【作者】 杨文超；

【导师】 余为一；

【作者基本信息】 安徽农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 E2蛋白是猪瘟病毒的跨膜结构糖蛋白,是最易发生变异的一种蛋白,能诱导感染动物产生保护性免疫和猪瘟病毒中和抗体,是一种免疫优势蛋白,其中A1、B、C区对介导中和抗体的产生很重要,用A1和B或A1和C的单抗可见协同中和反应。表达CSFV Brescia株E2蛋白重组伪狂犬病毒以及用亲和层析法从昆虫细胞中的基因工程E2蛋白均能保护免疫猪抵抗猪瘟病毒的感染,可以确定E2蛋白诱导的免疫反应足以保护免疫猪抵抗猪瘟病毒的感染。为制备猪瘟病毒E2蛋白的抗体,研究E2基因表达产物在免疫检测中的应用及E2蛋白的中和性抗原表位,我们进行了猪瘟病毒E2基因部分片段的克隆和原核表达。首先根据GenBank中登录的猪瘟病毒基因组序列,设计一对引物,利用聚合酶链式反应(Polymerase chain reaction,PCR)技术从本实验室保存的重组质粒(PGEX-4T-1-PE2)中扩增出大小约为622bp的基因;经单酶切鉴定,与已知的猪瘟病毒E2基因含有相同的DraⅠ酶切位点。PCR产物分离纯化后连接到pMD18-T载体,并转化宿主菌DH5α,经菌液PCR筛选出阳性重组克隆PMD18-T-TE2,结果表明本实验成功的克隆到猪瘟病毒TE2基因。其次,根据所克隆的猪瘟病毒TE2基因序列和原核表达载体PGEX-4T-1多克隆酶切位点设计另一对引物,应用PCR技术从重组质粒PMD18-T-TE2中扩增出长约622bp的猪瘟病毒TE2基因,编码包括CSFV E2囊膜糖蛋白主要抗原区域A、B、C和D。再将扩增的猪瘟病毒TE2基因克隆到原核表达载体PGEX-4T-1中,经质粒PCR和EcoRⅠ、SalⅠ双酶切鉴定,筛选出阳性重组克隆,构建原核表达重组质粒PGEX-4T-1-TE2。然后将该重组质粒转化大肠杆菌(EcoliRosetta(DE3)BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)表达出谷胱甘肽-S-转移酶(GST)与TE2的融合蛋白GST-TE2,融合蛋白的分子量约为47.0KD,与预期结果相符。通过优化原核表达条件,确定了原核表达的最佳诱导时间和诱导剂浓度。对表达蛋白的可溶性进行鉴定,结果表明表达产物GST-TE2融合蛋白以包涵体形式存在。我们用优化的原核表达条件对含有PGEX-4T-1-TE2质粒的Rosetta(DE3)BL21菌进行大量诱导表达,反复冻融和超声方法裂解诱导菌,用十二烷基肌氨酸钠加超声波的方法使包涵体充分溶解,Folin-酚法测定提取的融合蛋白GST-TE2浓度约为1.2mg/ml。应用提取的GST-TE2融合蛋白作为抗原免疫小鼠制备鼠抗猪瘟病毒TE2多克隆抗体。琼脂扩散实验表明制备的多抗具有良好的反应性,ELISA实验表明鼠多抗的有效稀释度可达1:25600,免疫印记(Western-blotting,WB)实验证实鼠抗猪瘟病毒GST-TE2多抗含有抗GST抗体和抗融合蛋白GST-TE2抗体。综上所述,本研究成功地克隆了猪瘟病毒TE2基因,成功构建了原核表达质粒PGEX-4T-1-TE2并进行了原核表达,提取了融合蛋白GST-TE2,并且制备了效价高、反应性强的鼠源抗猪瘟病毒GST-TE2多克隆抗体,这些为进一步研究猪瘟病毒E2蛋白免疫学功能及其应用提供了试验材料。更多还原

【Abstract】 E2 protein is transmembrane glycoprotein of Classical Swine Fever Virus(CSFV), prone to mutation, a kind of immunologic preponderant protein, and can induce infected animals to protective immunity and neutral antibody of CSFV,section A1、B、C is very important for engendering neutral antibody, cooperative neutral reaction is happened when using monoclonal antibody A1 and B or A1 and C. Expression of recombining CSFV Brescia strain E2 gene to Pseudorabies Virus and purification of gene engineering E2 protein by affinity chromatography from insects cells both protect immune pigs from infection of CSFV. In order to prepare antibody against CSFV E2 protein, do research on application of expressed production of E2 gene to immunologic detection and neutral antigen epitopes of E2 protein, partial fragment of CSFV E2 gene was cloned and expressed.Firstly, specific primers for CSFV E2 gene were designed according to the CSFV genomic sequence registered in GeneBank.Using PCR technology, one gene fragment about 622bp was amplified from a recombinant plasmid PGEX-4T-1- PE2 which was conserved in our experiment. The result of endonuclease Dra I digesting PCR product showed that this gene fragment has the same restriction enzyme site as known CSFV E2 gene. Gene fragments were ligated to pMD18-T vector after purification, the recombinant plasmid pMD18-T-TE2 was transformed into Escherichia coli (E.coli)DH5α,the recombinants were analyzed after they were identified by PCR, subsequently. This suggests that the CSFV TE2 gene has been successfully cloned.Secondly, gene-specific primers were designed based on the cloned TE2 nucleotide sequences and multi-clone restriction enzyme sites of prokaryotic expressive vector pGEX-4T-l, one gene fragment which encode major antigenic domains of E2(A、B、C、D) about 622bp was amplified from a recombinant plasmid pMD18-T-TE2 by PCR. Then the cDNA fragment which encode major antigenic domains of E2(A、B、C、D) was cloned into pGEX-4T-l,the recombinant plasmid PGEX-4T-1-TE2 was identified by PCR and endonuclease EcoR I +Sal I digestion. The PGEX-4T-1-TE2 was transformed into E.coli Rosetta BL21 DE3 and GST-TE2 fusion protein was induced to express by isopropylthio-β-D-galactoside(IPTG). The MW of the fusion protein was about 47 000 as analyzed by SDS-PAGE.After optimizing prokaryotic expression conditions, we determined the optimum inducement time and concentration of IPTG. The solubility of GST-TE2 fusion protein was identified and the result indicated that expressed GST-TE2 protein existed as inclusion bodies. The recombinant plasmid was induced to express large-scale GST-TE2 fusion protein under the optimal condition, the induced recombinant bacteria was lysed by freeze-thaw and sonication. We obtained the GST-TE2 inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. The concentration of fusion protein GST-TE2 was about 1.2mg/ml by folin-hydroxybenzene.Finally, using the GST-TE2 fusion protein as antigen, polyclonal antiserum to CSFV E2 was derived from mouse. Results of agar diffusion assay demonstrated that the polyclonal antibody could well react to TE2 protein. ELISA indicated that the immunized rabbits had antibody titers of 1:25600.Western-blotting showed that polyclonal antibody against CSFV GST-TE2 from mouse contain antibody against GST and antibody against GST-TE2 fusion protein.In conclusion, we have successfully cloned、expressed CSFV TE2 gene, and obtained the polyclonal antibody against CSFV TE2.A11 these provide some experimental materials for the future studies on the immunological function of CSFV E2.更多还原