禽戊型肝炎病毒衣壳蛋白多克隆抗体的制备及其在乳酸乳球菌中的表达

Preparation of polyclonal antibodies against avian hepatitis E virus capsid protein and its expression in Lactococcus lactis

将编码禽戊型肝炎病毒(a HEV)衣壳蛋白的ORF2基因片段克隆入原核表达载体p ET-30a(+)中,构建重组质粒p ET-30a(+)-ORF2。质粒经鉴定正确后转化入大肠杆菌Rosseta中筛选阳性菌,阳性菌经IPTG诱导后检测目的蛋白表达情况。用表达的重组蛋白免疫新西兰白兔制备多克隆抗体,应用间接ELISA检测抗体效价。将衣壳蛋白主要抗原区域ORF2Δ250(251~606 aa)基因片段克隆入乳酸菌表达载体p TX8048中获得阳性质粒p TX8048-ORF2Δ250,质粒电转化入宿主菌Lactococcus lactis NZ9000中筛选阳性菌p TX8048-ORF2Δ250/L.lact is NZ9000。阳性菌经Nisin诱导后采用West ern-blot检测目的蛋白表达情况。结果表明,a HEV衣壳蛋白在大肠杆菌中以包涵体形式表达,制备的多克隆抗体效价为1∶216。重组菌p TX8048-ORF2Δ250/L.lactis NZ9000经Nisin诱导后,Western-blot检测到约55 ku的目的蛋白。上述研究结果将为鸡戊型肝炎乳酸菌疫苗研究提供参考。

The ORF2 gene fragment coding the capsid protein of avian hepatitis E virus（a HEV） was cloned into prokaryotic expression vector p ET-30a（＋）to construct recombinant plasmid p ET-30a（＋）-ORF2.The characterized plasmid p ET-30a（＋）-ORF2 was transformed into Escherichia coli competent cells to screen positive strains.The secretory expression of protein was induced by IPTG,and was then detected by Western-blot.The expressed protein was used to produce polyclonal antibodies by immunizing New Zealand white rabbits.The antibody titer was tested by indirect enzyme-linked immunosorbent assay.The gene fragment of antigen domain（between 251 and 606 aa） in ORF2 gene（ORF2Δ250） was cloned into the vector p TX8048,and the positive plasmid p TX8048-ORF2Δ250 was screened,and then the plasmid was electrotransformed into host strains Lactococcus lactis NZ9000 to produce the positive bacteria p TX8048-ORF2Δ250/L.lactis NZ9000.The expression of objective protein was detected by Western-blot after the strains were induced by Nisin.The results showed that the capsid protein was expressed in E.coli strain.The titer of the prepared polyclonal antibodies was 1 ∶ 216.The molecular weight of expressed protein in the strains pT X8048-ORF2Δ250/L.lactis NZ9000 was 55 ku in size.This study provides important references for the study of vaccines against avian hepatitis.