Construction and expression of vector of tumstatin T7 of tumstatin T7 peptide and derivative T7-NGR thereof

A technology of T7-NGR and tumorstatin, which is applied in the fields of molecular biology and oncology, can solve the problems of low pertinence, easy drug resistance, and patient damage

Inactive Publication Date: 2009-09-02
INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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Traditional radiotherapy and chemotherapy methods not only have low perti...

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  • Construction and expression of vector of tumstatin T7 of tumstatin T7 peptide and derivative T7-NGR thereof
  • Construction and expression of vector of tumstatin T7 of tumstatin T7 peptide and derivative T7-NGR thereof
  • Construction and expression of vector of tumstatin T7 of tumstatin T7 peptide and derivative T7-NGR thereof

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Embodiment 1

[0092] 1. Construction of the cloning vector pMD-T7 of the tumorstatin T7 peptide gene sequence

[0093] The pET28a plasmid containing tumstatin sequence stored in the laboratory, namely pET28a-tumstatin, was used as a template. Using Escherichia coli preferred codons, design a pair of primers for amplifying the T7 gene as follows:

[0094] The upstream primer is 5'- CATATG ACCATGCCGTTCTTATTCTGC-3',

[0095] The downstream primer is 5'- AAGCTT ACAGCCAGTAGGAGTA ATCGTTTC-3' (synthesized by TaKaRa Corporation).

[0096] The Nde I restriction site is introduced upstream, and the HindIII restriction site and stop codon are introduced downstream.

[0097] The PCR reaction amplifies the sequence of interest. PCR reaction conditions: After denaturation at 94°C for 5 min, cycle 35 times according to the following parameters: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s. Finally, extend at 72°C for 5 min. Using the TA cloning method, the...

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Abstract

The invention constructs recombinant plasmid vector containing tumstatin T7 peptide and gene sequence of derivative T7-NGR thereof and obtains recombinant T7 peptide and T7-NGR fusion protein. The amino acid sequence of the derivative T7-NGR is TMPFLFCNVNDVCNFASRNDYSYWLGSCNGRCVSGCAGRC. The invention combines the generation activity of tumstatin T7 peptide for restricting the tumor neovascularization effectively and high specificity of NGR guide peptide, so as to lead the generation activity of T7 peptide for restricting the tumor neovascularization to be greatly strengthened. The recombinant cloned vectors are respectively identified by sequencing: the direction, position and sequence of integration are correct. The expression vector is transformed into competent E.coli BL21 (DE3) and then is expressed by the induction of IPTG; electrophoresis is carried out on the thalli after cracking; and by Western-Blot identification, the expression product is in accordance with the expected. The pET7N expression vector fusion protein T7-NGR has potential application prospect in the diagnosis and treatment of tumor.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and oncology, and relates to gene recombination technology in the field of molecular biology. The specific gene is directional cloned into a corresponding plasmid vector, constructed into a cloning vector and an expression vector, and a fusion protein is prepared. Background technique [0002] Cancer has always been a threat to human health, and it is also the number one killer of various diseases. Diagnosis and treatment of tumors, especially the prevention and diagnosis and treatment of recurrence and metastasis of tumors is a major issue faced by human beings in the fight against tumors. Traditional radiotherapy and chemotherapy methods not only have low pertinence, but also bring great damage to patients, and are prone to drug resistance. Since Folkman put forward in 1971 that "tumor growth and metastasis are dependent on the formation of new blood vessels", inhibiting the formatio...

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Application Information

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IPC IPC(8): C12N15/70C12N15/12C12N15/62C07K14/78C07K19/00A61K38/39A61K38/10A61P35/00
Inventor 宋娜玲贺欣赵启仁颜廷东张春明王德芝刘鉴峰胡亚楠褚丽萍温镭
Owner INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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