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Method for preparing and producing tissue type activator of plasminogen from mutant of transgene goat by nuclear transplantation

A plasminogen and nuclear transfer technology, applied in the fields of botanical equipment and methods, plant genetic improvement, biochemical equipment and methods, etc., can solve the problem of "position effect" that cannot be overcome by transgene expression.

Inactive Publication Date: 2005-08-31
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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Problems solved by technology

Most scholars believe that the "position effect" of transgene expression cannot be overcome even if all biological elements with known functions are used to construct breast tissue-specific expression vectors or all current gene transfer technologies are used

Method used

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  • Method for preparing and producing tissue type activator of plasminogen from mutant of transgene goat by nuclear transplantation
  • Method for preparing and producing tissue type activator of plasminogen from mutant of transgene goat by nuclear transplantation
  • Method for preparing and producing tissue type activator of plasminogen from mutant of transgene goat by nuclear transplantation

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Embodiment Construction

[0019] Materials and methods

[0020] 1. Reagents and medium: All reagents were purchased from Sigma-Aldrich Company; endonucleases and molecular biology reagents used were purchased from Promega Company; medium was purchased from Gibco Company.

[0021] 2. Construction of random integration vector: PCR primers were synthesized from goat homologous sequences, and the regulatory sequence at the 5' end of the Guanzhong dairy goat β-casein gene was cloned in segments. The total length was 6200bp, and the 3' end was the stop codon of exon 7 The last 6Kb, the 5' end and the 3' end introduce restriction sites, and introduce two 2.4Kb β-globin insulator sequences before the 5' end, construct a pGBC vector, cut the site behind the 3' end, Klenow Enzyme filling and dephosphorization treatment. The Xhol site in pLoxP was cut, filled in and self-ligated to remove the Xhol site. Then ploxP-neo was excised with Not1 and Sal1, blunt-ended and ligated with pGBC vector. The Xhol site in th...

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Abstract

A process for preparing transgenic she-goat to secrete human histotypic plasminogen activator mutant by nuclear transplant method features that the human histotypic plasminogen activator mutant regulated by the regulator sequence of beta-casein can be expressed in the milk gland of transgenic she-goat. It includes configuring carriers pGBCN-tPAm, pGBC2-tPAm and pGBC4-tPAm, transfecting the fibroblast of milk she-goat fetus, and cloning by nuclear transplanting.

Description

technical field [0001] The invention discloses a method for producing medicinal protein by using transgenic animals, and particularly discloses a method for producing human tissue-type plasminogen activator mutant transgenic dairy goats in milk by nuclear transplantation. Background technique [0002] Human tissue-plasminogen activator (t-PA) mutant (t-PAm) is a new type of protein that has been patented by the applicant (Institute of Bioengineering, Academy of Military Medical Sciences: Chinese Patent No. ZL93109234. 5). It has a high affinity for the fibrin-plasminogen complex in the thrombus, can rapidly form the fibrin-t-PAm-plasminogen complex, activate the plasminogen to form plasmin, and hydrolyze the plasminogen in the thrombus. Fibrin (Liu Shihui, Huang Peitang, etc., Construction, expression and characteristic analysis of tissue-type plasminogen activator mutants. Chinese Science (B), 1995, 4: 399). However, due to the complex structure of t-PAm itself, there are...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N5/16C12N15/06C12N15/12C12N15/58C12N15/62C12N15/85
Inventor 沈伟杨正田邓继先潘庆杰
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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