PCDH10基因慢病毒载体构建及其在MDA-MB-231细胞中的表达  

Construction of PCDH10 Gene Lentiviral Vector and Its Expression in MDA-MB-231 Cells

作　　者：张微 岳丽玲 张延勇[2] 韩翠翠[3] 徐天娇 朱文斌 刘立琨 ZHANG Wei;YUE Li-ling;ZHANG Yan-yong;HAN Cui-cui;XU Tian-jiao;ZHU Wen-bin;LIU Li-kun(Qiqihar Medical College, Institute of Medical Sciences, Qiqihar, Heilongjiang Province, 161006 China;The Third Affiliated Hospital of Qiqihar Medical College, Qiqihar, Heilongjiang Province, 161006 China;School of Pharmacy, Qiqihar Medical College, Qiqihar, Heilongjiang Province, 161006 China)

机构地区：[1]齐齐哈尔医学院医药科学研究院,黑龙江齐齐哈尔161006 [2]齐齐哈尔医学院第三附属医院,黑龙江齐齐哈尔161006 [3]齐齐哈尔医学院药学院,黑龙江齐齐哈尔161006

出　　处：《系统医学》2019年第16期27-29,38,共4页Systems Medicine

基　　金：齐齐哈尔市科技局项目(SGFF-201632)。

摘　　要：目的构建原钙黏蛋白10(PCDH10)基因慢病毒表达载体并建立稳定过表达PCDH10的MDA-MB-231细胞系。方法 2018年6—11月,根据PCDH10基因序列设计引物,应用PCR法扩增PCDH10全长片段,连接线性化的pLV-EF1α-GFP-Puro载体,获得pLV-PCDH10慢病毒重组质粒,测序正确后进行PCDH10基因慢病毒载体的包装及滴度测定。将重组质粒pLV-PCDH10转染MDA-MB-231细胞,应用反转录聚合酶链反应(RT-PCR)和Western blot分别检测MDA-MB-231细胞中PCDH10的表达情况。结果测序结果证实成功构建慢病毒表达载体pLV-PCDH10,病毒滴度为5×10^8TU/mL;转染MDA-MB-231细胞后,RT-PCR和Western blot结果显示PCDH10的m RNA和蛋白表达较对照组高表达。结论成功构建pLV-PCDH10慢病毒载体并建立稳定过表达PCDH10的MDA-MB-231细胞系,为深入研究PCDH10基因在乳腺癌发生发展中的功能提供实验基础。Objective To construct a lentiviral expression vector of protocadherin 10 (PCDH10) gene and establish a MDA-MB-231 cell line stably overexpressing PCDH10. Methods From June to November 2018, primers were designed based on the PCDH10 gene sequence, and the full -length PCDH10 fragment was amplified by PCR. The linearized pLV-EF1α-GFP-Puro vector was ligated to obtain the pLV -PCDH10 lentiviral recombinant plasmid. The PCDH10 gene lentiviral vector was packaged and titered. The recombinant plasmid pLV-PCDH10 was transfected into MDAMB-231 cells, and the expression of PCDH10 in MDA-MB-231 cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Results The sequencing results confirmed that the lentiviral expression vector pLV-PCDH10 was successfully constructed, and the virus titer was 5×108 TU/mL. After transfection of MDA-MB-231 cells, RT-PCR and Western blot showed that the mRNA and protein expression of PCDH10 was higher than that of the control group. Conclusion The pLV-PCDH10 lentiviral vector was successfully constructed and the MDA-MB-231 cell line stably overexpressing PCDH10 was established, which provided an experimental basis for further study of the function of PCDH10 gene in the development of breast cancer.

关 键 词：PCDH10基因 慢病毒载体 载体构建 MDA-MB-231细胞 

分 类 号：R373[医药卫生—病原生物学]