锦鲤疱疹病毒囊膜蛋白ORF59的克隆、分析及其主要B细胞表位区的原核表达

Cloning,analysis of Koi herpesvirus envelope protein ORF59 and prokaryotic expression of major B cell epitope domain

从感染锦鲤疱疹病毒(Koi herpesvirus,KHV)的锦鲤(Cyprinus carpiokoi)肾脏组织中提取DNA,通过PCR扩增了KHVORF59基因。该基因全长411bp,所编码的蛋白包含136个氨基酸,分子量14.3kDa,等电点(PI)6.91,有12个潜在的O糖基化位点。此研究克隆的KHVORF59基因第130位碱基由G突变为A,使其编码的第44位氨基酸由Ala突变为Thr。采用DNAStar程序,在综合分析二级结构柔性区、蛋白的亲水性、表面可能性和抗原性指数的基础上,预测了KHVORF59蛋白主要B细胞表位,并将其区段的编码序列与KHVORF59完整编码序列分别克隆入原核表达载体pET-32a(+),构建重组质粒pET32a-ORF59S和pET32a-ORF59C,转入大肠杆菌Rosetta菌株,IPTG诱导表达。SDS-PAGE及WesternBlot分析显示,pET32a-ORF59S可以高效表达,表达的截短KHVORF59蛋白主要以可溶性形式存在,采用HisBindResin填料,层析纯化了该截短蛋白。

We amplified Koi herpesvirus ORF59 gene by PCR using template DNA extracted from the kidney of Cyprinus carpio koi infected with Koi herpesvirus （KHV）.The full length of the gene is 411 bp encoding an envelope protein which consists of 136 amino acids.The predicted molecular weight of KHV ORF59 is 14.3 kDa and its estimated isoelectric point is 6.91.Altogether 12 potential O-glycosylation sites are found in this protein.There is a mutation from G to A at 130^th site of cloned KHV ORF59 gene and it causes an amino acids substitution from Ala to Thr.With the software DNA Star and based on the analysis of the flexibility in second structure,hydrophilicity,surface probability and antigenic index of KHV ORF59 protein,the B cell epitopes are predicted.The sequence encoding the major epitope domain and the complete coding sequence of KHV ORF59 gene were subcloned into pET-32a（＋） vector to construct recombinant plasmids named pET32a-ORF59S and pET32a-ORF59C,which then were transformed into E.coli Rosetta respectively and expressed by IPTG inducement.SDS-PAGE and Western blot results show that pET32a-ORF59S can be highly expressed and the truncated KHV ORF59 protein is mainly expressed as soluble.With His Bind Resin filling,the truncated protein is purified.