Prokaryotic expression of the nucleocapsid protein gene in bovine coronavirus and its preliminary application
10.3969/j.issn.1002-2694.2010.01.019
- VernacularTitle:牛冠状病毒N基因的原核表达及初步应用
- Author:
Heyi LIU
;
Liyun YU
;
Xilin HOU
;
Liuxia SUN
;
Yulong ZHOU
;
Jinyi WANG
;
Shuangyi LIU
;
Fanze PIAO
- Publication Type:Journal Article
- Keywords:
bovine coronavirus;
N gene;
cloning;
sequence analysis;
prokaryotic expression
- From:
Chinese Journal of Zoonoses
2010;(1):76-80
- CountryChina
- Language:Chinese
-
Abstract:
To obtain and analyze the sequence of the nucleocapsid gene from bovine coronavirus, and to produce the fusion protein of the N gene in E.coli in order to use this recombinant protein for the study of bovine coronavirus. The N gene of BCV-DQ strain was amplified by RT-PCR, in which the primers were designed on the basis of N gene sequence of BCV-Mebus strain. The PCR products of 1 347 bp in length were cloned and sequenced, and then inserted into the prokaryotic vector pET30a. The recombinant plasmids were then transformed into Escherichia coli BL21 and identified by SDS-PAGE and Western blot assay. ELISA assay was optimized of N protein as the coating antigen to detect the viruses in the clinical samples. In comparison with 6 BCV strains in GenBank, the sequence identity was proved to be more than 98.3%. Result in SDS-PAGE showed that the fusion protein had a molecular weight of 60 ku, and could be specifically recognized by mouse serum against BCV. The indirect ELISA was used to test 256 serum samples collected from Heilongjiang province and 65.23% samples were positive. On testing field samples, an overall agreement of 95.31% was generated between the the neutralization test of viruses (VN) and indirect ELISA. It is apparent that the N gene was highly conservative and is expressed in E. coli in high level,also the prokaryotic expression products of this gene show a fine reactiongenicity in immune responses. It was also suggested that the N protein may be a useful antigen for sero-diagnosis and epidemiological investigation of BCV.
