Recombinant fusion resisting tomur attack and transfer

A fusion protein, anti-tumor technology, applied in the biological field, can solve the problems of short survival period and poor prognosis

Inactive Publication Date: 2005-11-02
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many malignant tumor cells highly express uPA and uPAR, and tumor patients with high expression of uPA / uPAR have poor prognosis and short survival time

Method used

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  • Recombinant fusion resisting tomur attack and transfer
  • Recombinant fusion resisting tomur attack and transfer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Expression in Escherichia coli

[0020] 1. Construction of recombinant pGEM-ATF-PAI2CD fusion protein gene clone

[0021] 1) Amplify the PAI2CD gene by PCR: use the plasmid pZT-PAI2CD as a template, and use primers P1 and P2 to perform PCR amplification. cycle, 72°C 5mim. Take 2 μl of the PCR reaction solution, identify it by agarose gel electrophoresis, and observe a single band of 1.1 kb, which is consistent with the length of the complete PAI-2CD gene. The amplified product (about 1100bp in length) was recovered and inserted into the KpnI and BamHI sites in pGEM3zf(+) after double digestion with KpnI and BamHI to obtain the recombinant plasmid pGEM-PAI2CD, which was analyzed by restriction endonuclease zymogram. The characteristic enzyme cutting site of PAI-2CD, the reading frame of nucleotide sequence analysis is correct.

[0022] 2) Amplify the ATF coding sequence by RT-PCR

[0023] Extract and isolate total mRNA from the highly metastatic lung cancer...

Embodiment 2

[0040] Example 2 Expression in Yeast

[0041] 1. Construction of recombinant pGEM-ATF-PAI2CD fusion protein gene clone

[0042] Method is with embodiment 1.

[0043] 2. Construction of recombinant fusion protein gene expression plasmid

[0044] The recombinant plasmid pGEM-ATF-PAI2CD was digested with BamHI, the incision was filled in by klenow, and then digested with EcoRI to recover the 1.6kb ATF-PAI2CD cDNA target fragment. The fragment was connected to the pPIC9K vector, and the pPIC9K vector was digested with NotI, klenow filled in the cut, and EcoRI to obtain the recombinant expression plasmid pZWY-ATF-PAI2CD.

[0045] 3. Transformation of GS115 with pZWY-ATF-PAI2CD and screening of positive transformants

[0046] The recombinant expression plasmid pZWY-ATF-PAI2CD linearized by SalI was used to electrotransform GS115, and the bacterial solution was spread on MD / His - Select the plate, culture it at 30°C for 2 days, and screen out His-Mut + phenotype transformants. ...

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Abstract

A recombinant fusion protein able to resist against tumor invasion and transfer is composed of the recombinant urokinase fragment and type-2 mutant as plasminogen activator inhibitor. The fusion protein gene cDNA is amplified by PCR method, and is cloned to expression vector to obtain fusion gene expression plasmid, which is used to transform host bacteria. The methanol or temp can induce the expression of said fusion protein. After purified, a high-purity (95%) expression product is obtained.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a gene recombination anti-tumor invasion and metastasis new drug, in particular to a fusion protein of a recombinant urokinase fragment and a plasminogen activator inhibitor-2 mutant. technical background [0002] Tumor cell invasion and metastasis is the main cause of death in patients with malignant tumors. So far, there is no specific and effective anti-tumor cell invasion and metastasis drug. It is known that the urokinase-type plasminogen activator and its receptor (uPA / uPAR) system plays a key role in tumor invasion and metastasis. Many malignant tumor cells highly express uPA and uPAR, and tumor patients with high expression of uPA / uPAR have poor prognosis and short survival period. However, the physiological inhibitor of u-PA, plasminogen activator inhibitor-2 (PAI-2), has a low expression level in most highly invasive and metastatic tumors, and the prognosis is good w...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61P35/00C07K19/00C12N15/62C12P21/00
Inventor 朱运松王霞李平谭理张宇清孙兴会侯敏
Owner FUDAN UNIV
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