草鱼Ⅲ型呼肠孤病毒外衣壳蛋白VP38的表达分析及多克隆抗体制备

Expression Analysis and Polyclonal Antibody Preparation of Outer Shell Protein VP38 Encoded by Genotype Ⅲ Grass Carp Reovirus

关键词:  草鱼Ⅲ型呼肠孤病毒  外衣壳蛋白  表达分析  多克隆抗体  免疫学检测

Key words:  Genotype Ⅲ grass carp reovirus  outer shell protein  expression analysis  polyclonal antibodies  immunological detection

摘要:

为进一步开展草鱼Ⅲ型呼肠孤病毒外衣壳蛋白VP38的生物学功能研究准备实验材料，同时探讨并构建一种草鱼Ⅲ型呼肠孤病毒的免疫学检测方法。实验构建了VP38的原核表达质粒pET28a-VP38，转化至BL21感受态细胞后利用IPTG（Isopropyl β-D-Thiogalactoside）诱导表达，8 mol/L尿素溶解重组蛋白后免疫小鼠，制备鼠抗VP38多克隆抗体；利用制备的抗体探究Grass carp reovirus-104（GCRV-104）感染后VP38在翻译水平的表达动力学；利用Western blot、间接免疫荧光分析（IFA）对抗体进行评估；构建VP38的真核表达载体pEGFP-N1-VP38，转染至草鱼性腺（Grass carp ovary，GCO）细胞内进行亚细胞定位分析。结果显示：重组VP38蛋白在原核表达系统中以包涵体形式存在；制备的鼠抗VP38多克隆抗体既能够识别重组VP38蛋白，也能够识别GCO细胞感染GCRV-104后表达的VP38蛋白；GCRV-104感染后72 h VP38主要分布在细胞质中，与亚细胞定位结果一致；VP38在感染的前期微量表达，感染中后期大量表达。本研究制备的鼠抗VP38多克隆抗体具有较高的效价和较好的特异性，为构建草鱼Ⅲ型呼肠孤病毒的免疫学检测方法提供了较好的技术路线。

Abstract:

To prepare experimental materials for further research on the biological function of VP38 and build an immunological detection method of genotype Ⅲ grass carp reovirus,in the experiment the prokaryotic expression plasmid pET28a-VP38 was constructed and transformed into BL21 competent cells,the expression of recombinant fusion protein was induced by IPTG and dissolved in 8 mol/L urea,the mice were immunized with recombinant protein to prepare mouse anti-VP38 polyclonal antibodies.Western blot and indirect immune-fluorescence analysis (IFA) were carried out to evaluate the antibody.The expression kinetics of VP38 at the translation level after GCRV-104 infection was investigated with the prepared antibodies.The antibody was evaluated by Western blot and indirect immunofluorescence assay (IFA).The eukaryotic expression vector pEGFP-N1-VP38 was constructed and transfected into grass carp GCO cells for sub-cellular localization analysis.The results showed that the recombinant VP38 protein existed as an inclusion body in the prokaryotic expression system.The prepared mouse anti-VP38 polyclonal antibody could recognize both the recombinant VP38 protein and the VP38 protein expressed by GCO cells infected with GCRV-104.VP38 was mainly distributed in the cytoplasm 72 h after GCRV-104 infection,which was consistent with the results of sub-cellular localization.VP38 was expressed in a small amount in the early stage of infection and in a large amount in the middle and late stage of infection.The mouse anti-VP38 polyclonal antibody prepared in this study has higher titer and better specificity,and provides a better technical route for constructing the immunological detection method of genotype Ⅲ grass carp reovirus.