旋毛虫成虫DNaseⅡ—T3223-7基因的克隆及其表达特性鉴定

发布时间：2018-05-14 11:38

  本文选题：旋毛虫 + 原核表达　； 参考：《中国兽医学报》2017年01期

【摘要】：利用RT-PCR技术从旋毛虫成虫得到T3223-7基因,并进行扩增克隆到原核克隆载体pMD18-T中,将重组质粒转入克隆菌DH5α,提取质粒进行酶切和测序鉴定,连接至pET-28a表达载体,最后转入表达菌Rosetta(DE3)。用1mmol/L IPTG诱导培养重组表达菌,对菌体裂解物进行SDS-PAGE分析,发现重组蛋白以包涵体的形式表达,约为40 000,与理论值相符。将纯化后的重组蛋白免疫家兔,制备兔抗T3223-7蛋白多克隆抗体,间接ELISA测定多抗效价达1∶320 000,Western blot检测表明制备的多克隆抗体能与T3223-7抗原发生特异性反应,并能识别和结合旋毛虫成虫排泄分泌物(ES)。
[Abstract]:T3223-7 gene was obtained from adult Trichinella spiralis by RT-PCR technique, and cloned into prokaryotic clone vector pMD18-T. The recombinant plasmid was transferred to DH5 伪. The recombinant plasmid was digested and sequenced and ligated to pET-28a expression vector. Finally, the recombinant plasmid was transferred into the expression strain Rosetta-DE3. The recombinant expression bacteria were induced by 1mmol/L IPTG and analyzed by SDS-PAGE. It was found that the recombinant protein expressed in the form of inclusion body was about 40 000, which was consistent with the theoretical value. Rabbit anti-T3223-7 polyclonal antibody was prepared by immunizing rabbits with purified recombinant protein. Indirect ELISA assay showed that the polyclonal antibody could react specifically with T3223-7 antigen. It can recognize and bind the excretory secretion of Trichinella spiralis.
【作者单位】： 吉林大学人兽共患病研究所/人兽共患病研究教育部重点实验室;中国疾病预防控制中心寄生虫病预防控制所;河南科技大学动物疫病与公共安全重点实验室;
【基金】：国际(地区)合作与交流项目(31520103916) 国家自然科学基金资助项目(31402185)
【分类号】：S852.7
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本文编号：1887704