【作者】 刘珂；

【导师】 黎满香； 陈溥言；

【作者基本信息】 湖南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 本实验利用含猪瘟病毒石门株(CSFV Shimen strain)全基因的质粒为模板,扩增囊膜糖蛋白E2中B/C抗原区和A╱D抗原区的线性化B-Cell表位。扩增了两个单独的基因片段(E2蛋白A╱D区和B/C区基因序列)与一个以上两基因片段融合的基因片段(E2蛋白A/D区连接B/C区基因序列),将扩增的三个片段分别插入原核表达载体pET-32a(+)中,构建成重组表达质粒pTA,pTB和pTC。将构建成功的阳性质粒转化原核表达菌BL21(DE3),对转化重组菌用IPTG进行诱导表达并对诱导表达条件进行优化,使目的蛋白得到高效表达。应用Ni-NTA亲和层析柱纯化三种重组蛋白,纯化产物经SDS-PAGE电泳鉴定其纯化效果,并用Western-blot检验重组蛋白对猪瘟阳性血清特异的反应原性。在此基础上,将三种纯化的蛋白定量后等体积混合弗氏佐剂免疫猪。经过两次免疫后检测免疫动物的猪瘟特异性抗体消长和免疫相关细胞因子变化情况。结果表明,三种蛋白均可刺激机体产生体液免疫应答和细胞免疫应答,pTC蛋白的免疫效果明显优于另两种蛋白,并且非常接近商品化细胞疫苗的免疫效果,pTB和pTC刺激机体产生的猪瘟抗体均达到阳性。三种重组蛋白诱导的细胞因子应答符合免疫应答规律。说明三种重组蛋白具有良好的免疫原性和免疫效果。更多还原

【Abstract】 Three recombinant prokaryotic vectors, pTA, pTB and pTC were constructed containing classical swine fever virus C strain E2 linear B-cell epitope gene(A/D antigenic domain and B/C antigenic domain) using pET 32a(+) expressing vectors. Their expressing characteristics were compared and it was found that pTA, pTB and pTC in BL21(DE3) can present high production of recombinant protein. Then, expression of protein were optimized by inducing in different time and different concentration of IPTG. After that, the recombinant protein was purified by Ni-NTA affinity chromatography. Purified protein was quantified and detected by SDS-PAGE and Western-blot. The purified proteins mixing with Freud’s adjust and inoculated to porcines. After vaccination, specificity antibody titers against CSFV and relative cytokines production were detected by ELISA.更多还原