Novel recombinant human blood coagulation factor VIII and production method thereof

A blood coagulation factor and gene recombination technology, applied in the direction of blood coagulation/fibrinolytic factor, VII factor, recombinant DNA technology, etc., can solve the problems of difficulty, FVIII difficulty, FVIII expression level can not meet the needs of treatment, etc., to improve screening efficiency, The effect of simplifying the screening process

Inactive Publication Date: 2012-01-11
SHANGHAI TONEKER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Novel recombinant human blood coagulation factor VIII and production method thereof
  • Novel recombinant human blood coagulation factor VIII and production method thereof
  • Novel recombinant human blood coagulation factor VIII and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Obtaining of coagulation factor VIII gene sequence

[0020] (1) Preparation of DNA and RNA

[0021] High molecular weight DNA is extracted from the peripheral blood cells of a normal person according to conventional methods. Total RNA was extracted from fresh embryonic tissue using a guanidine isothiocyanate-cesium chloride gradient method, and mRNA was isolated using an oligodeoxyadenylate (Oligo-dT) cellulose column.

[0022] (2) Design of oligonucleotide primers and polymerase chain reaction

[0023] Seven pairs of primers were designed and synthesized. In order to facilitate the cloning of the whole molecule, the fragments amplified by each pair of primers contained corresponding restriction endonuclease sites on both sides. The primers are amplified by reverse transcription polymerase chain reaction (RT / PCR), wherein the second primer of each pair of primers is used as a primer for the reverse transcription reaction. Reverse transcription reaction syst...

Embodiment 2

[0029] Example 2 Construction of coagulation factor VIII B domain deletion cDNA (FVIII(ΔB))

[0030] Four oligonucleotide primers were designed, and most of the B domains of FVIII were deleted and mutated by PCR (polymerase chain reaction). The obtained PCR products A and B respectively correspond to 1868-2334bp and 4974-5164bp of FVIII. After the products A and B are denatured and re-annealed, 16 bp of nucleic acid sequences can be hybridized. This hybridization product can be used as a template to amplify to obtain product C (658 bp), corresponding to 1868-2334 bp and 4974-5164 bp of the FVIII gene. PCR product C and FVIII gold long cDNA were restricted with BamHI and PstI, and PCR product C was used to replace the corresponding fragment of FVIII full-length cDNA, thus producing FVIII cDNA with B domain deletion (see figure 2 ), named as FVIII(ΔB). Compared with the full-length FVIII cDNA, this FVIII (ΔB) lacks the base sequence of 2334-4974 bp in the FVIII cDNA. ( fig...

Embodiment 3

[0031] Construction of embodiment 3FVIII cDNA expression vector and expression in mammalian cells

[0032] The above FVIII cDNAs (FVIII full-length cDNA, FVIII(ΔB) cDNA, FVIII-HL) were cloned into the expression vector pIRES-dhfr. figure 2 is a schematic diagram of the construction of different FVIII expression plasmids, image 3 It is a schematic diagram of cloning different FVIII expression plasmids into pIRES-DHFR / GS respectively, Figure 4 It is a schematic diagram of FVIII-HC / FVIII-LC double expression plasmid, such as figure 2 , image 3 , Figure 4As shown, different FVIII expression plasmids were constructed, including B domain deletion and FVIII heavy and light chain co-expression plasmids, and FVIII full-length cDNA (FVIII-W) was cloned. On this basis, various molecular reconstructions were constructed, including B Domain-deleted FVIII (FVIII-ΔB), heavy chain portion of FVIII (FVIII-HC) and light chain portion of FVIII (FVIII-LC), and cloned into the A multiple...

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Abstract

The invention provides a novel recombinant human blood coagulation factor VIII and a production method thereof. Particularly, the invention relates to reconstruction of a human blood coagulation factor VIII gene. A novel expression vector containing a weakened internal ribosome entry site (IRES for short) sequence and double screening marks is constructed. The vector can be used for high-efficiency recombinant expression of the human blood coagulation factor VIII, so that the expression of the vector in a mammalian cell expression system is increased.

Description

technical field [0001] The present invention relates to a novel recombinant blood coagulation factor VIII (abbreviated as "FVIII", also known as antihemophilic factor (AHF)) and a production method thereof. Background technique [0002] Hemophilia A is a common X-linked hereditary bleeding disorder with an incidence rate of 1:10,000 in males and accounts for 85% of all hemophilias. The pathogenic factor is due to insufficient quantity or abnormal quality of coagulation factor VIII (FactorVIII-related Antigen, referred to as "FVIII"). Coagulation factor VIII is a plasma polymer glycoprotein consisting of 2332 amino acid residues. Coagulation factor VIII plays a very important role in the endogenous coagulation system. It is a cofactor for activating coagulation factor IX. The FVIII gene is located at Xq28, with a full length of 186kb, consisting of 26 exons and 25 introns. [0003] The current mainstay of treatment for the disease is a concentrated FVIII preparation extract...

Claims

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Application Information

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IPC IPC(8): C07K14/755C12N15/67C12N15/85C12N5/10C12P21/00
Inventor 王志强李昂何胜祥
Owner SHANGHAI TONEKER BIOTECH
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