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目的构建和表达小鼠精子蛋白Sp17与白介素5(IL-5)融合蛋白,并对其进行纯化和免疫原性鉴定。方法采用PCR技术从质粒pGEM-1-IL-5中扩增IL-5基因片段,将其克隆至pET/Sp17原核表达载体中与Sp17基因融合,构建重组质粒pET/Sp17-IL-5,经酶切鉴定后转化大肠杆菌BL21(DE3),IPTG诱导表达,采用Ni2+-NTAAgarose纯化,SDS-PAGE检测、N端测序及Western blot;重组蛋白Sp17-IL-5免疫小鼠后ELISA检测小鼠血清特异性抗体。结果成功构建小鼠精子蛋白Sp17与IL-5融合蛋白的高效表达质粒pET/Sp17-IL-5,重组工程菌pET-28a(+)-Sp17-IL-5/BL21经IPTG诱导目的蛋白表达率约28%,PAGE初步测定目的蛋白相对分子量(Mr)约39000,纯化后蛋白纯度达91%,免疫后小鼠血清中检测到特异性抗体。结论Sp17-IL-5蛋白经基因克隆获得了较高的表达量,并初步显示了较好的免疫活性,为新型Sp17避孕疫苗的研制奠定基础。
Objective To construct and express mouse sperm protein Sp17 and interleukin 5 (IL-5) fusion protein and purify them and identify their immunogenicity. Methods The IL-5 gene fragment was amplified by PCR from the plasmid pGEM-1-IL-5, cloned into pET / Sp17 prokaryotic expression vector and fused with Sp17 gene to construct recombinant plasmid pET / Sp17-IL-5. The recombinant plasmids were transformed into E.coli BL21 (DE3) after induced by IPTG. The recombinant plasmids were induced by IPTG and purified by Ni2 + -NTAAgarose, SDS-PAGE, N-terminal sequencing and Western blot. Specific antibodies. Results The recombinant plasmid pET / Sp17-IL-5 was successfully constructed and the recombinant protein pET-28a (+) - Sp17-IL-5 / BL21 was induced by IPTG About 28%. The relative molecular weight (Mr) of the target protein was about 39,000 by PAGE and the protein purity was 91% after purification. Specific antibodies were detected in the serum of the mice after immunization. Conclusion The higher expression level of Sp17-IL-5 protein was obtained by gene cloning, and initially showed good immunocompetence, laying a foundation for the development of new Sp17 contraceptive vaccine.