云南猪株旋毛虫43 ku抗原基因的原核表达

发布时间：2018-01-14 03:35

本文关键词：云南猪株旋毛虫43 ku抗原基因的原核表达　出处：《中国病原生物学杂志》2015年03期 　论文类型：期刊论文

更多相关文章： 旋毛虫 ku抗原基因 克隆 原核表达 纯化

【摘要】：目的构建云南猪株旋毛虫43ku抗原基因原核表达载体,诱导其表达目的蛋白,为研究其功能奠定基础。方法收集云南株旋毛虫成虫,提取总RNA,采用RT-PCR获得43ku抗原基因。将PCR产物插入克隆载体pMD18-T中,重组质粒经酶切、PCR及测序鉴定正确后,将目的片段插入原核表达载体pET20b中并转化大肠埃希菌BL21,通过IPTG诱导表达目的蛋白,经镍柱亲和层析纯化后进行SDS-PAGE鉴定。结果 RT-PCR扩增出云南猪株旋毛虫43ku抗原基因,其基因序列全长1 134bp,与GenBank中注册的序列同源性最高为98.8%。重组表达质粒pET20b-Ts43经双酶切得到1 000bp和3 700bp左右两条片段,与预期结果相符。表达产物经SDS-PAGE鉴定,其分子质量单位为43ku。纯化蛋白经SDS-PAGE分析为单一43ku蛋白带。结论成功构建了云南猪株旋毛虫43ku抗原基因的原核表达载体,表达并纯化了43ku蛋白,为其功能研究奠定了基础。
[Abstract]:Objective to construct the prokaryotic expression vector of 43 ku antigen gene of Trichinella spiralis in Yunnan and induce it to express the target protein. The 43 ku antigen gene was obtained by RT-PCR. The PCR product was inserted into the cloned vector pMD18-T. The recombinant plasmid was confirmed by restriction endonuclease digestion and sequencing. The target fragment was inserted into prokaryotic expression vector pET20b and transformed into Escherichia coli BL21. The target protein was induced by IPTG. Results the 43 ku antigen gene of Trichinella yunnanensis was amplified by RT-PCR and its gene sequence was 1134 BP. The highest homology with the registered sequence in GenBank was 98.8. The recombinant expression plasmid pET20b-Ts43 was digested with two enzymes to obtain 1 000 BP and 3 BP. About 700bp two fragments. The expressed product was identified by SDS-PAGE. The purified protein was identified as a single 43 ku protein band by SDS-PAGE. Conclusion the prokaryotic expression vector of 43 ku antigen gene of Trichinella spiralis in Yunnan was successfully constructed. The 43 ku protein was expressed and purified, which laid a foundation for the study of its function.
【作者单位】：承德医学院病原生物学实验中心;
【基金】：河北省高等学校科学技术研究重点项目(No.ZH2011125)
【分类号】：R383.15
【正文快照】： 旋毛虫病(trichinellosis)是由旋毛虫(Trichinellaspiralis)引起的一种严重的人兽共患寄生虫病,其临床表现复杂多样,在诊断及治疗方面仍面临着诸多问题。肌肉活检查见囊包或幼虫是旋毛虫病最准确的诊断方法,但在早期或轻度感染者往往容易漏诊,而因取材的局限性晚期患者活检阳

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