牛疱疹病毒Ⅰ型VP22和猪繁殖与呼吸综合征病毒GP5融合基因DNA疫苗的免疫效应

为了提高表达GP5的猪繁殖与呼吸综合征病毒（PRRSV）DNA疫苗的免疫效应,将具有蛋白转导功能的牛疱疹病毒1型（BHV-1）VP22基因插入到经过修饰具有更好免疫原性的PRRSV修饰型ORF5基因（ORF5M）上游,构建VP22和ORF5M融合表达的真核表达质粒pCI-VP22-ORF5M。经间接免疫荧光试验（IFA）和Westernblot检测证实体外表达后,免疫BALB/c小鼠,检测小鼠免疫后的GP5特异性ELISA抗体、抗PRRSV中和抗体和脾淋巴细胞增殖反应,并与非融合的真核表达质粒pCI-ORF5M进行比较。结果显示,融合表达VP22-GP5的DNA疫苗 pCI-VP22ORF5M诱导的体液免疫和细胞免疫反应均明显高于非融合表达的DNA疫苗pCI-ORF5M,表明蛋白转导相关蛋白BHV-1 VP22能显著增强表达GP5的PRRSV DNA 疫苗的免疫效应,有效发挥了基因免疫佐剂效应;这为研制PRRSV高效DNA疫苗奠定了基础,同时也为其它疾病的高效新型疫苗研究提供了思路。     

共表达PRRSV GP5蛋白和CSFV E2蛋白的“自杀性”DNA疫苗在小鼠体内诱导的免疫应答

为了获得新型双价"自杀性"DNA疫苗,将猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratorysyndrome virus,PRRSV)GP5基因克隆于此前构建的表达猪瘟病毒(Classical swine fever virus,CSFV)E2基因的甲病毒复制子载体疫苗pSFV1CS-E2中.为了增强免疫效果,在密码子优化的GP5基因中插入了泛DR表位(PADRE),在CSFV E2基因后融合伪狂犬病病毒(PrV)UL49基因,获得了6种重组质粒.间接免疫荧光试验显示,PRRSV GP5和CSFV E2基因在瞬时转染的293T细胞中得到同时表达,将6种重组质粒和空载体pSFV1CS分别免疫BALB/c小鼠,用间接ELISA方法检测血清抗体水平,通过基于CSFE/WST-8的淋巴细胞增殖试验和细胞因子ELISA评价疫苗诱导的细胞免疫.结果显示,除pSFV1CS组外,从各疫苗组小鼠血清中均可检测到低水平的针对GP5和E2蛋白的抗体;各疫苗组小鼠脾细胞经CSFV和PRRSV刺激后均能诱导特异性的淋巴细胞增殖:部分疫苗组小鼠脾细胞经CSFV和PRRSV刺激后可分泌较高水平的IFN-γ和IL-4;引入UL49的疫苗组细胞免疫应答显著高于其它疫苗组.结果表明,这些共表达GP5和E2蛋白的自杀性DNA疫苗可以诱导体液免疫和细胞免疫,PrV UL49可以增强其细胞免疫应答.     

MDV-1 VP22增强鸡传染性法氏囊炎病毒VP2 DNA免疫作用

马立克氏病病毒（Marek’s disease virus,MDV）被膜成分中的VP22具有蛋白转导功能．将鸡传染性法氏囊炎病毒（infectious bursal disease virus,IBDV）VP2与MDV—VP22同时插入pcDNA3．1构建融合蛋白表达载体,在COS-1细胞中进行暂态表达．结果表明：VP22并不能促进VP2向周围未转染细胞转导,即VP22对VP2没有明显的蛋白转导作用,相反,VP2影响VP22在细胞内的正常定位．然而,通过小鼠股四头肌免疫注射PEI包裹的裸质粒DNA进行DNA免疫,以间接ELISA测定VP2抗体效价,FACS检测T细胞亚类．发现,虽然VP22没有明显促进VP2的体液免疫应答,但脾脏淋巴细胞分群结果表明,VP22-VP2融合蛋白表达载体免疫组和VP2质粒免疫组的CD3^＋T细胞数量显著高于eGFP-VP2组和对照组（P〈0．05）,融合蛋白表达载体免疫组小鼠CD8^＋／CD4^＋T细胞的比例与VP2质粒免疫组相比,明显增强（P〈0．05）,与对照组相比差异极显著（P〈0．01）．这表明VP22对VP2的特异性细胞免疫反应具有一定增强作用．     

牛疱疹病毒Ⅰ型ul49（VP22）基因的序列分析及其表达

以牛疱疹病毒Ⅰ型(BHV—Ⅰ)国内地方分离株感染的细胞培养物制备PCR模板,扩增出大小约0．77kb的ul49基因完整编码区片段,将扩增片段克隆到pMDl8—T载体中,获得含ul49基因的重组质粒pMD—VP22。采用双脱氧末端终止法进行序列测定,发现与国外Cooper株ul49基因序列完全一致,说明ul49基因相当保守。进一步将BHV—Ⅰul49完整编码区片段插入原核表达载体pET—28a和真核表达载体pEGFP—C1中,获得分别与6xHis融合的原核表达质粒pET—28aVP22和与EGFP融合的真核表达质粒pEGFPVP22。将pET—28aVP22转化大肠杆菌BL21(DE3),经IPTG诱导,SDS—PAGE电泳检测发现在38kDa处有一条特异性的表达带。利用脂质体介导,将pEGFPVP22转染PK—15细胞,经G418加压筛选,获得稳定表达VP22的细胞克隆。在倒置荧光显微镜直接检测未固定的活细胞,发现pEGFPVP22转染细胞能发出很强的荧光并主要集中在细胞核中,而对照载体pEGFP—C1转染细胞的荧光分布于脑浆。     

MDV-1 CVI988/Rispens疫苗毒体外感染期间VP22的表达及细胞间转导作用

利用CVI988 VP22蛋白C端94~243 aa片段作为抗原制备特异性抗体, 并用于检测MDV-1不同毒株感染鸡胚成纤维细胞(CEF)时, 在不同感染时间内的表达情况. 结果发现, 当感染量PFU=50时, MDV各不同致病力的毒株(GA, RB1B和CVI988株)VP22最早在3 h时开始向病毒感染细胞的邻近细胞核内扩散; 8 h时, VP22几乎扩散到所有细胞的核内. 这说明VP22在感染细胞形成空斑之前就已经开始表达, 并高效转导入其他正常的细胞核内. 实验中发现血清I型马立克氏病病毒(MDV-1)弱毒株CVI988/Rispens的VP22蛋白缺失²⁰¹TKSERT²⁰⁶. 结果证实这一缺失对VP22蛋白转导功能并没有明显影响, 即CVI988 VP22仍具有高效的细胞内转导作用. 这为深入研究和开发MDV CVI988株的VP22蛋白转导功能奠定了基础.     

增强恶性疟原虫DNA疫苗免疫反应的研究

构建了含有恶性疟原虫抗原基因 ( AWTE)及白介素 2基因的重组质粒 p CMV- AWTE以及p CMV- IL2、p CMV- IL2 - AWTE、p RSV- AWTE。将纯化的质粒混合后免疫小鼠 ,3次免疫后比较其诱导机体产生的免疫应答的水平 ,发现 IL- 2可以明显地提高机体的细胞免疫 ,而对体液免疫的影响甚微。麻醉剂、蔗糖、免疫剂量等因素也可以不同程度地提高机体的免疫应答水平 ,RSV启动子与 CMV启动子对免疫应答水平无明显的影响     

HSV-1间层蛋白VP22转录调控功能的分析

HSV-1间层蛋白是一类重要的功能蛋白,在病毒复制的多个环节中具有重要意义．为了解主要间层蛋白VP22在病毒复制过程中的生物学特性,本实验利用氯霉素乙酰转移酶系统,分析了VP22对病毒启动子的转录调控功能,结果表明VP22对HSV-1觯,TK和gC基因启动子明显具有剂量效应关系的转录抑制作用;VP22也能抑制病毒不同转录调控因子（VP16和ICP0）对启动子的转录激活作用,尤其明显抑制ICP0对TK和gC基因启动子的转录激活作用;VP22能明显抑制组蛋白乙酰转移酶PCAF对ICP0转录激活的促进作用,此抑制作用较VP22抑制ICP0的转录激活作用更为显著．VP22对其他病毒启动子的转录抑制效应,在内对照实验β-gal活性分析中也得到了证明．     

治疗型VP22△-mE6△/mE7重组蛋白疫苗的表达与免疫学初步分析

制备16型人乳头瘤病毒mE6Δ/mE7蛋白与I型人单纯疱疹病毒VP22Δ蛋白的治疗型分子内佐剂融合蛋白疫苗,并检测其免疫原性和抗肿瘤相关生物活性。通过克隆HSV-1 VP22Δ及HPV-16 mE6Δ/mE7基因,构建pET28a-VP22Δ-mE6Δ/mE7原核表达载体。重组质粒在Rosetta（DE3）宿主菌中进行诱导表达,表达蛋白经分离、复性后,通过镍离子亲和层析进行纯化,纯化蛋白经SDS-PAGE、Western blot 鉴定,并免疫BalB/C及C57BL/6小鼠,检测其免疫原性和抗肿瘤活性。结果显示,VP22Δ-mE6Δ/mE7蛋白以包涵体形式表达,分子量约为34kDa,表达量约占菌体总蛋白的45%。该蛋白免疫小鼠后血清特异性IgG、特异性淋巴细胞增殖效果及对TC-1致瘤小鼠的肿瘤治疗效果均高于无佐剂单一重组蛋白疫苗。以上结果说明,所获得的重组融合蛋白具有较好的免疫原性和抗肿瘤活性,为治疗型HPV分子内佐剂疫苗的进一步研究奠定了基础。     

MDV-1 VP22羧基端在大肠杆菌中高效可溶性表达

VP22是血清I型马立克氏病病毒(MDV-1)的复制和传播必不可少的组分。本研究从MDV-1无致病性的CVI988/Rispens疫苗感染的鸡胚成纤维细胞DNA中扩增得到VP22基因,并在大肠杆菌中表达其C端功能区。SDS-PAGE电泳发现,VP22C端得到高效可溶性表达,大小为42kDa左右。将获得的阳性条带经切胶免疫和细菌超声波裂解的上清作为抗原免疫6周龄Balb/C小鼠,均能诱导产生特异性抗VP22C端抗体。通过免疫荧光试验,检测到VP22在感染MDVCEF所有细胞核中呈特异性表达。这一抗体对深入研究VP22蛋白转导功能起到重要的作用。     

MDV-1 VP22羧基端在大肠杆菌中高效可溶性表达

VP22是血清Ⅰ型马立克氏病病毒(MDV-1)的复制和传播必不可少的组分.本研究从MDV-1无致病性的CVI988/Rispens疫苗感染的鸡胚成纤维细胞DNA中扩增得到VP22基因,并在大肠杆菌中表达其C端功能区.SDS-PAGE电泳发现,VP22 C端得到高效可溶性表达,大小为42kDa左右.将获得的阳性条带经切胶免疫和细菌超声波裂解的上清作为抗原免疫6周龄Balb/C小鼠,均能诱导产生特异性抗VP22 C端抗体.通过免疫荧光试验,检测到VP22在感染MDV CEF所有细胞核中呈特异性表达.这一抗体对深入研究VP22蛋白转导功能起到重要的作用.     

伪狂犬病毒VP22蛋白C-端系列缺失突变体的构建及亚细胞定位分析

以绿荧光蛋白(GFP)为标记,构建了一系列伪狂犬病毒VP22蛋白的C-端缺失突变体与GFP融合表达的真核表达质粒,脂质体介导转染Hela细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,发现伪狂犬病毒VP22蛋白与核定位有关的结构域在第60个到第90个氨基酸残基之间,第111个到第159个氨基酸残基有可能与形成细胞核内的颗粒有关,与微管蛋白结合有关的结构域可能在第187到第241个氨基酸残基之间.上述研究结果为进一步深入研究伪狂犬病毒VP22蛋白的结构与功能奠定了基础.     

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8 /CD4 T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.     

Evaluation of the VP22 gene adjuvant for enhancement of DNA vaccine against somatostatin in mice

Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. A novel SS-VP22 fused vaccine, pEGS2SS-V, was constructed from pEGS2SS plasmid with a VP22 gene fragment. Two times of immunization with pEGS2SS-V-induced anti-SS antibodies in mice. Compared with mice immunized with pEGS2SS and 0.85% saline, the growth performance of mice immunized with pEGS2SS-V was increased by 14.1% (P < 0.05) and 48.4% (P < 0.01) on the 2nd week after the first vaccination, respectively. The results indicated that the effects of the somatostatin DNA vaccine could be improved effectively by VP22 gene adjuvant.     

融合表达牛疱疹病毒1型VP22及猪繁殖与呼吸综合征病毒GP5重组伪狂犬病毒TK-/gE-/VP22GP5+的构建

猪繁殖与呼吸综合征(porcine reproductive andrespiratory syndrome,PRRS)和伪狂犬病(pseudora-bies,PR)是两种严重危害养猪业的重要病毒性传染病。而引发PR的伪狂犬病病毒(pseudorabiesvirus,PRV)是构建基因工程疫苗优良的活病毒载体[1]。已有报道将表达猪瘟病毒(hog cholera virus,HCV)囊膜蛋白E1基因的重组PRV(rPRV)二价基因工程疫苗免疫猪后,能同时抵抗HCV和PRV的强毒攻击[2],显示了PRV作为活病毒载体的可行性和“一针防两病”的独特优点。由ORF5基因编码的囊膜糖蛋白GP5是猪繁殖与呼吸综合征病毒(porcine reproductive …     

伪狂犬病毒VP22蛋白C-端系列缺失突变体的构建及亚细胞定位分析

以绿荧光蛋白(GFP)为标记,构建了一系列伪狂犬病毒VP22蛋白的C-端缺失突变体与GFP融合表达的真核表达质粒,脂质体介导转染Hela细胞,通过荧光显微镜观察分析各个缺失突变体的亚细胞定位,发现伪狂犬病毒VP22蛋白与核定位有关的结构域在第60个到第90个氨基酸残基之间,第111个到第159个氨基酸残基有可能与形成细胞核内的颗粒有关,与微管蛋白结合有关的结构域可能在第187到第241个氨基酸残基之间。上述研究结果为进一步深入研究伪狂犬病毒VP22蛋白的结构与功能奠定了基础。     

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.     

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.