产气荚膜梭菌α-β_2-β_1融合基因的构建及表达

Construction and Expression of α-β_2-β_1 Fusion Gene of Clostridium perfringens

目的构建产气荚膜梭菌α-β2-β1融合蛋白基因表达载体,并对表达产物进行鉴定。方法利用PCR技术,从含α毒素基因的质粒pXCPA02中扩增出α毒素基因,经NcoⅠ酶切,回收0·95kb的α基因片段,再与经NcoⅠ酶切并含融合基因β1-β2的质粒pXETB1B2连接,获得重组质粒pXETAB2B1,经NcoⅠ、BamHⅠ和EcoRⅠ酶切鉴定,并对其表达产物进行SDS-PAGE和Western blot分析。结果重组质粒pXETAB2B1含有α-β2-β1融合基因。表达产物相对分子质量约为95000,并可被特异性抗体识别。结论已获得高效表达α-β2-β1的重组菌株。

Objective To construct the expression vector of α-β2-β1 fusion gene of Clostridium peorringens and identify the expressed product. Methods Amplify α--toxin gene from plasmid pXCPA02 containing Clostridium peorringens α-toxin gene by PCR,digest with Nco Ⅰ and recover a 0. 95 kb fragment. Digest plasmid pXETB1 B2 with Neo Ⅰ ,and ligate the obtained α-β2-β1 fusion gene to α- toxin gene. Identify the obtained recombinant plasmid pXETAB2B1 by restriction analysis and the expressed product by SDS-PAGE and Western blot. Results The recombinant plasmid pXETAB2B1 contained α-β2-β1 fusion gene. The expressed α-β2-β1 fusion protein, with a relative molecular weight of 95 000, was recognized by its specific antibody. Conclusion A recombinant bacterial strain for high expression of α-β2-β1 fusion gene of Clostridium peorringens was successfully constructed.