骨骼肌注射乙型肝炎表面抗原基因在小鼠体内诱发乙肝表面抗…

将含有乙型肝炎表面抗原（ＨＢｓＡｇ）基因的重组质粒ｐＡｃｔ－ＭＳ直接注入小鼠骨骼肌中，２周后加强免疫１次。免疫后４￣９周间，每周检测小鼠血清中ＨＢｓ。结果表明实验组６／８只小鼠ＨＢｓ为阳性，对照组８只小鼠均未检测到ＨＢｓ。本实验证实直接注射质粒ＤＮＡ外源基因能获得表达，并能引起免疫反应。     

用微弹轰击法介导的转基因对小鼠进行乙肝病毒遗传免疫研究

用包裹ＤＮＡ的金微弹轰击耳朵，将表达乙型肝炎病毒表面抗原（ＨＢｓＡｇ）基因的重组质粒转入小鼠皮肤细胞，在小鼠血清中检测ＨＢｓＡｇ及抗－ＨＢｓ抗体。实验组８只小鼠中，有３只在基因转移３天后检测到ＨＢｓＡｇ。１４天再轰击１次。１６天后２只鼠中检测到抗－ＨＢｓ抗体。２８天后４只鼠中检测出抗ＨＢｓ抗体。对照组４只小鼠中ＨＢｓＡｇ及抗－ＨＢｓ抗体均未检测出。本实验为今后进一步分析乙肝病毒的遗传免疫奠定了实验基础，提供了实验依据。     

用微弹轰击法介导的转基因对小鼠进行乙肝病毒遗传免？…

用包裹ＤＮＡ的金微弹轻击耳朵，将表达乙型肝炎病毒表面抗原（ＨＢｓＡｇ）基因的重组质粒转入小鼠皮肤细胞，在小鼠血清中检测ＨＢｓＡｇ及抗－ＨＢｓ抗体。实验组８只小鼠中，有３只在基因转移３天后检测到ＨＢｓＡｇ。１４天再轰击１次。１６天后２只鼠中检测到抗－ＨＢｓ抗体。２８天４只鼠中检测出抗ＨＢｓ抗体。对照组４只小鼠中ＨＢｓＡｇ及抗－ＵＨＢｓ抗体均未检测出。本实验为今后进一步分析乙肝病毒的遗传免疫奠定了实验     

应用重组质粒pAM－HBsAg在果蝇细胞中表达乙型肝炎病毒表面抗原及基因免疫的初步研究

应用果蝇（ＤＳ２）表达系统，构建了含有乙型肝炎病毒表面抗原（ＨＢｓＡｇ）、摄金蛋白启动子（ＭＴｎ－ｐｒｏｍｏｔｅｒ）的共表达质粒ｐＡＭ－ＨＢｓＡｇ，转染细胞，经克隆，存活细胞株的培养上清液经硫酸铵沉淀、氯化铯（ＣＳＣｌ）密度梯度离心沉淀，获得的抗原用酶联免疫吸附试验（ＥＬＩＳＡ）、放射免疫分析（ＲＩＡ）法检测抗体，免疫吸印法（Ｗｅｓｔｅｒｎｂｌｏｔ）和电泳凝胶银染显色证实分子量为２３０００和２７０００，免疫电镜观察显示表达产物为２２ｕｍ球型颗粒，通过重金属离子（ＣｕＳＯ４、ＺｎＳＯ４）的诱导可增加抗原的表达量。用共表达质粒ｐＡＭ－ＨＢｓＡｇ的ＤＮＡ，注射Ｂａｌｂ／Ｃ小鼠的股四头肌。经ＥＬＩＳＡ、ＲＩＡ检测抗体产生情况，结果免疫后的小鼠经硫酸锌喂养抗体高于普通喂养的小鼠。Ｓｏｕｔｈｅｒｎ杂交证实鼠肌肉细胞存在ＨＢｓＡｇ基因。小鼠免疫接种实验表明，ＤＳ２细胞表达的抗原与直接用ＤＮＡ含有ＨＢｓＡｇ的重组质粒）免疫小鼠均获抗ＨＢｓＡｇ的抗体。     

利用穿梭质粒快速构建含乙型肝炎病毒表面抗原（HBsAg …

目的 应用新型杆状病毒表达系统快速构建含有ＨＢｓＡｇ基因的重组杆状病毒，高效表达ＨＢｓＡｇ，为ＨＢＶ诊断试剂、疫苗及治疗研究提供依据。方法 构建含有ＨＢｓＡｇ基因的供体质粒ｐＦＢ－ＢＳ，转化Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达试剂盒中的ＤＨ１０Ｂａｃ致敏菌，利用其含有的细菌Ｔｎ７转座繁忙将ＨＢｓＡｇ基因重组至穿梭质粒Ｂａｃｍｉｄ上，快速筛选出含有ＨＢｓＡｇ基因的重组杆状病毒。结果 此重组病毒能在昆虫细     

乙肝核酸疫苗NV—HB／s接种后小鼠体液免疫应答的初步研究

乙肝核酸疫苗ＮＶ┐ＨＢ／ｓ接种后小鼠体液免疫应答的初步研究赵连三秦山李水仙唐红刘丽周思亮雷秉钧将乙肝病毒表面抗原（ＨＢｓＡｇ）主蛋白的编码基因Ｓ插入真核细胞表达质粒ｐＲｃ／ＣＭＶ／ＡＴＧ中的ＣＭＶ启动子下游，获得的重组质粒（ＮＶ－ＨＢ／ｓ）转染真核细...     

乙型肝炎病毒S基因129Leu和145Arg变异株的抗原表达？…

目的 研究我国发现的乙型病毒Ｓ基因突变株表达ＨＢｓＡｇ及ＤＮＡ免疫的特性。方法 用自乙肝疫苗免疫无保护婴儿血清中克隆的２株变异Ｓ基因片段,置换已知核苷酸序列并可表达及复制的ＨＢＶ１．２个拷贝全基因野毒株重组质粒Ｐ３．８Ⅱ的Ｓ基因,将重组质粒转染ＨｅｐＧ２细胞,用Ａｂｂｏｔｔ试剂和２４种单抗检测染细胞培养上清中ＨＢｓＡｇ的结合力。用重组质粒ＨｅｐＧ２细胞,用Ａｂｂｏｔｔ试剂和２４种单抗检测转染细胞培     

乙型肝炎病毒pre S基因真核表达质粒诱导小鼠特异性？…

本文构建了含有ＨＢｓＡｇ ｐｒｅ Ｓ区基因的真核表达载体ｐＣＭＶ４－ｐｒｅ Ｓ，经大量提取质粒并纯化后，肌肉注射Ｂａｌｂ／ｃ小鼠，共免疫３次，间隔两周，结果有３／７的小鼠血清抗ｐｒｅ Ｓ２抗体呈阳性。     

乙型肝炎病毒基因疫苗诱导小鼠产生特异免疫应答

构建编码乙型肝炎病毒（ＨＢＶ）表面蛋白Ｓ的重组质粒ｐＣＲ３．１－Ｓ。将之直接肌肉注射Ｂａｌｂ／ｃ小鼠,观察小鼠ＨＢＶ特异的免疫应答。以ＥＬＩＳＡ法检测小鼠血清,＾３Ｈ－ＴｄＲ掺入法测定淋巴细胞增殖,＾５１Ｃｒ４ｈ释放法检测淋巴细胞杀伤功能。结果表明,与空载体对照组相比较,基因疫苗诱发小鼠产生良了的抗ＨＢｓ反应及ＨＢＶ特异的细胞免疫应答（Ｐ〈０．０５）,提示基因疫苗ｐＣＲ３．１－Ｓ有可能成为控制ＨＢ     

基因重组干扰素治疗急性乙型肝炎疗效观察

目的　以基因重组干扰素治疗急性乙型肝炎病人,观察其减少急性乙肝转慢率的效果。方法　用基因重组α干扰素（３００ 万Ｕ,肌内注射,隔日１ 次,１２ 周为一个疗程） 治疗１９ 名急性乙肝病人,对治疗后未产生抗ＨＢｓ 者,加用乙肝疫苗（３０ μｇ,肌内注射,每周注射１ 次,连用３ 周）;对照组为病情相似的１５ 例病人,服用一般保肝药物。结果　治疗组１８ 例（９５－０％ ,１８１９）ＨＢｓＡｇ 阴转,但均未产生抗ＨＢｓ,再用乙肝疫苗后,１７ 例（９４－０％ ,１７１９） 产生抗ＨＢｓ。２４～２４０ 周随访期间,１８ 例ＨＢｓＡｇ 阴转者无复发;１ 例ＨＢｓＡｇ 未阴转者,至随访结束时仍为阳性。对照组８ 例（５３－０ ％ ,８１５）ＨＢｓＡｇ 阴转,同时,８７－５ ％（７８）的病例产生抗ＨＢｓ,另７ 例ＨＢｓＡｇ 未阴转者,在２４ ～２４０ 周的随访期间,仅１ 例ＨＢｓＡｇ阴转,余６ 例ＨＢｓＡｇ 持续阳性。结论　对于急性乙型肝炎发病８ 周后ＨＢｓＡｇ 仍未阴转者,采用干扰素合用乙肝疫苗,对防止转慢及ＨＢｓＡｇ 阴转后抗ＨＢｓ 的产生可能有一定作用     

HBsAg基因疫苗诱导小鼠抗体产生的观察

将携带编码乙型肝炎病毒(HBV)表面抗原主要蛋白的S基因的质粒,直接注射小鼠肌肉中,小鼠4周后产生抗-HBs,阳转率83.3%(5/6),加强组阳转率66.7%(2/3),未加强组阳转率为100%(3/3);两组小鼠抗体持续时间可达28周以上;4周以后小鼠体内未检测到HBsAg.     

Cloning, protein expression and immunogenicity of HBs-murine IL-18 fusion DNA vaccine

Hepatitis B is a global serious disease caused by hepatitis B virus (HBV). There is no known cure for hepatitis B. The best way to deal with the disease is by preventing with hepatitis B vaccine. However, the current protein-based vaccines made up of recombinant hepatitis B surface antigen (HBsAg) are ineffective in chronic HBV carriers and a significant number of the vaccinees do not mount the protective immune response. Novel DNA-based immunization may overcome the deficits of the protein-based immunization and may provide more effective prophylactic and therapeutic outcomes. In this study, we constructed a recombinant plasmid carrying gene encoding the HBV surface antigen (HBs) linked to DNA segment encoding full-length murine interleukin-18, i.e. pcDNA-HBs-IL-18. Immunogenicity of the DNA construct was carried out in BALB/c mice in comparison with mock, i.e. pcDNA3.1+ and vaccines comprised of pRc/CMV-HBs and pRc/CMV-HBs plus pcDNA-IL-18. All vaccinated mice revealed significant serum anti-HBs IgG response after two intramuscular injections of the vaccines at 28 day interval as compared to the level of mock. Co-administration of pRc/CMV-HBs and pcDNA-IL-18 elicited arbitrarily higher levels of anti-HBs IgG than the levels in mice immunized with pRc/CMV-HBs alone and mice that received pcDNA-HBs-IL-18 although not statistically different. Further experiments are needed to investigate the subisotypes of the IgG antibody, the kinetics of cytokine and the cell-mediated immune response. For this communication, the prototype HBs-IL-18 DNA vaccine was successfully constructed and the gene encoding murine IL-18 was successfully cloned. The latter can be co-injected with the antigen coding DNA or used as a fusion partner to the DNA for priming the immune response. The recombinant HBs and full-length IL-18 proteins have potential for other research purposes. They may be used also as standard proteins in the protein quantification assay.     

In vivo induction of cellular and humoral immune responses by hybrid DNA vectors encoding simian/human immunodeficiency virus/hepatitis B surface antigen virus particles in BALB/c and HLA-A2-transgenic mice

To improve the immunogenicity of epitopes derived from Gag proteins of simian immunodeficiency virus (SIV) and from the envelope (Env) protein of human immunodeficiency virus type 1 (HIV-1), we have designed hybrid DNA vaccines by inserting sequences encoding antigenic domains of SIV and HIV-1 into the hepatitis B virus envelope gene. This gene encodes the hepatitis B surface antigen (HBsAg) capable of spontaneous assembly into virus-like particles that were used here as carrier. Injections of hybrid vectors encoding B-cell epitopes from the gp41 and the gp120 envelope proteins of HIV-1 induced specific humoral responses in BALB/c mice. Furthermore, high frequencies of IFN-gamma-secreting CD8+ T cells specific for various antigenic determinants of SIV-Gag were observed after intramuscular injections of hybrid DNA vectors in BALB/c mice. Genetic immunization of HLA-A2.1-transgenic mice with HIV-Env/HBsAg-encoding DNA generated a strong CTL response and IFN-gamma-secreting CD8+ T lymphocytes specific for HIV-1 envelope-derived peptide. H-2d-restricted HBs-specific T-cell responses dominated over SIV-Gag responses in BALB/c mice whereas HLA-A2-restricted HIV-Env response was enhanced after fusion with HBsAg. These data demonstrate that different B and T-cell epitopes of vaccine-relevant viral antigens can be expressed in vivo as fusion proteins with HBsAg but that the optimal immunogenicity may differ strikingly between individual epitopes.     

CpG寡脱氧核苷酸增强免疫抑制小鼠对乙肝疫苗的免疫应答

目的 :探讨CpG寡脱氧核苷酸 (ODN)增强免疫抑制小鼠对乙肝疫苗的免疫应答效果。方法 :选用环磷酰胺 (CTX)所致免疫抑制模型小鼠 ,以乙肝疫苗和 2 0 μgCpGODN联合或单独左胫前肌肌注免疫C5 7BL/6小鼠。 2wk后以同样剂量加强免疫 1次 ,再过 3wk后摘除眼球取血 ,用ELISA法检测抗 HBsIgG抗体和IL 12的水平。同时 ,无菌取脾脏做HE染色 ,观察脾脏淋巴细胞的数量和细胞核的变化。结果 :CpGODN与疫苗联合注射组产生的绝对抗体量比单独注射疫苗组提高 1倍 ;注射组产生IL 12的水平较单独注射疫苗组也有明显升高。光镜下各组脾脏淋巴细胞的变化如下 :正常对照组脾脏可见大量的淋巴细胞 ;与正常对照组相比较 ,CTX组的淋巴细胞明显稀少 ;而CTX加CpG组的淋巴细胞数明显增多 ,细胞核也明显增大。结论 :CpGODN能增强免疫抑制小鼠对乙肝疫苗的免疫应答     

几种不同基因免疫途径的比较研究

以乙肝表面抗原基因疫苗为模型, 采用肌肉注射、腹腔注射、表皮划痕三种免疫途径和不同剂量的裸露质粒ＤＮＡ免疫小鼠, 以ＥＬＩＳＡ方法检测小鼠血清中抗ＨＢｓＡｇ 抗体变化。实验结果表明表皮划痕方式免疫小鼠免疫反应阳性率高,且在相同剂量下其抗体滴度水平最高; 肌肉注射方式免疫小鼠, 抗体反应可维持较长时间; 腹腔注射方式免疫小鼠, 抗体反应产生快, 但维持时间较短。     

Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice

Two plasmid DNA vectors, pCAGGS(S) encoding the genes of the major envelope protein of hepatitis B virus (HBV), and pCAGGS(S + preS2) encoding the genes of the middle envelope protein were used to study the mechanism and therapeutic potential of DNA-based immunization. Injection of these plasmids into the regenerating bilateral tibialis anterior muscle (TA) of normal C57BL/6 mice induced hepatitis B surface antigen (HBsAg)-specific humoral and cellular immune responses. Seventy-two hours after injection of pCAGGS(S), infiltrating cells including antigen-presenting dendritic cells (DC) were localized around the injection site and HBsAg was expressed by both muscle cells and infiltrating cells. Spleen DC from the mice were exposed to HBsAg for up to 32 weeks after a single injection of pCAGGS(S), because these DC induced the proliferation of HBsAg-specific memory lymphocytes in culture without exogenous HBsAg. A single injection of pCAGGS(S) or pCAGGS(S + preS2) resulted in the clearance of HBsAg in 28 out of 30 HBV-transgenic (Tg) mice. In contrast, more than 7 monthly injections of an HBsAg-based vaccine were required for the clearance of HBsAg in 6 out of 29 HBV-Tg mice. Infiltrating DC at the DNA vaccine injection site may have a role in initiating HBsAg-specific immune response, whereas the persistence of HBsAg exposed spleen DC may contribute to long-lasting immunity. This study also suggested that DNA-based vaccines may be a potent tool for treating chronic HBV carriers.     

A novel HBV DNA vaccine based on T cell epitopes and its potential therapeutic effect in HBV transgenic mice

DNA vaccination represents a novel therapeutic strategy for chronic hepatitis B virus (HBV) infection. Recently, some HBV DNA vaccines have been used in the preliminary clinical trials and exhibited exciting results in chronic HBV carriers. But these vaccines only encoded the single viral antigen, the S or the PreS2/S antigen. In this study, we designed a polytope DNA vaccine encoding multiple T cell epitopes. We found that it induced stronger CTL responses than the vaccine encoding the single antigen in H-2d and H-2b mice, although the CTL response to Ld-restricted epitope suppressed the CTLs to other epitopes in H-2d-restricted mice. Interestingly, heat shock protein 70 as an adjuvant not only enhanced CTL response to the viral antigen but also overcame this epitope suppression. Furthermore, the polytope DNA vaccine resulted in a long-term down-regulation of hepatitis B virus surface antigen and inhibition of HBV DNA replication in a HBV transgenic mouse model. Therefore, our research indicates that it is practicable and feasible to design a polytope DNA vaccine for chronic hepatitis B immunotherapy.     

Response of dairy calves to vaccinia viruses that express foreign genes.

Repeated intradermal inoculations of calves with wild-type vaccinia virus and recombinant vaccinia viruses expressing human hepatitis B virus surface antigen and herpes simplex virus, type 1, glycoprotein D produced characteristic pox lesions at each site of injection. In some instances, calves were inoculated as many as five times at intervals from 4 to 7 weeks. The lesions invariably were more severe after the second inoculation. Subsequent inoculations produced a less severe area of redness, swelling, necrosis, and scab formation. No other signs of illness, such as an elevation in temperature, were noted in the calves. Vaccinia virus was isolated in low titers from scabs taken at various times after inoculation. No lesions were formed at the sites injected with tissue culture fluid and cellular debris at the same time that virus inoculations were made. Calf contact controls remained normal through the 8-week exposure in isolation units with calves inoculated twice with vaccinia virus. No neutralizing antibody to vaccinia virus was detected in the contact controls. In contrast, the virus-inoculated calves developed neutralizing antibody to vaccinia virus and to herpes simplex virus glycoprotein D in serum. In all cattle, a second inoculation significantly enhanced the neutralizing antibody response within 1 week, suggesting that an anamnestic response had occurred. No antibody to hepatitis B virus surface antigen was elicited in calves after repeated inoculations with vaccinia recombinants that express hepatitis B virus surface antigen and are known to elicit in rabbits antibodies reactive with hepatitis B virus surface antigen.