我国人轮状病毒不同基因型NSP4的致病性研究

对我国轮状病毒流行株NSP4基因变异特点的分析表明,NSP4基因主要可分为Wa组和Kun组,在Wa组内可形成三个亚组,形成了4种NSP4基因型.为了进一步阐明人轮状病毒流行株NSP4基因变异与其致病性变化是否存在联系,我们首先利用杆状病毒载体对NSP4蛋白进行表达,获得了对应4种不同NSP4基因型的重组杆状病毒rvBac97B6,rvBac97S34,rvBac97S36和rvBac97SZ8.用这些病毒感染Sf-9细胞后,检测细胞内Ca2+浓度的变化,发现与野生型杆状病毒感染细胞相比,重组病毒感染细胞内的Ca2+浓度显著升高,但各个重组病毒之间无显著性差异.在此基础上,我们进一步在E.coli中分别表达纯化了代表Wa和Kun基因分组的97S34和97SZ8流行株的NSP4.分别用纯化的重组NSP4蛋白攻击乳鼠后,发现不同基因型的NSP4蛋白的致腹泻活性没有明显差异,这种作用可被NSP4抗体拮抗,但这种拮抗作用存在基因型特异性.上述结果表明人轮状病毒流行株NSP4氨基酸序列间的变异并没有使其钙调节及致腹泻能力产生改变,在致腹泻作用中发挥关键作用(或决定性作用)的氨基酸位点在不同NSP4基因型间可能是相对保守的.针对NSP4抗体的有效性也为新型轮状病毒疫苗和药物研究提供了线索.     

我国人轮状病毒不同基因型NSP4的致病性研究

对我国轮状病毒流行株NSP4基因变异特点的分析表明,NSP4基因主要可分为Wa组和Kun组,在Wa组内可形成三个亚组,形成了4种NSP4基因型。为了进一步阐明人轮状病毒流行株NSP4基因变异与其致病性变化是否存在联系,我们首先利用杆状病毒载体对NSP4蛋白进行表达,获得了对应4种不同NSP4基因型的重组杆状病毒rvBac97B6,rvBac97S34,rvBac97S36和rvBac97SZ8。用这些病毒感染Sf9细胞后,检测细胞内Ca2 浓度的变化,发现与野生型杆状病毒感染细胞相比,重组病毒感染细胞内的Ca2 浓度显著升高,但各个重组病毒之间无显著性差异。在此基础上,我们进一步在E.coli中分别表达纯化了代表Wa和Kun基因分组的97S34和97SZ8流行株的NSP4。分别用纯化的重组NSP4蛋白攻击乳鼠后,发现不同基因型的NSP4蛋白的致腹泻活性没有明显差异,这种作用可被NSP4抗体拮抗,但这种拮抗作用存在基因型特异性。上述结果表明人轮状病毒流行株NSP4氨基酸序列间的变异并没有使其钙调节及致腹泻能力产生改变,在致腹泻作用中发挥关键作用(或决定性作用)的氨基酸位点在不同NSP4基因型间可能是相对保守的。针对NSP4抗体的有效性也为新型轮状病毒疫苗和药物研究提供了线索。     

表达轮状病毒nsp4基因重组腺病毒的构建与免疫评价

目的:应用非复制腺病毒表达系统构建表达人轮状病毒非结构蛋白4(NSP4)的重组腺病毒,初步评价其免疫保护效果。方法:构建含野生轮状病毒NSP4基因的穿梭质粒pshuttle-NSP4,与腺病毒骨架质粒pAdeasy经同源重组后在Ad-293细胞中包装获得pAd-NSP4重组腺病毒颗粒。电镜、RT-PCR、免疫荧光等方法鉴定病毒特征及在体外细胞中的表达。肌肉注射及滴鼻方式免疫小鼠,检测小鼠血清抗体效价及其中和保护效果。结果:获得了滴度为108.25CCID50/ml的重组腺病毒pAd-NSP4,免疫荧光检测到特异性目的蛋白的表达。二次免疫后肌肉注射和滴鼻小鼠的ELISA血清平均效价分别为1∶320和1∶1436.8;中和抗体效价1∶45.3和1∶71.8。结论:表达轮状病毒NSP4蛋白的非复制型重组腺病毒颗粒具有良好的免疫原性。滴鼻途径比肌肉注射可更加有效地诱导小鼠的免疫应答。     

A组轮状病毒NSP1基因克隆表达及免疫学性质研究

轮状病毒（rotavirus, RV）非结构蛋白1（non structural protein 1, NSP1）在病毒与宿主的相互作用中发挥着重要的功能。运用基因克隆和表达技术在大肠杆菌中表达了TB-Chen株RV NSP1蛋白,进行了NSP1的免疫学性质和RV感染细胞中NSP1蛋白的合成与分布以及NSP1的系统进化和基因分型研究。结果表明,大肠杆菌BL21(DE3)能高效表达重组NSP1蛋白（rNSP1）,rNSP1表达量约占菌体总蛋白的34.4%。rNSP1能诱导免疫豚鼠产生特异性血清抗体。Western blot及免疫荧光检测结果表明,抗rNSP1血清抗体能特异性识别自身蛋白,对SA11、Wa株的NSP1蛋白有交叉反应性;免疫荧光结果还表明,SA11感染的MA104细胞中合成的NSP1蛋白在细胞质中区域化聚集形成辐射状排列的颗粒状结构,而Wa株的NSP1不能形成此样结构。至今发现的A组RV至少可以分为16个不同的NSP1基因型,TB-Chen株NSP1为A2型。不同基因型有独特的敏感宿主范围,同一基因型可能感染不同种动物,同一种动物也可能感染不同基因型。基因型A4型和A16型仅在鸟类病毒株中出现;而且鸟类中只有A4型和A16型。研究结果为进一步研究NSP1蛋白质的结构功能及其应用开发奠定了很好的基础。     

表达轮状病毒nsp4基因重组腺病毒的构建与免疫评价

目的：应用非复制腺病毒表达系统构建表达人轮状病毒非结构蛋白4（NSP4）的重组腺病毒,初步评价其免疫保护效果。方法：构建含野生轮状病毒NSP4基因的穿梭质粒pshuttle-NSP4,与腺病毒骨架质粒pAdeasy经同源重组后在Ad-293细胞中包装获得pAd-NSP4重组腺病毒颗粒。电镜、RT-PCR、免疫荧光等方法鉴定病毒特征及在体外细胞中的表达。肌肉注射及滴鼻方式免疫小鼠,检测小鼠血清抗体效价及其中和保护效果。结果：获得了滴度为10^8.25CCID50/ml的重组腺病毒pAd-NSP4,免疫荧光检测到特异性目的蛋白的表达。二次免疫后肌肉注射和滴鼻小鼠的ELISA血清平均效价分别为1：320 和1：1436.8;中和抗体效价1：45.3和1：71.8。结论：表达轮状病毒NSP4蛋白的非复制型重组腺病毒颗粒具有良好的免疫原性。滴鼻途径比肌肉注射可更加有效地诱导小鼠的免疫应答。     

A组人轮状病毒NSP3基因的克隆及其在大肠杆菌中的表达

目的:获得轮状病毒NSP3基因的表达产物及其抗血清。方法:将TB-Chen株轮状病毒NSP3基因插入质粒pETL,构建重组表达质粒pET-NSP3,并转化大肠杆菌BL21(DE3)表达重组蛋白NSP3;用凝胶分离回收的方法纯化该蛋白,免疫豚鼠制备该蛋白的抗血清。结果:构建了重组表达质粒pET-NSP3,并在大肠杆菌中高效表达了重组蛋白NSP3,目的蛋白表达量占菌体总蛋白量的28.6%;有效地纯化了目的蛋白并制备了该蛋白的抗血清,Western印迹表明该抗血清能与重组蛋白NSP3发生特异性免疫反应。结论:通过质粒pETL能高效表达轮状病毒NSP3蛋白,该重组蛋白具有较好的免疫反应性,为进一步研究其结构、功能及免疫学性质奠定了基础。     

用小鼠模型分析可溶性表达结核分枝杆菌Ag85A的免疫原性

【目的】可溶性表达结核分枝杆菌Ag85A蛋白,并评价其免疫原性。【方法】利用冷休克表达质粒和含有伴侣质粒的大肠杆菌对Ag85A蛋白进行可溶性原核表达,并进行纯化与鉴定,通过C57BL/6小鼠模型对Ag85A蛋白的免疫原性,包括诱导机体特异性体液免疫应答和细胞免疫应答水平进行分析。【结果】重组菌诱导后裂解上清中检测到可溶性Ag85A蛋白的表达,经过亲和层析纯化收获了纯度在90%以上的Ag85A蛋白,Western blot鉴定显示其具有较好的免疫反应性。Ag85A蛋白免疫小鼠后,血清中可以检测到高水平的Ig G抗体效价,其中Ig G2b水平要高于Ig G1。通过特异性多肽、蛋白刺激脾脏和腹股沟淋巴结细胞可分泌高水平的IFN-γ、TNF-α等Th1型细胞因子。【结论】实现了Ag85A蛋白的可溶性表达,免疫特性评价显示Ag85A蛋白可诱导机体产生强烈的特异性体液免疫应答及Th1型的细胞免疫应答,从而为其进一步免疫学功能的研究奠定了重要基础。     

A组轮状病毒NSP6蛋白表达及免疫学性质研究

轮状病毒(RV）NSP6与NSP5由同一基因片段编码,至今对NSP6 性质了解很少。用基因重组表达和免疫学方法,重组表达了A组人RV NSP6蛋白,进行了NSP6的动物免疫及其抗原反应性、免疫原性研究以及RV感染细胞中NSP6的合成及亚细胞分布研究。研究结果表明,NSP6可在原核系统中高效表达,表达蛋白占菌体总蛋白的34.2%;NSP6免疫豚鼠血清抗体可特异性识别菌体细胞中表达的NSP6和SA11及Wa病毒感染的MA104细胞中合成的NSP6蛋白;病毒感染细胞中合成的NSP6在感染后3h就可检测到,12h表达量达到最高;NSP6在病毒感染细胞质中呈弥散状分布,并主要积聚在细胞核的周围,未观察到毒质体样结构。研究结果对深入了解RV NSP6的结构与功能具有重要的意义,具有重要的潜在应用价值。     

鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达

目的:构建鼠肝炎冠状病毒(MHV)非结构蛋白1(NSP1)及其突变体(NSP1 mu)的原核重组表达质粒,在大肠杆菌中分别融合表达重组NSP1及NSP1 mu。方法:以现有质粒载体为模板,扩增编码NSP1及NSP1 mu的基因片段,并克隆至pMD18-T克隆载体;菌落PCR鉴定阳性克隆并测序分析;将阳性克隆的目的片段亚克隆至表达载体pET-28a,并转化大肠杆菌TOP10感受态细胞,PCR和双酶切鉴定转化菌落;将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞并加入IPTG诱导表达,SDS-PAGE和免疫印迹分析目的蛋白的表达。结果:PCR扩增得到表达NSP1及NSP1 mu的特异片段,并克隆到pMD18-T载体,测序结果正确无误;构建了NSP1和NSP1 mu的重组表达质粒,并在大肠杆菌BL21(DE3)中分别融合表达了重组NSP1及NSP1 mu,表达的目的蛋白均能与His单克隆抗体特异结合;用Ni-NTA琼脂糖试剂盒纯化重组蛋白,获得可溶性的NSP1及NSP1 mu,相对分子质量分别为27×103和28×103。结论:在大肠杆菌中分别表达并纯化获得了大量可溶性重组NSP1及NSP1 mu。     

人乳头瘤病毒(HPV)58型DNA疫苗免疫原性的初步分析

为了检测HPV 58型不同L1基因的DNA疫苗的免疫原性,以pcDNA3.1为载体分别构建含HFV 58型不同L1基因的DNA疫苗,命名为L1h、L1h△c、L1S、L1SM和L1wt.用免疫印迹法检测各DNA疫苗的体外表达情况;各重组质粒与pcDNA3.1-h58L2和pcDNA3.1-GFP共转染293FT细胞,检测其形成假病毒的能力;并将各DNA疫苗肌肉注射免疫小鼠,利用假病毒中和实验检测中和抗体水平,用ELISPOT检测细胞免疫情况.结果显示,本实验成功构建了五种DNA疫苗,L1h△c的体外表达量最高,L1S和L1SM的表达量次之,L1wt没有表达;重组质粒L1S能够形成假病毒,而其他四种重组质粒均不能形成假病毒.L1S和L1h均可在小鼠体内诱导中和抗体,但L1S诱导的中和抗体的平均滴度为1:6 400,明显高于L1h诱导的中和抗体水平(平均滴度为1:48),而其他疫苗在小鼠体内未产生中和抗体.对五种疫苗均未检测出特异性的细胞免疫反应.结果提示,体外能够组装成假病毒的DNA疫苗在免疫动物后可诱导高滴度的中和抗体,为今后DNA疫苗的筛选提供参考.     

新型呼肠病毒 S基因 DNA 疫苗在小鼠体内的免疫原性

目的：探讨新型呼肠病毒R4株S片段免疫小鼠后引发的免疫应答。方法构建4个不同S基因节段的重组真核表达质粒,并免疫小鼠;ELISA检测血清以研究R4特异性抗体升高水平,并对其抗体亚型进行鉴定;ELISPOT检测小鼠淋巴细胞INF-γ的表达情况。结果与对照组相比,4个重组质粒免疫的小鼠血清都有明显的R4特异性抗体升高,尤其以S1和S3基因免疫后抗体水平较高,且均以IgG2a占绝对优势;S1基因免疫组小鼠的细胞免疫应答最强。结论 S1基因重组质粒免疫小鼠后可同时引发较强的体液免疫和细胞免疫应答,是较为理想的疫苗备选基因片段。     

融合表达小鼠IgG2b-Fc可增强人类免疫缺陷病毒1型Gag DNA疫苗的免疫原性

为阐明小鼠IgG2b-Fc对DNA疫苗免疫原性的增强作用,首先构建表达人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)CN54Gag基因的DNA疫苗及表达Gag与小鼠IgG2b-Fc融合基因的DNA疫苗,限制性酶切和DNA测序结果表明这两个疫苗均构建成功,蛋白免疫印迹结果也显示其正确表达。然后,利用上述DNA疫苗接种C57BL/6小鼠,比较两个DNA疫苗所诱导的特异性体液免疫反应和特异性细胞免疫反应。结果显示,融合表达小鼠IgG2b-Fc对特异性细胞免疫和体液免疫反应均有增强作用,但只有对特异性体液免疫反应的增强作用有统计学意义。     

Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.     

C3d3通过促进脾脏B细胞高表达CXCR4增强hCGβ DNA免疫体液免疫效应

本文旨在探讨分子佐剂C3d3与hCGβ融合在基因免疫中增强抗hCGβ体液免疫效应的机制。分别用质粒pCMV4-hCGB-C3d3、pCMV4-hCGβ和pCMV4免疫BALB/c小鼠,间接ELISA法检测免疫小鼠外周血IgG/IgA类抗hCGβ抗体水平;ELISPOT分析免疫鼠脾脏组织IgG/IgA类抗体分泌细胞水平(ASC);RT-PCR分析免疫鼠脾脏B细胞趋化因子受体表达,RT-PCR和FCM分析CXCR4表达水平;RT-PCR和ELISA检测脾脏组织CXCL12表达水平。结果显示,pCMV4- hCGβ-C3d3免疫组外周血IgG类抗hCGβ抗体水平明显高于pCMV4-hCGβ免疫组;而IgA类抗hCGβ抗体水平在两组间无明显差异。pCMV4-hCGβ-C3d3免疫组脾脏组织IgG类ASCs水平明显高于pCMV4-hCGβ组;两组间IgA类ASCs水平无明显差异。经pCMV4-hCGB、pCMV4-hCGβ- C3d3免疫鼠脾脏B细胞CXCR4表达明显高于对照组;且pCMV4-hCGβ-C3d3组明显高于pCMV4-hCGβ免疫组。CXCR4~ 细胞与ASCs呈正相关,r=0.966,(P<0.05)。pCMV4-hCGβ-C3d3和pCMV4-hCGβ组脾脏组织CXC L12表达均显著高于对照组。结果表明,分子佐剂C3d3与hCGβ基因融合,在基因免疫小鼠后能够显著升调节脾脏ASCs CXCR4表达,从而可能增强抗hcGβ基因疫苗的体液免疫效应。     

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.     

DNA immunization with Plasmodium falciparum serine repeat antigen: regulation of humoral immune response by coinoculation of cytokine expression plasmid

We immunized mice with plasmid expressing the 47-kDa amino-terminal domain of the Plasmodium falciparum serine repeat antigen (SERA) using gene gun and investigated humoral immune response to SERA antigen. Significant SERA-specific IgG was observed in BALB/c mice after immunization three times with SERA expression plasmid. Furthermore, these levels were increased by the coinoculation of cytokine (IFN-gamma, IL-4, GM-CSF, or IL-12) expression plasmid. In respect to the SERA-specific Ig subclasses, coinoculation of IFN-gamma, GM-CSF, or IL-12 expression plasmid increased the levels of SERA-specific IgG2a, and these were much higher than that in mice immunized with SERA expression plasmid alone. In contrast to the SERA-specific IgG2a, coinoculation of any cytokine expression plasmid did not change the levels of SERA-specific IgG1. These results indicate that cytokine expression plasmid enhances and regulates humoral immune response elicited by SERA DNA immunization.     

Immune responses with DNA vaccines encoded different gene fragments of severe acute respiratory syndrome coronavirus in BALB/c mice

To analyze the immune responses of DNA vaccine encoded different gene fragments of severe acute respiratory syndrome coronavirus (SARS-Cov), SARS-Cov gene fragments of membrane (M), nucleocapsid (N), spike a (Sa), and spike b (Sb) proteins were cloned into pcDNA3.1 (Invitrogen) vector to form plasmids pcDNAM, pcDNAN, pcDNASa, and pcDNASb, respectively. After mice were immunized intramuscularly with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmid, blood was collected and serum was separated. Humoral immune response was detected with the enzyme-linked immunosorbent assay, and cellular immune response of SARS-Cov DNA vaccines was detected with lymphoproliferation assay and cytotoxic T lymphocyte assay. Results show that cellular and humoral immune responses can be detected after immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb plasmids in BALB/c mice. However, pcDNAM stimulated the highest cellular immune response than other plasmids, and pcDNASa-pcDNASb stimulated the highest humoral immune response in week 12. The present results not only suggest that DNA immunization with pcDNAM, pcDNAN or pcDNASa-pcDNASb could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also to illustrate that gene immunization with these SARS DNA vaccines different immune response characters.     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

免疫共刺激分子OX40L对乙型肝炎核酸疫苗的免疫佐剂作用

[目的]为了进一步增强HBV DNA疫苗的免疫反应,本研究将共刺激分子OX40L 作为HBV DNA疫苗的分子佐剂免疫小鼠,旨在探讨共刺激分子OX40L对HBV DNA疫苗诱导体液和细胞免疫应答的影响.[方法]我们将HBV DNA疫苗(pcDS2)单独或联合共刺激分子质粒pOX40L免疫C57BL/6小鼠;分别在第0,2,4周进行免疫,在第6周检测抗-HBs IgG、IgG1和IgG2a,T淋巴细胞增殖指数,细胞因子表达水平和体内细胞毒性T淋巴细胞杀伤作用(CTL)等免疫学指标.[结果]pceDS2联合pOX40L免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠的T淋巴细胞体外经乙型肝炎表面抗原(HBsAg)刺激后,联合免疫组刺激指数(SI)明显高于pcDS2组;联合免疫组CD4 + T淋巴细胞的IL-4和IFN-γ表达水平及CD8 + T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体内CTL高于对照组,其中联合免疫组小鼠的体内CTL杀伤作用最强.[结论]共刺激分子OX40L不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性细胞免疫反应,尤其增强体内CTL的杀伤活性,为HBV DNA疫苗的研究奠定了基础.     

牛乳源金黄色葡萄球菌EsxA蛋白的表达及免疫原性分析

为分析牛乳源金黄色葡萄球菌(Staphylococcus aureus)EsxA蛋白的免疫原性,构建EsxA-p ET-28a重组表达质粒,重组质粒经诱导表达后进行SDS-PAGE和Western blotting鉴定。用纯化后重组EsxA蛋白免疫小鼠,用间接ELISA检测免疫小鼠血清中的IgG、IgG1和IgG2a水平;免疫小鼠经S.aureus菌株攻击后,检测小鼠肝、脾、肾组织荷菌数和免疫保护率,观察S.aureus菌株攻击后小鼠肝、脾、肾病理组织学变化。结果表明,成功诱导表达了EsxA重组蛋白,该重组蛋白免疫小鼠后血清抗体效价可达1∶900,与对照相比,重组蛋白免疫后可减少小鼠肝、脾、肾组织的荷菌数,减轻这些脏器的病理损伤,对免疫小鼠保护率达75%。上述结果表明,该重组Esx A蛋白具有良好的免疫原性。