马疱疹病毒1型(EHV-1)gC蛋白的原核表达及其免疫原性分析  

Prokaryotic expression and analysis of the immunogenicity of the gC gene of equine herpes virus 1

作　　者：王美月[1] 胡哲[1] 何佳[1] 刘荻萩[1] 王晓钧[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150009

出　　处：《中国病原生物学杂志》2016年第10期876-880,共5页Journal of Pathogen Biology

摘　　要：目的马鼻肺炎由马疱疹病毒1型(EHV-1)和4型(EHV-4)引起,但是二者存在广泛的抗原交叉反应,给两型病毒感染的鉴别诊断带来困难。EHV-1gC糖蛋白(gC1)具有凝集马红细胞的功能,而EHV-4gC糖蛋白不具有此特性,因此获得纯化的gC蛋白有助于建立马鼻肺炎鉴别诊断方法以及进一步研究马疱疹病毒感染的免疫分子机制。方法提取病毒基因组DNA,用特异性引物对目的基因片段进行扩增,对正确扩增的基因片段以及克隆载体pET30a(+)分别进行BamHⅠ和EcoRⅠ双酶切,将所得目的基因片段插入表达载体pET30a(+)中,构建重组质粒pET30a-gC1。经双酶切和序列鉴定为阳性的重组质粒pET30a-gC1转化入大肠埃希菌表达菌株Rosseta中,经IPTG诱导表达目的蛋白并对表达条件进行优化,对表达产物进行SDS-PAGE和Western blot分析。结果重组质粒转化菌经IPTG诱导表达60ku的目的蛋白,大小与预期相符,且在诱导温度为25℃时蛋白呈可溶性表达,诱导温度为30℃时蛋白以包涵体的形式表达。将包涵体电泳后切胶纯化回收,纯化效率相对较好,纯度较高。Western blot检测目的蛋白能被EHV-1和EHV-2感染马血清识别。结论成功构建EHV-1gC基因表达载体,其表达产物具有反应原性,为进一步开展EHV感染的分子诊断奠定了基础。Objective Equine rhinopneumonitis is caused by equine herpesvirus 1（EHV-1）and 4（EHV-4）.Distinguishing between EHV-1and EHV-4infections is difficult due to their cross-reactivity.A previous study indicated that glycoprotein C（gC1）of EHV-1is able to agglutinate equine red blood cells while that of EHV-4is unable to do so.Therefore,the expression and purification of gC1 in vitro will help to diagnose equine rhinopneumonitis and help to elucidate the molecular mechanism of EHV infection. Methods A pair of specific primers for the gC gene of EHV-1was designed to amplify gC1 from the genome of EHV-1.gC1 was cloned into a pET30a（＋）vector using BamHI and EcoRI restriction enzyme sites,and the resulting recombinant plasmid was designated pET30a-gC1.The recombinant plasmid was identified through digestion and sequencing.The positive recombinant plasmid was transformed into E.coli Rosetta.Expression of gC1 protein was induced using different concentrations of IPTG at different temperatures.The products were analyzed with SDS-PAGE and Western blotting. Results The obtained protein with a molecular weight of about 60 ku was detected with SDS-PAGE.The soluble protein was produced at 25℃ while inclusion bodies were formed at 30℃..Large pure quantities of gC1 were harvested from the gel fraction.The purified gC1 was detected by anti-EHV-1serum and antiEHV-4serum according to Western blotting. Conclusion A prokaryotic expression plasmid for gC1 was constructed.The EHV-1gC1 protein was successfully expressed in E.coli Rosetta.The purified gC1 protein reacted with EHV-1-positive sera.This work has provided a useful basis for further research on the molecular diagnosis of EHV infection.

关 键 词：马疱疹病毒 糖蛋白C 原核表达 包涵体纯化 

分 类 号：R373.11[医药卫生—病原生物学]