猪巨细胞病毒四川株gB基因的克隆表达及抗原性分析

为研究猪巨细胞病毒(PCMV)SC株gB蛋白的免疫学活性,采用PCR扩增PCMV SC株gB全基因和不含跨膜区及信号肽的gB基因片段,将后者克隆至pET-30a(+)载体,转化Rosetta(DE3)感受态细胞,经IPTG诱导以获得高效表达。将纯化蛋白免疫家兔制备多克隆抗体,分别用琼脂扩散试验和Western-blot对多克隆抗体和复性的gB蛋白进行检测。结果显示,扩增的PCMV gB基因全长2 580bp,编码860个氨基酸。与国内外参考株的核苷酸和氨基酸序列相似性分别为97.8%～99.5%和96.6%～99.0%;与其他9株β疱疹病毒核苷酸和氨基酸序列相似性分别为33.3%～41.4%和13.3%～49.4%;系统进化分析显示,PCMV与人疱疹病毒6型和7型属于一个分支。gB肽链N端1～23位氨基酸为信号肽,729～751位氨基酸间含有跨膜区。构建的表达载体pET30a-gB-B在Rosetta(DE3)感受态细胞中经IPTG诱导后以包涵体形式表达出大小约80ku的蛋白。所制备的血清琼扩抗体效价为1∶16,Western-blot显示重组gB蛋白可与PCMV阳性猪血清反应。结果表明,PCMV gB基因较为保守,与国内外不同地区的PCMV毒株具有较高的相似性,所表达的重组蛋白具有很好的抗原性。

Cloning and prokaryotic expression of glycoprotein B(gB) gene of porcine cytomegalovirus strain SC and antigenicity analysis of the expressed product

In order to study the immunologic competence of gB from porcine cytomegalovirus(PCMV) strain SC,the full length sequence of gB gene of PCMV and the partial fragment of gB gene which knockout transmembrane region and signal peptide were amplified respectively.The product from partial fragment was cloned into the pET-30a(+) vector,and transformed into Escherichia coli Rosetta(DE3) with recombinant protein expressed by IPTG induction.The natural protein was used to immunize rabbits for the gB anti-serum preparation,and the antigenicity of the recombinant protein was identified by agar diffusion test and western-blot.The results indicated that the PCMV gB gene contained 2580 nucleotides in full length encoding a protein of 860 amino acid residues.The nucleotide and amino acid sequence similarity of the gB gene of the strain SC shared from 97.8% to 99.5% and 96.6% to 99.0% with nine reference sequences isolated from other places around the world,respectively. Compared to other 9 β herpes viruses,the nucleotide and amino acid sequence similarity shared from 33.3% to 41.4% and 13.3% to 49.4%,and phylogenetic analysis suggested that the gB genes of PCMV,human herpesvirus 6(HHV-6) and HHV-7 were clustered on a clade. It was predicted that the 1-23aa was signal peptide sequence,and the 729-751aa was transmembrane region.The recombinant expression plasmid pET30a-gB-B was successfully constructed. The recombinant protein was expressed in inclusion body with molecular weight of 80ku.Agar diffusion test showed that the antibody titer was up to 1∶16,and western-blot manifested that the positive anti-PCMV serum could bind particularly with the protein.The results revealed that the gB gene of PCMV was highly specific and conservative,and the recombinant protein manifested an efficient immunogenicity.