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Method for enhancing expression quantity of gene recombinant human coagulation factor 8

A human coagulation factor, gene recombination technology, applied in genetic engineering, plant gene improvement, recombinant DNA technology and other directions, can solve the problems of difficulty, difficulty of FVIII, and the treatment level of FVIII expression is relatively large, and achieves improved screening efficiency, The effect of simplifying the screening process

Inactive Publication Date: 2009-12-09
SHANGHAI TONEKER BIOTECH
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Problems solved by technology

This may be a reflection of the body's need for FVIII, but is a major obstacle to recombinant expression of FVIII in vitro
In addition, in the current international attempts of gene therapy for hemophilia, it is also found that it is difficult to maintain the expression of FVIII at the therapeutic level. The reason is that the expression of FVIII in the artificially constructed FVIII expression system cannot meet the therapeutic requirements required. This poses great difficulties for large-scale industrial production of high-quality, inexpensive FVIII

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  • Method for enhancing expression quantity of gene recombinant human coagulation factor 8
  • Method for enhancing expression quantity of gene recombinant human coagulation factor 8
  • Method for enhancing expression quantity of gene recombinant human coagulation factor 8

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Embodiment Construction

[0020] Around the problem of low expression level of FVIII, people have carried out a lot of experimental research in order to increase its expression level. The results of the study on the expression of FVIII and its derivatives in eukaryotes showed that the expression of FVIII (ΔB) in Cos-7 cells was higher than that of the whole molecule of FVIII, and higher than that reported in foreign literature; the expression of FVIII had certain tissue specificity, The expression level is the highest in liver cancer cell lines, followed by COS-7 and CHO cells, and the lowest expression level in lung cells; the co-expression of FVIII heavy and light chain molecules in eukaryotic CHO cells, the expression level of FVIII is significantly The increase was 3 times that of FVIII (ΔB); the expression of FVIII (ΔB) and vWF was co-expressed, and the expression of FVIII also increased significantly.

[0021] Weakening technology, that is, through purposeful modification, the translation initiat...

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Abstract

The invention establishes a novel expression carrier containing a weakened internal ribosome entry site (IRES for short) sequence and a double-sieving mark by a molecular biology technique. The carrier can be used for the recombinant expression of a human coagulation factor 8, and can effectively enhance the expression quantity of the coagulation factor 8 in the cells of mammals by the comprehensive sieving of a stable expression strain.

Description

technical field [0001] The invention relates to a method for increasing the expression level of recombinant human coagulation factor VIII, in particular to a method for increasing the expression level of recombinant human coagulation factor VIII in mammalian cells. Background technique [0002] Coagulation factor VIII (Factor VIII-related Antigen, referred to as "FVIII") is a plasma polymer glycoprotein, composed of 2332 amino acid residues, coagulation factor VIII plays a very important role in the endogenous coagulation system, it Is a cofactor that activates coagulation factor IX. The FVIII gene is located at Xq28, with a full length of 186kb, consisting of 26 exons and 25 introns. Insufficient quantity or abnormal quality of clotting factor VIII in the body will lead to hemophilia A. Hemophilia A is a common X-linked genetic bleeding disorder with an incidence of 1:10,000 in males. Point mutations, small gene deletions cause mild FVIII deficiency; stop codons, large g...

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Application Information

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IPC IPC(8): C12N15/67C12N15/12C12N15/85
Inventor 何胜祥王志强
Owner SHANGHAI TONEKER BIOTECH
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