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

发布时间：2018-10-29 21:55

【摘要】：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。
[Abstract]:The ORF2 gene fragment encoding (a HEV) capsid protein of avian hepatitis E virus was cloned into prokaryotic expression vector p ET-30a (), and the recombinant plasmid p ET-30a ()-ORF2. was constructed. The plasmid was correctly identified and transformed into E. coli Rosseta to screen the positive bacteria. The expression of the target protein was detected by IPTG induction of the positive bacteria. The polyclonal antibody was prepared by immunizing New Zealand white rabbits with the expressed recombinant protein and the titer of the antibody was detected by indirect ELISA. The main antigen region of capsid protein ORF2 螖 250 (251 aa) was cloned into the expression vector p TX8048 of lactic acid bacteria to obtain the positive plasmid p TX8048-ORF2 螖 250. The plasmid was electrotransformed into the host strain Lactococcus lactis NZ9000 to screen the positive strain p TX8048-ORF2 螖 250/L.lact is NZ9000.. Positive bacteria were induced by Nisin to detect the expression of target protein by West ern-blot. The results showed that, a HEV capsid protein was expressed as inclusion body in E. coli, and the titer of polyclonal antibody was 1: 216. After the recombinant strain p TX8048-ORF2 螖 250/L.lactis NZ9000 was induced by Nisin, the target protein of about 55 ku was detected by Western-blot. The above results will provide reference for the study of chicken hepatitis E lactic acid bacteria vaccine.
【作者单位】：东北农业大学动物医学学院;东北农业大学食品学院;西北农林科技大学动物医学学院;
【基金】：黑龙江省自然科学基金项目(C201422) 黑龙江省博士后启动基金项目(LBH-Q14019)
【分类号】：S852.65

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