Construction of eukaryotic expression vector of human CC10 gene and expression of CC10 protein in lung adenocarcinoma A549 cell line

Abstract

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 enzymesHind III andBamH I and the cDNA sequence was assayed by the Sanger dideoxymediated 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.

Keywords

CC10 / gene cloning / protein expression

References

[1] BernaardA, RoelsH, LauwerysR, et al. . Human urinary protein 1: evidence for identity with clara cell protein and occurrence in respiratory tract and urogenital tract secretions. Clin Chim Acta, 2002, 207: 239-239

[2] LinnoilaR I, SzaboE, DemayoF, et al. . The role of CC10 in pulmonary carcinogenesis: from a marker to tumor suppression. Ann NY Acad Sci, 2000, 923: 249-249

[3] JesudasanR A, RahmanR A, ChandrashekharappaS, et al. . Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell line. Am J Genet, 1995, 56: 705-705

[4] FlemingT P, WatsonM A. Mammaglobin, a breast-specific gene, and its utility as a marker for breast cancer. Ann NY Acad Sci, 2000, 923: 78-78

[5] PatiernoS R, ManyakM J, FernandezP M, et al. . Uteroglobin: a potential novel tumor suppressor and molecular therapeutic for prostate cancer. Clin Prostate Cancer, 2002, 1(2): 118-118

[6] ShijuboN, KawabataI, SatoN, et al. . Clinical aspects of clara cell 10-kDa protein/uteroglobin (secretoglobin 1A1). Current Pharm Des, 2003, 914: 1139-1139

[7] ShijuboN, ItohY, YamaguchiT, et al. . Serum and BAL Clara cell 10 kDa protein (CC10) levels and CC10-positive bronchiolar cells are decreased in smokers. Eur Respir J, 1997, 10: 1108-1108

[8] YangY, ZhangZ, MukherjeeA B, et al. . Increased susceptibility of mice lacking Clara cell 10-kDa protein to lung tumorigenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen in cigarette smoke. J Biol Chem, 2004, 279(28): 29 336-29 336

[9] SzaboE, GoheerA, WitschiH, et al. . Overexpression of CC10 modifies neoplastic potential in lung cancer cells. Cell Grow Differ, 1998, 9(6): 475-475