A型产气荚膜梭菌重组菌株BL21（DE3）

Optimization of Expression condition of the recombinant strain

关键词:  A型产气荚膜梭菌  α毒素基因  突变  优化表达

Key words:  clostridium perfringene type A  alpha-toxin  mutant  optimizing expression

摘要:

摘 要：目的：对已构建的含有A型产气荚膜梭菌α 毒素突变基因的重组菌株BL21(DE3)( pXMCPA02)进行鉴定和表达条件优化。方法：采用限制性核酸内切酶酶切鉴定含α 毒素突变基因的重组质粒，同时用SDS-PAGE检测不同条件下α 毒素突变基因的表达情况。结果：酶切鉴定证实重组质粒pXMCPA02含有α 毒素突变基因且基因序列和阅读框架正确。以IPTG为诱导剂诱导α 毒素突变基因表达的优化条件是：培养基pH 7.5，培养温度37℃，IPTG浓度0.8mmol/L，菌体生长密度OD600达到0.8时加入IPTG，诱导时间5h，此时重组菌株pXMCPA02蛋白表达量为35 %。结论：实现α 毒素突变基因的高效表达，为A型产气荚膜梭菌α 毒素基因工程菌苗的生产工艺研究提供了可靠的试验数据。

Abstract:

Abstract: AIM: To identify the recombinant strain BL21(DE3)( pXMCPA02) containing alpha toxin mutant gene of Clostridium perfringens type A and optimize its expressional condition. Methods: To identify the recombinant plasmid pXMCPA02 with restriction endonucleases digestion. To detect expressional level of differently conditional alpha toxin mutant gene by SDS-PAGE. Results: The recombinant expression plasmid pXMCPA02 contained alpha toxin mutant gene having positive reading frame. The expression optimization result indicated that the alpha toxin mutant gene expression optimized condition with IPTG induction is culture medium pH 7.5,culture temperature 37℃, joining IPTG to final concentration 0.8mmol/L when the recombinant strain growth density OD600 achieved 0.8, and induction time 5h. The expression level of the alpha toxin proteins were about 35.% of total cellular protein by SDS-PAGE and gel system analysis. Conclusion: The alpha toxin mutant gene is expressed high by recombinant strain BL21(DE3)( pXMCPA02). This provided experimental data to research productive technique of trivalent genetically engineered vaccine.