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High-activity recombinant human coagulation factor IX fusion protein and preparation method thereof

A human blood coagulation factor and fusion protein technology, which is applied in the field of highly active recombinant human blood coagulation factor IX fusion protein and its preparation, can solve the problems of unknown high expression level and achieve high specific activity and high-efficiency expression

Inactive Publication Date: 2019-03-08
SHANGHAI CHILDRENS HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no precedent for using mammary gland bioreactors to express FIX fusion proteins in any published reports, and it is unknown whether breast bioreactors expressing FIX fusion proteins still have FIX activity and can maintain high expression levels

Method used

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  • High-activity recombinant human coagulation factor IX fusion protein and preparation method thereof
  • High-activity recombinant human coagulation factor IX fusion protein and preparation method thereof
  • High-activity recombinant human coagulation factor IX fusion protein and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Fragment synthesis

[0031] 1) Amplification of human IgG1Fc cDNA fragment

[0032] The upstream primer Fc-F: 5'-GAGCCCAAATCTTGTGACAAAACT-3'; the downstream primer Fc-R: 5'-TTTACCCGGAGACAGGGAGAGGCT-3' were used to perform PCR amplification using human peripheral blood cell cDNA as a template. The reaction system is as follows:

[0033]

[0034] A total of 4 reaction tubes are made, and the reaction conditions are 94℃5min; (94℃45sec, 56℃45sec, 72℃3min)×30 cycles; 72℃10min. Amplify about 0.7kb hIgG1Fc cDNA fragment, mix the reaction solution, take 2μL and send it to Shanghai Meiji Biotechnology Company for sequencing, the remaining products are subjected to agarose electrophoresis, gel cut, and the 0.7kb hIgG1Fc cDNA fragment is recovered with the kit. Then the sequence obtained by sequencing was compared with the sequence AF150959.1 in the Genebank database, and the comparison found that there was a difference of 1 base. It was verified that the site was a SNP site and did...

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Abstract

The invention discloses a high-activity recombinant human coagulation factor IX fusion protein and a preparation method thereof. The high-activity recombinant human coagulation factor IX fusion protein comprises a promoter, a human coagulation factor IX protein gene, a human immunoglobulin IgG1 Fc fragment gene and a terminator. A carrier is used to transfer a human FIX-Fc fusion protein gene expression kit to mammalian genome to obtain a transgenic animal; human FIX-Fc fusion expression protein is attained from milk of the transgenic animal. According to the method herein, the human FIX expressed in the milk of the animal may reach 6 IU / ml in activity and 3000 IU / mg in average specific activity which is about 12 times as high as that of natural FIX, and is applicable to the production ofhuman coagulation factor IX.

Description

Technical field [0001] The invention belongs to the technical field of transgene, and specifically relates to a highly active recombinant human coagulation factor IX fusion protein and a preparation method thereof. Background technique [0002] Hemophilia B is an X-chromosome-linked recessive hereditary bleeding disorder. The incidence in male newborns is about 1 / 25000. The mutation of the coagulation factor IX (FIX, Factor IX) gene leads to FIX protein synthesis in the body. Due to deficiency, patients are prone to spontaneous bleeding. Severe patients often suffer from disability due to joint bleeding and cerebral hemorrhage, which affects the quality of life and life span. Currently, patients with hemophilia B mainly rely on intravenous infusion to supplement exogenous coagulation factor IX for treatment. Such products include blood-derived and recombinant-derived coagulation factor IX products. [0003] The Fc fragment of human immunoglobulin IgG can mediate the recycling proc...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C07K19/00
CPCC07K2319/30C12N9/644C12N15/85C12Y304/21022
Inventor 曾凡一黄淑帧曾溢滔严红
Owner SHANGHAI CHILDRENS HOSPITAL
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