日本血吸虫候选疫苗原肌球蛋白的表达载体构建、原核表达及免疫原性研究

Cloning and Expression of Schistosoma Japonicum Tropomyosin Protein in Escherichia coli and Its Immunogenicity Analysis

目的将日本血吸虫原肌球蛋白基因（tropomyosin）克隆到原核表达载体pET-28a（＋）载体上，在BL-21（DE3）型大肠埃希菌中表达其产物，并对其进行免疫原性分析。方法从日本血吸虫的Expressed Sequence Tag（EST）文库中筛选出包含tropomyosin全长基因的EST，然后用高保真酶PCR扩增，构建到pET-28a（＋）载体上，最终在BL-21（DE3）型大肠埃希菌中用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。表达的his—tropomyosin融合蛋白用镍螯合树脂蛋白纯化柱（Ni—NTA resin）亲和层析纯化，得到纯度较高的tropomyosin后，通过Western Blotting技术，用感染日本血吸虫的兔血清作为天然抗体来研究tropomyosi的免疫原性。结果pET-28a（＋）-tropomyosin重组质粒在BL-21（DE3）型大肠埃希菌中成功表达，使用SDS—PAGE分析得到实际Mr约为40000的his—tropomyosin融合蛋白，Ni—NTA resin的纯化效果很明显，获得的相对纯的目的蛋白经过Western Blotting方法检测显示：tropomyosin融合蛋白能够与感染日本血吸虫6w的兔血清有较强的免疫反应。结论Tropomyosin能够在BL-21（DE3）型大肠埃希菌中高效表达，且抗原免疫原性强，日本血吸虫感染的兔血清能够识剐体外表达的tropomyosin，具有与天然的tropomyosin相同的免疲原性。

Objective The gene of .Schistosoma Japonicum tropomyosin was subcloned into an expression vector pET-28a （＋） ,then the recombinant plasmid was transformed into Escherichia coli BL-21 cells （DE3）. In the presence of Isopropyl β-D-thiogalactoside （IPTG） ,the fusion protein was expressed. The immunogenicity of the fusion tropomyosin protein was analyzed by Western Blotting. Methods EST containing the whole ORF of Schistosoma Japonicum tropomyosin was choosed from the EST library. After PCR amplification, the gene was cloned into the expression vector of pET-28a（＋）. The fusion tropomyosin was expressed in Escherichia coli BL-21 cells （DE3） in the presence of IPTG. Through affinity chromatography for purification, comparably highly pure tropomyosin was gained from the expressed fusion histropomyosin. Then the immunogenicity of tropomyosin was analyzed using the serum of rabbit infected with Schistosoma Japonicum by Western Blotting. Results The recombinant pET-28a（＋）-tropomyosin plasmid was successfully expressed in Escherichia coli BL-21 cells （DE3）,resulting in 40 000 Dolton fused his-tropomyosin. Achieved a success on the purification with Ni-NTA resin chromatography. Western Blotting revealed that the fused tropamyosin had strong immunoreaction with the serum of rabbit after infected with Schistosoma Japonicum 6 weeks. Conclusion Tropomyosin could be efficiently expressed in Escherichia coli BL-21 cells（DE3）. Tropomyosin expressed in vitro had strong immunogenit that it could be recognized by the serum of rabbit infected with Schistosoma Japonicum. possessing the same immunogenicity with nature tropomyosin.