旋毛虫Serpin基因的体外表达及其特性鉴定

In Vitro Expression and Characterization of Serpin Gene of Trichinella spiralis

目的体外表达旋毛虫丝氨酸蛋白酶抑制因子(Serpin)基因,并检测其在旋毛虫不同发育时期mRNA转录、蛋白表达与分子定位及反应原性情况,为以其作为候选诊断抗原基因提供实验依据。方法根据旋毛虫Serpin基因序列,以重组质粒pBlue-script-WM5为模板,利用PCR技术去除其全长cDNA的信号肽序列,采用原核表达载体pET-28a构建不含该基因信号肽的、融合蛋白不含组氨酸标签的重组表达质粒pET-28a-WM5,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物纯化后,采用Westernblot法检测反应原性。免疫组织化学技术检测Serpin蛋白在旋毛虫不同发育时期的表达及定位情况。采用RT-PCR和实时定量RT-PCR技术检测Serpin基因在旋毛虫不同发育时期的转录水平。结果重组质粒pET-28a-WM5经PCR、双酶切及测序鉴定证明构建正确,Serpin基因能够在大肠杆菌中高效表达。重组蛋白主要以包涵体形式存在,约占菌体总蛋白的51.1%。纯化后的重组蛋白纯度可达98%,与猪抗旋毛虫60d抗血清有较好的反应原性,与26d猪抗血清无明显反应信号;RT-PCR与实时定量RT-PCR结果显示,该基因在旋毛虫不同发育时期均有转录,但转录水平有明显差异,有2个转录高峰期(Ad2/Ad3和ML)以及1个转录水平明显下降期(Ad5);免疫组化鉴定表明,Serpin天然抗原在旋毛虫肌幼虫时期18d开始表达,且在感染35d后大量表达和分泌。结论已获得较高纯度的旋毛虫Serpin体外表达融合蛋白,其具有较好的反应原性。旋毛虫Serpin基因具有期特异性表达的特点,且在肌幼虫期具有高转录与高表达水平,可作为捕获旋毛虫肌幼虫期循环抗体的候选诊断抗原基因。

Objective To express the serine proteinase inhibitor （Serpin） gene of TrichineUa spiralis （Ts） in vitro, determine its transcription, expression and molecular location of expressed product at various development stages of Ts as well as the reacto- genicity of expressed protein, and evaluate the possibility of Serpin as a candidate antigen gene for diagnosis. Methods The fulllength cDNA sequence of Serpin, in which the signal peptide was deleted, was amplified by PCR with a pair of designed primers using recombinant plasmid pBlue-script-WM5 as template, and cloned into prokaryotic expression vector pET28a without histidine tags. The constructed recombinant plasmid pET-28a-WM5 was transformed to E. coli BL21（DE3） for expression under induction of IPTG. The expressed product was purified by electro-elution process and analyzed for reactogenicity by Western blot. The transcription level of Serpin gene at various development stages of Ts was determined by RT-PCR and real-time quantitative RT-PCR, and the expression. and location of Serpin protein by immunohistochemical method. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pET-28a-WM5 was constructed con＇ectly, and Serpin gene was highly expressed in E. coll. The expressed Serpin protein mainly existed in a form of inclusion body, contained about 51. 1% of total somatic protein and reached a purity of 98% after purification. It showed good reactogenicity with the serum of pig 60 d after infection with Ts, while no significant reaction with that 26 d after infection. Both RT-PCR and real-time quantitative RT-PCR proved transcription of Serpin gene in the whole life cycle of Ts. However, the transcription levels at various development stages were significantly different, which reached peak values at Ad2/ Ad3 and ML stages and decreased significantly at AdS stage. Immunohistochemical analysis indicated that Serpin protein was expressed in a small quantity at early stage （day 18） of Ts infection, while in a large quantity on day 35 after infection. Conclusion Serpin fusion protein with high purity was expressed in vitro and showed good reactogenicity. The expression of Serpin gene was stage-specific, and high transcription and expression levels were observed in muscle larvae （ML） of Ts, indicating that Serpin gene might be used as a candidate antigen gene for capture of anti-Trichinella antibody in late infection at ML stage.