猪轮状病毒JL94株vp4全基因克隆及原核表达载体构建

根据GenBank已发表的猪轮状病毒vp4基因保守序列,设计一对引物扩增猪轮状病毒地方株JL94株vp4全基因;将扩增产物与载体pMD-18T连接进行序列测定并将测序结果同国外分离株进行序列比较。用限制性内切酶BamHI和SalI将vp4基因从pMD-18T-vp4切下;同样用BamHI和SalI双酶切表达载体pGEX-6P-1;将这2个双酶切产物连接并转化,通过酶切鉴定和PCR鉴定证明完成了vp4原核表达载体的构建。测序结果表明:vp4全基因长2362bp,JL94株同国外分离株BEN307株、Gottfried株vp4全基因片段核苷酸同源性分别为93.44%和69.43%;氨基酸同源性分别为96.06%和71.88%。说明JL94株同BEN-307株属同一VP4血清型,而同Gottfried株属于不同血清型。重组原核表达载体pGEX-6P1-vp4的构建为表达VP4蛋白,研制诊断性抗原和病毒重组活载体疫苗奠定了基础。

The Clone of vp4 Omni-Gene from Porcine Rotavirus JL94 and the Construction of Its Vector for Prokaryotic Expression

According to conservative sequence of vp4 omni-gene of porcine rotavirus (PRV) published on GenBank, a pair of primers was designed to amplify the omni-gene of local strain of PRV JL94 of the vp4, and the amplified products were linked to vector pMD-18T to carry out its sequence determination, then compare this sequence with sequence of strain isolated abroad. Restricted enzymes BamHI and SalI were used to cut down the vp4 gene from pMD-18T-vp4; similarly, using BamHI and SalI to double-cut pGEX-6P-1 expression vector and then link these two double-cut products together and transfer. The completion of the construction of the vp4 prokaryotic expression vector was proved through the identification of enzyme-cutting and PCR analysis. The results of the sequencing showed that the length of omni-gene of vp4 is 2 362 bp, and the nucleotide homology of omni-gene of JL94 with BEN-307 strain isolated abroad and vp4 of Gottfried strain was 96.06% and 71.88% respectively, suggested that JL94 belong to the same serotype with BEN-307, and belongs to different serotype with vp4 of Gottfried strain. The construction of recombinant prokaryotic expression vector of pGEX-6P-1-vp4 has laid the foundation of the expression of vp4 protein and the development of diagnostic antigen and virus recombinant life vector vaccine.