Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

猪A群轮状病毒VP6的表达及IgG抗体ELISA检测方法的建立与初步应用

为建立猪A群轮状病毒抗体间接ELISA检测方法,用于开展对该病毒感染的流行病学调查和免疫抗体评估,进行了猪A群轮状病毒TM-a株内衣壳VP6基因的表达,用SDS-PAGE和Western blot鉴定分析。目的蛋白得到表达,大小约70ku,Western blot表明,该蛋白与猪A群轮状病毒阳性血清反应。用该蛋白作为包被抗原,通过对条件优化,包被抗原浓度为2μg/mL,待检血清稀释倍数为1∶40倍,标记抗体的稀释倍数为1∶4 000倍,建立了检测猪轮状病毒血清IgG抗体的间接ELISA方法。用该方法检测临床样品142份,并与间接免疫荧光试验(IFA)对比,结果显示,两者阳性符合率为94.6%,阴性符合率为83.4%,总符合率为91.5%,表明该方法有效可行。用建立的方法检测临床上不同年龄阶段的猪血清639份,分析其临床感染情况。结果证明该方法可用于猪轮状病毒流行病学调查、猪群抗体水平评估。

A组轮状病毒SA11VP6基因的克隆和表达

从ＳＡ１１ＶＰ６基因全序列克隆开始，设计一对两端带有酶切位点的引物，逆转录ＰＣＲ扩增出ＶＰ６全基因ｃＤＮＡ。经酶切后插入ｐＵＣ１９，构建了ＶＰ６全基因克隆ｐＲＡ６。再经酶切后插入痘苗病毒载体质粒ｐＪＳＡ１１７５中。利用Ｌｉｐｏｆｅｃｔｉｎ寻人ＴＬ－１４３细胞，利用ＴＫ基因和Ｌａｃ基因作为重组病毒的筛选标记。表达产物用单克隆抗体ＥＬＩＳＡ法检测，发现细胞培养上清和细胞裂解液都是阳性。Ｗｅｓｔｅｒｎｂｌｏｔ显示，表达产物分子量大小与天然ＶＰ６一致。