针对在……中表达的包涵体的外壳蛋白42多克隆抗体的制备与部分纯化

Production and Partial Purification of the Coat Protein 42 Polyclonal Antibody Against Inclusion Body Expressed in .

作者信息

Shibaei Naeimeh, Majidi Jafar, Bashir Nemat Sokhandan, Karkhaneh Aliasghar, Razavi Khadijeh

机构信息

Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

出版信息

Iran J Biotechnol. 2018 Dec 12;16(4):e1525. doi: 10.21859/ijb.1525. eCollection 2018 Dec.

DOI:10.21859/ijb.1525

PMID:31457029

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6697837/

Abstract

BACKGROUND

Expression of virus coat protein (CP) in often leads to production of partially folded aggregated proteins which are called inclusion bodies. virus (GFLV) is one of the most serious and widespread grapevine virus diseases around the world and in Iran.

OBJECTIVE

The main objective of this study was to find a simple and brief method for producing polyclonal antibodies (PAbs) to be used for immunodiagnosis of GFLV.

MATERIAL AND METHODS

An antigenic determinant in GFLV CP gene was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain BL21 (DE3).The recombinant coat protein of GFLV (CP42) was expressed and characterized by SDS-PAGE and western blot analysis using commercial anti-GFLV antibody. Expression of the CP was detected in the form of inclusion bodies in insoluble cytoplasmic fraction. Then, the inclusion bodies were isolated from the bacterial cells and injected into rabbits for PAbs production. The reaction of the antiserum was checked by ELISA assay. In order to analyze efficiency of the produced PAbs, first the infected and uninfected grapevine samples were confirmed based on morphological symptoms then the indirect plate- trapped antigen Enzyme-linked Immunosorbent Assay (IPTA-ELISA) was applied using the commercial anti GFLV antibody. In the next ELISA assay, efficiency of the raised polyclonal antibody was compared with commercial one.

RESULTS

The expression of recombinant CP42 induced by IPTG was confirmed by the band of 42 kDa in SDS-PAGE and western blot. The antiserum of purified inclusion body immunized rabbit was reacted with CP42 and GFLV infected Grapevine samples. The results revealed an acceptable efficacy for prepared antibodies compared to that of commercial antibody.

CONCLUSIONS

It was evident that the recombinant coat protein in the form of inclusion bodies can be prepared and used as the antigen for immunizing animals in order to produce PAbs.

摘要

背景

病毒外壳蛋白（CP）的表达常常导致部分折叠的聚集蛋白产生，这些蛋白被称为包涵体。葡萄扇叶病毒（GFLV）是世界范围内以及伊朗最严重且分布广泛的葡萄病毒病害之一。

目的

本研究的主要目的是找到一种简单且简便的方法来制备用于葡萄扇叶病毒免疫诊断的多克隆抗体（PAbs）。

材料与方法

将葡萄扇叶病毒CP基因中的一个抗原决定簇插入pET-28a细菌表达载体，构建体（pET-28a CP42）被克隆到大肠杆菌BL21（DE3）菌株中。利用商业抗葡萄扇叶病毒抗体通过SDS-PAGE和western blot分析对葡萄扇叶病毒重组外壳蛋白（CP42）进行表达和鉴定。CP的表达以包涵体形式在不溶性细胞质部分中被检测到。然后，从细菌细胞中分离出包涵体并注射到兔子体内以产生多克隆抗体。通过ELISA检测抗血清的反应。为了分析所产生的多克隆抗体的效率，首先根据形态症状对感染和未感染的葡萄样品进行确认，然后使用商业抗葡萄扇叶病毒抗体进行间接板夹抗原酶联免疫吸附测定（IPTA-ELISA）。在下一次ELISA检测中，将产生的多克隆抗体的效率与商业抗体进行比较。