伪狂犬病病毒Ea株UL31基因克隆、序列分析及其在大肠杆菌中的表达

Cloning and sequence analysis of the UL31 gene of pseudorabies virus Ea strain and its expression in E.coli

以伪狂犬病病毒Ea株基因组DNA为模板,通过PCR扩增含UL31基因的1000bp片段,扩增产物克隆于pMD18＿T中,双脱氧末端终止法序列测定。通过OM1GA2 0软件包分析发现,Ea株UL31基因编码271个氨基酸,蛋白质分子量为30 38kD,与Ka株UL31基因核苷酸与氨基酸序列同源性均在98%以上。将α＿疱疹病毒亚科9个不同成员UL31同源基因编码的氨基酸序列进行多重比对分析,发现4个保守的功能性结构域。将该片段插入原核表达载体pGEX＿KG中GST下游,构建的原核表达质粒pGEX＿UL31在大肠杆菌BL32(DE3)中获得了高效表达,SDS＿PAGE结果显示,表达的融合蛋白质分子量为56kD。

A 1.0 kb DNA fragment of UL31 gene of pseudorabies virus(PRV) Ea strain was amplified by PCR technique and cloned into pMD18-T.The sequence of the fragment was obtained by Sanger's sequencing technique and analysed by the software of OMIGA 2.0.The UL31 gene of PRV Ea strain encoded a protein of 271 amino acids,and its molecular weight was 30.38 KD.Both the nucleotide sequences identity and amino acid sequences identity of Ea and Ka strains were more than 98 %.Multiple alignment of the nine herpesvirus UL31 gene homology sequences indicated that the UL31 gene had four structure domains.Then,the fragment was inserted into downstream of the GST of an expression vector,pGEX-KG,to yield the recombinant plasmid pGEX-UL31.After induction by IPTG,a high expression of fusion protein was obtained,SDS-PAGE analysis showed that the fusion protein was 56 KD.