University, Changchun 130041, China)

    Objective To observe the curative effect of recombinant human interferon α2b(rhIFNα2b) gel combined with aciclovir on herpes zoster. Methods Divide 122 patients with herpes zoster into rhIFNα2b gel + aciclovir and rhIFNα2b groups randomly. The 62 patients in rhIFNα2b gel + aciclovir group were treated with rhIFNα2b gel by directly smearing onto the suffered part, 4 times a day, combined with 0.2 g of aciclovir tablet by oral route, 5 times a day. The 60 patients in rhIFNα2b gel group were treated with rhIFNα2b gel alone. A course consisted of 10 d. Observe the curative effect of the 2 groups. Results The total effective rate of rhIFNα2b gel + aciclovir group (88.71%) was significantly higher than that of rhIFNα2b gel group (73.33%). Compared with those in rhIFNα2b gel group, the mean time for stopping blistering, drying the blister, relieving pain and recovery of patients in rhIFNα2b gel + aciclovir group were shortened significantly, and the adverse reactions were mild. Conclusion rhIFNα2b gel combined with aciclovir showed significantly curative effect on herpes zoster.

    2008 08 [Abstract][OnlineView][Download 20K]

  • Development of A Method for Identification of Receptor Binding Specificity of Influenza Virus

    SUN Pei-lu△, XIA Xian-zhu, HOU Xiao-qiang, et al (△College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

    Objective To develop a method for rapid identification of receptor binding specificity of influenza virus. Methods The receptors on surfaces of normal chicken red blood cells(RBCs), the chicken RBCs treated with α-2, 3 sialidase and sheep RBCs were tested by flow cytometry with phytoagglutinin labeled by digoxin, i.e. DIG-SNA and DIG-MAA. Determine the receptor binding specificity of influenza virus, as well as influence of hydroformylation of RBCs on receptor binding specificity, by hemagglutination(HA) test with RBCs with two types of receptors. Results Flow cytometry showed both SAα2, 6Gal and SAα2, 3Gal receptors on surface of chicken RBCs, SAα2, 3Gal receptor on surface of sheep RBCs, and SAα2, 6Gal receptor on surface of chicken RBCs treated with α-2, 3 sialidase. Both the determination results of human and avian influenza virus strains proved that the developed method may be used for rapid and accurate identification of receptor binding specificity of influenza virus. The hydroformylation of RBCs showed no significant influence on receptor binding specificity. Conclusion A method for identification of receptor binding specificity of influenza virus was successfully developed.

    2008 08 [Abstract][OnlineView][Download 204K]

  • Development of Direct Competitive ELISA with McAb against Paralytic Shellfish Poisoning GTX2,3

    LUO Hui-wu△, XIANG Jun-jian (△Department of Life Science, Huizhou College, Huizhou 516007, Guangdong Province, China)

    Objective To develop a direct competitive ELISA(dc-ELISA) for paralytic shellfish poisoning(PSP) GTX2,3 with the McAb against GTX2,3. Methods Couple GTX2,3 with horseradish peroxidase(HRP) by peroxidizing reaction. The dc-ELISA for GTX2,3 was developed using rabbit anti-mouse IgG PcAb as coating antibody and GTX2,3 as competitor of GTX2,3-HRP in binding to McAb against GTX2,3. Test the cross reaction of McAb against GTX2,3 with C2 toxin by the developed dc-ELISA. Results Direct ELISA proved that GTX2,3 was successfully coupled with HRP. The sensitivity and detection limit of developed dc-ELISA for GTX2,3 were 1.5 and 1.5 ～ 40 μg / ml respectively. The McAb against GTX2,3 showed no cross reaction with C2 toxin. The developed dc-ELISA showed good specificity. Conclusion The developed dc-ELISA was suitable for rapid determination of PSP.

    2008 08 [Abstract][OnlineView][Download 39K]

  • A Rapid Peptide Extraction Method for Peptidomic Profile Analysis of Body Fluid

    CHEN Yu, HAN Jin-xiang, CUI Ya-zhou(Key Lab for Biotech-Drugs of Ministry of Health, Shandong Medicinal Biotechnology Centre, Key Laboratory for Modern Medicine and Technology of Shandong Province, Jinan 250062, China)

    Objective To explore the significance of a peptide extraction method which was based on ZipTipC18 in clinical diagnosis. Methods Peptides were extracted from normal pancreatic juice, the pancreatic juice of patients with carcinoma of pancreas and normal blood by a peptide extraction method based on ZipTipC18 and magnetic beads for PMF analysis to display the peptidomic profiles. The acquired data were standardized and the peptides of interest were identified by TOF / TOF analysis. Results The peptidomic profiles reflecting the protein composition of samples were obtained by MS analysis. The acquired data were accurate, reliable and of good reproducibility. By means of TOF / TOF analysis, one peptide from serum sample was identified as fibrinogen α chain precursor. The peptidomic prolifle of pancreatic juice of patients with carcinoma of pancreas was significantly different from that of healthy person. Conclusion The peptide extraction method based on ZipTipC18 may be used for the assistant diagnosis of diseases, especially tumors.

    2008 08 [Abstract][OnlineView][Download 812K]

  • Preparation of Oxidative Low Density Lipoprotein and Its Effect on Proliferation of Vascular Smooth Muscle Cells

    QI Wen-qian△, LI Xiang-jun, REN Li-qun (△College of Basic Medicine, Jilin University, Changchun 130021, China)

    Objective To prepare oxidative low density lipoprotein (ox-LDL) and explore its effect on the proliferation of vascular smooth muscle cells (SMCs). Methods Extract LDL by heparin precipitation and prepare ox-LDL by copper ion oxidation. Determine the proliferation level of SMCs by MTT method. Results LDL was oxidized completely, and SDS-PAGE showed significant decrease of relative molecular mass of the obtained ox-LDL. The proliferation level of SMCs stimulated with 200 mg / L ox-LDL increased significantly. However, when the concentration of ox-LDL was not less than 600 mg / L, the proliferation level of SMCs decreased significantly. Conclusion ox-LDL was successfully prepared. The prepared ox-LDL at a certain concentration stimulated the proliferation of vascular SMCs significantly.

    2008 08 [Abstract][OnlineView][Download 45K]


@2012 Editorial Department of "Chinese Journal of Biologicals"