[日本脑炎病毒prM的原核表达、纯化及单克隆抗体的制备]

[Prokaryotic expression, purification of prM of JEV and preparation of monoclonal antibody].

作者信息

Ning Bei-fang, Zhu Huai-min, Zhou Xiao-jun, Cao Yi, Zhou Ai-guo

机构信息

Department of Etiology of Second Military Medical University, Shang hai 200433, China.

出版信息

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2008 Feb;22(1):65-7.

PMID:18414706

Abstract

OBJECTIVE

To prepare monoclonal antibody (mAb) against prM epitope.

METHODS

The gene encoding prM was isolated using RT-PCR from brain of JEV infected mouse and cloned into prokaryotic expression vector pET-32a. Recombinant plasmid was transformed into E.coli BL21/DE3/LysS, then the transformed cells were expressed with the induction of IPTG. The expression and purification of the prM protein was analyzed by SDS-PAGE. The BALB/c mice were immunized with purified prM protein. Hybridoma cell lines secreting monoclonal antibodies against prM were established after cell fusion of mouse splenic cell and P3-X63-Ag8.653 cells. The specificity of mAb was identified by ELISA, Western Blot and Immunohistochemistry assay.

RESULTS

mAb against prM epitope of JEV was prepared successfully.

CONCLUSION

The obtained prM specific mAb was valuable for the prevention and dignosis of Japanese encephalitis.

摘要

目的

制备针对prM表位的单克隆抗体（mAb）。

方法

利用RT-PCR从感染日本脑炎病毒（JEV）的小鼠脑中分离出编码prM的基因，并克隆到原核表达载体pET-32a中。将重组质粒转化到大肠杆菌BL21/DE3/LysS中，然后用异丙基-β-D-硫代半乳糖苷（IPTG）诱导转化细胞表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析prM蛋白的表达和纯化情况。用纯化的prM蛋白免疫BALB/c小鼠。将小鼠脾细胞与P3-X63-Ag8.653细胞进行细胞融合后，建立分泌抗prM单克隆抗体的杂交瘤细胞系。通过酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法（Western Blot）和免疫组织化学分析鉴定单克隆抗体的特异性。