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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。
Objective To construct the prokaryotic expression vector of 43ku antigen of Trichinella spiralis in Yunnan pigs and induce its expression target protein, which laid the foundation for the study of its function. Methods The adult Trichinella spiralis strains were collected and the total RNA was extracted. The 43ku antigen gene was obtained by RT-PCR. The PCR product was inserted into the cloning vector pMD18-T. After the recombinant plasmid was identified by restriction enzyme digestion, PCR and sequencing, the target fragment was inserted into prokaryotic expression vector pET20b and transformed into Escherichia coli BL21. The recombinant protein was induced by IPTG, Purification by column affinity chromatography followed by SDS-PAGE. Results The 43ku antigen of Trichinella spiralis strain was amplified by RT-PCR. The full-length cDNA of Trichinella spiralis 43ku gene was 1 134bp in length, which was 98.8% homologous to the registered sequence in GenBank. Recombinant plasmid pET20b-Ts43 was digested by double digestion to obtain 1 000bp and 3 700bp two fragments, and the expected results. The expressed product was identified by SDS-PAGE, the molecular mass unit is 43ku. The purified protein was analyzed by SDS-PAGE as a single 43 ku protein band. Conclusion The prokaryotic expression vector of Trichinella spiralis 43ku antigen gene was successfully constructed, and 43ku protein was expressed and purified, which laid the foundation for its function study.