[Cloning and expression of N-terminal protective domain of spaA gene from Erysipelothrix rhusiopathiae C43311].
The spaA gene was amplified by PCR from the genomic DNA of Erysipelothrix rhusiopathiae C43311 strain, and inserted into the pMD18-T vector and then sequenced. The N-terminal protective domain of the spaA gene was amplified by PCR from the recombinant plasmid pMD18-spaA, then cloned into the prokaryotic expression vector pGEX-6p-2 and expressed in E. coli BL21 (DE3) by IPTG induction. The expressed protein was identified by SDS-PAGE and Western blot. The sequence analyses showed that the coding region of the spaA gene of C43311 strain was 1881bp in length, and the nucleotide sequence homology of the spaA genes between the C43311 strain and the previously reported different serotype strains of E. rhusiopathiae was 93 to 99%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 64kDa, and the Western blot results showed that the GST-SpaA-N fusion protein was recognized specifically by an antiserum against the SpaA protein of C43311 strain, suggesting that the fusion protein of GST-SpaA-N possessed high immunoreactivity.
Nazierbieke
Wulumuhan
W
Laboratory of Bioengineering, College of Biology and Environmental Sciences, Jishou University, Hunan 41600, China. ulum@jsu.edu.cn
Liu
Zhuxiang
Z
Li
Ke
K
Chen
Yiguang
Y
Borrathybay
Entomack
E
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004888:Erysipelothrix / Q000737:chemistry / Q000276:immunology*; D004889:Erysipelothrix Infections / Q000276:immunology / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D051379:Mice; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C114457:SpaA protein, Erysipelothrix rhusiopathiae