摘要
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。
AIM: To express hCacyBP in E.coli and prepare mouse polyclonal antib ody against hCacyBP so as to study tissue distribution of hCacyBP and evaluate i ts role during formation of multidrug resistance to gastric cancer. MET HODS: hCacyBP gene was subcloned into an expression vector pET28a, and then the recombinant vector was transformed into E.coli BL21. The recombinan t protein was expressed in BL21 under IPTG induction. Using purified hCacyBP as immunogen, mouse polyclonal antibody against hCacyBP was prepared. hCacyBP was d etected by Western blot in 10 kinds of rabbit tissues. Expresseion and distribut ion of hCacyBP in SGC7901/VCR and SGC7901 cells were detected by Western blot an d immunohistochemical staining. RESULTS: hCacyBP was succes sfully expressed in E.coli. The Western blot analysis showed that hCacyBP wa s expressed in all 10 kinds of rabbit tissues, but expression of brain and liver tissues were higher as compared with other tissues. Expression and distribution of hCacyBP in both SGC7901/VCR and SGC7901 cells had no significant difference. CONCLUSION: CacyBP expressed widespreadly in varied tissues. Po lyclonal antibody against hCacyBP that we prepared has high specificity, which p rovides a powerful tool for studing the function of hCacyBP.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第2期206-209,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目 (No .30 0 30 1 40)
关键词
人钙周期素结合蛋白
表达
多药耐药性
human calcyclin-binding protein
expression
m ultidrug resistance
