摘要
乙型肝炎是由乙型肝炎病毒(HBV)引起的全球性传染病。HBV除了引起急性肝炎外,还可导致慢性肝炎和肝硬化,而且与肝癌的发生有着密切的关系。
曾在国内外广泛用于乙型肝炎预防的血源性乙肝疫苗(PDV)因血源供应有限和潜在危险性,己经逐步被基因工程疫苗所代替。中国仓鼠卵巢癌细胞(Chinese hamster ovary cell,CHO cell)经基因转染后分泌的重组乙肝表面S抗原,和乙肝病人血清来源的抗原同样具有系统的抗原活性,而且在灵长类动物的实验中发现CHO细胞重组乙肝抗原比血清抗原和酵母来源的抗原具有更高的免疫活性,表现为产生更多的抗体和更高的血清滴度。
本课题主要内容是建立CHO-K1细胞基因转染乙肝表面S抗原基因的方法,并筛选出稳定表达的CHO细胞株。首先采用分子生物学的方法制备了转染级别的包含乙肝表面S抗原基因的两种质粒pS-1和pcDNA-s-1。然后,用两种转染方法分别转染COS-1细胞和CHO-K1细胞,并用ELISA的方法对转染结果进行检测。经过实验得知,磷酸钙转染法本身受其它很多因素影响且很大,转染方法不稳定;PEI转染法可以有效的转染细胞,实验稳定结果较好,并且确定了最佳的PEI/DNA的质量比为6.7。本实验还对PEI的毒性进行了测定,发现它对CHO-K1细胞毒性不大,细胞存活最少的也有76.01%存活。最后,确定了G418的最佳筛选浓度为600ug/ml后,用PEI转染法转染CHO-K1细胞,经G418加压筛选后,用酶联和透射电镜检测均得到细胞可稳定表达乙肝表面抗原的结论。在本课题中,成功的构建了稳定表达乙肝表面S抗原的CHO细胞株6H3。
Hepatitis B (HB) is a severe global infectious disease caused by Hepatitis B virus (HBV). Due to shortage of blood sources and potential infection risk, widely used PDV has been replaced by genetically engineered HBsAg vaccines. CHO-derived recombinant HBsAg has the similar properties as PDV from serum of hepatitis B patients. Primate results also showed that CHO-derived recombinant HBsAg possessed higher immune activity, and produced higher titers of HBsAg antibodies.
In this research, CHO-K1 adherent cells were successfully transfected with plasmids expressing Hepatitis B surface antigen (HBsAg) S protein. A stable CHO cell line producing high level of HBsAg S protein was obtained. At the first step, HBsAg S protein expressing plasmids (pS-1 and pcDNA-S-1) were purified and were tested in transient transfection of COS-1 cell and CHO-K1 cell, respectively. HBsAg S protein expression level was monitored by HBsAg ELISA. Results showed that PEI method had good transfection efficacy, but Calcium Phosphate Co-Precipitation method was unstable and had low efficiency. The best PEI/DNA ratio is 6.7. PEI also had very low cytotoxicity to CHO-K1 cell line. Therefore, CHO-K1 cells were permanently transfected with pcDNA-S-1 plasmid by PEI method at PEI/DNA ratio 6.7, and selected with G418 (Geneticin, 600ug/ml based on pilot G418 killing curve). Secreted HBsAg in conditioned medium was detected by a commercial HBsAg ELISA kit. After eight passages in T25 flasks, a stably transfected high-producing CHO-K1 clone, 6H3, was identified. HBsAg S protein in 6H3 clone CM was also detected by Transmission Electron Microscope (TEM).
引文
[1] Beasley R. P. Hepatitis B virus: The major etiology of hepatocellular carcinoma. Cancer, 1988, 61(10): 1942-1956.
[2] Lau J. Y.; Wright T. L. Molecular Virology and pathogenesis of hepatitis B, Lancet, 1993, 342(8883): 1335-1340.
[3] Wang, J.; Dai Z.; Peng W. Loemology, Third Edition. Shanghai: science technology publishing company, l998: 246.
[4]刘水渠,乙肝病毒抗原抗体的若干研究状况,浙江临床医学,2006,8(11): 1121-1122。
[5] Pierre Tiollais; Christine Pourcel; Anne Dejean. The hepatitis B virus, Nature, 1985, 317(10): 489-495.
[6]张晋红,沉仕珠,姚茹等,预防性乙肝疫苗的研究进展,中国医学理论与实践,2006,16(8):1028-1030。
[7]夏国良,乙型肝炎疫苗预防效果和乙型肝炎病毒基因变异的研究,中华流行病学杂志,2002,23(3):225-226。
[8] CDC, Notice to Readers: FDA Approval for a Combined Hepatitis A and B Vaccine, MMWR, 2001, 50(37): 806-807.
[9]张庶民,马霄,侯启明等,吸附百白破乙肝(CHO)四联疫苗接种反应及血清学效果观察,中国生物制品学杂志,2004,17(5):323-326。
[10] Rottinghaus S. T.; Poland G. A.; Jacobson R. M., et a1. Hepatitis B DNA Vaccine induces protective antibody responses in human nonresponders to conventional vaccination, Vaccine, 2003, 21(31): 4604-4608.
[11] Vitiello A; Ishioka G. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection, Induction of a primary cytotoxic T lymphocyte response in humans, J Clin Invest, l995, 95(1): 341.
[12] Shi T. D.; Wu Y.Z.; Bian J., el a1. Therapeutic peptides based on HBcAg 18-27 epitope can induce CTL response in vitro and in vivo, Immunological Journal, 2003, ll9(3): l65-l69.
[13] Kapusta J.; Modelska A.; Pniewski T., el a1. Oral immunization of human with transgenic lettuce expressing hepatitis B surface antigen, Adv Exp Med Biol, 2001, 495: 299-303.
[14]刘丹,于海鹏,盛军等,乙肝病毒表面抗原基因在人参细胞中的表达,植物生理与分子生物学学报,2005,31(5)。
[15] Pol S.; Driss F.; Michel M. L., et a1. Specific vaccine therapy in chronic hepatitis B infection, Lancet, 1994: 344-342.
[16]骆利敏,李明,夏虎等,乙型肝炎病毒重组多表位抗原诱发的小鼠免疫应答,中国免疫学杂志,2005,21(6):427-434。
[17] Coursaget P.; Bringer L,; Sarr G., el a1. Comparative immunogenicity in children of mammalian cell-derived recombinant hepatitis B vaccine and plasma-derived hepatitis B vaccine, vaccine, 1992, 10(6): 379.
[18] Young M. D.; Schneider D. L.; Zuckerman A. J. Adult hepatitis B vaccination using a novel triple antigen recombinant vaccine, Hepatology, 2001, 34: 372-376.
[19]范秀嫒,孔德清,张茂金,重组乙型肝炎疫苗(CHO细胞)及其应用,中国计划免疫,2005,11(3):234-237。
[20] Kane J. F.; Hartley D. Formation of recombinant protein inclusion bodies in Escherichia coli, Tibtech, 1988 (6): 95-101.
[21] Verma R,; Boleti E.; George A. J. Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems, Immunol Methods, 1998, 216(1-2): 165-181.
[22] Vassileva A.; Chugh D. A.; Swaminathan S. Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter, Biotechnol, 2001, 88 (1): 21-35.
[23] Wum F. M. Production of recombinant protein therapeutics in cultivated mammalian cell cells, Nature biotechnol, 2004, 22(11): 1393-1398.
[24] Walsh G. Pharmaceutical biotechnology products approved within the European Union, Europ J Pharm biopharm, 2003, 55: 3-10.
[25] Alt F. W.; Kellems R. E.; Bertino J. R.., et al. Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells, J. Biol. Chem, 252 (1978): 1357-1370.
[26] Urlaub G.; Chasin L. A. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc. Natl. Acad. Sci. USA, 1980, 77(7): 4216-4220.
[27] Conradt H. S.; Nimtz M.; Dittmar K. E.; Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells, Bio Chem, 264(29): 17368-17373.
[28]申烨华,耿信笃,CHO细胞表达系统研究新进展,生物工程进展,2000,20(4):23-25。
[29]侯森,冯喜增,聚合物基因载体的设计与优化,中国临床康复,2006,10(33):125-129。
[30] Rolland A. P. From genes to gene medicines: recent advances in nonviral genedelivery, Crit. Rev. Ther. Drug Carrier Syst, 1998, 15, 143-198.
[31] Matsui H.; Johnson L. G.; Randell S. H. Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells, J. Biol. Chem. 1997, 272: 1117-1126.
[32] Sadler K.; Tam J. P. Peptide dendrimers: Applications and synthesis, J Biotechnol, 2002, 90(3-4): 195-229.
[33] Kircheis R.; Blessing T.; Brunner S., et al. Tumor targeting with surface-shielded ligand-polycation DNA complexes, J Control Release, 2001, 72(1-3): 165-170.
[34] Boussif O.; Lezoualch F.; Zanta M. A., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine, Proc Natl Acad Sci USA, 1995, 92 (16): 7297-7301.
[35] Fischer D.; Bieber L. Y., et al. A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: efect of molecular weight on transfection efficiency and cytotoxicity, Pharm Res, 1999, l6 (8): 1273-1279.
[36] Wightman L.; Kircbeis R.; Rossler V., et a1. Diffetent behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo, J Gene Med, 2001, 3(4): 362-372.
[37] Brunner S.; Furtbauer E.; Sauer, et al. Overcoming the nuclear barrier: cell cycle independent nonviral gene transfer with linear polyethylenimine or electroporation. Mol Ther, 2002, 5(1): 80-86.
[38] Behr J. P. The proton sponge: a trick to enter cells the viruses did not exploit, Chimia, 1997, 5l: 34-36.
[39] Fischer D.; Li Y.; Ahlemeyer B., et al. In vitor cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis, J, Biomaterials, 2003, 24(7): 1121-1131.
