Construction of eukaryotic expression vector of human CC10 gene and expression of CC10 protein in lung adenocarcinoma A549 cell line.
A mammalian expression plasmid pcDNA3. 1-hCC10 was constructed and identified, then CC10 protein expression in A549 lung cancer cell line was detected. A 273 bp cDNA fragment was amplified from the total RNA of normal lung tissue by using RT-PCR and cloned into expression plasmid cDNA3. 1, and the recombinant plasmid was identified by employing double digestion restriction enzymes Hind III and BamH I and the cDNA sequence was assayed by the Sanger dideoxy-mediated chain termination method. The segment was then transfected into the A549 lung cancer cell line. The protein expression of CC10 was detected by immunofluorescence and Western blot. Our results showed that the cDNA fragment included the entire coding region (273 bp). The recombinant eukaryotic cell expression vector of pcDNA3. 1-hCC10 was successfully constructed, and the sequence of the insert was identical to the published sequence. A549 cells line transfected with the pcDNA3. 1-hCC10 expressed high level of CC10 protein. The recombinant plasmid cDNA3. 1-hCC10 may serve as an effective tool for the study of tumorogenesis and tumor treatment.
Zhong
Sheng
S
Institute of Respiratory Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xu
Yongjian
Y
Zhang
Zhenxiang
Z
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014598:Uteroglobin / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; C556389:SCGB1A1 protein, human; D014598:Uteroglobin