Hactb - Hf Ⅶ Heterozygous Loci Of The Building And Identification Of Expressed In Hek293 Cells

Coagulation factor Ⅶ (FVH) is important cofactor in the clotting process. It is the first protease in the extrinsic coagulation pathway. FVD converted to activated factor Ⅶ (FVIIa) and started blood coagulation process by the action of Ca2+and combinating with tissue factors.Changes in the coagulation factor Ⅶ have clinical significance of prevention, treatment and prognosis of Sepsis with shock, hyperlipidemia, cardiovascular diseases and liver diseases. Coagulation factor Ⅶ is commonly used to correct the coagulopathy of liver disease and treated hemophilia. Its raw materials is human endogenous plasma, the situation of plasma is a great shortage, it makes the coagulation factor Ⅶ is hard to find, it have delayed treatment of some patients, even endangered the patient’s life. Even if the FⅦ domestic situation of current is seriously short, Our country does not intend to lift the ban of restriction concerning the important of blood products to avoid the potential virus infection of blood sources. Therefore, the production of recombinant human coagulation factor Ⅶ using mammalian cell lines have a positive effect not only on the solution of the above problems but also providing an alternative way for large-scale production of hFVII.The key of efficient and stable expression of FⅦ is to construct efficient expression vector, the mWAP-hLF hybrid gene locus of our laboratory were high effectively expressed in Mouse mammary. Therefore, we used the regulatory region of the human ACTB to construct the hACTB-hFⅦ heterozygous gene locus in order to obtain efficient expression of hFVII in mammalian cell lines.ACTB gene belonged to the housekeeping genes This gene encodes one of six different actin proteins. Actins are highly conserved proteins that are involved in cell motility, structure, and integrity. Actin is a ubiquitous globular protein that is one of the most highly-conserved proteins known. The environmental factors influenced The expression level of actin hardly.In most organizations of the various growth stages individual can continuously express, even if there are changes, it is very small. Its expression do not be affected by any adjustment mechanism in addition to the interaction with RNA polymerase and promoter. In this study, we constructed a gap-repair vector by inserting six joint homologous arms firstly. The first time, homologous arms5and6is used to subclone the10Kb3’flanking region of the hACTB gene from the Bacterial artificial chromosome which harbors the hACTB gene locus (hACTB BAC), The secod time, homologous arms3and4is used to subclone the13Kb hFVH genomic sequence from the ATG code to the TAG code from the hFⅦ BAC, and the last time, homologous arms1and2is used to subclone the20Kb5’flanking region of the hACTB gene from the hACTB BAC. The result was confirmed by PCR、 restiction enzyme digestion and sequencing, we successfully constructed the50Kb hACTB-hFⅦ hybrid locus.Then we use the nucleofection technique, electric shocked expression vector to the nucleus of HEK293cells which is human embryonic kidney cells and observed the situation expression. First of all, we extracted genomic DNA of transfected cells and identified by PCR to detection the situation of genomic integration of hybrid locus. Then, we extracted total RNA of transfected cells and identified by RT-PCR to detection the situation, That is whether the hybrid locus transcribed to the hFⅦ gene correctly. Finally, we determine recombinant human coagulation factor Ⅶ expression in HEK293by the ELISA method.After pressure filter, we got the cell clones of stably transfected with the human coagulation factor Ⅶ, by identification of PCR and RT-PCR, we found that hACTB-hF Ⅶ hybrid locus of cell clones successfully integrated into the genome of the host HEK293cells and transcribed to the hFⅦ gene correctly, ELISA results showed that the expression level of recombinant human coagulation factor Ⅶ of positive cell clones was7.74ng/ml.Therefore, we have proved for the first time:regulatory region of hACTB gene can be used as regulatory sequence, Guiding its expression in mammalian cells HEK293.