鸡白痢沙门氏菌SpiC蛋白的原核表达及应用

[Prokaryotic expression and application of SpiC protein of Salmonella pullorum].

作者信息

Geng Shizhong, Liu Huan, Jiao Xinan, Pan Zhiming, Qian Shanshan, Guo Rongxian, An Shumin, Xue Ying

机构信息

Jiangsu Provincial Key Laboratory of Zoonoses, Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.

出版信息

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Feb;31(2):149-52, 158.

PMID:25652851

Abstract

OBJECTIVE

To express the SpiC protein of Salmonella pullorum and establish an indirect ELISA method with SpiC protein as antigen.

METHODS

The 384 bp spiC gene of Salmonella pullorum was amplified by PCR from the genomic DNA and cloned into pET30a vector. The recombinant plasmid pET30a-spiC was transformed into competent E.coli BL21(DE3) cells and induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Indirect ELISA based on purified SpiC protein was applied to detect 144 clinical serum samples.

RESULTS

SDS-PAGE and Western blotting confirmed that a soluble recombinant His-SpiC protein of 19.2 ku was expressed in BL21(DE3) cells. SPF chicken antibodies against GST-SpiC could recognize His-SpiC, indicating that His-SpiC had a good immunogenicity. The indirect ELISA that we established using His-SpiC protein as coating antigen for detecting antibodies against SpiC could differentiate infected from vaccinated animals (DIVA).

CONCLUSION

The recombinant His-SpiC was successfully expressed and the indirect ELISA with it as coating antigen could be used as DIVA method for the related vaccine of pullorum disease.

摘要

目的

表达鸡白痢沙门氏菌的SpiC蛋白，并建立以SpiC蛋白为抗原的间接ELISA方法。

方法

通过PCR从基因组DNA中扩增鸡白痢沙门氏菌384 bp的spiC基因，并克隆到pET30a载体中。将重组质粒pET30a-spiC转化到感受态大肠杆菌BL21(DE3)细胞中，用IPTG诱导表达。通过SDS-PAGE和Western印迹分析表达产物。应用基于纯化SpiC蛋白的间接ELISA检测144份临床血清样本。

结果

SDS-PAGE和Western印迹证实，在BL21(DE3)细胞中表达了19.2 ku的可溶性重组His-SpiC蛋白。SPF鸡抗GST-SpiC抗体能够识别His-SpiC，表明His-SpiC具有良好的免疫原性。我们建立的以His-SpiC蛋白为包被抗原检测抗SpiC抗体的间接ELISA能够区分感染动物和免疫动物（鉴别诊断）。