CAJ | 학술논문

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 v73′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，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 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-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∶5000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-1v7。
목적：험증재HeLa세포계중시부존재Zwint-1 v7선택성전절아형적mRNA화단백산물。방법：재결실편단량측설계인물，이용PCR화극륭측서험증HeLa세포중시부존재결실편단적mRNA；구건Zwint-1 v73′단특이구단(Z7)적GST융합표체재체pGEX-KG-GST-Z7，전화입BL21균충유도표체，균체경초성파쇄화2 mol/L뇨소세조순화，융합단백경SDS-PAGE전영병절효회수후작위항원여좌제혼합유화병면역C57BL/6소서，수획다극륭항혈청Anti-Z7，채용Dot blot검측다극륭항체적효개，Western blot검측HeLa세포전단백중적Z7단백산물。결과：PCR화측서결과표명존재결실37 bp적핵산편단；SDS-PAGE전영현시재30.7 kDa처출현명현적단백조대；Dot blot결과표명1∶5000희석적다극륭항체능검출12 ng GST-Z7；Western blot결과표명，1∶200희석적다극륭항체가식별HeLa세포중Zwint-1 v7단백。결론：본연구재핵산수평화단백수평초보험증료HeLa세포중존재Zwint-1v7。
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.