【作者】 李小青；

【导师】 张国成； 许东亮；

【作者基本信息】 第四军医大学 ， 儿科学， 2008， 博士

【摘要】 人细小病毒B19(Human parvovirus B19,又称人微小病毒B19,简称B19病毒)是迄今发现的能感染人类的最小的单链线状DNA病毒之一。自1990年我们在国内首次证实我国存在B19病毒感染以来,越来越多的研究揭示我国B19病毒感染可诱发急性再障危象、急性血小板减少性紫癜及孕妇非免疫性流产等疾病,尤其对于免疫功能低下或缺陷患者,因缺乏有效治疗药物,B19病毒持续感染可致慢性贫血,甚至危及生命。因此,在我国乃至世界都需要建立有效的B19病毒防治体系。B19病毒基因存在较大的变异。目前B19病毒分为三种基因型:1型(原型)、2型(类A6-和LaLi-)、3型(V9)。不同基因型的分布存在着地理的差异。欧美等西方国家流行的病毒株大多属于1型,2、3型少见;3型多见于西非。此三型其DNA序列差异约为10%,其中大于20%序列差异位于p6启动子区域。在编码VP1/VP2蛋白的开放阅读框内,2、3型与1型比较,DNA序列变异分别为9%、12%,但氨基酸变异仅为1.1%、1.4%。我们在国家自然科学基金课题(39870021)的资助下,采用基因克隆测序技术,对收集到的中国大陆B19病毒流行株(XA株)进行基因分析,发现我国B19病毒流行株的VP1基因独特区有明显的变异,其基因组一级结构的差异,导致所编码氨基酸的改变,可能影响到B19病毒抗原位点及免疫特性的变化。B19病毒基因的变异可导致病毒毒力、致病机制的改变。VP1基因表达的蛋白与病毒的免疫原性及机体感染后产生保护性抗体密切相关,为建立我国B19病毒感染的防治技术,必须深入研究我国病毒流行株变异基因区表达的蛋白的结构和功能位点的变化。因此本实验通过在原核及真核系统表达B19-XA2株VP1蛋白,研究其空间结构和功能位点的变化及其变化对抗原性的影响,以揭示变异的VP1基因表达蛋白的生物学活性,促进B19病毒分子流行病学、致病机理及预防疫苗的研究。研究目的:本实验目的在于扩增获得B19-XA2 VP1基因全长,构建其原核表达载体,表达融合蛋白。同时构建VP1穿梭载体,在昆虫细胞Sf9中表达VP1蛋白,并以原核及真核表达蛋白为免疫原,免疫动物,制备具有一定敏感性和特异性的抗VP1蛋白的多克隆抗血清,用于检测VP1蛋白的免疫原性。分别以原核及真核表达蛋白为抗原,采用ELISA方法与临床患儿的血清反应,并与德国Parvovirus B19 IgM ELISA Kit检测结果比较,分析表达产物VP1蛋白的反应原性,从而为以后研究VP1蛋白的生物学功能,和B19病毒的防治奠定基础。实验方法:1.获得目的基因自行设计引物,按巢式PCR筛选阳性B19感染标本,自阳性标本中按照常规PCR方法扩增获得长度为2345bp的HPV B19 VP1全长基因(nt2623-nt4968)。2.原核表达重组蛋白将VP1基因克隆入载体pET28(a),构建原核重组表达载体VP1-PET28(a),并在E.coli BL21(DE3)中扩增,通过IPTG诱导表达VP1融合蛋白,SDS-PAGE和Western-blot技术分析蛋白表达情况及初步鉴定表达蛋白。3.真核表达VP1蛋白将VP1基因及载体pFastbac1双酶切、连接、转化入DH10Bac,用抗生素及蓝白斑表型筛选阳性重组体,PCR鉴定,获得含目的基因的重组杆状病毒转移质粒VP1-Bacmid,用脂质体介导法将阳性重组体转入Sf9细胞,扩增病毒,循环冻融和裂解,超速离心,获溶解的蛋白质上清。4.以原核及真核表达的重组蛋白为抗原,免疫家兔,制备抗VP1蛋白的多克隆抗血清,ELISA法检测抗体效价;用重组菌超声裂解上清及转染后Sf9细胞裂解后上清(均含重组VP1蛋白)包被ELISA板,分别与临床患儿的血清反应,并与德国ELISA试剂盒检测结果比较,分析表达产物VP1蛋白的反应原性。实验结果:1.巢式PCR筛选到2份阳性B19感染标本,按照常规PCR方法扩增此2份标本,自其中1份标本获得B19 VP1全长基因(2345bp),将其命名为B19-XA2株。2.原核表达载体VPl-PET28(a)经SalⅠ和XbaⅠ双酶切,琼脂糖凝胶电泳观察酶切片段分子量与预期结果相符(2345bp),测序结果证实序列完全正确。3.将重组菌VP1-PET28(a)-BL21(DE3)经诱导剂IPTG诱导,可表达分子量约为87kDa的蛋白,经Western-blot鉴定,可与6-His抗体结合,证实为VP1融合蛋白。4.构建穿梭载体VP1-Bacmid,通过抗生素及蓝白斑表型双重筛选阳性重组体,经PCR鉴定,获得含目的基因的重组杆状病毒转移质粒VP1-Bacmid,用脂质体介导法将阳性重组体转入Sf9细胞,细胞出现典型病变,收集病变细胞,裂解后SDS-PAGE显示有分子量87kDa的蛋白表达,与预期分子量一致。5. ELISA检测原核系统表达蛋白刺激后产生抗VP1多克隆抗体效价可达1:12800。真核系统表达蛋白刺激后产生抗体效价可达1:50000。6.以重组杆状病毒表达的VP1蛋白作抗原,与B19病毒阳性血清有反应,其OD值略低于德国ELISA试剂盒检测结果。而大肠杆菌系统表达的重组VP1蛋白作为抗原,与B19病毒感染后阳性血清无反应。结论:1.成功构建了重组人细小病毒B19-XA2株VP1全长基因的E.coli.:VP1-PET28(a)-BL21(DE3)。2.成功构建了人细小病毒B19-XA2株VPl全长基因重组杆状病毒转移质粒VP1-Bacmid,并在昆虫细胞Sf9中成功表达VP1蛋白。3.两种表达系统表达的重组VP1蛋白作为抗原有诱导特异性抗体产生的能力,即具有良好的免疫原性。4. Bac-to-Bac系统表达的VP1蛋白其生物活性更接近天然产物,可用作抗原检测人感染B19病毒后的血清抗体,且与进口试剂盒进行平行比较,初步达到同类试剂盒水平。更多还原

【Abstract】 Human parvovirus B19(HPV B19) was one of the smallest single chain DNA virus which confirmed to cause diseases in human. Since 1990 we confirmed that there were B19 virus infection for the first time in our country, more and more researches indicated that B19 virus was contributed to some disesases, such as acute aplastic crisis, acute thrombocytolytic purpura and nonimmune abortion. Especially in immunocompromided patients, the persistent HPV B19 infection could result in chronic anemia due to deficiency of effective therapy, even endanger life. Therefore, it was necessary to establish effective preventing and curing system of B19 virus in our country and even the world.B19 virus genome existed variation and was now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). There were different distribution of genotype due to regional discrepancy, such as human parvovirus B19 type 1 circulated in European and American , whereas type 2 and 3 encountered infrequently in this area. The type 3 attained commonly in western African. In overall DNA sequence, the three virus types differed by 10%. The most striking DNA dissimilarity, of >20%, was observed within the p6 promoter region. Within the open reading frame(ORF) encoding the VP1/VP2 proteins, the majority of nucleotide substitutions were synonymous: at the nucleotide level, genotypes 2 and 3 differed from the prototype by 9 and 12%, respectively, but at the amino acid level they differed by only 1.1 and 1.4%. We analyzed DNA sequence of the prevalent strain(XA strain) of B19 virus from chinese using the gene cloning and sequencing technology by assistance of the national natural sciences fundation(39870021) and discovered the VP1 unique region of B19-XA strain had the obvious variation in our country. The primary structure differences of its genome could cause alteration of coding amino acids, which might be contributed to the change of viral antigen site and immunal characteristic.The variation of B19 virus gene might result in changes of viral toxicity and pathogenic mechanism. The VP1 protein was closely correlation with viral immunogenicity and generation of protective antibodies after infection. So it was necessary to investigate thoroughly variation of structure and functional position of proteins which were expressed by the region of gene mutation in order to establish the preventing and controlling technology for B19 virus infection in our country. Accordingly, this experiment intended to constructe expressive vectors and express VP1 fusion protein of B19-XA2 strain in procaryon and eukaryon systems, then study change of the protein spatial structure and its functional position, as well as influence to antigenicity so as to reveal biologic activity of the VP1 protein and promote the investigation of B19 virus molecular epidemiology, pathogenic mechanism and preventive vaccine.Objective: Aim of this experiment lies in amplifying the whole VP1 gene of B19-XA2, constructing recombinant procaryotic expressive vector and the shuttle vector (VP1-Bacmid), expressing the fusion protein in E.coli. and Sf9 cells, respectively. In order to investigate immunogenicity of the fusion VP1 protein, rabbits were immunized by the fusion VP1 proteins from procaryon and eukaryotic expressive systems and produced the multiclone antiserum of anti-VP1 protein possessing a certain sensitivity and specificity. In addition, to analyze reactionogenicity of the VP1 fusion protein, the clinical serological detecting were respectively carried out using two kinds of ELISA on the basis of different antigen: one antigen was the VP1 fusion protein from procaryon and eukaryotic expressive systems, the other was parvovirus B19 IgM ELISA Kit of Germany. Above all, this experiment established the substructure for the further investigation of VP1 protein biological function and protection of B19 virus infection.Methods:1. To obtain the interested gene The positive B19 specimen were screened by nested PCR with primers which were independently designed. Then the VP1 whole gene of HPV-B19 (nt2623-nt4968) was amplified from the above positive specimen by conventional PCR assay.2. To express the recombinant protein in procaryon system The VP1 gene was inserted into the multiclone site of the pET28(a) expressive vector, afer that the recombinants were transformed into competent E.coli BL21(DE3). The VP1 fusion protein was expressed by IPTG inducing, analyzed by SDS-PAGE, and identified by Western blot.3. To express the VP1 protein in eukaryon system The VP1 gene and the pFastbac1 donor vector were digestted by SalⅠa nd XbaⅠrestrictive enzyme, connected each other with T4 DNA ligase, and then the recombinant vectors were emerged. Meanwhile the recombinant vectors were transformed into competent E.coli DH10Bac. The positive recombinants--the baculovirus shuttle vectors (VP1-Bacmid) containing the interested gene, were screened by antibiotics and phenotype of Blue-White spot and verified via PCR. In the subseqence testing, the VP1-Bacmid were transfected into Sf9 cells by a cationic lipid(Cellfectin reagent) mediation, then the recombinant baculoviruses were performed and amplified in Sf9 cells. Sf9 transfected cells were freezed repeatly and disintegrated with lysate, and in the end the supernatant containing the VP1 protein was procured by ultracentrifugation.4. Domestic rabbits were respectively immunized by the fusion VP1 proteins from procaryon and eukaryotic expressive systems and produced the multiclone antibody of anti-VP1 protein which valence was detected by ELISA. In the other hand, to analyze reactionogenicity of the VP1 fusion protein, the clinical serological detection were respectively carried out using two kinds of ELISA on the basis of different antigen: one antigen was the VP1 fusion protein from the supernatant of recombinant bacteria disintegrated by ultrasonic wave and the supernatant of transfected Sf9 cells after disintegration by lysate, the other was parvovirus B19 IgM ELISA Kit of Germany.Results:1. The VP1 whole gene of B19 were successfully amplified from one of the positive B19 specimen screened with nested PCR, and be named as B19-XA2 strain.2. The VPl-PET28(a) vectors were digested by SalⅠa nd XbaⅠrestrictive enzyme, and then we observed that the length of fragment matched with anticipative results by agarose gel electrophoresis. Sequencing result confirmed that the sequence of recombinant was entirely accurate. 3. As the recombinant bacteria VP1-PET28(a)-BL21(DE3) were propagated by IPTG induction, the fusion protein were expressed and exactly proved to be the VP1 fusion protein using Western-blot, which molecular weight was approximately 87kDa and could conjugate with 6-His antibody.4. The recombinant baculovirus shuttle vectors (VP1-Bacmid) which were screened by antibiotics and phenotype of Blue-White spot, successfully constructed and verified via PCR. Meanwhile, the VP1-Bacmid were transfected into Sf9 cells by cellfectin reagent, then the recombinant baculovirus were amplified in Sf9 cells. Sf9 cells emerging typical pathological changes were assembled and disintegrated. The 87kDa interested protein which molecular weight was consistent with the anticipated was found by SDS-PAGE.5. As antigen, both of the VP1 fusion proteins from procaryon and eukaryotic expressive systems could make rabbits to produce the multiclone antibodies of anti-VP1 protein. Its valence was detected by ELISA, and the results showed that valence of the former was 1:12800, while the later 1:50000.6. The VP1 protein producing in recombinant baculovirus system could well react to the positive sera of patients after B19 virus infection, moreover its OD value was lower than that of the German ELISA Kit. However, the VP1 protein producing from recombinant E.coli. system didn’t exist immunologic reaction with these positive sera.Conclusion:1. We successfully constructed the recombinant E.coli. VP1-PET28(a)- BL21(DE3).2. We successfully constructed the recombinant baculovirus shuttle vectors(VP1-Bacmid), and effectively expressed the VP1 protein in Sf9 insect cells. 3. The fusion VP1 proteins from either procaryon or eukaryotic expressive systems could stimulate animals to generate the specific antibody, so both of them possesed immunogenicity.4. The VP1 protein expressing in Bac-to-Bac system, which bioactivity was much similarly to native protein, could be used to detect specific antibody after B19 virus infection, but also its valence attained the parallel level compared with abroad kit.更多还原