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目的原核表达并纯化人CD96胞外区蛋白,免疫家兔制备抗体。方法构建pET32-CD96重组质粒,转化大肠埃希菌BL21(DE3),以IPTG诱导表达,Ni2+-NTA树脂纯化蛋白并免疫家兔,以间接ELISA检测抗体效价,Westernblot鉴定抗体特异性。结果质粒pET32-CD96经测序鉴定,通过SDS电泳显示能在BL21(DE3)中高效表达,相对分子质量约37000;Westernblot实验结果显示制备的多克隆抗体可与HL-60细胞提取蛋白及原核表达的人CD96蛋白胞外区特异性结合。结论成功构建并表达人CD96胞外区蛋白,免疫家兔获得高滴度抗血清,为后续研究奠定了基础。
Objective To prokaryotic express and purify human CD96 extracellular domain protein and immunize rabbits to prepare antibodies. Methods The recombinant plasmid pET32-CD96 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was induced by IPTG. The protein was purified by Ni2 + -NTA resin and immunized rabbits. The antibody titer was detected by indirect ELISA. The antibody specificity was identified by Western blot. Results The plasmid pET32-CD96 was identified by sequencing and expressed by SDS-PAGE in BL21 (DE3) with a molecular weight of about 37000. The results of Western blot showed that the prepared polyclonal antibody could bind to the protein extracted from HL-60 cells and express in prokaryotic Human CD96 protein extracellular region specifically binds. Conclusion The successful construction and expression of human CD96 extracellular domain protein and immunization of rabbits to obtain high titer antiserum, which laid the foundation for further study.