伪狂犬病病毒IE180基因的克隆及其在大肠杆菌中的表达

Cloning of the Immediate-early Gene IE180 of Pseudorabies Virus Ea Strain and Its Expression in E.coli

提取伪狂犬病病毒国内地方分离株 Ea株基因组 DNA,Bam H 酶切后回收 4 .8kb左右的片段 ,克隆到 p UC1 8中 ,酶切分析和部分序列测定筛选到含 IE1 80基因的重组质粒 p UCIE。进一步将长约 1 .8kb的 IE1 80基因 5′端部分编码区亚片段克隆到原核表达载体 p ET- 2 8a中 ,使其置于 p ET- 2 8a的 T7启动子下游并同 6× His(多聚组氨酸标签 ) -Tag融合 ,重组表达质粒 p ET1 .8转化 BL2 1 (DE3) ,在 IPTG诱导下获得高效表达 ,表达产物以包涵体形式存在 ,相对分子质量为 6 2 0 0 0 ,同预期大小相当 ,并能同抗 6×

The genomic DNA of Pseudorabies virus(PRV) Ea strain was extracted,purified and digested with BamHⅠ.The 4 8 kb fragments were recovered and cloned into the vector pUC18,resulting in lots of recombinant plasmids consisted of different 4 8 kb BamHⅠ fragments.A recombinant plasmid pUCIE contained the complete IE180 gene was selected by restriction analysis,sequence determination and comparison with the IE180 gene of other PRV strain.Then,a 1 8 kb fragment encoding the N terminus of IE180 was subcloned into downstream of the T7 promoter of the expression vector,pET 28a,to yield the recombinant expression plasmid pET1 8.After transformed into E.coli BL21(DE3),fusion protein was expressed under the induction with IPTG.The results of SDS PAGE analysis showed that the expressed protein was 62 000 in size and formed inclusion body.The specificities of the fusion protein was also confirmed to react with the antiserum against 6×His by Western immunoblotting assay.The expression products would be useful to further study the structure and function of the IE180 of PRV.