摘要
为研究抗α型魏氏梭菌α毒素ScFv蛋白的生物学功能,利用PCR技术克隆抗α毒素1D5单抗的ScFw基因,构建含SCFQ基因表达质粒pHOG-lD5的重组表达菌株XL1-BLUE(pHOG-lD5)。酶切鉴定和序列分析证实构建的PHOG-1D5重组表达质粒含有ScFw基因,且基因序列和阅读框架正确。ELISα检测结果表明在重组菌株XL1-BLUE(PHOG-1D5)培养上清和细胞周质中均检测到目的蛋白。SDS-PαGE分析表明目的蛋白表达量占菌体总蛋白相对含量的29.2%。同时对ScFv蛋白生物学活性进行检测,结果表明其能中和α毒素的卵磷脂酶活性且能够对致死性腹腔攻击α毒素的小鼠产生良好的被动保护作用,从而为临床治疗α毒素中毒奠定了坚实基础。
In order to study the biological function of ScFv protein of anti-alpha toxin of Clostridium welchii type A, the ScFv gene of anti-alpha toxin 1D5 monoclonal antibody was cloned by PCR. The recombinant expression strain XL1- BLUE (pHOG-lD5) containing the expression plasmid pHOG-lD5 was established. The recombinant strain XL1-BLUE (pHOG-lD5) containing ScFv gene was established. The recombinant plasmid pHOG-lD5 contained ScFv gene which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The ELISA assay showed that the target protein was detected in both the culture supernatant and the periplasm of the recombinant strain XL1- BLUE (pHOG-lD5). The SDS-PAGE analysis showed that the expression level of the ScFv proteins was about 29.2% of total cellular protein. The biological activity assay showed that the expressed ScFv protein not only had the activity of neutralizing lecithinase, but also had a good passive protective effect on the mice suffering lethal intraperitoneal attack of alpha toxin, thus provided a solid foundation for clinical treatment of alpha toxin poisoning.
作者
许崇利
许崇波
林一民
XU Chongli;XU Chongbo;LIN Yimin(College of Medical Technology , Chongqing Medical and Pharmaceutical College , Chongqing 401331, China;Yingdong College of Life Sciences , Shaoguan University , Shaoguan 512005, China;Chongqing Cancer Institute ,Chongqing 400030, China)
出处
《扬州大学学报（农业与生命科学版）》
CAS
北大核心
2019年第2期47-53,共7页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
广东省自然科学基金资助项目(2018B0303110009)’
重庆医药高等专科学校自然科学技术研究项目(2019)
