幽门螺杆菌ureI核酸疫苗免疫活性的初步研究

目的 观察幽门螺杆菌ureI核酸疫苗诱导C57BL/6小鼠产生体液免疫应答及细胞免疫应答水平.方法 重组质粒pcDNA3.1(+)/ureI、空质粒pcDNA3.1(+)及PBS分别肌注免疫C57BL/6小鼠.ELISA法检测小鼠血清中特异性IgG抗体水平以及脾淋巴细胞培养上清中IL-4和IFN-γ含量,MTT比色法检测脾淋巴细胞增殖反应,PCR和免疫荧光组化法检测ureI基因和蛋白表达.结果 pcDNA3.1(+)/ureI能够诱导小鼠产生特异性IgG抗体,该组小鼠脾淋巴细胞经UreI重组蛋白刺激后,其刺激指数和培养上清中IL-4、IFN-γ含量明显升高; ureI基因可在小鼠肌细胞中有效表达.结论 pcDNA3.1(+)/ureI核酸疫苗能刺激C57BL/6小鼠产生较强的体液免疫应答和细胞免疫应答,为进一步研究该疫苗的免疫保护作用奠定了基础.     

乙型肝炎病毒S基因与C基因免疫小鼠诱生的免疫应答

目的 观察小鼠对HBV S基因和基因疫苗的应答。方法 用已构建的HBV S基因疫苗(pCR3.1-S)和C基因疫苗(pCR3.1-C)分别给Balb/c小鼠多点肌肉注射,2wk后追加免疫一次,用ELISA法及MTT法检测小鼠血清抗体及脾细胞对HBsAg或HBcAg的特异性增殖反应。结果 免疫接种2wk后小鼠血清抗体滴度明显高于对照组,pCR3.1-C组的刺激指数明显高于pCR3.1-S注射组。结论 HBV S和C基因疫苗诱导较强的体液和细胞免疫应答强度;C基因尤以细胞免疫增高明显。     

沙眼衣原体MOMP基因真核表达载体免疫小鼠的研究

目的：用pcDNA3-MOMP真核表达质粒直接免疫小鼠，观察小鼠对沙眼衣原体MOMP基因疫苗的免疫应答。方法：大量制备重组质粒pcDNA3-MOMP,给小鼠肌肉注射，每3周免疫1次，共免疫3次。用微量免疫荧光及MTT法检测小鼠血清抗体及脾细胞对沙眼衣原体的特异性增殖反应。结果：初次免疫接种后小鼠血清中检测出特异性抗体，加强免疫后抗体水平明显上升，免疫荧光滴度最高达1：256。脾细胞对沙眼衣原体的刺激指数明显高于对照组（P＜0．01）。结论：pcDNA3-MOMP在小鼠体内既可诱导体液免疫应答，又可诱导特异性细胞免疫应答。     

乙型肝炎病毒基因疫苗诱导小鼠产生免疫应答的效应

目的 :构建编码乙型肝炎病毒 (HBV)表面蛋白S的重组质粒pCR 3 .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。方法 :以ELISA法检测小鼠血清 ,3H TdR掺入法测定淋巴细胞增殖 ,51 Cr 4h释放法检测淋巴细胞杀伤功能。结果 :与空载体对照组相比较 ,基因疫苗诱发小鼠产生良好的抗HBs反应及HBV特异的细胞免疫应答 (P <0 .0 5)。结论 :基因疫苗pCR 3 .1 S可诱导balb/c小鼠产生全面的免疫应答     

不同载体及靶基因对乙型肝炎病毒DNA疫苗免疫效果的影响

目的探讨不同DNA载体及靶基因对DNA疫苗免疫效果的影响.方法用已构建的不同载体HBV S基因疫苗(pCR3.1-S,pcDNA3-S)和HBVC基因疫苗(pCR3.1-C)分别给Balb/c小鼠多点肌肉注射,2 wk后追加免疫一次,用ELISA法及MTT法分别检测小鼠血清抗-HBs及抗HBc及脾细胞对HBsAg或HBcAg的特异性增殖反应.结果免疫接种2 wk后小鼠血清抗-HBs及抗HBc抗体均明显高于对照组,载体pCR3.1的表达效力稍高于pcDNA3,但同一基因不同载体间无显著差异.不同靶基因血清抗体滴度及脾细胞对HBsAg或HBcAg的刺激指数均明显高于对照组,刺激指数pCR3.1-C组明显高于单纯pCR3.1-S注射组.结论两种载体均可以诱导较强的体液免疫应答;但同一基因不同载体间无显著差异.HBV S和C基因疫苗均可诱导较强的体液和细胞免疫应答强度;C基因以细胞免疫增高为主.     

细粒棘球绦虫Eg95抗原基因疫苗体外瞬时表达及对小鼠诱导的免疫应答

目的　检测细粒棘球绦虫Eg95抗原基因疫苗(pcDNA3 Eg95)体外瞬时表达,探讨其诱导小鼠的体液和细胞免疫效果。　方法　pcDNA3 Eg95经脂质体转染HeLa细胞瞬时表达。逆转录聚合酶链反应(RTPCR)检测Eg95抗原信使RNA(mRNA)在HeLa细胞中的表达,酶联免疫吸附测定(ELISA)和蛋白质印迹法(Westernblot ting)检测Eg95蛋白的瞬时表达情况。pcDNA3 Eg95基因疫苗肌肉注射免疫BALB/c小鼠,ELISA检测IgG和IgG2a水平,四甲基偶氮唑盐试验(MTT法)检测免疫小鼠T淋巴细胞增殖反应。　结果　RTPCR检测结果显示,pcD NA3 Eg95瞬时表达组有Eg95抗原基因mRNA表达,ELISA和Westernblotting检测结果表明,可在HeLa细胞中特异性表达Eg95蛋白。用pcDNA3 Eg95基因疫苗免疫BALB/c小鼠,第3周出现特异性IgG免疫应答,持续升高至第10周,显著高于对照组。从第2周开始,小鼠血清IgG2a应答即为阳性,且长时间(至第10周 )维持较高水平,与pcD NA3空质粒组比较,其差异具有非常显著性意义(P<0.01)。用原核表达的Eg95重组蛋白刺激免疫小鼠脾细胞,有明显的T细胞增殖反应。pcDNA3 Eg95基因疫苗免疫组刺激指数明显高于pcDNA3空质粒组(P<0.01)。结论　pcDNA3 Eg95基因疫苗可诱发小鼠产生特异的体液免疫和细     

细粒棘球绦虫Eg95基因疫苗和重组抗原诱导小鼠免疫应答的比较研究

目的观察比较细粒棘球绦虫Eg95重组抗原和基因疫苗诱导小鼠的免疫应答状况。方法实验组和对照组小鼠分别注射Eg95重组抗原(rEg95)、费氏佐剂(FCA)、pcDNA3-Eg95基因疫苗、pcDNA3质粒和生理盐水,收集各组血清用酶联免疫吸附(ELISA法)检测抗体IgG和IgG2a亚类水平;采集脾细胞用四甲基偶氮唑盐试验(MTT法)检测免疫小鼠的脾淋巴细胞增殖反应。结果rEg95免疫组小鼠在第二次免疫后开始检测到抗Eg95抗原的IgG,并随着免疫次数的增多,血清抗体效价升高,在第1次免疫后第10周时,免疫抗体滴度可达到1∶25,600。Eg95基因疫苗免疫的小鼠产生抗体滴度随免疫次数的增加而升高,最高可达1∶3,200,但是低于Eg95重组蛋白免疫小鼠产生的抗体滴度水平。pcDNA3-Eg95免疫组产生IgG2a亚类抗体水平明显高于对照组和rEg95组。在第四次免疫后,进行淋巴细胞转化试验,MTT法检测证实rEg95和pcD-NA3-Eg95免疫的小鼠,其脾细胞均可在体外被特异性刺激增生。结论细粒棘球绦虫Eg95重组抗原和基因疫苗均可诱发小鼠产生特异性免疫应答。     

多发性骨髓瘤MUC1-2VNTR基因疫苗诱导小鼠免疫应答的实验研究

目的观察黏蛋白1两串联重复区（MUC1-2VNTR）基因疫苗诱导BALB／c小鼠体液及细胞免疫应答的效果。方法将含MUC12VNTR的重组质粒peDNA3．1-2VNTR／myc-hisB免疫小鼠,免疫后采用ELISA动态检测血清中抗VNTR特异性抗体的水平,乳酸脱氢酶法检测细胞毒性T细胞（CTL）反应活性,MTS法检测脾淋巴细胞增殖活性。结果重组质粒免疫组小鼠血清中检出了MUC1-2VNTR抗原特异性抗体。用重组质粒免疫BALB／c小鼠后,在效靶比为80：1时可显著地诱导特异性CTL应答。在MUC1-2VNTR抗原肽的刺激下,免疫小鼠脾淋巴细胞得到增殖;与空质粒对照组比较,差异有统计学意义（P〈0．01）。结论MUC1-2VNTR基因疫苗可诱导小鼠产生特异性细胞及体液免疫应答,对多发性骨髓瘤的疫苗治疗有重要意义。     

IL-15表达质粒联合HPV基因疫苗诱导细胞免疫应答的实验研究

目的研究白细胞介素15(IL-15)真核表达质粒,对人乳头瘤病毒(HPV)16E7基因疫苗所诱导的小鼠特异性细胞免疫应答的影响。方法构建含IL-15的真核表达质粒pcDNA3.1-IL-15。将该质粒与HPV16 E7基因疫苗通过肌肉注射方式免疫雌性BALB/c小鼠。基因免疫后测定其血清γ-干扰素(IFN-γ)水平;并制备脾淋巴细胞悬液,经体外E7蛋白再刺激后用MTT法检测其T淋巴细胞增殖情况。结果pcDNA3.1-IL-15与pcD-NA3.1-E7共同注射,可以提高免疫小鼠血清中IFN-γ水平至414.1pg/ml,与E7 空质粒组、E7组比较差异有统计学意义(P<0.01)。pcDNA3-1-IL-15与pcDNA3.1-E7共同注射,可以增强特异性T细胞增殖反应,OD570差值为1.313,与其他各组比较差异均有统计学意义(P<0.01)。结论IL-15真核表达质粒可以提高HPV16 E7基因疫苗的免疫原性,增强小鼠细胞免疫应答。     

复合鼠疫疫苗F1+ rV的免疫学研究

目的通过由提取的鼠疫F1抗原和重组鼠疫V抗原免疫小鼠,评价鼠疫亚单位疫苗的免疫效果,为鼠疫疫苗研制奠定基础。方法将60只小鼠随机分为3组实验组,免疫后不同时间点进行血清ELISA检测、ELISPOT、MTT 细胞增殖以及流式细胞分析。结果ELISA检测结果显示F1+rV+Al(OH)3组有较强的体液免疫应答;ELISPOT、MTT 细胞增殖结果显示F1+rV+Al(OH)3组在不同时间点经F1、rV抗原刺激后分泌IFN γ的脾脏淋巴细胞数均高于其余各组。结论鼠疫双组分疫苗能引起小鼠明显的体液免疫和细胞免疫,Al(OH)3佐剂能够显著提高其应答水平。     

表达口蹄疫病毒复合多表位基因的重组鸡痘病毒以及DNA疫苗在豚鼠中的免疫原性研究

目的评价本课题组构建的共表达FMDV复合多表位基因以及猪白细胞介素18基因的非复制型重组鸡痘病毒活载体疫苗vUTA2-OAT以及DNA疫苗pVRI-OAT诱导FMDV模型动物豚鼠产生特异性体液免疫和细胞免疫的能力。方法将上述2种重组疫苗分别单独或联合接种豚鼠,然后测定FMDV特异性结合抗体、中和抗体和T淋巴细胞增殖反应,并用250ID50的FMDV进行攻击,观察其保护效果。结果这2种重组疫苗均能诱导豚鼠产生特异性的体液免疫及细胞免疫应答。其中以pVRI-OAT/vUTA2-OAT的联合免疫方式的效果最好,其诱导的抗体水平接近于常规灭活疫苗,而细胞免疫水平则比后者高得多。攻击保护结果显示3个重组疫苗组均有一定保护效果,完全保护率和部分保护率分别为1/4和2/4。而PBS对照组发病率为4/4。结论证实了这2种重组疫苗在豚鼠体内具有良好的免疫原性。上述研究结果为FMDV基因工程疫苗的研究奠定了基础。     

Combined Immunization of Mice with DNA, rMVA and rAd5 Expressing HIV-1 Structural Genes from Different Subtypes

The objective of present paper is to study the immunogenicity of combinations of multiple vector vaccines expressing HIV-1 structural genes from different subtypes. Mice were vaccinated with DNA (B'/C) and rMVA (B'/C) vaccines expressing B'/C recombinant subtype gag-pol and env genes, DNA (B') and rAd5 (B') vaccines expressing subtype B' gag gene with different combination schemes. HIV-1 Gag-specific cellular immune responses and P24- specific IgG levels were analyzed by IFN-γ enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) respectively. ELISPOT results indicated that the Gag-specific cellular immune responses induced by combination of three vaccines were much higher than that induced by combination of two vaccines. Among the groups of mice immunized with two vaccines, the groups with rAd5 booster elicited higher cellular immune responses compared with the groups with rMVA booster. All the test groups of three vaccines in combination could induce similar level of cellular immune responses, which did not correlate with the immunization order. ELISA results showed that p24- specific IgG induced by combination of three vaccines were much higher than that induced by combination of two vaccines. It indicates that the combination scheme of multiple vector vaccines maybe a promising AIDS vaccine strategy.     

Vaccination with DNA vaccines encoding MPB70 or MPB83 or a MPB70 DNA prime-protein boost does not protect cattle against bovine tuberculosis

SETTING: Bovine tuberculosis is a problem in a number of countries and protection of cattle by vaccination could be an important control strategy. OBJECTIVES: To determine the ability of DNA vaccines, which express the mycobacterial antigens MPB83 and MPB70 and a DNA prime-protein boost strategy to stimulate immune responses in cattle and protect against bovine tuberculosis. DESIGN: Groups of cattle (n=10) were vaccinated with MPB83 DNA, MPB70 DNA, or MPB70 DNA followed by MPB70 protein or injected with BCG or control plasmid DNA. Animals were challenged intratracheally with virulent Mycobacterium bovis at 13 weeks and protection assessed 17 weeks later at postmortem. RESULTS: In contrast to the strong cellular immune responses induced by BCG, the DNA vaccines induced minimal interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) responses. Cattle primed with MPB70 DNA and boosted with MPB70 protein induced a strong antibody response and a weak IFN-gamma response. BCG gave significant reduction in four pathological parameters of disease while the DNA vaccines and MPB70 DNA/protein did not protect animals against challenge with M. bovis. Moreover, cattle vaccinated with MPB70 DNA/protein had a significantly higher proportion of animals with severe lung lesions (>100 lesions) than the MPB70 DNA alone or the control group. Increased bovine PPD-specific IL-4 mRNA expression in cattle, post-challenge, correlated with the presence of tuberculous lung lesions. CONCLUSION: Vaccination of calves with MPB70 or MPB83 DNA vaccines or with a more immunogenic MPB70 DNA prime-protein boost strategy did not induce protection against bovine tuberculosis.     

DNA vaccines

BACKGROUND AND OBJECTIVES: Humoral and cellular immune responses to protein antigens can be efficiently primed by nucleic acid or DNA vaccination. In DNA-based vaccination, immunogenic proteins are expressed with correct posttranslational modification, conformation or oligomerization; this ensures the integrity of epitopes that stimulate neutralizing antibody (B cell) responses. DNA (or RNA) immunization is exceptionally potent in stimulating T cell responses because antigenic peptides are efficiently generated in (endogenous or exogenous) processing pathways (without interference by viral proteins) from intracellular or extracellular protein antigens expressed after transient in vivo transfection. Both features are difficult to achieve with recombinant subunit vaccines produced in eukaryotic or prokaryotic expression systems. The current state of vector designs, strategies for delivery of DNA vaccines, priming humoral and cellular immune responses by DNA vaccines, experimental strategies facilitated by DNA vaccines, unique advantages of DNA vaccination, experience of DNA vaccination in preclinical animal models and clinical trials, and potential risks of DNA vaccination are discussed. Excellent reviews on DNA-based vaccination have been published recently [1-3].     

Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

INTRODUCTION Hepatitis C continues to be a severe health threat to a large population with about 123 million people being affected globally[1]. The etiologic agent, hepatitis C virus (HCV)[2] is able to establish persistent infections in up to 85% of infe…     

DNA immunization with fusion genes encoding different regions of hepatitis C virus E2 fused to the gene for hepatitis B surface antigen elicits immune responses to both HCV and HBV

AIM：Both Hepatitis B virus(HBV)and Hepatitis C virus (HCV) are major causative agents of transfusion-associated and community-acquired hepatitis wordwide.Development of a HCV vaccine as well as more effective HBV vaccines is and urgent task.DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against viral infection.The aim of this study is to achieve immune responses against both HCV and HBV by DNA immunization with fusion constructs comprising various HCV E2 gene fragments fused to HBsAg gene of HBV.METHODS:C57BL/6 mice were immunized with plasmid DNA expressing five fragmets of HCV E2 fused to the gene for HBaAg respectively.After one primary and one boosting immunizatios,antibodies against HCV E2 and HBsAg weretested and subtyped in ELISA.Splenic cytokine expression of IFN-γ and IL-10 was analyzed using an RT-PCR assay Post-immune mouse antisera also were tested for their ability to capture HCV viruses in the serum of a hepatitis C patient in vitro.RESULTS:After immunization,antibodies against both HBsAg and HCV E2 were detected in mouse sera,with Ig2a being the dominant immunoglobulin sub-class.Highlevel expression of INF-γwas detected in cultured splenic cells Mouse antisera against three of the five nfusion constructs were able to capture HCV viruses in an in vitro assay.CONCLUSION:The results indicate that these fusion constructs could efficiently elicit humoral and Th1 dominant cellular immune responses against both HBV S and HCV E2 antigens in DNA-immunized mice.They thus could serve as candidates for a bivalent vaccine against HBV and HCV infection.In addition,the capacity of mouse antisera against three of the five fusion constructs to capture HCV viruses in vitro suggested that neutralizing epitopes may be present in other regions of E2 besides the hypervariable region1.     

日本血吸虫混合DNA疫苗pBK-CMV-Sj26/Sj32诱导小鼠的免疫应答

目的　观察含日本血吸虫候选疫苗分子谷胱甘肽S -转移酶 (Sj2 6 )和天冬酰胺肽链酶 (Sj32 )的真核载体 pBK-CMV -Sj2 6 /Sj32诱导小鼠的免疫应答情况。 方法　将质粒按 5 0 μg/只在 0w ,3w ,5w免疫小鼠股四头肌 ,第一次免疫后取小鼠脾细胞作T淋巴细胞转化实验 ;在 0w、3w和 6w经小鼠尾静脉取血 ,ELISA检测血清中抗体效价及阳性反应率。结果　淋巴细胞转化实验示血吸虫成虫抗原能特异性刺激免疫鼠脾细胞 ;第一次免疫后特异性抗体效价开始升高。抗体最高效价为 1∶2 0 ,3次免疫后有 91.7%小鼠血清呈阳性反应。结论　真核载体 pBK -CMV -Sj2 6 /Sj32可诱导小鼠体液及细胞免疫应答     

日本血吸虫混合DNA疫苗pBK-CMV-Sj26/Sj32诱导小鼠的免疫应答

目的　观察含日本血吸虫候选疫苗分子谷胱甘肽S -转移酶 (Sj2 6 )和天冬酰胺肽链酶 (Sj32 )的真核载体 pBK-CMV -Sj2 6 /Sj32诱导小鼠的免疫应答情况。 方法　将质粒按 5 0 μg/只在 0w ,3w ,5w免疫小鼠股四头肌 ,第一次免疫后取小鼠脾细胞作T淋巴细胞转化实验 ;在 0w、3w和 6w经小鼠尾静脉取血 ,ELISA检测血清中抗体效价及阳性反应率。结果　淋巴细胞转化实验示血吸虫成虫抗原能特异性刺激免疫鼠脾细胞 ;第一次免疫后特异性抗体效价开始升高。抗体最高效价为 1∶2 0 ,3次免疫后有 91.7%小鼠血清呈阳性反应。结论　真核载体 pBK -CMV -Sj2 6 /Sj32可诱导小鼠体液及细胞免疫应答     

采用流式细胞仪检测艾滋病病毒1型膜蛋白小鼠免疫后IFN-γ的表达

目的通过流式细胞技术检测艾滋病病毒1型(HIV-1)Gp120膜蛋白免疫小鼠后的IFN-γ的表达。方法 构建表达HIV-1gp120基因的复制型重组痘苗病毒。与DNA疫苗联合免疫小鼠,用流式细胞仪检测小鼠脾淋巴细胞中IFN-γ的表达。结果 获得表达中国HIV-1流行株gp120基因的重组痘苗病毒rVVgp120、rVVM304,能正确表达Gp120蛋白。与DNA疫苗p120-VRC、pM304-VRC联合免疫小鼠后,用流式细胞仪检测到小鼠脾淋巴细胞能特异性的表达IFN-γ。结论 用流式细胞检测技术成功检测到免疫小鼠的脾淋巴细胞特异性的表达IFN-γ,用此方法可方便快捷地检测小鼠的细胞免疫效果。     

血吸虫疫苗的研究进展

血吸虫蛋白疫苗以血吸虫的抗原为主要成分,诱导机体产生抗体。重组蛋白疫苗融合多种血吸虫抗原,诱导机体产生多种抗体,因此可提高机体对血吸虫的抵抗力。热休克蛋白疫苗以HSP为主要成分,既是血吸虫的抗原物,又可增强机体细胞免疫水平,提高机体的免疫力。DNA疫苗能同时诱发体液免疫和细胞免疫,具有众多传统疫苗无法比拟的优点。抗日本血吸虫DNA疫苗的研究在实验动物已取得了一定进展,多价疫苗、联合疫苗为未来疫苗的研究提供了发展方向。