牛副流感病毒3型HNex蛋白表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.01.029

摘要/Abstract

摘要： 本研究旨在获得牛副流感病毒3型（BPIV3）的HNex蛋白及其多克隆抗体。以提取的BPIV3细胞毒的RNA为模板，利用RT-PCR方法扩增包含血凝素神经氨酸酶（HN）的基因片段，然后以此为模板扩增编码HN蛋白膜外区片段（HNex基因），并进行氨基酸序列测定。将HNex基因插入克隆载体pEASY-Blunt Simple中，经双酶切连接至pET-30a（+）表达载体中，构建重组原核表达载体pET-30a-BPIV3-HNex，转化大肠杆菌Rosetta^TM（DE3）pLysS感受态细胞，IPTG诱导后，用SDS-PAGE和Western blotting方法鉴定表达产物。经亲和层析方法纯化的HNex蛋白作为免疫原，制备兔抗BPIV3-HNex多克隆抗体。结果显示，本研究成功克隆BPIV3 HNex基因。原核表达蛋白结果表明，53 ku处有特异条带出现，并且可以与鼠抗His发生特异性免疫反应。免疫兔的血清中BPIV3 HNex抗体效价为1：819 200。Western blotting结果表明，制备的兔抗BPIV3 HNex多克隆抗体能与BPIV3蛋白发生特异性反应。总之，本研究利用原核表达系统表达BPIV3 HNex蛋白，并获得兔抗BPIV3 HNex多克隆抗体，为进一步探索BPIV3 HN蛋白的功能及亚单位疫苗研制提供参考。

关键词: 牛副流感病毒3型(BPIV3); HNex蛋白; 原核表达; 多克隆抗体

Abstract: The purpose of this study was to obtain the HNex protein of Bovine parainfluenza virus (BPIV3) and its polyclonal antibody.The extracted BPIV3 cytotoxic RNA was used as the template,and the gene fragment containing HN was amplified by RT-PCR,and then using this as the template,the HN protein outer region (HNex gene) was amplified and the nucleotide sequence was determined.The HNex gene was inserted into the cloning vector pEASY-Blunt Simple,and then double-digested into the pET-30a(+) expression vector to construct a recombinant prokaryotic expression vector pET-BPIV3-HNex and transformed into competent Rosetta^TM(DE3) pLysS.After IPTG induction,the expression products were identified by SDS-PAGE and Western blotting.HNex protein purified by affinity chromatography was used as immunogen to prepare rabbit anti-BPIV3-HNex polyclonal antibody.Sequencing results showed that this study successfully cloned the BPIV3 HNex gene.The prokaryotic expressed protein results showed that a specific band appeared at the 53 ku position,and it could specifically react with murine anti-His.The ELISA titer of BPIV3-HNex antibody in the serum of immunized rabbits was 1:819 200.The Western blotting results showed that the rabbit anti-BPIV3-HNex polyclonal antibody could specifically react with BPIV3 protein.In summary,this study used the prokaryotic expression system to express the BPIV3 HNex protein and obtained rabbit anti-BPIV3 HNex polyclonal antibodies,providing references for further exploration of BPIV3 HN protein function and the development of subunit vaccines.

Key words: Bovine parainfluenza virus type 3 (BPIV3); HNex protein; prokaryotic expression; polyclonal antibody

中图分类号:

S823

李明珠, 曲哲会, 宋志峰, 李臣锋, 于越洋, 高明春, 王君伟. 牛副流感病毒3型HNex蛋白表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2021, 48(1): 265-272.

LI Mingzhu, QU Zhehui, SONG Zhifeng, LI Chenfeng, YU Yueyang, GAO Mingchun, WANG Junwei. Expression of Bovine Parainfluenza Virus Type 3 HNex Protein and Preparation of Its Polyclonal Antibodies[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 265-272.

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