猪轮状病毒vp4基因的克隆及其在昆虫细胞中的表达

本研究扩增猪轮状病毒中国分离株JL94株VP4蛋白主要抗原编码区基因(1-756bp)，将测序结果与国外分离株进行比较；将该基因片段同载体pMel BacA连接后，与杆状病毒DNA共转染入昆虫细胞Sf9，经蚀斑筛选纯化重组病毒并再感染Sf9细胞获得vp4基因的表达，对表达的VP4蛋白进行Western blot分析和血清中和抗体试验。结果表明：JL94株VP4主要抗原编码区基因与国外分离株CRW-8株、Gottfried株该基因片段氨基酸同源性分别为96．43％和67％，说明JL94株与CRW-8株属同一VP4血清型，而与Gottfried株属不同血清型。JL94株vP4主要抗原编码区氨基酸最大变异处位于aa81-aa207。vp4基因在昆虫细胞中表达量占细胞总蛋白的20％，Western Blot证实表达蛋白有良好的生物学活性。所表达的蛋白免疫小鼠产生中和抗体，阻断JL94在MA104细胞上引起的细胞病变。

Cloning of vp4 Gene from Porcine Rotavirus JL94 Isolate and Expression in Baculovirus System

A pair of primers was designed to amplify vp4 gene of major antigen site (1-756 bp) of JL94 isolate. The sequence was analyzed in comparison with the VP4 amino acid sequences of two reference porcine rotavirus. The amino acid sequences were 96.43% and 67% correspondingly. Notably, the most divergence of amino acid sequence is located in a region delimited by aa81-aa207. The vp4 gene was inserted into expression plasmid pMel BacA. pMel BacA and Bac-N-blue DNA were cotransinfected insect cell Sf9. After 3 time plaque, reconstitution virus affected Sf9 and expresed in the cell. It indicated that the 30kDa product of the vp4 gene was 20% of total cell protein. Western blotting and neutralization test continmed that product had a nice biological activity. This study provides the basis for PRV identification, molecular epdiemiology investigation, and research of diagnostic reagent and genetic engineering vaccine.