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Recombinant human neovascularization inhibin and pharmaceutical composition thereof

A new blood vessel and inhibin technology, applied in the field of genetic engineering, can solve the problems of weakening drugs to inhibit endothelial cell proliferation, expensive drug production costs and prices, and incomplete retention of biological activity, so as to improve the efficiency of refolding and purification, Increased refolding and purification yields, reduced time and labor-intensive effects

Inactive Publication Date: 2014-10-01
赵进
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the production of recombinant human angiostatin by yeast has a mature three-dimensional conformation, which can avoid high failure rate and refolding in vitro, the yield of yeast expression is extremely low, and the highest yield does not exceed 5 mg / L. The production cost is very high, which is not suitable for clinical application. cause great inconvenience
[0006] The recombinant human angiostatin expressed by Escherichia coli currently on the market inserts 5 histidines at the N-terminus of wild-type human angiostatin, although this improves the renaturation rate to a certain extent, but Its overall weight folding yield is still low, which directly leads to very expensive production costs and selling prices of drugs, and its biological activity is not fully retained
Because the N-terminal is the biologically active region of human neoangiostatin protein, it is mainly because the N-terminal region is rich in histidine, and the domain formed by histidine can be combined with Zn 2+ Therefore, the insertion of an additional histidine sequence at the N-terminal will affect the normal structure of the N-terminal active region, and affect the relationship between the N-terminal active domain and the Zn 2+ The role of the protein, which in turn affects the drug activity of the protein, weakens the effect of the drug on inhibiting the proliferation of endothelial cells

Method used

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  • Recombinant human neovascularization inhibin and pharmaceutical composition thereof
  • Recombinant human neovascularization inhibin and pharmaceutical composition thereof
  • Recombinant human neovascularization inhibin and pharmaceutical composition thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0059] Cloning of the gene for expression of recombinant human neoangiostatin with C-terminal and N-terminal additional amino acids.

[0060] According to the C-terminal and N-terminal amino acid sequences of the novel recombinant human neoangiostatin of the present invention, primers are designed, and the DNA sequence encoding recombinant human neoangiostatin is amplified from a human liver cDNA library by standard RT-PCR technology. Primers used in PCR are:

[0061] Upstream primer (SEQ ID No.5): 5'-CGCCATATGCGGGGATCCCACAGCCACCGCGACTTC-3', containing an Nde I site;

[0062] Downstream primer (SEQ ID No. 6): 5'-GTCTCAAGCTTAATGGTGATGGTGATGGTGCTTGGAGGCAGTCATGAA-3', including a stop codon followed by a Hind III site.

[0063] The PCR reaction conditions were: 30 PCR cycles, denaturation at 94°C for 1 minute, annealing at 57°C for 1 minute, and extension at 72°C for 1.5 minutes.

Embodiment 2

[0065] Construction of recombinant human neoangiostatin expression strain with C-terminal and N-terminal additional sequences.

[0066] The 598bp PCR amplification product was treated with two restriction endonucleases Nde I and Hind III, and then enzyme-linked with the pET22b vector digested with Nde I and Hind III for 10 hours under the action of T4 ligase to obtain Expression vector rhEnS-h-pET22b. The expression vector rhEnS-h-pET22b was transformed into competent cells of Escherichia coli M15 and JM109 strains, and then spread on solid medium plates containing ampicillin resistance, and the transformants were screened for high protein expression strains and constructed Gene identification. Finally, a highly expressed strain of rhEnS-h (hereinafter referred to as E.coli M15 (rhEnS-h)) was successfully obtained from the E. coli M15 transformant, in which the expression of rhEnS-h accounted for about 50% of the total protein of the bacteria; sequence identification The res...

Embodiment 3

[0068] Production, refolding and purification of recombinant human neoangiostatin (rhEnS-h) with additional C- and N-terminal sequences.

[0069] (1) Preparation of engineering bacteria for industrial production

[0070] Pick the engineered strain E.coli M15 (rhEnS-h) and inoculate it into the TYP (recipe: sodium chloride 5g / L, peptone 11g / L, yeast extract 11g / L) plate medium with ampicillin resistance Incubate at 37°C. Pick 10 colonies and inoculate them into 10ml of TYP liquid medium with ampicillin resistance, shake culture until the OD600 is 0.9, add a final concentration of 1.5mM IPTG to all cultures to induce protein expression, and continue to culture for 5 hours , using SDS-PAGE with a gel concentration of 15% to detect the expression level of rhEnS-h, and select the one with the highest expression level as the seed culture medium (the expression level of rhEnS-h accounts for about 50% of the total bacterial protein).

[0071] (2) Preparation of fermented seed liquid...

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Abstract

The invention discloses a recombinant human neovascularization inhibin and a preparation method thereof, and especially relates to a preparation method adopting a novel refolding mode to produce the recombinant human neovascularization inhibin. The preparation method greatly improves the refolding rate of recombinant human neovascularization inhibin through in-vitro refolding on the basis of high expression efficiency, thus a breakthrough is achieved in the high density in-vitro refolding of recombinant human neovascularization inhibin, at the same time the subsequent purification process is simplified, and the bioactivity of the refolded protein is increased. The preparation method greatly reduces the production cost of human neovascularization inhibin which is massively required in preclinical detection and clinical tests and prominently increases the curative effect at the same time.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant human neoangiostatin and its preparation and application. Background technique [0002] Angiogenesis is the process of forming new capillaries from already formed blood vessels. Normal neovascularization is involved in many physiological processes, such as embryonic development, wound healing, and endometrial reconstruction during the female menstrual cycle. On the other hand, abnormal neovascularization is a critical step in many pathological processes, especially tumor growth and metastasis. Angiogenesis is a multi-step process involving endothelial cell proliferation, migration and differentiation, extracellular matrix degradation, microtubule formation, and new capillary sprouting, and a series of regulatory factors are involved. [0003] The occurrence and growth of tumors depend on the blood vessels around the tumor tissue to provide n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70A61K38/17A61P35/00
CPCC07K14/47A61K38/00
Inventor 赵进
Owner 赵进
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