1株猪腺病毒3型的分离培养及其fiber蛋白多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.02.024

摘要/Abstract

摘要： 本研究旨在对临床1份腹泻病料进行病原的分离培养并探究该病原编码的主要抗原蛋白的生物学特点。采用巢式PCR检测病料猪腺病毒3型（Porcine adenovirus type 3，PADV3）核酸并进行病毒分离培养，采用免疫过氧化物酶单层细胞染色法（IPMA）进行病毒血清学鉴定，PCR扩增fiber基因，将其ORF区克隆至pET-28a（+）载体并转化大肠杆菌BL21（DE3）感受态细胞，IPTG诱导重组fiber蛋白表达，采用SDS-PAGE和Western blotting鉴定重组蛋白，将纯化的重组蛋白免疫家兔，制备多克隆抗体并测定抗体免疫活性。结果表明，病料为PADV3核酸阳性，感染ST细胞产生典型的"葡萄串"细胞病变效应（CPE），P5~P11代次中的PADV3核酸稳定，分离的毒株P5代为PADV3型血清学阳性，fiber基因开放阅读框（ORF）为1 296 bp（GenBank登录号：MT774498），与PADV3毒株的核苷酸相似性最高（86.1%），与其他毒株相似性均低于40%；原核表达的重组fiber蛋白分子质量为45.2 ku，fiber蛋白多克隆抗体与PADV3感染的细胞呈特异性的细胞核染色。本试验成功分离得到1株PADV3毒株，命名为PADV3-HY1812，所制备的fiber蛋白多克隆抗体具有良好的免疫活性。

关键词: 猪腺病毒3型; 分离培养; fiber基因; 原核表达; 抗体

Abstract: The purpose of this study was to isolate and culture the pathogen of a clinical diarrheal disease and to explore the biological characteristics of the main antigen proteins encoded by the pathogen.The nucleic acid of Porcine adenovirus type 3 (PADV3) was detected by nested PCR and the virus was isolated and cultured,and was identified by immunoperoxidase monolayer staining (IPMA).The ORF region of fiber gene was amplified by PCR and cloned into pET-28a(+) vector and transformed into E.coli BL21 (DE3) competent cells.Recombinant fiber protein (recfiber) was induced by IPTG and was identified by SDS-PAGE and Western blotting.The purified recombinant protein was immunized to rabbits to prepare polyclonal antibody and determine its immune activity.The results showed that the pathogen was positive for PADV3 nucleic acid,and infected ST cells produced typical "grapevine" cytopathic effect (CPE).The nucleic acid of PADV3 was stable in the passages from P5 to P11,and the P5 of the isolated strain was serologically positive for PADV3.The open reading frame (ORF) of fiber gene was 1 296 bp (GenBank accession number:MT774498),which had the highest nucleotide similarity (86.1%) with PADV3 strain and less than 40% similarity with other strains.The molecular weight of the recombinant fiber protein expressed in prokaryotic cells was about 45.2 ku.The polyclonal antibody of fiber protein showed specific nuclear staining with PADV3 infected cells.A PADV3 strain named PADV3-HY1812 was successfully isolated in this experiment and the polyclonal antibody against fiber protein had good immune activity.

Key words: Porcine adenovirus type 3; isolation and culture; fiber gene; prokaryotic expression; antibody

中图分类号:

S852.65

陈果亮, 唐青海, 杨海, 杨灿, 王芳宇. 1株猪腺病毒3型的分离培养及其fiber蛋白多克隆抗体的制备[J]. 中国畜牧兽医, 2021, 48(2): 619-630.

CHEN Guoliang, TANG Qinghai, YANG Hai, YANG Can, WANG Fangyu. Isolation and Culture of a Porcine Adenovirus Type 3 Strain and Preparation of Polyclonal Antibody Against Its fiber Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(2): 619-630.

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