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Clone and expression of OPN associated protein pro-MMP-9 gene

A pro-mmp-9, -pro-mmp-9 technology, applied in the field of cloning and expression of OPN downstream associated protein matrix metalloproteinase 9 zymogen gene, can solve the problem of high cost, complicated operation, low direct purification yield, etc. problem, to achieve the effect of simple and feasible preparation method and high purity

Inactive Publication Date: 2014-08-27
NANJING YOKO PHARMA
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AI Technical Summary

Problems solved by technology

[0004] As a secreted protein, matrix metalloproteinase-9 zymogen acts extracellularly with very little content, low yield of direct purification from organisms, cumbersome operation and high cost Also high, can not meet the needs of industrial production

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 pro-MMP-9 cloning, expression and protein purification

[0037] A method for cloning, expressing and protein purification of a matrix metalloproteinase-9 zymogen, which is mainly divided into the following steps:

[0038] 1. Construction of recombinant cloning vector TA-pro-MMP-9

[0039] (1) Primer design for pro-MMP-9

[0040] Using pET28a(+)-pro-MMP-9 as a template, pro-MMP-9 was amplified by splicing PCR, and four primers pro-MMP-9 p1, pro-MMP-9p2, pro-MMP-9p1-G were designed -3', pro-MMP-9p2-G-5'.

[0041]The upstream primer pro-MMP-9p1 contains endonuclease site Nde I, and the downstream primer pro-MMP-9p2 contains endonuclease site Hind III,

[0042] pro-MMP-9p1:5'-CG CATATG GCCATGGTGTACTGAG-3'

[0043] pro-MMP-9p1-G-3':5'-GTATCATAATGCTCCGGATACATTAGA-3'

[0044] pro-MMP-9p2-G-5': 5'-AGCCTGATGAAACTCGCAATTCTGTA-3'

[0045] pro-MMP-9p2:5'-CG AAGCTT TGCGATGATAGATGTCACAC-3'

[0046] (2) Splicing PCR to amplify the pro-MMP-9 gene fragment

[00...

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Abstract

The invention mainly relates to a clone, an expression and a protein purification method of an OPN associated protein pro-MMP-9 zymogen. The method includes the steps of recombination of the structure of a cloning vector TA-pro-MMP-9, recombination of the structure of an expression plasmid pET-28a(+)-pro-MMP-9 and induced expression and identification of pro-MMP-9 protein. A prokaryote genetic expression system is built, so that a lot of pro-MMP-9 can be expressed, high-purity protein purification procedures are built, and pro-MMP-9 protein which is large in amount, high in purity and simple and feasible in preparing method is obtained.

Description

technical field [0001] The invention belongs to the technical field of gene cloning and expression, and in particular relates to a method for cloning and expressing an OPN downstream associated protein matrix metalloproteinase 9 zymogen gene. Background technique [0002] Pancreatic cancer is a highly malignant tumor. In my country, the incidence of pancreatic cancer is gradually increasing, and it is one of the top ten malignant tumors that cause population death. The one-year survival rate of untreated patients is about 20%, and the five-year survival rate is less than 4%. Invasion and metastasis are the main causes of treatment failure in pancreatic cancer patients. Osteopontin (OPN) is a secreted calcium-binding phosphorylated glycoprotein with multiple functions. Recent studies have found that OPN is highly expressed in pancreatic cancer tumor cells and is closely related to the invasion and metastasis of pancreatic cancer. Therefore, the research on its mechanism of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N15/70
CPCC12N9/6491C12N15/70C12Y304/24035
Inventor 陈爱萍蒋晟梁静顾传虎姚洛芫
Owner NANJING YOKO PHARMA
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