摘要
目的:克隆及表达猪链球菌2型(S.suis2)人源分离株Habb截短溶菌酶释放蛋白(MRP)基因。方法:根据S.suis2mrp基因的序列设计引物,克隆和分析江苏海安患者分离株Habb截短mrp基因(tmrp),构建原核表达质粒pGEX4T-2-mrp。在大肠杆菌中诱导带有谷胱甘肽转移酶(GST)标签的融合蛋白tMRP-GST表达;经亲和层析法纯化后,用凝血酶酶切去除重组蛋白中的GST,制备纯化的截短MRP(tMRP)抗原。用Westernblot检测重组抗原的活性。结果:序列分析表明,获得的tmrp基因长957bp;原核表达的融合蛋白tMRP-GST的相对分子质量(Mr)约61000;凝血酶处理的tMRP抗原的Mr约为35000。Westernblot分析显示,MRP-GST、tMRP蛋白均可与MRP多克隆抗血清发生特异性反应。结论:成功地克隆人源分离株Habb截短的mrp基因,并在原核系统实现高效表达,制备的纯化抗原tMRP具有良好的抗原性,为开展相关免疫学研究奠定了基础。
AIM： To clone and express the truncated Habb mrp gene of human Streptococcus suis type 2 （ S. suis 2） and detect its activity. METHODS： A pair of primers based on S. suis 2 mrp gene were schemed out. The turncated mrp （tmrp） gene of S. suis 2 strain Habb isolated from diseased person in Haian, Jiangsu province was cloned and analyzed. The prokaryotic expression plasmid pGEX4T-2- tmrp was constructed. The expression of recombinant protein with glutathione S-transferase （GST） was induced in E. coil TG1. The fusion protein （tMRP-GST） was purified by affinity chromatography, and the GST was cut from tMRP-GST with thrombin protease to gain the truncated MRP （tMRP） antigen. The activity of recombinant protein was analyzed by Western blot. RESULTS： Sequence analysis showed that the length of the truncated mrp was 957 bp. The prokaryotic expressed production was a fusion protein, whose molecular weight was about 61 kD, and the molecu- lar weight of the purified tMRP protein was about 35 kD. Western blot analysis showed that tMRP-GST and tMRP were detected specifically by MRP antfserum on nitrocellulose membrane. CONCLUSION. The truncated mrp gene of human S. suis 2 strain Habb is successfully cloned, and the high expression of the functional recombinant protein is achieved in the prokaryotic system, which facilitates the further studies on the bio-function and immunology.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2006年第2期178-180,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家"十五"重大传染病科技攻关课题(No.2003BA712A03-05)
