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目的运用基因工程方法制备Caveolin-1重组蛋白及其抗体,为研究Caveolin-1的功能奠定基础。方法构建原核表达载体pGEX-5X-1-Caveolin-1,酶切鉴定后,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot方法鉴定基因片段的正确性及表达蛋白的特异性后,进行纯化。纯化的重组蛋白免疫Balb/c小鼠3次后,ELISA法测定兔多克隆抗体滴度。结果克隆得到的目的基因片段大小与预期相符。重组Caveolin-1蛋白相对分子质量约为48000,以包涵体形式存在Caveolin-1蛋白N端融合GST标签便于纯化及鉴定。获得的高纯度Caveolin-1融合蛋白,免疫动物后,可产生特异的高滴度抗体(ELISA滴度达1:10000)。结论构建了高表达Caveolin-1蛋白的原核表达系统,制备的Caveolin-1重组蛋白免疫动物,获得了特异、高效价的抗体,为研究Caveolin-1基因的生物学活性提供了实验材料和依据。
Objective To prepare Caveolin-1 recombinant protein and its antibody by genetic engineering and lay the foundation for the study of the function of Caveolin-1. Methods The prokaryotic expression vector pGEX-5X-1-Caveolin-1 was constructed and identified by restriction enzyme digestion. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot analysis of the correctness of the gene fragment and protein specificity, purification. Rabbit polyclonal antibody titers were measured by ELISA after immunized Balb / c mice three times with purified recombinant protein. Results The size of the target gene cloned was in line with the expectation. The relative molecular mass of recombinant Caveolin-1 protein is about 48000. The N-terminal fusion GST tag of Caveolin-1 protein exists in the form of inclusion body for purification and identification. High-purity Caveolin-1 fusion protein was obtained and the animals were immunized to produce specific high-titer antibodies (ELISA titer 1: 10000). Conclusion The prokaryotic expression system of Caveolin-1 protein was constructed. The specific anti-Caveolin-1 recombinant protein was immunized to get the specific and high titer antibody, which provided experimental materials and basis for studying the biological activity of Caveolin-1 gene.