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Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity

An endothelial growth and inhibitory protein technology, applied in the field of genetic engineering drugs, can solve the problem of limited inhibitory effect on endothelial cells

Active Publication Date: 2013-06-19
上海普洛创智医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the inhibitory effect of endostar on endothelial cells is relatively limited, so further research is needed in this field to develop improved drugs

Method used

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  • Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity
  • Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity
  • Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Embodiment 1, the acquisition of gene sequence

[0087] SEQ ID NO: 1 and SEQ ID NO: 2 show the gene and protein sequence of EDI-8t protein. Take fresh human umbilical cord, use Trizol reagent to extract total RNA, dissolve it in RNase-inactivated sterile double-distilled water, use this as a template, and use Tiangen Company’s reverse transcription kit for RT-PCR, according to the kit instructions operate. Then with the obtained reverse transcription product as a template, primer 1 (sequence is ATCG CTCGAGAAAAGAGAGGCTGAAGCTGGCAAGAATGGAGGGCCA (SEQ ID NO: 4), wherein the underlined part is the introduced XhoI cutting point) and primer 2 (the sequence is ATC GAATTC ATTA GTGATGGTGATGGTGATG CATGGGATACAATAAATA (SEQ ID NO: 5), where the underlined part is the introduced EcoRI cleavage point) is used as the upstream and downstream primers for PCR, and the PCR product with XhoI and EcoRI restriction sites at both ends is obtained. The DNA electrophoresis verification resul...

Embodiment 2

[0090] Embodiment 2, expression of gene in Pichia pastoris

[0091] Extract the above-mentioned recombinant plasmid pPIC-EDI-8t, digest it with BglII (Takara) and extract it, then electrotransform Pichia pastoris expression strain GS115 (purchased from Invitrogen Company), spread the transformed product on an MD plate, and incubate at 30°C for 3- 5 days.

[0092] The recombinant expression strain was identified by PCR method, and the recombinant expression strain Gp-EDI-8t was obtained by shaking flask screening after obtaining positive clones.

[0093] After the expression strain was fermented and cultured, the electrophoresis of the supernatant was as follows: figure 2 shown.

Embodiment 3

[0094] Embodiment 3, recombinant protein purification

[0095] Centrifuge the fermentation broth, perform ultrafiltration, nickel column affinity chromatography under alkaline conditions, anion exchange, and gel filtration, and finally obtain the target protein with a 6×His Tag tag at the carboxyl terminal. The electrophoresis verification results are as follows: image 3 shown.

[0096] According to the results of electrophoresis and Western blot, it was speculated that the target protein contained some dimer forms.

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Abstract

The invention relates to a recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity. The EDI-8t protein is separated for the first time, and the endothelial cell growth inhibiting function and the tumor growth inhibiting function of the EDI-8t protein are verified. The EDI-8t protein has a good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering drugs, and in particular relates to a recombinant endothelial cell growth inhibitor, its genetic engineering expression and clinical application. Background technique [0002] Collagen (Collagen), also known as collagen, is a polymer that makes up various extracellular matrix. form exists. In the composition of the human body, collagen accounts for about 33% of the total protein, which can ensure and connect various tissues to support the structure of the human body. [0003] In recent years, the research on the inhibition of angiogenesis derived from collagen is gradually becoming a hot topic. In 1997, Professor Folkman of Harvard Medical School and his team discovered the first collagen-derived endothelial cell growth inhibitor angiostatin (O'Reilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, Lane WS et al..Cell, 1994, 79(2):315-328), and then another inhibitory molecule, endost...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02A61K38/17A61P35/00A61P35/02
Inventor 周庆玮甘人宝杜鹏钱悦张倩冯宝山丁红珍
Owner 上海普洛创智医药科技有限公司
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