【摘要】 间隙连接蛋白31(Connexin31,Cx31)是间隙连接蛋白(Connexin)家族的一员,目前对于Cx31的功能及其调节方式知之甚少。本实验利用Fmoc固相多肽合成的方法合成Cx31羧基端一个多肽片段(250 266AA),经HPLC纯化后偶联到匙孔槭血蓝蛋白,免疫新西兰雄兔后采血检测、并纯化,采用Cx31myc表达蛋白进行Westernblotting、细胞免疫荧光染色、免疫沉淀实验,证实得到的抗体为抗间隙连接蛋白31的特异抗体。更多还原

【Abstract】 Connexin 31 is a member of connexins family.In this work,c-terminal immunogenic peptide of connexin31 was synthesized by using solid phase synthesis.The purified peptide was coupled to the hemocyanin from Keyhole Limpets(KLH).The peptide-KLH solution was mixed with freund’s complete or incomplete adjuvant and immunized rabbit to prepare specific polyclonal antibody against the topology of connexin31.A full-length human connexin31 gene was cloned into expression vector,pcDNA3.1/myc-his(-)B,and transformed into HT1080 cell line stably or into Hela cell transiently.The expressed protein was used to test the polyclonal antibody.Western blotting analysis and immunoprecipitation suggested that the polyclonal antibody recognized the fusion protein of the connexin31-myc.Cell immunofluorescence staining showed that the connexin31 localized at the site of the cell-cell contact gap junction.The antiserum against connexin31 prepared by differential immunogenic peptide-KLH has good specificity.更多还原