人CC10基因在肺腺癌A549细胞中的表达

Expression of Human CC10 Gene in Lung Adenocarcinoma A549 Cell

目的构建及鉴定人CC10基因真核细胞表达载体。方法采用RT-PCR法,从人肺组织的总RNA中扩增出273bp的人CC10cDNA片段,利用DNA重组技术将其克隆到真核细胞表达载体pcD-NA3.1中,脂质体法转染pcDNA3.1-hCC10到肺腺癌A549细胞,荧光免疫细胞化学法及Westernblot法检测CC10蛋白的表达。结果用酶切重组质粒并行序列鉴定,人CC10基因已经正确克隆到真核细胞表达载体pcDNA3.1中,体外染pcDNA3.1-hCC10的肺腺癌A549细胞中CC10蛋白表达明显增高。结论成功构建了CC10基因的真核细胞表达载体pcDNA3.1-hCC10,并获得表达。

Objective To construct and identity human the expression plasmid pcDNA3. 1-hCC10. Methods A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by RT-PCR,then inserted it into expression vector pcDNA3. 1 by DNA recombinant techniques. We transfected pcDNA.3. 1- hCC10 in A549 cells by liposome-mediated gene transfer method. The protein expression of CC10 was detected by the techniques of irnrnunofluorescence and Western blot. Results After the identification of restriction enzymes analysis and sequencing, the recombinant plasmidpcDNA3. 1-hCC10 was confirmed which contained the correct and entire nucleotide sequence of the CC10 DNA in pcDNA3. 1. The expression of CC10 in protein level ascend in A549 cells transfected with reconstructive plasmid. Conclusion The pcDNA3. 1-hCC10 expression plasmid was successfully constructed, acquired stably protein expression in A549 lung cancer cell line.