HeLa细胞中Zwint-1选择剪接亚型v7的表达鉴定  

Identification of the alternatively spliced variant v7 of Zwint-1 in HeLa cells

作　　者：苏景华 李蕊[1] 刘俊[1] 周艳琳[1] 陈小璇[1] 代剑平[1] 王革非[1] 李康生[1] 

机构地区：[1]汕头大学医学院微生物学与免疫学教研室,广东汕头515041

出　　处：《癌变．畸变．突变》2014年第2期117-122,共6页Carcinogenesis,Teratogenesis & Mutagenesis

基　　金：国家自然科学基金资助项目(31300761)

摘　　要：目的:验证在HeLa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pGEXKG-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,融合蛋白经SDS-PAGE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,采用Dot b lot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-PAGE电泳显示在30.7 kDa处出现明显的蛋白条带;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:本研究在核酸水平和蛋白水平初步验证了HeLa细胞中存在Zwint-1 v7。OBJECTIVE：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 chosen basen 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 SDS-PAGE gels,GST-Z7 was used as antigens and emulsified with adjuvants to immunize 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 evaluated by Western blot with Anti-Z7. RESULTS：The PCR and sequencing results showed mRNA with 37 bp deletions in HeLa cells. SDS-PAGE confirmed that GST-Z7 was correctly expressed in BL21 in the inclusion body. 12 ng GST-Z7 could be detected by the 5 000 times diluted Anti-Z7. Zwint-1 v7 from HeLa cells could be detected by the 200 times diluted Anti-Z7. CONCLUSION：We confirmed that Zwint-1 v7 protein existed in HeLa cells at the levels of translations of nucleic acid and protein structure.

关 键 词：HELA细胞 Zwint-1v7蛋白 原核表达 多克隆抗体制备 

分 类 号：Q786[生物学—分子生物学]