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Method for preparing recombinant human antithrombase III protein using mammary gland biological reactor

A technology of bioreactor and antithrombin, applied in the field of antithrombin III, can solve the problems of increased consumption and increased consumption of antithrombin III

Inactive Publication Date: 2007-04-11
青岛森淼生物技术研究所
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  • Claims
  • Application Information

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Problems solved by technology

[0007] d) Increased consumption of antithrombin III: such as increased blood coagulation caused by various reasons, antithrombin III neutralizes activated coagulation factors, resulting in increased consumption

Method used

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  • Method for preparing recombinant human antithrombase III protein using mammary gland biological reactor
  • Method for preparing recombinant human antithrombase III protein using mammary gland biological reactor
  • Method for preparing recombinant human antithrombase III protein using mammary gland biological reactor

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Embodiment 1

[0094] In this example, taking the Laoshan dairy goat (Capra hircus) as an example, a transgenic vector based on the goat casein gene (β-casein) was constructed with the human antithrombin III (AT-III) protein gene, and finally obtained The human antithrombin III (AT-III) protein gene is a functional gene in a Laoshan dairy goat mammary gland bioreactor.

[0095] The specific operation procedure is as follows:

[0096] Acquisition of functional genes

[0097] The human antithrombin III (AT-III) protein gene uses human hepatocyte cDNA as the PCR substrate, and then establishes ligature sites at both ends by PCR amplification. The PCR product is connected to pUC19, and then DNA sequence analysis; the DNA sequence of the cloned AT-III gene is the same as that of the published AT-III protein gene.

[0098] The specific method is: the preparation of human antithrombin III (AT-III) gene is to design PCR primers according to the published DNA sequence of AT-III (GeneBank: D29832 and ...

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Abstract

The process of preparing recombinant human antithrombase III protein with mammary gland bioreactor includes the following steps: selecting mammalian lactoprotein gene as the control region and expression frame of the transgene, establishing unique cleavage site on the frame, connecting human AT-III protein gene function part, lactoprotein secretion sequence and enterokinase protein cleaving sequence gene to the expression frame, and connecting one screening gene to the end of the vector; transferring the expression vector to in vitro cultured animal somatic cell to obtain cell strain containing human AT-III protein gene; transferring the transgenic cell strain to mature denucleated oocyte of goat to fuse the nucleus donor cell and the denucleated oocyte; transferring the cloned embryo to yow oviduct to obtain transgenic goat; detecting transgenic milk and purifying; animal experiment and human body experiment; and applying recombinant AT-III protein in human body.

Description

technical field [0001] The invention belongs to a method for producing recombinant human antithrombin III using a non-human mammalian mammary gland bioreactor, in particular to a method for preparing recombinant human antithrombin III protein using a nonhuman mammalian mammary gland bioreactor Methods. Background technique [0002] Antithrombin III (antithrombin III, referred to as AT-III) is an important anticoagulant factor in human plasma. It plays an important role in the balance of coagulation and anticoagulation. It is an important serine protease inhibitor in normal human plasma. Its main function is to inactivate thrombin, such as combining with thrombin Xa and XIa. And mainly synthesized by the liver. [0003] It is the anticoagulant system that is opposite to the function of the blood coagulation system in the human body. Under normal circumstances, the two maintain a dynamic balance. AT-III mainly reflects the function of the body's anticoagulation system. The...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/85C12N15/12C12N5/06C12N15/06A61K38/17
Inventor 袁三平邹贤刚鲜建
Owner 青岛森淼生物技术研究所
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