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Production of gene recombinant fusion protein K1-EN

A fusion protein, gene recombination technology, applied in recombinant DNA technology, chemical instruments and methods, hybrid peptides, etc.

Inactive Publication Date: 2007-01-17
SHANGHAI UNIV
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  • Abstract
  • Description
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Problems solved by technology

Third, it does not induce drug-hanging properties

Method used

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  • Production of gene recombinant fusion protein K1-EN
  • Production of gene recombinant fusion protein K1-EN

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Experimental program
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Embodiment Construction

[0022] The invention mainly includes three parts: the construction of the recombinant yeast gene engineering bacterium pPICZαA-K1-EN / GS115, the expression and purification of the recombinant fusion protein K1-EN, and the measurement of the biological activity of the recombinant fusion protein K1-EN.

[0023] The specific plan is described as follows:

[0024] 1 Construction and identification of recombinant plasmid pPICZαA-K1-EN

[0025] 1.1 Construction of recombinant plasmid pPICZαA-K1

[0026] Design upstream and downstream primers of K1 according to the nucleotide sequence of K1:

[0027] Upstream primer: 5'-GGG GAA TTC GTG TAT CTC TCA GAG-3';

[0028] Downstream primer: 5'-TTT GGT ACC GCC ACC TTC CTC TTC ACA C-3'.

[0029] Introduce a tether (Gly) into the downstream primer of K1 3 nucleotide sequence, so that about 300bp of COOH end with connecting arm (Gly) can be directly obtained after PCR amplification 3 K1 fragment. Recover and purify the amplified product.

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Abstract

Production of gene-recombinant fused protein K1-EN is carried out by constructing for recombinant plasmid pPICZ alpha A-K1-EN, constructing for recombinant yeast gene engineering bacterial pPICZ alpha A-K1-EN / GS115, expressing for recombinant fused protein K1-EN and purifying recombinant fused protein K1-EN. Pichia is eukaryotic expression system with methanol as the only carbon resource; pPICZ alpha A contains Paox1, which is secretory expression plasmid and expresses accidental protein under methanol induction. It is cheap and no need for externally heating source and has large scale.

Description

technical field [0001] The invention relates to a preparation method of gene recombination fusion protein K1-EN. Background technique [0002] Malignant tumor, namely cancer, is one of the most serious diseases threatening human health. Conventional treatments for malignant solid tumors include surgical extirpation, chemotherapy, and radiotherapy. These treatments are prone to recurrence, have large toxic and side effects on the human body, and induce drug resistance, etc., which are not ideal. Breakthroughs in the basic research of life sciences and the development of biotechnology have provided other more effective and feasible means for the ultimate victory of malignant tumors. [0003] In the early 1970s, Dr. Folkman put forward the view that "tumor proliferation is dependent on blood vessels", that is, the volume exceeds 1mm 3 ~2mm 3 Some tumors need new blood vessels to maintain the supply of nutrients and oxygen and excrete metabolites, and also need new blood vess...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/81C12N1/19C12P21/02C07K19/00C07K1/14
Inventor 黄筱颖陈宇光
Owner SHANGHAI UNIV
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