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目的原核表达曼氏血吸虫虫卵白细胞介素-4诱导因子(IL-4-inducing principle of schistosoma eggs,IPSE),并制备小鼠多克隆抗体。方法人工合成IPSE基因,采用PCR技术克隆其成熟体开放阅读框,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-IPSE。转化E.coli BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE和Western blot分析后,应用组氨酸标签蛋白纯化柱纯化包涵体蛋白,免疫BALB/c鼠,制备多克隆抗体。结果克隆的IPSE基因与GenBank中登录的基因序列同源性为100%;重组表达质粒pET-28a-IPSE经PCR及双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约为16 000,以包涵体形式表达,表达量约占菌体总蛋白的30%,可与鼠抗His单抗特异性结合;用纯化的包涵体蛋白免疫BALB/c小鼠后,获得的特异性抗血清效价为2×10-4,该血清可与IPSE蛋白发生特异性反应。结论已原核表达、纯化了曼氏血吸虫IPSE蛋白,并制备了高滴度的特异鼠抗血清,为进一步从日本血吸虫中筛选相似蛋白及研究其功能奠定了基础。
Objective To express the interleukin-4 inducing principle of schistosoma eggs (IPSE) in prokaryotic cells and to prepare mouse polyclonal antibodies. Methods The artificial IPSE gene was synthesized and inserted into the prokaryotic expression vector pET-28a by PCR. The recombinant plasmid pET-28a-IPSE was constructed. E.coli BL21 (DE3) was transformed into E.coli BL21 and induced with IPTG. The expressed product was analyzed by SDS-PAGE and Western blot. The protein of inclusion body was purified with histidine-tagged protein purification column and immunized BALB / c mice to prepare polyclonal antibody. Results The sequence of the cloned IPSE gene was 100% homologous with that of GenBank. The recombinant plasmid pET-28a-IPSE was confirmed by PCR and restriction enzyme digestion. The recombinant protein had a molecular weight of about 16 000, Expressed in the form of inclusion body. The expression amount of the recombinant protein was about 30% of the total bacterial protein, which could specifically bind to the mouse anti-His monoclonal antibody. After the BALB / c mice were immunized with the purified inclusion body protein, the specific antiserum efficacy The price is 2 x 10-4, and this serum specifically reacts with the IPSE protein. Conclusions Prokaryotic expression has been carried out to purify the IPSE protein of Schistosoma mansoni and to prepare high titer anti-serum of mice, which lays the foundation for further screening of Schistosoma japonicum proteins and their functions.