重组乙型肝炎前S2S疫苗的研究
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摘要
乙型肝炎是由乙型肝炎病毒(HBV)感染引起的一种传染性疾病。我国现有乙肝疫苗仅含S蛋白,尚有5 %～10 %的人群无反应或弱反应。研究发现, preS2 (前S2)蛋白有很强的免疫原性,可强化同存的S蛋白;前S2抗体产生早,利于快速预防;前S2有多聚人血清白蛋白受体(PHSA-R),可介导与HBV竞争结合肝细胞,从而阻断HBV的感染,具有免疫原性强、快速预防和治疗等多重作用。本课题完成含前S2(120-146)免疫表位重组质粒pcs-S2S的构建,并转染中国仓鼠卵巢细胞(CHO cells),获得能稳定分泌表达前S2S的重组CHO细胞株;经DNA测序和杂交证明前S2S基因重组到CHO细胞中;表达产物经电泳及免疫印迹证明有前S2蛋白的表达;免疫效力实验证明有S和前S2抗体的产生,并能诱导细胞免疫应答。该研究为研制预防和治疗双重作用的新型乙肝疫苗做了有益的尝试。
Hepatitis B is an infectious disease caused by hepatitis B virus (HBV). HBV is responsible to acute and chronic hepatitis, as well as hepatocirrhosis, liver cancer. Hepatitis B vaccine inoculation is the most effective way to prevent the disease. The hepatitis B vaccine presently used in China comprises major S protein only. Its disadvantages are: (1) 5%-10% of the recipients raise only little or even no response; (2) three inoculations are needed to obtain essential antibody level; (3) it is used only in prevention, but not therapy. So to prevent quickly and enhance protection still are the problem that a novel Hepatitis B vaccine needs solve. Nowadays researchers found that pre-S protein, with high antigenisity, can enhance the immunoresponse of S protein; production of pre-S2 antibody is earlier than S antibody, it plays an important role on early prevention. And pre-S2 can adhere to hepatocytes via poly human serum albumin (PHSA). Therefore vaccine containing pre-S2 can prevent HBV from infecting liver cells by competitive joining. So it may cure hepatitis. More and more date show that a novel hepatitis B vaccine with pre-S can overcome the disadvantage of only S-protein vaccine.
In terms of vaccine designing strategy, the antigen molecule should be large, the formed polymer or particle is more, effect of immunogenicity is better. But in genetic engineering, the product molecule should be small to obtain high expression and to decrease the side effects. So it is necessary to consider the characteristic of vaccine and genetic engineering products on design genetic engineering vaccine.
The topic leads in a new concept of epitope -based vaccine design, reasonably retains the whole S gene, chooses 26 amino acids sequence of pre-S2 (120-146) that contains rich T and B cell epitope, removes 29 amino acids unnecessary sequence of pre-S2 and makes the size of recombinant protein suitable and express easily. The designed vaccine by aboved method not only retains necessary antigen epitope of antigen stimulates, but also reduces influence on protein secretion because of the expressed large molecule protein. The preliminary results showed the vaccine had expressed S2S protein, the expression level and immunity effect surpassed current S vaccine.
In the study, the whole S gene adding the epitope gene of pre-S2 (120-146) was cloned into a eukaryotic expression plasmid pcs and the recombinant plasmid pcs-S2S was constructed by the recombinant DNA technology . the plasmid pcs-S2S was transformed into CHO dhfr–cell successfully, totally screened 2400 cells, obtained recombinant CHO cell A7 strain. The A7 strain was subcultured for 50 passages and change medium for 30 times continuously, It is established that the stability of preS2S protein was good. The results of S and S2 proteins detected in CHO cells A7 strain were strong positive by DNA bot hybridization; expressed gene was detected by PCR, then sequenced, the length of S gene is 678 bp, the length of preS2 gene is 78 bp, the results of SDS-PAGE and sequencing were correct. It was verified that A7 strain cell contained S and S2 gene. Expressed protein preS2S was identified By SDS-PAGE and Western blot.
The genetic engineering products in Europe and America are expressed mainly by CHO cell, about 40%~70%, there are 22 kinds products in America which has been authorized to go on the market, but only two kinds in China. This is major gap between China and Europe and America on Biological drugs manufacture. This topic chose CHO cell expression system, we tried to overcome many technique problems in transfection and screening, groped and solved the problem of clone and amplification of monoplast and successfully obtained recombinant CHO cell A7 strain that can express recombinant protein. It is a benefic attempt on CHO cell expression system.
In the past antibody level was used to estimate a vaccine. But present research showed that cellular immunity plays an important role in those diseases caused by intracellular infection, e.g. hepatitis B. At present, vaccines for person using are still aluminium adjuvant vaccine primarily, we choose aluminium adjuvant to prepare S2S vaccine. In addition, on the basic of another study (patent), we use BCG to prepare S2S vaccine, which obtained better effect on humoral immunity and cell immunity.
S2S-aluminium adjuvant vaccine was detected by RIA method, Immunity efficacy of S2S vaccine (relative efficacy were 4.1, 2.0, 1.97) was higher than S vaccine (relative efficacy was 1.57). Anti-HBs and anti-preS2 was detected by ELISA method, anti- HBs induced by S2S vaccine was higher than S vaccine, p<0.05, it showed significant difference. Although the level of anti-preS2 induced by S2S vaccine was higher than S vaccine, p >0.05, there was no significant difference. what is more, preS2 antibody induced by S2S vaccine reaches the highest level in second week, it is significant on early prevention.
The humoral immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine , there was no statistical difference, but cell immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine, it showed significant difference. IL-2, lymphocyte stimulation indices and CD4+/CD8+ value of S2S-BCG vaccine were obviously higher than S2S-aluminium adjuvant vaccine. BCG can induce humoral immunity and cell immunity, but aluminium adjuvant has not this function, it recruits the deficient that current Hepatitis B vaccine only induces humoral immunity, the novel Hepatitis B recombination vaccine has potential application value on prevention and therapy Hepatitis B.
In a word, It is shown to be good prospect for study of recombinant pre-S2S hepatitis B vaccine produced by CHO cells on enhancing immunogenicity and inducing cellular immunity. It can lay a foundation for further developing new type hepatitis B vaccine for prevention and therapy.
Hepatitis B is an infectious disease caused by hepatitis B virus (HBV). HBV is responsible to acute and chronic hepatitis, as well as hepatocirrhosis, liver cancer. Hepatitis B vaccine inoculation is the most effective way to prevent the disease. The hepatitis B vaccine presently used in China comprises major S protein only. Its disadvantages are: (1) 5%-10% of the recipients raise only little or even no response; (2) three inoculations are needed to obtain essential antibody level; (3) it is used only in prevention, but not therapy. So to prevent quickly and enhance protection still are the problem that a novel Hepatitis B vaccine needs solve. Nowadays researchers found that pre-S protein, with high antigenisity, can enhance the immunoresponse of S protein; production of pre-S2 antibody is earlier than S antibody, it plays an important role on early prevention. And pre-S2 can adhere to hepatocytes via poly human serum albumin (PHSA). Therefore vaccine containing pre-S2 can prevent HBV from infecting liver cells by competitive joining. So it may cure hepatitis. More and more date show that a novel hepatitis B vaccine with pre-S can overcome the disadvantage of only S-protein vaccine.
In terms of vaccine designing strategy, the antigen molecule should be large, the formed polymer or particle is more, effect of immunogenicity is better. But in genetic engineering, the product molecule should be small to obtain high expression and to decrease the side effects. So it is necessary to consider the characteristic of vaccine and genetic engineering products on design genetic engineering vaccine.
The topic leads in a new concept of epitope -based vaccine design, reasonably retains the whole S gene, chooses 26 amino acids sequence of pre-S2 (120-146) that contains rich T and B cell epitope, removes 29 amino acids unnecessary sequence of pre-S2 and makes the size of recombinant protein suitable and express easily. The designed vaccine by aboved method not only retains necessary antigen epitope of antigen stimulates, but also reduces influence on protein secretion because of the expressed large molecule protein. The preliminary results showed the vaccine had expressed S2S protein, the expression level and immunity effect surpassed current S vaccine.
In the study, the whole S gene adding the epitope gene of pre-S2 (120-146) was cloned into a eukaryotic expression plasmid pcs and the recombinant plasmid pcs-S2S was constructed by the recombinant DNA technology . the plasmid pcs-S2S was transformed into CHO dhfr–cell successfully, totally screened 2400 cells, obtained recombinant CHO cell A7 strain. The A7 strain was subcultured for 50 passages and change medium for 30 times continuously, It is established that the stability of preS2S protein was good. The results of S and S2 proteins detected in CHO cells A7 strain were strong positive by DNA bot hybridization; expressed gene was detected by PCR, then sequenced, the length of S gene is 678 bp, the length of preS2 gene is 78 bp, the results of SDS-PAGE and sequencing were correct. It was verified that A7 strain cell contained S and S2 gene. Expressed protein preS2S was identified By SDS-PAGE and Western blot.
The genetic engineering products in Europe and America are expressed mainly by CHO cell, about 40%~70%, there are 22 kinds products in America which has been authorized to go on the market, but only two kinds in China. This is major gap between China and Europe and America on Biological drugs manufacture. This topic chose CHO cell expression system, we tried to overcome many technique problems in transfection and screening, groped and solved the problem of clone and amplification of monoplast and successfully obtained recombinant CHO cell A7 strain that can express recombinant protein. It is a benefic attempt on CHO cell expression system.
In the past antibody level was used to estimate a vaccine. But present research showed that cellular immunity plays an important role in those diseases caused by intracellular infection, e.g. hepatitis B. At present, vaccines for person using are still aluminium adjuvant vaccine primarily, we choose aluminium adjuvant to prepare S2S vaccine. In addition, on the basic of another study (patent), we use BCG to prepare S2S vaccine, which obtained better effect on humoral immunity and cell immunity.
S2S-aluminium adjuvant vaccine was detected by RIA method, Immunity efficacy of S2S vaccine (relative efficacy were 4.1, 2.0, 1.97) was higher than S vaccine (relative efficacy was 1.57). Anti-HBs and anti-preS2 was detected by ELISA method, anti- HBs induced by S2S vaccine was higher than S vaccine, p<0.05, it showed significant difference. Although the level of anti-preS2 induced by S2S vaccine was higher than S vaccine, p >0.05, there was no significant difference. what is more, preS2 antibody induced by S2S vaccine reaches the highest level in second week, it is significant on early prevention.
The humoral immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine , there was no statistical difference, but cell immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine, it showed significant difference. IL-2, lymphocyte stimulation indices and CD4+/CD8+ value of S2S-BCG vaccine were obviously higher than S2S-aluminium adjuvant vaccine. BCG can induce humoral immunity and cell immunity, but aluminium adjuvant has not this function, it recruits the deficient that current Hepatitis B vaccine only induces humoral immunity, the novel Hepatitis B recombination vaccine has potential application value on prevention and therapy Hepatitis B.
In a word, It is shown to be good prospect for study of recombinant pre-S2S hepatitis B vaccine produced by CHO cells on enhancing immunogenicity and inducing cellular immunity. It can lay a foundation for further developing new type hepatitis B vaccine for prevention and therapy.
引文
[1] Em C, Xiang Z. Novel Vaccine Approach [J]. J Immumol,1996,156(10):3579-3584.
[2] Wu Y Z, Shi T D.EBVD: an new approach to molecular design of vaccine[J]. Immunologist,1998,(suppl):1487-1491.
[3]冯立,胡显文,陈惠鹏.治疗性乙型肝炎疫苗研究进展[J].生物技术通讯,2004,5(3):289-291.
[4] 庄辉. 乙型肝炎疫苗免疫[J].中华肝脏病杂志,2003,11(4):197-198.
[5] Kane M. Global program for control of hepatitis B infection[J].Vaccine, 1995,13(S1)S47.
[6]庄辉.乙型肝炎流行病学研究进展[J].国外医学流行病学传染病学分册,2004,31(3):133-138.
[7] Julian Bilous, Steven Wiersmg. 世界卫生组织乙型肝炎控制目标和策略[J].中国计划免疫, 2004,10(3):178-179.
[8] 郭文. 乙型肝炎疫苗市场概况[J].世界临床药物, 2005,26(1):60-62.
[9] 施光峰. 病毒性肝炎. 翁心华主编.传染病学.第 3 版[M].上海:复旦大学出版社,2003:25-52.
[10] 施理, 张宜俊, 易学瑞, 等. 小鼠免疫接种乙型肝炎病毒表面 S 疫苗引起的细胞毒 T 淋巴细胞反应[J].中华医院感染学杂志, 2003,13(7):612-615.
[11] Ganem D, Prince AM. Hepatitis B virus infection-Natural history and clinical consequences [J].N Engl J Med,2004,350(11):123-133.
[12] Wen YM, Liu HJ, Chen HZ, et al. Studies on intrahepatic hepatitis B virus DNA in 98 viral hepatitis B patients[J].Chin Med J,1986,99:861-865.
[13] 秦刚, 施光峰. 治疗性乙型肝炎疫苗研究进展[J].中国抗感染化疗杂志, 2005,5(2):124-127.
[14] Pol S, Nalpas B, Driss F, et al. Efficacy and limitations of a specific immunotherapy inchronic hepatitis B[J].J Hepatol, 2001,34: 917-921.
[15] Pol S, Driss F, Michel ML, etal. Specific vaccine therapy in chronic hepatitis B infection[J]. Lancet,1994,344:342.
[16] Zhang S, Chan JY,YEO W, et al. Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues[J]. J Viral Hepat,2000,7:115-123.
[17] Vietheer PT, Netter HJ, Sozzi T, et al. Failure of the lamivudine-resistant rtM2041 hepatitis B virus mutants to efficiently support hepatitis delta virus secretion[J].J Virol, 2005,79: 6570-6573.
[18] 刘崇柏主编. 病毒性肝炎[M].北京:化学工业出版社,2003,11: 28-67.
[19] Yukio F, Kuroda S, Van Eerd PlVl, et a1. Protective efficacy of a novel hepatitis B vaccine consisting of M (pre-s2+s) protein particles (a third generation vaccine)[J].Vaccine,1990,8:192-197.
[20] Krugman S, Giles JP, Hammond J.Hepatitis Virus: effect of heat on the infectivity and antigenicity of the MS-1and MS-2 strains[J].J Infect Dis, 1970,122(5):432-436.
[21] 刘崇柏, 苏崇鳌. 乙型肝炎疫苗免疫及存在的问题[J].中国计划免疫,2004,10(3):159-162.
[22] 吴维寿, 孙超美, 姜铭波, 等. 乙型肝炎免疫预防持续效果探讨(18 年随访结果) [J].中国计划免疫, 2005,11(3):204-207.
[23] 汪春义. 基因工程乙肝疫苗糖基化程度对其性质的影响.长春生物制品研究所硕士研究生论文.1995.
[24] 刘文军, 杨芙蓉, 阮力, 等. 乙型肝炎表面抗原 S-蛋白糖化位点序列的人工变异及其哺乳动物细胞表达产物的形状研究[J]. 病毒学报,1997,13(1):33-40.
[25] 储瑞银, 江福美, 吕鸿声, 等. 杆状病毒运载的乙型肝炎病毒表面抗原基因(preS2-S)在家蚕体内的高效表达[J].科学通讯, 1991,36:859-862.
[26] 尚继军, 项金忠, 谢云, 等. 乙型肝炎病毒表面抗原中蛋白基因在昆虫细胞中的表达及其初步鉴定[J].中国生物制品学杂志, 1997,10(4):196-199.
[27] 任贵方, 阮力, 张一鸣, 等. 几种乙型肝炎病毒 DNA 重组质粒的改造及其在哺乳动物细胞高效表达乙型肝炎表面抗原[J].微生物学报, 1984,24(4):326-334.
[28] 张一鸣, 任贵方, 王申, 等. adw 与 adr 亚型乙型肝炎表面抗原基因在哺乳动物细胞中的表达[J].病毒学报, 1985,1(3):210-215.
[29] 任贵方, 张一鸣, 阮力, 等. 乙型肝炎病毒表面抗原基因在中国地鼠卵巢细胞中高效表达及纯化抗原免疫原性的研究[J].病毒学报, 1987,3(4):313-319.
[30] 任贵方, 阮薇琴, 田淑芳, 等. 乙型肝炎病毒表面抗原主蛋白基因转基因细胞系的建立与细胞性质的研究[J].病毒学报, 1991,7(2):112-119.
[31] Belew M, Mei YF, Li B, et al. Purification of recombinant hepatitis B surface antigen produced by transformed Chinese hamster ovary(CHO) cell line grown in culture[ J].Bioseparation.1991,1:397-408.
[32] 谢秋玲, 陈小佳, 戴云, 等. 中国仓鼠卵巢细胞表达系统在疫苗研制中的应用[J].中国公共, 2004 ,20(7):873-874.
[33] 艾智武. 中国仓鼠卵巢细胞重组乙型肝炎疫苗的研究进展[J].国外医学 预防、诊断、治疗用生物制品分册, 1996,19(2):49-51.
[34] 郭秀婵, 仪忠军, 毕静平. 我国中华地鼠卵巢(CHO)细胞表达重组乙肝疫苗发展现状[J].疾病监测, 1999,14(6):238-239.
[35] 梁争论, 李成明, 荆庆, 等. 乙型肝炎基因重组疫苗接种小学生抗体反应比较[J].中华预防医学杂志, 1998,32(3):165-167.
[36] 胡显文, 陈惠鹏, 汤仲明, 等. 美国、欧盟和中国生物技术药物的比较[J].中国生物工程杂志, 2005,25(2):82-94.
[37] 申 烨 华 , 耿 信 笃 . CHO 细 胞 表 达 系 统 研 究 新 进 展 [J]. 生 物 工 程 进 展 , 2000,20(4):23-25.
[38] Coursaget P, Bringer L, Sarr G, et al. Comparative immunogenicity in children of mammalian cell-derived recombinant hepatitis B vaccine and plasma-derived hepatitis B vaccine[J]. vaccine, 1992,10(6):379.
[39] Pol S. Nalpas B. Efficacy and limitations of a specific immunotherapy in chronichepatitis B[J].J Hepatol, 2001,34(6):917.
[40] Soulie JC, Devillier P, Santarelli J, et al. Immunogenicity and safety in new borns of a new recombinant hepatitis B vaccine containing the S and preS2 antigens [J]Vaccine, 1991, 9:135-137.
[41] Madalinski K, Sylvan SP. Hellstrom U, et al. Antibody responses to preS components after immunization of children with low dose of BioHepB[J]. Vaccine, 2001,20:92-97.
[42] Pride MW. Evaluation of B and T-cell responses in chimpanzes immunized with Hepagens, a hepatitis B vaccine containing pre-S1,preS2 gene products [J]Vaccine, 1998,16:543-550.
[43] Mahony FJ, Kane M. Hepatitis B vaccine.In:Plotkin, SA etal.Vaccines .3rd ed[M].Philadelphia,WB:Saunders Company,1999,158-182.
[44] Valenzuela P, Angellica M, Willian JR. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast [J].Nature, 1982,298-347.
[45] Del Canbo. Immunogenicity of 20 micrograms of recombinant DNA hepatitis B vaccine in healthy neonates: a comparison of three different vaccination schemes[J].J Med Virol, 1993,41(1):30.
[46] Jtephenne J. Development and production aspects of a recombinant yeast-derived hepatitis B vaccine[J]. Vaccine, 1990,8(Suppl):69.
[47] Young MD, Schneider DL, Zuckerman AJ, et al. Adult hepatitis B vaccination using a novel triple antigen recombinant vaccine[J].Hepatology, 2001,34:372-376.
[48] 冯宗铭, 钟武威, 吴雪, 等. 含 preS2 的乙肝表面抗原基因的表达[J].生物化学与生物物理学报,1987,19(5):428-431.
[49] 储瑞银, 宓怡德, 吕鸿声, 等. 乙型肝炎病毒表面抗原基因(preS2-S)在家蚕培养细胞中的表达[J].生物化学与生物物理学报, 1990,22(4):385-389.
[50] 田淑芳, 杨安道, 阮薇琴, 等. 乙型肝炎病毒表面抗原中蛋白基因在中国地鼠卵巢细胞中的高效表达及其产物活性的初步检测[J].病毒学报, 1993,9(1):7-13.
[51] Yamada T, Iwabuki H, Kanno T, et a1. Physicochemical and immunologicalcharacterization of hepatitis B virus envelope particles exclusively consisting of the entire L(pre-S1+pre-S2+S) protein [J].Vaccine, 200l, 30:3154-3163.
[52] Hui J, Li G, Kong Y , et al. ExpreS2ion and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.[J].Journal of Biotechnology ,1999,72:49-59.
[53] Yerushalmi B, Raz R., Blondheim O, et al. Safety and immunogenicity of a novel mammalian cell-derved recombinant hepatitis B vaccine containing Pre-S1 and Pre-S2 antigens in neonates[J]. Pediatr Infect Dis J, 1997,16:587-592.
[54] Pride MW, Bailey CR, Muchmore E, et al. Evaluation of B and T-cell responses in chimpanzees immunized with Hepagene,a hepatitis B vaccine containing pre-S1 and pre-S2 and S gene products[J]. Vaccine, 1998,16: 543-550.
[55] Cho SP, Lee B, Min MK. Recombinant polioviruses expreS2ing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes[J]. Vaccine, 2000,18:2878-2885.
[56] Mandl S, Sigal LJ, Rock KL, et al. Poliovirus vaccine vectors elicit antigen-specific cytotoxic T cell and protect mice against lethal challenge with malignant melanoma cells expreS2ing a model antigen[J].Proc Natl Acad Sci USA , 1998,95:8216-8221.
[57] Tang S, Rij RV, Silver D, et al .Toward a poliovirus-based Simian Immunodifiency virus vaccine.[J]. J Virol, 1997,71:7841-7850.
[58] Zanetti M, Croft M, Xiong S, et al. T cell immunity elicited via somatic transgene immunization[J].FASEB J, 1998,12:A626-633.
[59] Gerloni M, Minter KL, Xiong S, et al. Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory[J]. J Immunol, 1999,162:3782-3789.
[60] Xiong S, Gerlori M, Zanetti M. In vivo role of B lymphocytes in somatic transgene immunization[J].Proc Natl Acad Sci USA, 1997,94:6352.
[61] Xiong S, Gerloni M, Zanetti M. Engineening vaccines with heterologous B and T cell epitopes using immunoglobulin genes[J].Nature Biotechnol, 1997,15:882.
[62] 骆利敏, 李明, 夏虎, 等. 乙型肝炎病毒多表位治疗性抗原基因的合成、表达及抗原性研究[J].第一军医大学学报,2003,23(8):802.
[63] 甘慧, 王海平, 周勇, 等. 乙型肝炎病毒多表位嵌和蛋白的表达、纯化和鉴定[J].军事医学科学院院刊,2003,27(1):40.
[64] Hopp TP. Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier[J]. Mol Immunol, 1984,21: 13-16.
[65] BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines— yesterday, today, and tomorrow[J]. THE LANCET Infectious Diseases, 2002,2: 425-431.
[66] Livinston BD,Crimi C,Grey H,et al. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable tothose elicited by acute viral infection[J]. J Immunol, 1997,159(3): 1383 -1392.
[67] SHI Tong-dong, WU Yu-zhang, BIAN Jiang, et al. Therapeutic peptides based on HBcAgl8- 27 epitope can induce CTL response in vitro and in vivo[J]. J Immunol,,2003,19(3):165-169.
[68] Moyle PM, Olive C, Ho M, et al. Towards the synthesis of a highly pure, multiepitopic, mucosal group A streptococcal lipopeptide vaccine[J]. International Congress Series, 2006,1289:324-328.
[69] Rottinghaus ST, Poland GA, Jacobson RM, et al. Hepatitis B DNA Vaccine induces protective antibody responses in human nonresponders to conventional vaccination[J]. Vaccine, 2003,21(31):4604-4608.
[70] Peterson R K D , Arntzen C J . On risk and plant-based biopharmaceuticals[J]. Trends in Biotechnology, 2004,22(2) :64-66.
[71] Cho SP, Lee B, Min MK. Recombinant polioviruses expreS2ing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes[J]. Vaccine, 2000,18:2878-2885.
[72] Reinhild Prange, Margaret Werr. DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response[J].Vaccine, 1999,17:617-623.
[73] Christopher D, Jones, Mark Page, Andrew Bacon, et al. T-cell and antibody response characterisation of a new recombinant pre-S1, pre-S2 and SHBs antigen-containing hepatitis B vaccine; demonstration of superior anti-SHBs antibody induction in responder mice[J].Vaccine, 1999,17:2528-2537.
[74] Roy MT, Wu MS, Barr LJ, et al. Powderject Vaccines In the Induction of antigen-specific CD8+T cells, T helps cells and protective Levels of antibody in humans by particles-mediated administration of a hepatitis B virus DNA vaccine[J]. Vaccine, 2000,19(7-8): 764-778.
[75] Kattner G, Kramer A, Schmidtke G, et a1. Characterization of neutralizing anti-pre-Sl and anti-pre-S2 (HBV)monoclonal antibodies and their fragments.[J]Molecular Immunology, 1999,36:669-683.
[76] Oka Y, Akbar S M F, Horiike N et al. Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice[J]. Immunology, 2001,103:90-97.
[77] Rabinovich NR, Pamela M, David LK. Vaccine technologyies:view to the future[J]. Science, 1994,265:1401.
[78] Wu Y Z, Shi T D. EBVD:an new approach to molecular design of vaccine[J]. Immunologist, 1998,(suppl):1487—1491.
[79] Ferriar C, Bertiletti A, Penna A, et al. Identification of immunodominant T cell epitope of the hepatitis B virus nuclecapid antigen[J].J Clin Invest, 1991,88:214-219.
[80] J.萨姆布鲁克, E.F.弗里奇, T.蔓尼阿蒂斯. 分子克窿[M]第 2 版.北京:科学技术出版社, 1992:787-793.
[81] 中国生物制品标准化委员会编《中国生物制品规程》.北京:化学工业出版社,2000.
[82] 国家药典委员会编《中华人民共和国药典》三部. 北京:化学工业出版社.,2005.
[83] Downing LA, Bernstein JM, Walter A. Active respiratory syncytial virus Purifled by ion-exchange chromatography: characterization of binding and elution requirements[J]. J Virol Methods, 1992,38(2):215-228.
[84] Kremer L, Estaquier J, Wolowczuk I, et al. Ineffective cellular immune response associated with T-cell apoptosis in susceptible Mycobacterium bovis BCG-infected mice[J].Infect Immun, 2000,68:4264-4273.
[85] Ishikawa T, Kono D. Polyclonality and multispecificity of the CTL response to a single viral epitope [J]. J Immunol, 1998,261(11):5842.
[86] Kutose K, Akbar SH. Production of antibody to hepatitis B surface antigen (anti-HBs) by murine hepatitis B virus carriers: neonatal tolerance versus antigen presentation by dendritic cells [J]. Immunology,1997,92(4):494.
[87] Leroux-Roels G, Desombere I., De Tollenaere G, et al. Hepatitis B vaccine containing surface antigen and selected pres1 and preS2 sequences. 1.safety and immunogenicity in young, healthy adults[J]. Vaccine,1997,15:1724-1731.
[88] Schldel F, Soussan P,Sato Y, et al. HBV molecular biology and immunology[J]. Biotest Bulletin, 1990,4: 13-83.
[89] Davis HL, Mt Sinai. DNA vaccines for prophylactic or therapeutic immunization against hepatitis B virus[J]. J Med,1999,66:84-90.
[90] Soussan P, Pol S, Garreau F, Brechot C, Kremsdorf D. Vaccination of chronic hepatitis B virus. carriers with preS2/S envelope protein is not associated with the emergence of envelope escape. mutants [J]. J Gen Virol, 2001,82:367-371.
[91] Neurath AR, Kent SB, Parker K, Prince AM, Strick N, Brotman B, Sproul P. Antibodies to a synthetic peptide from the preS 120-145 region of the hepatitis B virus envelope are virus neutralizing[J]. Vaccine, 1986, 4(1):35–37.
[92] Prange R, Werr M. DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response[J]. Vaccine, 1999,17: 617-623.
[93] Davis HL, McCluskie MJ, Gerin JL, et al. DNA vaccine for hepatitis B: evidence for immuogenicity in chimpanzees a comparison with other vaccines[J]. Proc Natl Acad Sci USA, 1996,93:7213-7218.
[94] Jungers P, Chauveau P, Couroucé AM, et al. Immunogenicity of the recombinant GenHevac B Pasteur vaccine against hepatitis B in chronic uremic patients[J]. J Infect Dis, 1994,169: 399 -402.
[95] Shimizu Y, Guidotti LG, Fowler P, et a1.Dendritic cell immunization breaks cytotoxic T lymphocyte tolerance in hepatitis B virus transgenic mice[J]. J Immunol, 1999,18:4520-4529
[96] Budkoska A, Leroux-Roels G, Cupps TR, et a1. Detection of antibodies directed against the PreS2 gene product of hepatitis B virus: relationship between anti-PreS response and recovery[J]. Ann Inst Pasteru Immunol, 1985:136-157.
[97] George P. S. Filamentous fusion phage: novel expreS2ion vector that display cloned antigens on the virion surface [J]. Science, 1985,228: 1315-1317.
[98] Cheng J, Zhong Y W, Liu Y, et al. Screening and identification of a humanized single chain variable region antibody for hepatitis C virus non-structural 3 protein [J]. Chin J Immunol, 2000,16(4):246.
[99] Itoh Y, Takai E, Ohnuma H, et al. A synthetic peptide vaccine involving the product of the pre-S(2)region of hepatitis B virus DNA: protective efficacy in chimpanzees[J].Proc AatlAcad Sci USA, 1986,83(23): 9174-9178.
[100] Milich DR, Mclachlan A, Chisari FV, et al. Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues[J]. J Immuno, 1986,137(7-8): 2703-2710.
[101] 金立杰, 赵小琳, 方艳秋, 等. 乙型肝炎疫苗-卡介苗联合疫苗细胞免疫的研究[J].中国生物制品杂志, 2006,19(2):171-173.
[102] 杨丽华,王志, 赵小琳,等. 乙型肝炎疫苗-卡介苗联合疫苗在动物中的免疫应答[J].中国生物制品杂志, 2006,19(4):378-380.
[103] 金立杰, 赵小琳, 罗璇, 等. 乙型肝炎卡介苗联合疫苗与单价乙型肝炎疫苗免疫效果的比较[J].中国免疫学杂志, 2006,22(10):14-17.
[104] Keisari Y, Seger M, Lency J, et al . IL-1、TNF-α and IL-2 production by peritonealand spleen cell from schistosom a mansonin infect mice and its potentation by perimmunization with schistosomal antigen and immunostimulants[J]. Immunobiol, 1993, 188:446-459.
[105] Tamura M, Yoo YC, Yoshimatsu K et al, Effects of muramyl dipeptide derivatives as adjuvants on the induction of antibody response to recombinant hepatitis sueface antigen[J].Vaccine, 1995,13(1):77-82.
[2] Wu Y Z, Shi T D.EBVD: an new approach to molecular design of vaccine[J]. Immunologist,1998,(suppl):1487-1491.
[3]冯立,胡显文,陈惠鹏.治疗性乙型肝炎疫苗研究进展[J].生物技术通讯,2004,5(3):289-291.
[4] 庄辉. 乙型肝炎疫苗免疫[J].中华肝脏病杂志,2003,11(4):197-198.
[5] Kane M. Global program for control of hepatitis B infection[J].Vaccine, 1995,13(S1)S47.
[6]庄辉.乙型肝炎流行病学研究进展[J].国外医学流行病学传染病学分册,2004,31(3):133-138.
[7] Julian Bilous, Steven Wiersmg. 世界卫生组织乙型肝炎控制目标和策略[J].中国计划免疫, 2004,10(3):178-179.
[8] 郭文. 乙型肝炎疫苗市场概况[J].世界临床药物, 2005,26(1):60-62.
[9] 施光峰. 病毒性肝炎. 翁心华主编.传染病学.第 3 版[M].上海:复旦大学出版社,2003:25-52.
[10] 施理, 张宜俊, 易学瑞, 等. 小鼠免疫接种乙型肝炎病毒表面 S 疫苗引起的细胞毒 T 淋巴细胞反应[J].中华医院感染学杂志, 2003,13(7):612-615.
[11] Ganem D, Prince AM. Hepatitis B virus infection-Natural history and clinical consequences [J].N Engl J Med,2004,350(11):123-133.
[12] Wen YM, Liu HJ, Chen HZ, et al. Studies on intrahepatic hepatitis B virus DNA in 98 viral hepatitis B patients[J].Chin Med J,1986,99:861-865.
[13] 秦刚, 施光峰. 治疗性乙型肝炎疫苗研究进展[J].中国抗感染化疗杂志, 2005,5(2):124-127.
[14] Pol S, Nalpas B, Driss F, et al. Efficacy and limitations of a specific immunotherapy inchronic hepatitis B[J].J Hepatol, 2001,34: 917-921.
[15] Pol S, Driss F, Michel ML, etal. Specific vaccine therapy in chronic hepatitis B infection[J]. Lancet,1994,344:342.
[16] Zhang S, Chan JY,YEO W, et al. Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues[J]. J Viral Hepat,2000,7:115-123.
[17] Vietheer PT, Netter HJ, Sozzi T, et al. Failure of the lamivudine-resistant rtM2041 hepatitis B virus mutants to efficiently support hepatitis delta virus secretion[J].J Virol, 2005,79: 6570-6573.
[18] 刘崇柏主编. 病毒性肝炎[M].北京:化学工业出版社,2003,11: 28-67.
[19] Yukio F, Kuroda S, Van Eerd PlVl, et a1. Protective efficacy of a novel hepatitis B vaccine consisting of M (pre-s2+s) protein particles (a third generation vaccine)[J].Vaccine,1990,8:192-197.
[20] Krugman S, Giles JP, Hammond J.Hepatitis Virus: effect of heat on the infectivity and antigenicity of the MS-1and MS-2 strains[J].J Infect Dis, 1970,122(5):432-436.
[21] 刘崇柏, 苏崇鳌. 乙型肝炎疫苗免疫及存在的问题[J].中国计划免疫,2004,10(3):159-162.
[22] 吴维寿, 孙超美, 姜铭波, 等. 乙型肝炎免疫预防持续效果探讨(18 年随访结果) [J].中国计划免疫, 2005,11(3):204-207.
[23] 汪春义. 基因工程乙肝疫苗糖基化程度对其性质的影响.长春生物制品研究所硕士研究生论文.1995.
[24] 刘文军, 杨芙蓉, 阮力, 等. 乙型肝炎表面抗原 S-蛋白糖化位点序列的人工变异及其哺乳动物细胞表达产物的形状研究[J]. 病毒学报,1997,13(1):33-40.
[25] 储瑞银, 江福美, 吕鸿声, 等. 杆状病毒运载的乙型肝炎病毒表面抗原基因(preS2-S)在家蚕体内的高效表达[J].科学通讯, 1991,36:859-862.
[26] 尚继军, 项金忠, 谢云, 等. 乙型肝炎病毒表面抗原中蛋白基因在昆虫细胞中的表达及其初步鉴定[J].中国生物制品学杂志, 1997,10(4):196-199.
[27] 任贵方, 阮力, 张一鸣, 等. 几种乙型肝炎病毒 DNA 重组质粒的改造及其在哺乳动物细胞高效表达乙型肝炎表面抗原[J].微生物学报, 1984,24(4):326-334.
[28] 张一鸣, 任贵方, 王申, 等. adw 与 adr 亚型乙型肝炎表面抗原基因在哺乳动物细胞中的表达[J].病毒学报, 1985,1(3):210-215.
[29] 任贵方, 张一鸣, 阮力, 等. 乙型肝炎病毒表面抗原基因在中国地鼠卵巢细胞中高效表达及纯化抗原免疫原性的研究[J].病毒学报, 1987,3(4):313-319.
[30] 任贵方, 阮薇琴, 田淑芳, 等. 乙型肝炎病毒表面抗原主蛋白基因转基因细胞系的建立与细胞性质的研究[J].病毒学报, 1991,7(2):112-119.
[31] Belew M, Mei YF, Li B, et al. Purification of recombinant hepatitis B surface antigen produced by transformed Chinese hamster ovary(CHO) cell line grown in culture[ J].Bioseparation.1991,1:397-408.
[32] 谢秋玲, 陈小佳, 戴云, 等. 中国仓鼠卵巢细胞表达系统在疫苗研制中的应用[J].中国公共, 2004 ,20(7):873-874.
[33] 艾智武. 中国仓鼠卵巢细胞重组乙型肝炎疫苗的研究进展[J].国外医学 预防、诊断、治疗用生物制品分册, 1996,19(2):49-51.
[34] 郭秀婵, 仪忠军, 毕静平. 我国中华地鼠卵巢(CHO)细胞表达重组乙肝疫苗发展现状[J].疾病监测, 1999,14(6):238-239.
[35] 梁争论, 李成明, 荆庆, 等. 乙型肝炎基因重组疫苗接种小学生抗体反应比较[J].中华预防医学杂志, 1998,32(3):165-167.
[36] 胡显文, 陈惠鹏, 汤仲明, 等. 美国、欧盟和中国生物技术药物的比较[J].中国生物工程杂志, 2005,25(2):82-94.
[37] 申 烨 华 , 耿 信 笃 . CHO 细 胞 表 达 系 统 研 究 新 进 展 [J]. 生 物 工 程 进 展 , 2000,20(4):23-25.
[38] Coursaget P, Bringer L, Sarr G, et al. Comparative immunogenicity in children of mammalian cell-derived recombinant hepatitis B vaccine and plasma-derived hepatitis B vaccine[J]. vaccine, 1992,10(6):379.
[39] Pol S. Nalpas B. Efficacy and limitations of a specific immunotherapy in chronichepatitis B[J].J Hepatol, 2001,34(6):917.
[40] Soulie JC, Devillier P, Santarelli J, et al. Immunogenicity and safety in new borns of a new recombinant hepatitis B vaccine containing the S and preS2 antigens [J]Vaccine, 1991, 9:135-137.
[41] Madalinski K, Sylvan SP. Hellstrom U, et al. Antibody responses to preS components after immunization of children with low dose of BioHepB[J]. Vaccine, 2001,20:92-97.
[42] Pride MW. Evaluation of B and T-cell responses in chimpanzes immunized with Hepagens, a hepatitis B vaccine containing pre-S1,preS2 gene products [J]Vaccine, 1998,16:543-550.
[43] Mahony FJ, Kane M. Hepatitis B vaccine.In:Plotkin, SA etal.Vaccines .3rd ed[M].Philadelphia,WB:Saunders Company,1999,158-182.
[44] Valenzuela P, Angellica M, Willian JR. Synthesis and assembly of hepatitis B virus surface antigen particles in yeast [J].Nature, 1982,298-347.
[45] Del Canbo. Immunogenicity of 20 micrograms of recombinant DNA hepatitis B vaccine in healthy neonates: a comparison of three different vaccination schemes[J].J Med Virol, 1993,41(1):30.
[46] Jtephenne J. Development and production aspects of a recombinant yeast-derived hepatitis B vaccine[J]. Vaccine, 1990,8(Suppl):69.
[47] Young MD, Schneider DL, Zuckerman AJ, et al. Adult hepatitis B vaccination using a novel triple antigen recombinant vaccine[J].Hepatology, 2001,34:372-376.
[48] 冯宗铭, 钟武威, 吴雪, 等. 含 preS2 的乙肝表面抗原基因的表达[J].生物化学与生物物理学报,1987,19(5):428-431.
[49] 储瑞银, 宓怡德, 吕鸿声, 等. 乙型肝炎病毒表面抗原基因(preS2-S)在家蚕培养细胞中的表达[J].生物化学与生物物理学报, 1990,22(4):385-389.
[50] 田淑芳, 杨安道, 阮薇琴, 等. 乙型肝炎病毒表面抗原中蛋白基因在中国地鼠卵巢细胞中的高效表达及其产物活性的初步检测[J].病毒学报, 1993,9(1):7-13.
[51] Yamada T, Iwabuki H, Kanno T, et a1. Physicochemical and immunologicalcharacterization of hepatitis B virus envelope particles exclusively consisting of the entire L(pre-S1+pre-S2+S) protein [J].Vaccine, 200l, 30:3154-3163.
[52] Hui J, Li G, Kong Y , et al. ExpreS2ion and characterization of chimeric hepatitis B surface antigen particles carrying preS epitopes.[J].Journal of Biotechnology ,1999,72:49-59.
[53] Yerushalmi B, Raz R., Blondheim O, et al. Safety and immunogenicity of a novel mammalian cell-derved recombinant hepatitis B vaccine containing Pre-S1 and Pre-S2 antigens in neonates[J]. Pediatr Infect Dis J, 1997,16:587-592.
[54] Pride MW, Bailey CR, Muchmore E, et al. Evaluation of B and T-cell responses in chimpanzees immunized with Hepagene,a hepatitis B vaccine containing pre-S1 and pre-S2 and S gene products[J]. Vaccine, 1998,16: 543-550.
[55] Cho SP, Lee B, Min MK. Recombinant polioviruses expreS2ing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes[J]. Vaccine, 2000,18:2878-2885.
[56] Mandl S, Sigal LJ, Rock KL, et al. Poliovirus vaccine vectors elicit antigen-specific cytotoxic T cell and protect mice against lethal challenge with malignant melanoma cells expreS2ing a model antigen[J].Proc Natl Acad Sci USA , 1998,95:8216-8221.
[57] Tang S, Rij RV, Silver D, et al .Toward a poliovirus-based Simian Immunodifiency virus vaccine.[J]. J Virol, 1997,71:7841-7850.
[58] Zanetti M, Croft M, Xiong S, et al. T cell immunity elicited via somatic transgene immunization[J].FASEB J, 1998,12:A626-633.
[59] Gerloni M, Minter KL, Xiong S, et al. Activation of CD4 T cells by somatic transgenesis induces generalized immunity of uncommitted T cells and immunologic memory[J]. J Immunol, 1999,162:3782-3789.
[60] Xiong S, Gerlori M, Zanetti M. In vivo role of B lymphocytes in somatic transgene immunization[J].Proc Natl Acad Sci USA, 1997,94:6352.
[61] Xiong S, Gerloni M, Zanetti M. Engineening vaccines with heterologous B and T cell epitopes using immunoglobulin genes[J].Nature Biotechnol, 1997,15:882.
[62] 骆利敏, 李明, 夏虎, 等. 乙型肝炎病毒多表位治疗性抗原基因的合成、表达及抗原性研究[J].第一军医大学学报,2003,23(8):802.
[63] 甘慧, 王海平, 周勇, 等. 乙型肝炎病毒多表位嵌和蛋白的表达、纯化和鉴定[J].军事医学科学院院刊,2003,27(1):40.
[64] Hopp TP. Immunogenicity of a synthetic HBsAg peptide: enhancement by conjugation to a fatty acid carrier[J]. Mol Immunol, 1984,21: 13-16.
[65] BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines— yesterday, today, and tomorrow[J]. THE LANCET Infectious Diseases, 2002,2: 425-431.
[66] Livinston BD,Crimi C,Grey H,et al. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable tothose elicited by acute viral infection[J]. J Immunol, 1997,159(3): 1383 -1392.
[67] SHI Tong-dong, WU Yu-zhang, BIAN Jiang, et al. Therapeutic peptides based on HBcAgl8- 27 epitope can induce CTL response in vitro and in vivo[J]. J Immunol,,2003,19(3):165-169.
[68] Moyle PM, Olive C, Ho M, et al. Towards the synthesis of a highly pure, multiepitopic, mucosal group A streptococcal lipopeptide vaccine[J]. International Congress Series, 2006,1289:324-328.
[69] Rottinghaus ST, Poland GA, Jacobson RM, et al. Hepatitis B DNA Vaccine induces protective antibody responses in human nonresponders to conventional vaccination[J]. Vaccine, 2003,21(31):4604-4608.
[70] Peterson R K D , Arntzen C J . On risk and plant-based biopharmaceuticals[J]. Trends in Biotechnology, 2004,22(2) :64-66.
[71] Cho SP, Lee B, Min MK. Recombinant polioviruses expreS2ing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes[J]. Vaccine, 2000,18:2878-2885.
[72] Reinhild Prange, Margaret Werr. DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response[J].Vaccine, 1999,17:617-623.
[73] Christopher D, Jones, Mark Page, Andrew Bacon, et al. T-cell and antibody response characterisation of a new recombinant pre-S1, pre-S2 and SHBs antigen-containing hepatitis B vaccine; demonstration of superior anti-SHBs antibody induction in responder mice[J].Vaccine, 1999,17:2528-2537.
[74] Roy MT, Wu MS, Barr LJ, et al. Powderject Vaccines In the Induction of antigen-specific CD8+T cells, T helps cells and protective Levels of antibody in humans by particles-mediated administration of a hepatitis B virus DNA vaccine[J]. Vaccine, 2000,19(7-8): 764-778.
[75] Kattner G, Kramer A, Schmidtke G, et a1. Characterization of neutralizing anti-pre-Sl and anti-pre-S2 (HBV)monoclonal antibodies and their fragments.[J]Molecular Immunology, 1999,36:669-683.
[76] Oka Y, Akbar S M F, Horiike N et al. Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice[J]. Immunology, 2001,103:90-97.
[77] Rabinovich NR, Pamela M, David LK. Vaccine technologyies:view to the future[J]. Science, 1994,265:1401.
[78] Wu Y Z, Shi T D. EBVD:an new approach to molecular design of vaccine[J]. Immunologist, 1998,(suppl):1487—1491.
[79] Ferriar C, Bertiletti A, Penna A, et al. Identification of immunodominant T cell epitope of the hepatitis B virus nuclecapid antigen[J].J Clin Invest, 1991,88:214-219.
[80] J.萨姆布鲁克, E.F.弗里奇, T.蔓尼阿蒂斯. 分子克窿[M]第 2 版.北京:科学技术出版社, 1992:787-793.
[81] 中国生物制品标准化委员会编《中国生物制品规程》.北京:化学工业出版社,2000.
[82] 国家药典委员会编《中华人民共和国药典》三部. 北京:化学工业出版社.,2005.
[83] Downing LA, Bernstein JM, Walter A. Active respiratory syncytial virus Purifled by ion-exchange chromatography: characterization of binding and elution requirements[J]. J Virol Methods, 1992,38(2):215-228.
[84] Kremer L, Estaquier J, Wolowczuk I, et al. Ineffective cellular immune response associated with T-cell apoptosis in susceptible Mycobacterium bovis BCG-infected mice[J].Infect Immun, 2000,68:4264-4273.
[85] Ishikawa T, Kono D. Polyclonality and multispecificity of the CTL response to a single viral epitope [J]. J Immunol, 1998,261(11):5842.
[86] Kutose K, Akbar SH. Production of antibody to hepatitis B surface antigen (anti-HBs) by murine hepatitis B virus carriers: neonatal tolerance versus antigen presentation by dendritic cells [J]. Immunology,1997,92(4):494.
[87] Leroux-Roels G, Desombere I., De Tollenaere G, et al. Hepatitis B vaccine containing surface antigen and selected pres1 and preS2 sequences. 1.safety and immunogenicity in young, healthy adults[J]. Vaccine,1997,15:1724-1731.
[88] Schldel F, Soussan P,Sato Y, et al. HBV molecular biology and immunology[J]. Biotest Bulletin, 1990,4: 13-83.
[89] Davis HL, Mt Sinai. DNA vaccines for prophylactic or therapeutic immunization against hepatitis B virus[J]. J Med,1999,66:84-90.
[90] Soussan P, Pol S, Garreau F, Brechot C, Kremsdorf D. Vaccination of chronic hepatitis B virus. carriers with preS2/S envelope protein is not associated with the emergence of envelope escape. mutants [J]. J Gen Virol, 2001,82:367-371.
[91] Neurath AR, Kent SB, Parker K, Prince AM, Strick N, Brotman B, Sproul P. Antibodies to a synthetic peptide from the preS 120-145 region of the hepatitis B virus envelope are virus neutralizing[J]. Vaccine, 1986, 4(1):35–37.
[92] Prange R, Werr M. DNA-mediated immunization to hepatitis B virus envelope proteins: preS antigen secretion enhances the humoral response[J]. Vaccine, 1999,17: 617-623.
[93] Davis HL, McCluskie MJ, Gerin JL, et al. DNA vaccine for hepatitis B: evidence for immuogenicity in chimpanzees a comparison with other vaccines[J]. Proc Natl Acad Sci USA, 1996,93:7213-7218.
[94] Jungers P, Chauveau P, Couroucé AM, et al. Immunogenicity of the recombinant GenHevac B Pasteur vaccine against hepatitis B in chronic uremic patients[J]. J Infect Dis, 1994,169: 399 -402.
[95] Shimizu Y, Guidotti LG, Fowler P, et a1.Dendritic cell immunization breaks cytotoxic T lymphocyte tolerance in hepatitis B virus transgenic mice[J]. J Immunol, 1999,18:4520-4529
[96] Budkoska A, Leroux-Roels G, Cupps TR, et a1. Detection of antibodies directed against the PreS2 gene product of hepatitis B virus: relationship between anti-PreS response and recovery[J]. Ann Inst Pasteru Immunol, 1985:136-157.
[97] George P. S. Filamentous fusion phage: novel expreS2ion vector that display cloned antigens on the virion surface [J]. Science, 1985,228: 1315-1317.
[98] Cheng J, Zhong Y W, Liu Y, et al. Screening and identification of a humanized single chain variable region antibody for hepatitis C virus non-structural 3 protein [J]. Chin J Immunol, 2000,16(4):246.
[99] Itoh Y, Takai E, Ohnuma H, et al. A synthetic peptide vaccine involving the product of the pre-S(2)region of hepatitis B virus DNA: protective efficacy in chimpanzees[J].Proc AatlAcad Sci USA, 1986,83(23): 9174-9178.
[100] Milich DR, Mclachlan A, Chisari FV, et al. Two distinct but overlapping antibody binding sites in the pre-S(2) region of HBsAg localized within 11 continuous residues[J]. J Immuno, 1986,137(7-8): 2703-2710.
[101] 金立杰, 赵小琳, 方艳秋, 等. 乙型肝炎疫苗-卡介苗联合疫苗细胞免疫的研究[J].中国生物制品杂志, 2006,19(2):171-173.
[102] 杨丽华,王志, 赵小琳,等. 乙型肝炎疫苗-卡介苗联合疫苗在动物中的免疫应答[J].中国生物制品杂志, 2006,19(4):378-380.
[103] 金立杰, 赵小琳, 罗璇, 等. 乙型肝炎卡介苗联合疫苗与单价乙型肝炎疫苗免疫效果的比较[J].中国免疫学杂志, 2006,22(10):14-17.
[104] Keisari Y, Seger M, Lency J, et al . IL-1、TNF-α and IL-2 production by peritonealand spleen cell from schistosom a mansonin infect mice and its potentation by perimmunization with schistosomal antigen and immunostimulants[J]. Immunobiol, 1993, 188:446-459.
[105] Tamura M, Yoo YC, Yoshimatsu K et al, Effects of muramyl dipeptide derivatives as adjuvants on the induction of antibody response to recombinant hepatitis sueface antigen[J].Vaccine, 1995,13(1):77-82.