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Construction process of pig serum albumin locus targeting vector for producing foreign protein

A technology of human serum albumin and gene targeting, applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc.

Inactive Publication Date: 2005-12-14
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

Provides a useful tool for the production of human serum albumin using transgenic animals

Method used

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  • Construction process of pig serum albumin locus targeting vector for producing foreign protein
  • Construction process of pig serum albumin locus targeting vector for producing foreign protein
  • Construction process of pig serum albumin locus targeting vector for producing foreign protein

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Embodiment Construction

[0014] 1 Reagents, plasmids and strains used

[0015] In the implementation of the present invention, the following reagents have been used: random primer labeling kit, pGEM-7Zf cloning vector, T carrier kit, pGEM-T Vector, pig genome library, [a-32p]dCTP (from Yahui Company ), E.coli DH10β, pGEM-7Zf plasmid, Ploxp plasmid, p5ZF-BGHpolyA plasmid (preserved in our laboratory), E.coli DH5α competent cells and DNA standard Marker (product of Tianwei Times), T4 DNA polymerase (product of Huamei Company) , Pyrobest DNA polymerase, T4 DNA polymerase, Klenow large fragment, T4 DNA ligase (product of TAKARA company), various endonucleases (products of Biolab and Takara company); various primers were synthesized by the National Biomedical Analysis Center. There are also some kits: QIAprep Spin Miniprep Kit (50) (gene company product), Gel Extraction Mini Kit (50) (Shanghai Huashun Bioengineering Company), plasmid extraction kit, PCR purification kit (Vitejie company product) .

[001...

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Abstract

The present invention constitutes three kinds of targeting vector capable of being located and integrated onto pig serum albumin locus and makes it possible to produce human serum albumin with pig serum. First, the 35 kb, including the 14 kb before translation starting site, all the exons and introns, and 3'-end translation 4 kb, of pig albumin genome are cloned and sequence analyzed. Then, two kinds of targeting vector capable of locating and integrating human serum albumin cDNA onto pig serum albumin and one kind of targeting vector capable of locating and integrating human serum albumin cDNA onto pig serum albumin locus are constituted. These targeting vectors are transfected pig fetus fibroblast or other cells, hitted cells are screened for nuclear transplantation of animal cells, and transgenic pig capable of producing human serum albumin in its serum is obtained.

Description

technical field [0001] The invention relates to a method for constructing three types of targeting vectors using pigs to produce exogenous proteins, especially human serum albumin (human serum albumin, HAS), so that the targeting vectors constructed by the method can be used to produce HAS in pig serum for easy purification. These targeting vectors are transfected into pig fetal fibroblasts or other cells, and the targeted cells are screened out for nuclear transplantation animal cloning, so as to obtain transgenic pigs capable of producing HSA in serum. Background technique [0002] It is an ideal method to produce HSA with pig blood. Currently, for the production of HSA, the raw materials are all from human body. Due to the large demand and the large number of donors, the composition of the donors is complex, and false negatives are unavoidable even if strict screening tests are adopted. lead to contamination of blood-derived products. If HSA is produced by animals, it ...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 孙世蕙邓继先周艳荣卢建申吴晓洁
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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