表达EB病毒主要膜蛋白gp350／220的非复制型重组痘苗病毒的构建

利用非复制痘苗病毒质粒载体pNEOCK11β75及pNEOCK,改造了表达EB病毒主要膜蛋白gp350/22的复制型重组痘苗病毒VMA,构建了非复制型重组痘苗病毒VMA△CK。该病毒能在鸡胚原代成纤维细胞（CEF）中正常繁殖,而在人源细胞中不能正常繁殖。在CEF中连续传代至第25代,经PCR证明,该病毒符合非复制型重组痘苗病毒的特征。经免疫荧光及免疫酶斑法证实,VMA△CK可稳定表达gp350/220,且表达水平与VAM无明显差异。VMA△CK经腹腔免疫Balb/C小鼠,4周后能诱生一定水平的抗gp350/220特异性抗体,加强免疫2周后该抗体水平明显升高。这一结果类似于VMA免疫Balb/C小鼠的结果,初免后,VMA△CK且抗痘苗抗体水平明显低于VMA免疫组;加强免疫2周后,两组小鼠的抗痘苗抗体水平趋于一致。上述结果证明,所构建的非复制痘苗病毒不影响目的抗原的表达,也不影响该抗原的免疫原性,但导致病毒毒力下降,而且用该病毒免疫小鼠后小鼠抗痘苗病毒载体的免疫反应明显下降。     

表达EB病毒主要膜蛋白gp340/220不同蛋白区段的非复制型重组痘苗病毒的构建

Epstein-Barr病毒(EBV)主要膜蛋白gp340/220,是研究EBV亚单位疫苗的主要抗原.为了更详细地研究gp340/220的免疫原性,用PCR法获得不同区段的EBV BLLF1基因,并将其克隆入表达载体pNeock11β7.5中,通过重组质粒与非复制痘苗病毒在鸡胚成纤维细胞中同源重组,获得3株表达EBV gp340/220不同区段的重组痘苗病毒.PCR和Southern blot结果证实,各个重组病毒基因组中有相应外源基因的整合.Western blot结果表明,3种重组病毒感染人源细胞后都能够稳定表达蛋白,表达蛋白均为糖蛋白.免疫荧光结果表明,3种蛋白分布在细胞的不同部位.重组痘苗病毒的成功构建为更好地研究gp340/220的免疫原性提供了实验基础.     

双表达狂犬病毒基因的非复制型痘苗病毒改建及免疫效果

为减少重组病毒非必需外源基因,进一步提高非复制重组痘苗病毒狂犬病疫苗的安全性,本研究改建不含报道基因LacZ的双表达狂犬病毒aG株G、N的非复制重组痘苗病毒VTKRGΔCKRN。采用G418-neo富集、蓝白斑及免疫蚀斑筛选等方法,以表达狂犬病毒糖蛋白(RG)基因的非复制重组痘苗病毒VTKRG△CKlacZ作为亲本株,利用同源重组原理,删除C与K片断间lacZ,并将狂犬病毒核蛋白(RN)基因插入C-K片段间。经核酸及蛋白水平检测表明VTKRGΔCKRN能同时稳定有效地表达狂犬病毒G和N,并具有非复制病毒生长特性。重组病毒裸鼠毒力实验表明VTKRG△CKRN较复制型重组痘苗病毒VTKRG病毒毒力显著减弱。VTKRGΔCKRN免疫小鼠可诱生较高有效中和抗体,并能保护小鼠免于致死剂量狂犬病毒攻击。以1.6×106PFU较低免疫剂量、仅一次免疫狗可保护狗经致死量中国狂犬病毒街毒株SBD株攻击后存活,诱生具有保护性中和抗体。非复制狂犬-痘苗重组病毒VTKRGΔCKRN免疫效果好、更具安全性。     

表达乙型脑炎病毒抗原的非复制型重组痘苗病毒能在小鼠中诱发免疫保护

使用2×107PFU乙脑病毒野毒株GSS抗原preM-E-NS1-NS2a的非复制型重组痘苗病毒NTV-JEV免疫3周龄BALB/c小鼠,小鼠体内乙脑病毒特异性抗体滴度1∶71,IgG2a/IgG1为1.5,中和抗体滴度为1∶8,免疫组小鼠产生特异性补体介导的细胞杀伤作用。以10 LD50乙脑P3株病毒对小鼠进行了脑内攻击,NTV-JEV免疫组小鼠存活率为100%,与乙脑减毒活疫苗SA14-14-2免疫组存活率相同。100 LD50P3株病毒攻击后NTV-JEV免疫组存活率28.6%,与SA14-14-2组免疫效果无明显差别。存活小鼠体内乙脑病毒特异性抗体升高为1∶179,IgG2a/IgG1比值为1.9,中和抗体大于1∶16。以30 LD50GSS株病毒进行攻击后,NTV-JEV免疫组和SA14-14-2组保护率均为8.3%。上述结果表明,NTV-JEV与乙脑减毒活疫苗SA14-14-2株具有相近的保护力,NTV-JEV不仅可以保护小鼠抵抗同株病毒GSS的攻击,而且可以抵抗异株病毒P3的攻击。     

IL-5在非复制重组痘苗病毒中的表达及其对重组病毒免疫效果的影响

利用非复制型痘苗病毒表达载体 pNEOCK11β75IL5和重组病毒RVJ12 3,通过两步重组构建了能同时表达IL 5和乙型肝炎病毒HBsAg的非复制型重组痘苗病毒RVJ12 3Δ11β75IL5。Southern blot证实 ,痘苗病毒C K片段间基因缺失的同时伴有IL 5基因的插入。鼻腔吸入分别免疫Balb/c小鼠和新西兰白兔 ,ELISPOT实验证实 ,免疫后两周小鼠肺淋巴细胞的抗HBsAgIgA抗体分泌细胞 (ASC)数比对照组 (RVJ12 3Δ11β75 )增加约 2倍 ,而同时小鼠肺淋巴细胞的抗HBsAgIgG抗体分泌细胞 (ASC)数与对照组无差别。可在小鼠血液、肺浸出液以及新西兰白兔血液、肺浸出液、其它分泌液样品中检测到抗乙型肝炎病毒HBsAg的特异性的IgA、IgG抗体 ,与对照组相比 ,IgA抗体阳转率及抗体滴度提高 ,而IgG则无差异。本实验说明 :IL 5可在体内选择性地增强机体的粘膜IgA反应。提示非复制载体疫苗中 ,表达的该细胞因子可有效的增强疫苗的粘膜免疫反应 ,为粘膜疫苗的发展策略提供了新的途径     

IL—5在非复制重组痘苗病毒中的表达及其对重组病毒免疫效效果的影响

利用非复制痘苗病毒表达载体pNEOCKβ751IL5和重组病毒RVJ123,通过两步重组构建了能同时表达IL－5和乙型肝炎病毒HBsAg的非复制型重组痘苗病毒RVJ123△11β75IL5。Southern-blot证实,痘苗病毒C－K片段间基因缺失的同时伴有IL－5基因的插入,鼻腔吸入分别免疫Balb/c小鼠和新西兰白兔。ELISPOT实验证实,免疫后两周小鼠肺淋巴细胞的抗HBsAgIgA抗分分泌细胞（ASC）数比对照组（RVJ123△11β75）增加约2倍,而同时小鼠肺淋巴细胞的抗HBsAgIgG抗体分泌细胞（ASC）数与对照组无差别。可在小鼠血液、肺浸出液以及新西兰白兔血液、肺浸出液、其它分泌液样品中检测到抗乙型肝炎病毒HBsAg的特异性的IgA,IgG抗体,与对照组相比,IgA抗体阳性转率及抗体滴度提高,而IgG则无差异。本实验说明：IL－5可在体内选择性地增强机体的粘膜IgA反应。提示非复制载体疫苗中,表达的该细胞因子有效的增强疫苗的的粘膜免疫反应,为粘膜疫苗的发展策略提供了新的途径。     

双表达狂犬病毒基因的非复制型痘苗病毒改建及免疫效果

为减少重组病毒非必需外源基因,进一步提高非复制重组痘苗病毒狂犬病疫苗的安全性,本研究改建不含报道基因LacZ的双表达狂犬病毒Ag株G、N的非复制重组痘苗病毒VTKRG△CKRN.采用G418-neo富集、蓝白斑及免疫蚀斑筛选等方法,以表达狂犬病毒糖蛋白(RG)基因的非复制重组痘苗病毒VTKRG△CKlacZ作为亲本株,利用同源重组原理,删除C与K片断间lacZ,并将狂犬病毒核蛋白(RN)基因插入C-K片段间.经核酸及蛋白水平检测表明VTKRG△CKRN能同时稳定有效地表达狂犬病毒G和N,并具有非复制病毒生长特性.重组病毒裸鼠毒力实验表明VTKRG△CKRN较复制型重组痘苗病毒VTKRG病毒毒力显著减弱.VTKRG△CKRN免疫小鼠可诱生较高有效中和抗体,并能保护小鼠免于致死剂量狂犬病毒攻击.以1.6×106PFU较低免疫剂量、仅一次免疫狗可保护狗经致死量中国狂犬病毒街毒株SBD株攻击后存活,诱生具有保护性中和抗体.非复制狂犬-痘苗重组病毒VTKRG△CKRN免疫效果好、更具安全性.     

非复制重组痘苗病毒天坛株C-K缺失区表达载体的构建及重组病毒生物学性状的研究

将反向串联的痘苗病毒启动p11k和p7.5k表达结构引入非复制痘苗病毒质粒载体,得到非复制痘苗病毒表达载体pNEOCK11β75、pNEOCK7511β、pNEOCK1175和pNEOCK7511。利用载体pNEOCK11β75、pNEOCK7511β及pNEOCK11β75IL6和RVJ123重组病毒进行同源重组,分别得到非复制重组痘苗病毒RVJ123△11β75、RVJ123△7511β和RVJ123△11β75IL6。Southern-blot证实：重组病毒RVJ123△11β75 C－K片段间大片段基因的稳定缺失,同时β－半乳糖苷酸基因插入到缺失区内并稳定表达,不影响另外非必要区外源基因的表达,缺失区内两外源基因的表达夫相互干扰,并且缺失区内外源基因的转录方向对其DNA复制及蛋白表达以及另外非必要区基因的表达无影响。     

表达粒细胞巨噬细胞集落刺激因子的非复制痘苗病毒的抗肿瘤效果

构建了表达GM－CSF的非复制型重组痘苗病毒RV△11β75GMCSF。Southern blot证实重组病毒C－K片段间基因缺失,同时插入GM－CSF 基因,GM－CSF可以分泌形式良好表达。Wistar大鼠Walker‘s瘤内注射重组病毒RV△11β75GMCSF后,可以延长荷瘤鼠的生存时间,重组病毒RV△11β75GMCSF修饰的肿瘤细胞裂解物（WRC256）的肿瘤免疫治疗,可以抑制荷瘤大鼠的肿瘤生长,瘤重较对照组减轻,此结果提示,非复制重组痘苗病毒表达的GM－CSF可作为有效的肿瘤免疫治疗佐剂。     

表达小鼠粒细胞-巨噬细胞集落刺激因子的重组非复制型痘苗病毒的构建及鉴定

目的:以非复制型痘苗病毒天坛株为载体表达小鼠粒细胞-巨噬细胞集落刺激因子(GM-CSF),并体外鉴定其生物学活性。方法:构建含有小鼠GM-CSF的重组痘苗病毒质粒,与非复制型痘苗病毒进行同源重组,筛选表达小鼠GM-CSF的重组痘苗病毒rNTVGMCSFLacZ,对目的基因及蛋白的表达进行鉴定,并在体外检测目的蛋白的生物学活性。结果:构建的重组病毒rNTVGMCSFLacZ中正确插入了小鼠GM-CSF基因,Western印迹结果表明其能正确表达GM-CSF,且在体外证明rNTVGMCSFLacZ表达的小鼠GM-CSF能分泌到细胞外,具有生物学活性。结论:构建了一株能分泌表达有生物学活性的小鼠GM-CSF的重组非复制型痘苗病毒。     

Induction of humoral and cellular immune responses in mice by a recombinant fowlpox virus expressing the E2 protein of bovine viral diarrhea virus

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK-/gE-/GP5+expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV),based on the PRV genetically depleted vaccine strain TK-/gE-/LacZ+,scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene (the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK-/gE-/LacZ+,resulting in a PRV recombinant named TK-/gE-/GPSm+,which expressed the modified GPSm protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK-/gE-/GPSm+and TK-/gE-/GP5+expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK-/gE-/GPSm+against PRRSV were higher than that induced by TK-/gE-/GP5+.Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.     

Induction of Epstein-Barr virus lytic replication by recombinant adenoviruses expressing the zebra gene with EBV specific promoters

The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.     

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus (PRV), an alpha-herpesvirus, has been developed as a live viral vector for animal vaccines. However, the PRV recombinant virus TK⁻/gE⁻/GP5⁺ expressing GP5 of porcine reproductive and respiratory syndrome virus (PRRSV), based on the PRV genetically depleted vaccine strain TK⁻/gE⁻/LacZ⁺, scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV. To develop a booster-specific immune response of such PRV recombinants, the ORF5m gene (the modified ORF5 gene having better immune responses) was substituted for the ORF5 gene and introduced into PRV TK⁻/gE⁻/LacZ⁺, resulting in a PRV recombinant named TK⁻/gE⁻/GP5m⁺, which expressed the modified GP5m protein. The recombinant virus was confirmed using PCR, Southern blotting and Western blotting. TK⁻/gE⁻/GP5m⁺ and TK⁻/gE⁻/GP5⁺ expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses. The results indicated that the protecting neutralization antibodies (the 3/6 vaccinated mice obtained 1:16) and cell immune responses induced by TK⁻/gE⁻/GP5m⁺ against PRRSV were higher than that induced by TK⁻/gE⁻/GP5⁺. Thus, the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV. Translated from Journal of Biotechnology, 2005, 21(6): 858–864 [译自: 生物工程学报]     

Construction of a combinatorial immune library of human single-chain antibodies to orthopoxviruses and selection of antibodies to recombinant prA30L of the variola virus

A combinatorial immune library of human single-chain antibodies (scAbs) was constructed using the genes coding for the variable domains of the heavy and light chains of human immunoglobulins. The genes were cloned from lymphocytes of four subjects vaccinated with the vaccinia virus (VACV). The library included 3 · 10⁷ independent clones. After enrichment with clones producing scAbs against a recombinant analog of the variola virus envelope protein prA30L, the library was used to select a panel of scAbs binding both prA30L and VACV. All scAbs selected were tested for virus-neutralizing activity, and two scAbs proved to suppress VACV plaque formation in monolayers of Vero E6 cells. The specificity of antigen binding was verified by ELISA and Western blotting. The amino acid sequences of the virus-neutralizing scAbs were determined by sequencing their genes.     

Comparison of the murine humoral immune response to recombinant simian virus 40 large tumor antigen: Epitope specificity and idiotype expression

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (SV40 T-Ag) was used to immunize inbred strains of mice to compare the humoral immune responses. Specifically we examined the epitope specificities and idiotype (Id) expression on anti-(SV40 T-Ag) responses induced in BALB/c and C57BL/6 inbred strains of mice. The predominant SV40 T-Ag epitopes recognized by the anti-(SV40 T-Ag) responses appeared to differ between these two inbred strains, this being based on the ability of sera to inhibit the binding of several murine monoclonal antibodies specific for SV40 T-Ag. In addition, anti-(SV40 T-Ag) responses produced in C57BL/6 mice failed to express a previously described cross-reactive Id expressed in the anti-(SV40 T-Ag) response in BALB/c mice. This cross-reactive Id is detected by a mouse monoclonal anti-Id, designated 58D, which has been shown to represent a potential focal point for manipulating the humoral immune response to SV40-induced tumors in BALB/c mice. Together, these data indicate that the functional duality of the humoral immune response, as assessed by epitope recognition and Id expression, differs between these two inbred strains of mice when immunized with a recombinant SV40 T-Ag.     

Generation of recombinant adenovirus expressing siRNA against the L mRNA of measles virus and subacute sclerosing panencephalitis virus

Subacute sclerosing panencephalitis (SSPE) is a fatal neurodegenerative disease caused by prolonged persistent infection of the central nervous system with a measles virus (MV) mutant called SSPE virus. At present, there is no effective treatment to completely cure SSPE and development of a new therapeutic measure(s) against this fatal slow virus infection is needed. We previously reported that replication of MV and SSPE virus was effectively inhibited by small interfering RNA (siRNA), either chemically synthetic or plasmid-driven ones, that were targeted against different sequences of the mRNA for the L protein of MV. In this study, we have generated recombinant adenovirus expressing the siRNAs (rAd-siRNA-MV-L2, -L4 and -L5) and demonstrated that these rAd-siRNAs efficiently inhibited replication of MV and SSPE virus in a dose-dependent manner. Due to their high capacity for gene delivery to nerve cells and the potential to inhibit SSPE virus replication, the rAd-siRNAs could be a good candidate for a novel therapeutic measure against SSPE.     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

表达猪繁殖与呼吸综合征病毒GP5和M融合蛋白的重组腺病毒的构建及其对小鼠的免疫原性

利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽将猪繁殖与呼吸综合征病毒(PRRSV)的GP5和M蛋白编码基因串联,置于复制缺陷型腺病毒载体的表达盒中,通过一次转录和翻译,可以同时实现2个蛋白的表达,以发挥GP5蛋白的病毒中和优势和M蛋白的细胞免疫优势作用。分别利用RT-PCR、间接免疫荧光和Western blotting等方法,对获得的重组腺病毒(rAd-GP5-2A-M)进行检测,结果均证明GP5-2A-M蛋白不仅获得了正确表达,而且能自我裂解为GP5和M蛋白。以单独表达GP5(rAd-GP5)、M(rAd-M)和融合表达GP5-M(rAd-GP5-M)的重组腺病毒为对照,研究该重组腺病毒在小鼠体内诱导免疫应答情况,结果表明,虽然4种重组腺病毒均能诱导小鼠产生特异性抗体和细胞免疫反应,但重组腺病毒rAd-GP5-2A-M所诱导产生的体液免疫和细胞免疫应答水平最高。本研究结果提示,利用具有自动裂解功能的FMDV2A多肽构建PRRSV复合基因工程疫苗是一种切实可行的新策略,也为构建其他动物病毒病的基因工程疫苗提供了新思路。