摘要
目的构建ST8Sia1基因过表达载体,建立稳定过表达ST8Sia1的细胞株,检测ST8Sia1的表达及对细胞增殖的影响。方法通过PCR反应扩增ST8Sia1基因片段,双酶切目的基因片段和pEGFP-C1载体以及pHBLV-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro载体,分别将目的基因片段和不同载体连接,得到ST8Sia1-pEGFP-C1重组载体和重组慢病毒载体。采用PCR方法和DNA序列进行鉴定。分别用不同载体转染黑色素瘤细胞WM451,筛选建立稳定过表达ST8Sia1的细胞株,Real-time PCR检测稳转细胞ST8Sia1 mRNA水平;Western blot法检测稳转细胞中ST8Sia1蛋白表达水平;CCK-8法检测稳转细胞的生长增殖活性;软琼脂克隆形成实验分析稳转细胞的克隆形成能力。结果成功构建ST8Sia1-pEGFP-C1重组质粒及ST8Sia1过表达慢病毒载体。发现ST8Sia1过表达慢病毒载体转染效率较高,建立稳定过表达ST8Sia1的WM451细胞株,发现ST8Sia1过表达促进肿瘤细胞增殖和克隆形成。结论成功构建了ST8Sia1过表达载体,并发现ST8Sia1过表达能够促进黑色素瘤细胞WM451的增殖、克隆形成能力。
Aim To construct different over-expression vectors of ST8Sia1 gene and establish a cell line with stable ST8Sia1 over-expression,and to check the proliferation of cells with ST8Sia1 over-expression. Methods Polymerase chain reaction (PCR) was used to amplify the code region of ST8Sia1.The amplified ST8Sia1 fragment and pEGFP-C1 vector,pHBLV-CMV-MCS-3flag-EF1-ZsGreen-T2A-Puro vector were digested with restriction enzymes.The target gene fragment and the different vectors were ligated to obtain the ST8Sia1-pEGFP-C1 recombinant vector and the recombinant lentiviral vector, respectively. PCR and DNA sequencing were used to identify recombinant vectors.Melanoma cells WM451 were transfected with different vectors.Cell line with stable ST8Sia1 over-expressing was established.ST8Sia1 mRNA level was checked by real-time PCR.ST8Sia1 protein expression level was checked by Western blot.Proliferation of the stable cells was assayed by CCK-8 method.The clony formation of stable cells was also performed. Results Both ST8Sia1-pEGFP-C1 recombinant plasmid and ST8Sia1 over-expression lentiviral vectors were successfully constructed.The transfection efficiency of ST8Sia1 over-expression lentiviral vector was much higher than that of ST8Sia1-pEGFP-C1 recombinant plasmid.A WM451 cell line with stable ST8Sia1 over-expression lentiviral vectors was established.Results showed that the over-expression of ST8Sia1 promoted the proliferation and colony formation of cells. Conclusions ST8Sia1 over-expression vectors are successfully constructed.The over-expression of ST8Sia1 promotes the proliferation and colony formation of WM451 cells.
作者
刘金宜
富炜琦
杜冠华
王金华
杨淬
LIU Jin-yi;FU Wei-qi;DU Guan-hua;WANG Jin-hua;YANG Cui(School of Ethnic Medicine,Key Lab of Chemistry in Ethnic Medicinal Resources,State Ethnic AffairsCommission & Ministry of Education,Yunnan Minzu University,Kunming650500,China;Institute of MateriaMedica,Chinese Academy of Medical Sciences & Peking Union Medical College,Drug Targets &Screening Center-the Key Lab of Beijing,Beijing100050,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2019年第5期733-739,共7页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81573454
81760655)
北京自然科学基金资助项目(No 7172142)
中国医学科学院医学与健康科技创新工程(No 2016-I2M-3-007)
国家科技部十三五重大新药创制项目(No 2018ZX09711001-005-025)
