People Eef1a1 - Clotting Factor Of Constructing Hybrid Loci And Expressed In 293 Ft Cells Of Identification

Human coagulation factor Ⅶ is a single-chain glycoprotein synthesis in a liver. The blood tissue factor (TF) exposed FⅦ When the regulation of tissue damaged by combining with the formation of the TF-FⅦ complex to stimulate the extrinsic coagulation pathway, and start the endogenous pathway of coagulation by FVII.Coagulation factor VII in the treatment of congenital and acquired FⅦ lack of disease, and surgery to stop bleeding and serious accidents, trauma, and other aspects of a very wide range of applications.However, FⅦ is trace plasma protein in the human body which normal content is about only200～400ng/ml.Therefore,in the present study,we use the Three-step ’Gap-repair’ Method to achieve the Construction of human EEF1A1-FⅦ heterozygous loci in order to obtain the high expression of FⅦ and the subsequent large-scale preparation,and provide a exploratory study for mammalian cell lines expressing vector.This study included the following two aspects:1.Construction of human EEF1A1-FⅦ heterozygous lociWe described here a successive three-step gap-repair method. First, a gap-repair vector based on pBR322vector backbone by inserting six joint homologous arms was constructed. Then using gap-repair method mediated by RED recombination system of A,-prophage in Escherichia coli, in the first step, the10kb3’ flanking region of the hEEF1A1gene was subcloned from the bacterial artificial chromosome(BAC) which harbors the hEEFlAl gene locus into the gap-repair vector; in the second step, the13kb hFⅦ genomic sequence from the ATG code to the TAG code was subcloned from the hFⅦ BAC;in the third step, the20kb5’ flanking region of the hEEFlAl gene was subcloned from the hEEFlAl BAC.A11these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a50kb hEEF1A1-hFⅦ hybird locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing.2.The expression of human EEF1A1-FⅦ heterozygous loci in293FT cell linesWe transfected the linearized EEF1A1-FⅦ heterozygous loci to obtain the resistant cells through the pressure of600mg/ml of G418.G418-resistant filter to get the hybrid cell clone the genomic and mRNA of the extraction, application of PCR detection of the target gene integration and transcription of the situation, the results show that the constructed human EEF1A1-human FⅦ heterozygous loci integrated the293FT cell genome, and transcript successfully.In the present study, we successfully constructed the human EEF1A1-FⅦ heterozygous loci, and FⅦ expression in mammalian cells, and laid the foundation for future mammalian cell lines expressing the large carrier.