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目的原核表达LHRH-TAT-DNaseⅡα融合基因(LTD),并制备多克隆抗体。方法采用PCR法克隆LTD基因片段,插入p ET-28a载体,构建重组原核表达质粒p ET-28a-LTD,转化大肠埃希菌BL21(Rossetta),经IPTG诱导表达。融合蛋白经电洗脱分离纯化后,免疫獭兔,制备LTD蛋白多克隆抗体,采用ELISA法检测抗血清效价。结果构建的重组原核表达质粒经PCR、双酶切及测序鉴定证明构建正确;纯化得到的融合蛋白相对分子质量为39 270,浓度为0.5 mg/ml,可被兔抗LTD抗体识别。血清抗体效价可达1∶6 400。结论成功原核表达了DNaseⅡ融合蛋白LTD,并制备了多克隆抗体,为进一步研究DNaseⅡ蛋白体内外抗肿瘤活性及作用机制奠定了基础。
Objective To express LHRH-TAT-DNaseⅡα fusion gene (LTD) in prokaryotic cells and prepare polyclonal antibody. Methods The LTD gene fragment was cloned by PCR and inserted into p ET-28a vector to construct recombinant prokaryotic expression plasmid p ET-28a-LTD. The recombinant plasmid was transformed into E. coli BL21 (Rossetta) and induced by IPTG. After purified by electroelution, the fusion protein was immunized with Rex rabbit to prepare LTD protein polyclonal antibody. The antiserum titer was detected by ELISA. Results The constructed recombinant prokaryotic expression plasmid was verified by PCR, double enzyme digestion and sequencing. The purified fusion protein showed a correct molecular weight of 39 270 at a concentration of 0.5 mg / ml and could be recognized by rabbit anti-LTD antibody. Serum antibody titers up to 1: 400. Conclusion The successful expression of DNase Ⅱ fusion protein LTD and the preparation of polyclonal antibody have laid the foundation for further study on antitumor activity and mechanism of DNase Ⅱ protein in vitro and in vivo.