伪狂犬病病毒Fa株gE基因在大肠杆菌中的表达及gE-ELISA鉴别诊断方法的建立

Expression of the gE gene of PRV Fa strain and development of the gE-ELISA for identification of PRV infection from vaccination

试验以pPGE DNA为模板,用1对分别含有EcoRⅠ和Bam HⅠ酶切位点的伪狂犬病病毒gE基因特异性引物,扩增出约1.7kb的含完整gE基因的DNA片段;目的片段经EcoRⅠ和Bam HⅠ双酶切后,插入原核表达载体pBV220得到重组表达质粒pBVgE,并转化大肠杆菌DH5α;对温敏诱导表达所获产物进行SDS-PAGE、Western-Blot和琼脂双扩散检测。结果表明,gE糖蛋白得到了高效表达,表达产物约占总蛋白的17%。为了鉴别伪狂犬病疫苗免疫猪和自然感染猪,用表达纯化的gE蛋白为抗原,建立了GE-ELISA方法。选定的反应条件包括GE抗原包被浓度为6.6mg/L,血清最适稀释度为1∶40。测试结果:与PRV、HCV、PRRSV、JEV、SA215阳性血清呈阴性反应,而与PRV标准阳性血清呈阳性反应。这一结果表明该法特异性强、敏感性高、重复性好,可用于猪伪狂犬病的临床诊断,尤其疫苗免疫猪和自然感染猪的鉴别。

A 1.7 kb fragment encoding gE of PRV Fa strain was obtained by PCR from pPGE plasmid DNA us- ing a pair of primers containing EcoR I and BamH I sites. The DNA fragment digested with EcoR I and BamH I was inserted into the expression plasmid pBV220. The recombinant plasmid was named pBVgE. The recombinant E. coli were induced at 42 C for 4-6 h, incubated at 30 C for 3 h later. A unique protein band was characterized in the lysate of the transformed bacteria by SDS-PAGE electrophoresis. The expression level was up to 17% of the total bacterial proteins. Western-blot analysis showed the expressed protein was specific to antisera against PRV Fa strain. To identify whether the pseudorabies infected from vaccination or field virus, the gE-ELISA was devel oped using the expressed gE protein as antigen. Before the serum samples were added, the wells were pre-incubated with the extracts of pBV220 recombinant E.coli for 30 min at room temperature. The gE-ELISA method was established by selecting the conditions with the coating concentration of gE antigen of 6.6 mg/L. The optimal dilution of serum to be 1 ： 40. It revealed a negative reaction with positive sera of PRV,HCV,PRRSV,JEV and SA215 and a positive reaction with PRV standard positive serum. The results showed that the gE-ELISA method should be advanced by its high sensibility,strong specificity and good repeatability and could be used to identify the PRV infection from vaccination.