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目的 :原核表达hCacyBP并制备小鼠抗hCacyBP多克隆抗体。观察该蛋白组织分布情况 ,研究其可能在胃癌多药耐药形成中的作用。方法 :将hCacyBP基因的全编码区克隆进原核表达载体pET2 8a中 ,转化E .coli,以IPTG诱导重组目的蛋白的表达。以纯化的重组目的蛋白作为免疫原免疫小鼠 ,制备相应的多克隆抗体。以Westernblot法检测hCacyBP在兔 10种组织的表达情况 ;以Westernblot和免疫组化染色法检测hCa cyBP在胃癌耐药细胞SGC790 1/VCR及其亲本药敏细胞SGC790 1中的表达和分布。结果 :成功地表达重组目的蛋白并制备了小鼠抗hCacyBP的多克隆抗体 ,Westernblot分析显示 ,hCacyBP在兔的 10种组织中均有表达 ,在脑和肝组织中表达略高 ;hCacyBP在胃癌耐药细胞SGC790 1/VCR及其亲本药敏细胞SGC790 1中的表达未见明显差异。结论 :CacyBP是在多种组织中广泛表达的一种蛋白质。制备的小鼠抗hCa cyBP多克隆抗体特异性较高 ,为进一步研究hCacyBP的功能提供了工具。
OBJECTIVE: Prokaryotic expression of hCacyBP and preparation of mouse anti-hCacyBP polyclonal antibody. Observe the distribution of the protein, and study its possible role in the multidrug resistance of gastric cancer. Methods: The full coding region of hCacyBP gene was cloned into prokaryotic expression vector pET2 8a and transformed into E.coli. The recombinant protein was induced by IPTG. The purified recombinant protein was used as immunogen to immunize mice to prepare the corresponding polyclonal antibody. The expression of hCacyBP in 10 tissues of rabbits was detected by Western blot. The expression and distribution of hCa cyBP in gastric cancer cell line SGC790 1 / VCR and its parental drug sensitive cell line SGC7901 were detected by Western blot and immunohistochemistry. Results: The recombinant protein was successfully expressed and the mouse polyclonal antibody against hCacyBP was prepared. Western blot analysis showed that hCacyBP was expressed in 10 tissues of rabbits and slightly higher in brain and liver tissues. The expression of hCacyBP in gastric cancer There was no significant difference in the expression of SGC790 1 / VCR and its parental drug sensitive cell SGC790 1. Conclusion: CacyBP is a widely expressed protein in many tissues. The prepared mouse anti-hCa cyBP polyclonal antibody is highly specific, which provides a tool for further studying the function of hCacyBP.