Introduction to Coagulation Factor Ⅷ

Posted November 29, 2019 by Bonnibelle

Recombinant mouse anti factor VIII antibody G99 is capable of binding to coagulation factor VIII C2 domain, expressed in Chinese Hamster Ovary cells (CHO).

Recombinant mouse anti factor VIII antibody G99 is capable of binding to coagulation factor VIII C2 domain, expressed in Chinese Hamster Ovary cells (CHO).

What is coagulation factor Ⅷ?

Human coagulation factor Ⅷ (FⅧ) is an important protein in human plasma, which is the main component of human endogenous coagulation pathway. The low level of FⅧ in blood will lead to different degrees of coagulation dysfunction, that is, hemophilia A (HA).

The structure of human coagulation factor Ⅷ

The FⅧ gene consists of 26 exons and 25 introns, of which 14 and 26 exons are the largest, 3106 bp and 1958 bp, respectively, with a total length of 186 kb, located at the end of the long arm of X chromosome, accounting for about 0.1% of the whole X chromosome. After transcription and cleavage, the FⅧ gene is about 9 kb long and encodes a precursor containing 2351 amino acid residues. After the signal peptide was hydrolyzed in endoplasmic reticulum, a mature FⅧ single chain protein with 2332 amino acid residues and 6 domains was formed. According to the homology of the internal sequence of FⅧ, FⅧ can be divided into three domains: A, B and C. Among them, the A domain can be divided into three types: A1, A2 and A3, each containing about 350 amino acid residues, with 30% homology. A1 and A2 are located in the heavy chain, and A3 in the light chain. The B domain is located in the middle of the molecule, with 908 amino acid residues. The C domain is located in the C end of the light chain, with C1 and C2, each containing about 150 amino acid residues, with 40% homology. The A3 region of FⅧ is located in the phospholipid bilayer of cell membrane and adjacent to C1 and C2 regions; the long axis of C1 region is approximately parallel to the cell membrane plane and almost perpendicular to C2; there are two highly conserved βbarreled domains between A1, A2 and A3 regions with small angles to each other. The whole molecule is arranged as a1-a2-b-a3-c1-c2. Through the complex interaction with various structural proteins and enzymes inside and outside the cell, it affects the protein activity, transmembrane transport and gene expression of FⅧ.

Expression vector and system of recombinant FⅧ

There are three kinds of common vectors: bacterial plasmids, phages and animal and plant viruses. They are self-replicating DNA or RNA molecules that transfer the target gene to the receptor cell. In the construction of recombinant FⅧ vector, these three types have been used, and the application of bacterial plasmids and animal viruses is more common. Virus vectors have been widely used for their advantages of wide host range, large capacity and simple operation. Adenovirus, adeno-associated virus, retrovirus and herpes simplex virus have been reported. In the expression system, prokaryotic expression system is easy to operate, but there are some defects in the expression of eukaryotic recombinant FⅧ protein, such as the wrong link of disulfide bond, the ineffective folding of peptide chain and the low efficiency of glycosylation, so it is not widely used.

Gene therapy

The main pathogenesis of hemophilia A is the inversion and missense mutation of intron 22. With the thorough research on the structure of FⅧ gene and protein by scientists, and the low requirement of individuals on the normal physiological level of FⅧ, many organs can produce active FⅧ. Therefore, hemophilia A is the first choice for somatic gene therapy.

In vitrotreatment
In vitro treatment, the cells of a certain tissue are taken out from the patient's body first. After in vitro expansion and culture, the vector carrying the recombinant FⅧ gene is transfected into the recipient cells, and the successful transfected cells are selected to be transplanted into the patient's body, and then the target of treatment is achieved by the expression of FⅧ in these transplanted cells. Because of the insecurity of virus vector, tissue cells such as fibroblasts and myoblasts are used in clinic.

In vivotherapy
In vivo treatment refers to the direct injection of FⅧ expression vector into the patient's body. At present, the safest treatment is to inject naked DNA without carrier directly into the patient, and to treat by transfection of liver cells in vivo and self -expression of FⅧ, which is an effective treatment in vivo. In addition, through the efficient integration of transposons with mammalian genes, the eukaryotic transposon vector containing FⅧ gene was introduced into the receptor to express f Ⅷ. However, due to the uncertainty of transposon integration gene location, the safety of this method needs to be further verified.

It has to be mentioned that in recent years, with the birth of a new gene editing technology CRISPR/Cas9, scientists only need to design a snippet of CRISPR with dozens of bases, using target specific RNA to bring Cas9 nuclease to specific targets of the genome, which can cut specific gene sites and lead to mutations. This technology has been applied in gene knockout, gene editing related diseases, gene activation and expression and many other fields. CRISPR/Cas9 can also be used as a precise gene cutting tool in eukaryotic transcription regulation. The application of CRISPR/Cas9 in the study of recombinant f Ⅷ can not only simplify the steps of gene editing, but also greatly improve the efficiency of recombination, reduce the residue or deletion of gene fragments caused by cutting, which can improve the success rate of transfection. In the same expression system, CRISPR/Cas9 can correctly fold the expressed recombinant f Ⅷ, thus improving the yield of recombinant FⅧ. CRISPR/Cas9 also has a revolutionary application prospect in the gene therapy of hemophilia A. It is believed that the application of CRISPR/Cas9 in the study of recombinant FⅧ will have profound significance.
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Categories Biotech
Last Updated November 29, 2019