Construction And Expression Of Recombinant Eukaryotic Expression Plasmid Containing Human Gene Brca1

Background:Breast cancer is one of the most common malignances in women. The incidence is increased as the living standard improved. In some place, breast cancer is the first death caused by malignancy. Some researches indicated that breast cancer is caused by the interaction of multi-factors and genes, and have a family genetic predisposition. In the high incidence of breast cancer family, there is one or more autosomal dominant breast cancer susceptibility gene, in which the most common is BRCA1 and BRCA2.Related researches indicated BRCA1 could inhibit the growth of cells, also participate in the regulation of cell cycle,gene transcription,DNA damage repair and apoptosis and many cell activities. They play an important role in the maintenance of genome stability. Mutation in BRCA1 accounts for12.8% in hereditary breast cancer and ovarian cancer family history in worldwide, compared 3.8% in the analysis of 130 female breast cancers in China. More than 80%hereditary breast cancer and some sporadic breast cancer is closely related to the abnormal structure and function of BRCA1 gene. Approximately 40-50% of hereditary breast cancer is caused by BRCA1 mutations. To explore the mechanism of BRCA1 acts, providing new targets for early diagnosis and clinical treatment for breast cancer.Objective:Constructed the BRCA1 gene eukaryotic expression plasmid , and screened out . the breast cancer cell lines with high expression of BRCA1 gene, To make the foundation of observing the biological characteristics and cell cycles of cells high expressing BRCA1 protein. Methods:Extracted total RNA from the placenta as primers, amplified cDNA sequences by RT-PCR, connected the amplified product with pMD18-T vector, and transformed into E.coli JM109,then picked cloning, extracted plasmid, Select the correct clone plasmid ,cut by X ho I and BamH I, Agarose gel recovered fragments; eukaryotic expressed vector inserted in pcDNA3.1 (+),cut by double enzyme, Agarose gel recovered linear carrier fragments .Connected these tow common fragments and T4DNA enzyme. Constructed recombinant plasmid pcDNA3.1-BRCA1, and transformed into E.coli JM109,Extracted endotoxin-free recombinant plasmid pcDNA3.1-BRCA1, and transfected the breast cancer cell lines MDA- MB-231. Screened the transfected cell lines by G418 and obtained stable cell lines with high expression of BRCA1.Extracted the total protein before and after transfection, and identified the expression of BRCA1 with Western blot. Use Statistical analysis software of SPSS13.0(P<0.01).Results:933 base pairs in the N terminal of BRCA1 gene ware successfully cloned. Sequencing and extracting plasmid restriction enzyme digestion confirmed the pcDNA3.1-BRCA1 contained the right 933 base pairs in the N terminal of BRCA1 gene with corerect insert direction, fragment size and DNA sequence. The leval of BRCA1 expression after transinfection was higher than pre-transfection.Conclusion:1,BRCA1 gene was successfully cloned.2,The sequencing inserted in the BRCA1 cloning was already confirmed.3,The pcDNA3.1-BRCA1 eukaryotic expression plasmid was successfully produced.4,Western blot confirmed BRCA1 gene expressed high in Breast cancer.