Cloning and expression of recombinant truncated SElX protein and evaluation on the related emetic activities
10.3760/cma.j.cn112338-20191010-00724
- VernacularTitle:重组缩短的葡萄球菌类肠毒素X蛋白克隆表达及催吐活性评价
- Author:
Tong WANG
1
;
Xiaoxia TAO
;
Fanliang MENG
;
Xinpeng LI
;
Duo WANG
;
Dongliang HU
;
Jianzhong ZHANG
;
Guoqing WANG
;
Xiaomei YAN
Author Information
1. 北华大学医学技术学院，吉林 132013；传染病预防控制国家重点实验室，感染性疾病诊治协同创新中心，中国疾病预防控制中心传染病预防控制所，北京 102206
- Keywords:
Enterotoxin;
Recombinant protein;
Inclusion body;
Renaturation;
Emetic activity
- From:
Chinese Journal of Epidemiology
2020;41(4):567-570
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To analyze the amino acid polymorphism of truncated Staphylococcal enterotoxin-like toxin X (tSElX), and to evaluate its related emetic activities.Methods:Sequence of tselx was compared with both the genome sequence of 145 CC398 strains completed in our research group and the NCBI database. Primers were designed to amplify the target gene of tselx, and the fragment was recombined into pMD18-T vector and sequenced. PCR product was digested with BamHⅠ and EcoRⅠ, and constructed into plasmid pGEX-6P-1 and pET-28a (+). After recombinant plasmid was identified, the protein expression was induced by IPTG. Proteins expressed in the form of inclusion bodies were denatured and renatured, then purified by affinity chromatography and ultrafiltration. Purified tSElX protein was then fed to common marmosets with the dose of 250 μg/kg to observe the vomiting reaction. Results:tselx gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0 %, while the homology with the four American strains were 97.8 %(1) and 98.9 %(3), respectively. Through two sets of expression systems and different induction conditions, tSElX was expressed in the form of inclusion bodies. The high purity soluble recombinant tSElX was thus obtained by denaturated and renaturated processes. At the dose of 250 μg/kg, tSElX protein did not cause vomiting in common marmosets. Conclusions:Results of this study showed that the amino acid sequence of tSElX was highly conserved and was universally present in a particular clone group. We obtained soluble recombinant tSElX protein with high purity. We also noticed that tSElX did not have the animal emetic activity at a dose of 250 μg/kg.
