猪轮状病毒vp4基因的克隆及其在昆虫细胞中的表达  

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

作　　者：宋岩[1] 师东方[1] 樊琛[1] 刘胜旺[2] 魏华[3] 李一经[1] 

机构地区：[1]东北农业大学动物医学院病原分子生物学实验室,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所生物技术国家重点实验室,黑龙江哈尔滨150001 [3]东南大学医学院微生物学教研室,江苏南京210009

出　　处：《中国病毒学》2005年第1期61-64,共4页Virologica Sinica

基　　金：黑龙江省"十五"攻关项目 (GC01B510)

摘　　要：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(1 756 bp),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。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.

关 键 词：猪轮状病毒 VP4基因 主要抗原编码区基因 克隆 真核表达 

分 类 号：S852.65[农业科学—基础兽医学]