HeLa细胞中Zwint-1选择剪接亚型v7的表达鉴定.docVIP

HeLa细胞中Zwint-1选择剪接亚型v7的表达鉴定 苏景华，李 蕊*，刘 俊，周艳琳，陈小璇，代剑平，王革非， 李康生 （广东 汕头 515041,汕头大学医学院微生物学与免疫学教研室） 【摘要】 目的：验证在肿瘤细胞系中是否存在Zwint-1 v7选择性剪切亚型的mRNA和蛋白产物。 方法：在缺失片段两侧设计引物，利用PCR和克隆测序验证HeLa细胞中是否存在缺失片段的mRNA；构建Zwint-1 v7 3’端特异区段（Z7）的GST融合表达载体pGEX-KG-GST-Z7，转化入BL21菌种诱导表达，菌体经超声破碎和2 mol/L尿素洗涤纯化后，融合蛋白经SDS电泳并切胶回收后作为抗原与佐剂混合乳化并免疫C57BL/6小鼠，收获多克隆抗血清Anti-Z7，进行Dot blot检测多克隆抗体的效价，Western blot检测HeLa细胞全蛋白中的Z7蛋白产物。 结果：PCR和测序结果表明存在缺失37 bp的核酸片段；SDS电泳显示融合蛋白GST- Z7主要以包涵体的形式表达；Dot blot结果表明1﹕5 000稀释的多克隆抗体能检出12 ng GST-Z7；Western blot结果表明，1﹕200稀释的多克隆抗体可识别HeLa细胞中Zwint-1 v7蛋白。 结论：核酸水平和蛋白水平初步验证了HeLa细胞中存在Zwint-1 v7。 【关键词】免疫学；Zwint-1/Zwint-1 v7蛋白；原核表达；多克隆抗体制备 Identification of the Alternatively Spliced Variant v7 of Zwint-1 in HeLa SU Jinghua, LI Rui*, LIU Jun, ZHOU Yanlin, CHEN Xiaoxuan, DAI Jianping, LI Kangsheng （Department of Microbiology and Immunology , The Key Immunopathology Laboratory of Guangdong Province, Shantou University Medical College, Shantou 515041, Guangdong, China） 【A】 : The aim was to identify whether the mRNA and protein expression of Zwint-1 v7 existed in human cancer cell line. Methods: To identify the mRNA with the deleted fragment in HeLa cells, we designed primers on the each side of the deleted fragment for PCR and sequencing. Therefore, we constructed prokaryotic expression vector pGEX-KG-GST-Z7, the inserted fragment Z7 was chosed based on the different sequences between Zwint-1 and Zwint-1 v7. This vector was transformed into BL21 strain for the expression of fusion protein GST-Z7. After ultrasonication and purified from the SDSgels, GST-Z7 was used as antigens and emulsified with adjuvants to immune C57BL/6 female mice for the preparation of polyclonal antibody Anti-Z7. The serum titer was validated by Dol blot test. The Zwint-1 v7 protein in the total proteins of HeLa cells was detected by Western blot with Anti-Z7. Results: The PCR and sequencing results showed that there existed mRNA with 37 bp deletions in HeLa cells, SDSshowed that GST-Z7 was correctly expressed in BL21