人血白蛋白的原核表达及应用

Prokaryotic Expression and Application of Recombinant Human Serum Albumin

旨在构建人血白蛋白(HSA)基因的原核表达载体pGEX-4T-1-rHSA,诱导表达后纯化重组HSA蛋白,制备多克隆抗体并对其进行鉴定。根据GenBank发表的HSA(NM_000477.5)的核苷酸序列设计合成1对特异性引物,以人胚肺二倍体细胞的总RNA为模板,利用RT-PCR扩增HSA基因,将其克隆至pGEX-4T-1载体,构建重组原核表达质粒pGEX-4T-1-rHSA,经PCR、酶切及测序鉴定正确后转化大肠杆菌BL21(DE3),进行IPTG诱导表达,表达产物经变性、复性、亲和层析纯化后作为免疫原制备多克隆抗体,应用间接ELISA和Western-blot方法对所得抗体进行检测。结果表明,重组蛋白主要以包涵体的形式存在,分子质量为92 kD;重组蛋白具有良好的反应原性和免疫原性;应用其制备的抗血清效价达到1∶100 000;纯化的多克隆抗体与血源人血清白蛋白和重组人血白蛋白均能产生特异性结合,具有良好的生物学活性。

This study was aimed to express and purify the protein of pGEX-4T-I-rHSA in prokaryocytes and prepare anti-HSA polyclonal antibody. The HSA gene was amplified from human embryo lung diploid total RNA by RT-PCR, with primers based on the published HSA （ NM_00047.5 ） sequence in GenBank, then it was cloned into prokaryotic expression vector pGEX-4T-1 to construct a recombinant expression vector pGEX-4T-I-rHSA. The pGEX-4T-I-rHSA was transformed into E. coli BL21 （ DE3 ） . The purified expressed protein was injected into a rabbit and the polyclonal antibody was then prepared, which were identified by the indirect ELISA and Western-blot analysis. Results showed that the recombinant protein mainly exists in the inclusion body, about 92 kD and it has good reactogenicity and immunogenicity. The ELISA titer of the antiserum was approximately 1 ： 100 000. Western-blot analysis revealed that the purified polyclonal antibody against pGEX-dT-i-rHSA had a specific affinity for natural human serum albumin and the expressed protein.