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目的原核表达并纯化伪旋毛虫丝氨酸蛋白酶抑制剂(Trichinella pseudospiralis serine protease inhibitor,Tpserpin)基因,并鉴定其抗原性。方法从伪旋毛虫肌幼虫提取总RNA,RT-PCR扩增Tp-serpin基因,插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-Tp-serpin,转化大肠埃希菌(E.coli)Rosetta gami(DE3),IPTG诱导表达,表达产物经SDS-PAGE分析后,用Ni-NTA Agarose亲和层析纯化,纯化产物经SDS-PAGE分析纯度,Western blot鉴定反应原性。结果重组表达质粒pET-28a-Tp-serpin经PCR、双酶切及测序鉴定证明构建正确,与GenBank中登录的Tp-serpin基因相似性达99%。表达的重组Tp-serpin蛋白相对分子量约43 000,表达量约占菌体总蛋白的40%,主要以可溶性形式存在;纯化后的重组Tp-serpin蛋白纯度达95%以上,可被伪旋毛虫感染60 d的猪血清特异性识别,具有较好的反应原性。结论成功构建了重组表达质粒pET-28a-Tp-serpin,并在E.coli Rosetta gami(DE3)中表达了重组蛋白,为旋毛虫病的血清学诊断候选抗原的研制及开发提供了科学依据,也为阐明serpin在伪旋毛虫入侵时期调节宿主免疫反应的作用奠定了基础。
Objective To express and purify Tpserpin gene in E. coli and identify the antigenicity of Tpserpin. Methods Tp-serpin gene was amplified by RT-PCR and inserted into prokaryotic expression vector pET-28a (+). The recombinant plasmid pET-28a-Tp-serpin was constructed and transformed into Escherichia coli (E.coli) Rosetta gami (DE3) and induced by IPTG. The expressed product was analyzed by SDS-PAGE and purified by Ni-NTA Agarose affinity chromatography. The purity of the purified product was analyzed by SDS-PAGE. . Results The recombinant plasmid pET-28a-Tp-serpin was constructed correctly by PCR, double enzyme digestion and sequencing. The results showed that the recombinant plasmid pET-28a-Tp-serpin was 99% identical to the Tp-serpin gene registered in GenBank. The expressed recombinant Tp-serpin protein has a relative molecular weight of about 43 000 and an expression level of about 40% of the total bacterial protein, mainly in a soluble form. The purity of the purified recombinant Tp-serpin protein is over 95% Pig serum 60 days after infection was specifically recognized and had good reactionogenicity. Conclusion The recombinant plasmid pET-28a-Tp-serpin was constructed successfully and the recombinant protein was expressed in E.coli Rosetta gami (DE3), which provided a scientific basis for the development and research of the serological diagnostic candidate antigen of Trichinella. It also laid the foundation for elucidating the role of serpin in regulating host immune responses during the invasion of pseudotuberculosis.