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Kringle 5 mutant protein, and preparation method and application thereof

A mutant and protein technology, applied in the fields of molecular biology and medicine, can solve the problems of weak anti-angiogenic effect, difficult preparation cost, systemic toxicity, etc.

Inactive Publication Date: 2013-03-13
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, several angiogenesis inhibitors are being developed for the treatment of angiogenic diseases, but these compounds have some associated disadvantages, for example, suramin is a potent inhibitor, but at doses required for antitumor activity Causes severe systemic toxicity in humans; compounds such as retinoids, interferons, and antiestrogens are safe for human use but have weak antiangiogenic effects; others may be difficult or expensive to prepare

Method used

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  • Kringle 5 mutant protein, and preparation method and application thereof
  • Kringle 5 mutant protein, and preparation method and application thereof
  • Kringle 5 mutant protein, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Acquisition of Kringle5 mutant protein coding gene (N sequence does not exist but C sequence exists, in vitro synthesis + PCR method).

[0027] If the C sequence is His-His-His-His-His-His, the following gene fragments can be designed and synthesized by Shanghai Sangong Company:

[0028] SEQ ID NO: 1 (76 bp): GCGAATTCCATGGACTGTACTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCA

[0029] SEQ ID NO: 2 (78 bp): CCACCTACATCACCATCAGGGTTACGGCAGTAATTTTTTTTC CAGACCCGCCCGTGGATTTGTCTCTGGAGTGAAAATG

[0030] SEQ ID NO: 3 (73 bp): TGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAAC TTTACGAC TACTGTGATGTCCCTCAGTGTGCG

[0031] SEQ ID NO: 4 (49 bp): GTCCCTCAGTGTGCCCACCACCACCACCACTAATAGCGC ACACTGA

[0032] Restriction sites, start codons, and stop codons were introduced into the above-mentioned gene fragments, and the 5' of the synthesized nucleic acid fragments were phosphorylated, routinely extracted with phenol and chloroform, and conventional PCR reaction operations were performed. Th...

Embodiment 2

[0035] Acquisition of mKringle5 protein coding gene (both N sequence and C sequence exist, in vitro synthesis + PCR method).

[0036] If the N sequence is Phe-Glu, and if the C sequence is His-His-His-His-His-His, the following gene fragments can be designed and synthesized by Shanghai Sangon:

[0037] SEQ ID NO: 6 (82 bp): GCGAATTCCATGTTTGAAGACTGTACTGGGACGCCATGCC AGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCA

[0038] SEQ ID NO: 6 (78 bp): CCACCTACATCACCATCAGGGTTACGGCAGTAATTTTTTTTC CAGACCCGCCCGTGGATTTGTCTCTGGAGTGAAAATG

[0039] SEQ ID NO: 8 (73 bp): TGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGAAAAC TTTACGAC TACTGTGATGTCCCTCAGTGTGCG

[0040] SEQ ID NO: 9 (49 bp): GTCCCTCAGTGTGCCCACCACCACCACCACTAATAGCG CACACTGA

[0041] A restriction site, a His-tag tag, a start codon, and a stop codon were introduced into the above gene fragment. Phosphorylate the 5' of the synthesized nucleic acid fragments, and routinely perform phenol and chloroform extraction. Carry out routine PCR reaction operat...

Embodiment 3

[0043] The 5′ and 3′ ends of the mKringle5 protein gene (abbreviated as mKringle5 gene) were cut with restriction endonucleases, and the empty vector (such as pBV220) planned to be used was also cut with the same enzyme, and then ligated in vitro ( That is, recombination), the vector containing the gene sequence of the Kringle5 mutant protein can be obtained. Next, the preparation of the pBV220 / mKringle5 mutant protein recombinant plasmid expression vector will be described in detail.

[0044] PCR primer design: According to the gene sequence of Kringle5 mutant protein, the following primers can be designed: primer 1: 5′-GCGAATTCCATGGACTGTACTGGGACG–3′, primer 2: 5′-GCGGATCCCCTATTAGTGG TGGTGGT GGTGGTGGGCACACTGAGGGAC–3′. Among them, GAATTC and GGAT CC are restriction endonucleases EcoRI and BamHI restriction sites; ATG is a start codon; TAATAG is a stop codon; GTGGTGGTGGT GGTGGTG is a sequence of 6 histidines; 5′GC is a protection sequence .

[0045] PCR amplification of Kring...

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Abstract

The invention relates to a Kringle 5 mutant protein (mKringle 5 protein) which is characterized by comprising the following amino acid sequence: N sequence-DCGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTTNPKLYDYCDVPQCA-C sequence, wherein the N sequence does not exist, or is any other amino acid sequence with undefined length; and the C sequence does not exist, or is any other amino acid sequence with undefined length. The invention also relates to an expression vector and cloning vector of the mKringle 5 protein, a cell strain converted from a prokaryotic cell or eukaryotic cell by an mKringle 5 protein related vector, and a preparation method and application of the mKringle 5 protein. The preparation method can be used for massively and efficiently preparing the Kringle 5 mutant protein. The angiogenesis inhibitor prepared by the method has high development activity and low toxicity, and the activity for inhibiting angiogenesis of the angiogenesis inhibitor is obviously higher than that of the Kringle 5 protein.

Description

technical field [0001] The invention belongs to the fields of molecular biology and medicine, and relates to a sequence of recombinant human plasminogen Kringle5 mutant protein (mKringle5 protein), a preparation method and application of the protein. Background technique [0002] Human plasminogen (human Plasminogen, hPgn) is composed of five Kringle ring domains with basically the same amino acid composition, each domain is composed of about 80 amino acids, Kringle5 is the fifth of plasminogen domain. In 1994, O'Reilly extracted Angiostatin from the serum and urine of tumor-bearing mice, an endogenous protein that can specifically inhibit the proliferation of vascular endothelial cells. Studies have shown that Kringle5 has the biological activity of inhibiting endothelial cell proliferation, and its inhibitory activity is stronger than that of Angiostatin. Kringle5 also has the biological activity of inhibiting endothelial cell migration. Because of its high activity, ce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/68C12N15/09C12P21/02C12N15/63A61K38/48A61P9/14
Inventor 陈显久王军解军牛勃赵荣瑞
Owner SHANXI MEDICAL UNIV
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