IBDV南京野毒株VP2结构蛋白基因克隆与表达

CLONING AND EXPRESSION OF VP2 STRUCTURAL PROTEIN GENE OF CHINESE FIELD VIRULENT IBDV IN E.COLI

用微机辅助设计合成了一对引物，用于扩增传染性法氏囊病病毒（ＩＢＤＶ）中国地方株ＶＰ２基因片段。ＲＴＰＣＲ扩增出一１３２１ｂｐ的目的片段，分子杂交鉴定为ＶＰ２基因。扩增产物经双酶切后插入含噬菌体ＰＲＰＬ双强启动子的ｐＣＹＴＥＸＰ１表达质粒，构建了ＶＰ２基因克隆ｐＣＶＰ２１，经ＤｏｔＥＬＩＳＡ筛选出４个ＶＰ２表达阳性克隆。ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ显示有一约３２０００的目的蛋白带，且能与ＩＢＤＶ阳性血清结合。

A set of primers were designed to amplify the VP2 gene fragmnet of IBDV from China. A fragment of 1 321 bp was generated by RT PCR, resulting positive while detected by hybridization assay with VP2 nucleic acid probe. The pCVP21 was contructed by inserting the amplified VP2 gene fragment into expression vector pCYTEXP1, containing bacteriophage promoters P R and P L in tandem preceded by the cIts 857 repressor gene. Four colons, expressing VP2 protein, were selected by Dot ELISA with IBDV antiserum. SDS PAGE and Western blot analyses indicated that a 32 000 protein was expressed as expected.