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Human cystathionine beta-synthetase recombinant protein and use

A cystathionine and synthase technology, which is applied to human cystathionine β-synthase recombinant protein and application fields, can solve the problems of unseen CBS enzyme cloning and expression, research on medicinal application, reduction of human CBS enzyme activity, and the like, To achieve the effect of stable process, good activity and high purity

Inactive Publication Date: 2008-12-17
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, many studies have shown that CBS plays a vital role in the occurrence of homocysteinemia, and its activity loss or point mutation can lead to the reduction of human CBS enzyme activity, which is related to high homocysteine. The occurrence of cystinemia is closely related, and the medicinal research of CBS in homocysteinemia has not been reported, but its molecular structure and biological characteristics have been quite profoundly understood; The human CBS gene has been cloned, but there is no CBS enzyme clone expression and medicinal application research in China

Method used

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  • Human cystathionine beta-synthetase recombinant protein and use
  • Human cystathionine beta-synthetase recombinant protein and use
  • Human cystathionine beta-synthetase recombinant protein and use

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Embodiment 1

[0028] A method for preparing recombinant human cystathionine β-synthetase (rhCBS), characterized in that the human CBS gene is cloned, and the rhCBS prokaryotic expression system is separately constructed to obtain the rhCBS protein with biological enzyme activity. The specific steps include: constructing pET-32a-CBS expression plasmid; transformation of BL21 host bacteria; expression, isolation and purification of rhCBS protein with enzymatic activity.

Embodiment 2

[0030] Acquisition of CBS gene clones:

[0031]In view of the difference between Escherichia coli's preference for amino acid codons and human cells, the present invention designed and synthesized a DNA fragment containing Escherichia coli's preferred codons at the 5' end of the human gene, which can be used without changing the amino acid composition and sequence of the product human CBS. On the basis of improving the expression level of CBS in Escherichia coli; based on the full cDNA sequence of human CBS reported by Genbank, two pairs of primers were designed in the non-coding region and coding region of the upstream and downstream of the CBS gene, the non-coding region primer 1 P1 (upstream primer) P2 (downstream primer); design and synthesize primer 2P3 (upstream primer) P4 (downstream primer) in the coding region, and use this pair of primers to amplify the CBS target gene containing the initiation codon ATG. The primer P3 (upstream primer) introduces the enzyme cutting ...

Embodiment 3

[0047] Expression and sequence analysis of human CBS gene in Escherichia coli

[0048] The expression vector can be constructed by using cDNA as a template to amplify the full length of the CBS gene, and the restriction endonuclease EcoR

[0049] I and BamH I double-digest the target gene product and the expression vector plasmid pET-32a, separate and recover the two DNA fragments by agarose gel electrophoresis, and obtain an equimolar mixture of the two DNA fragments. 32a-CBS recombinant plasmid transformed into CaCl 2 The treated E.coli BL21 were selected and cultured by ampicillin. Pick a single colony, prepare a plasmid, and identify it by restriction endonuclease digestion. Those containing human CBS gene fragments and inserted in the correct direction are the target engineering bacteria; the CBS gene sequence is determined, and compared with Blast, it is 100% matched with Genbank. See figure 1 . It can be highly expressed in Escherichia coli BL21, so that CBS account...

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Abstract

The invention discloses the application of a recombinant human cystathionine Beta-synthase (rhCBS) in the preparation of drugs for curing or preventing hyperhomocysteinemia and related diabetes and cardiovascular diseases and a preparation method of the rhCBS. The preparation method of the invention mainly adopts an E.coli prokaryotic expression system which is optimized by expression conditions to obtain highly active rhCBS. The rhCBS can significantly reduce the HCY concentration in a cell-culture medium and improve the survival rate of myocardial cells and endothelial cells at the high HCY level. In an overall animal model with hyperhomocysteinemia, compared with traditional folic acid, VitB6 and VitB12 for curing the hyperhomocysteinemia, the rhCBS can greatly reduce the HCY level in plasma of a rat fed by homomethionin and alleviate the damage to cardiac muscle and vessels and has extremely obvious curative effects on the hyperhomocysteinemia. In addition, the rhCBS can be applied to the preparation of drugs for curing or preventing the hyperhomocysteinemia and related cardiovascular diseases and has broad market prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a prokaryotic high-efficiency expression technology of recombinant human cystathionine β-synthetase (rhCBS) and the application of recombinant protein in the preparation of drugs for preventing and treating cardiovascular diseases. The complete human CBS cDNA is cloned , independently constructed the prokaryotic expression system of human CBS, and expressed recombinant CBS protein with wild activity, and determined the therapeutic effect of recombinant CBS protein as a drug by using the in vitro culture model of cardiovascular cells and rat hyperhomocysteinemia model. Background technique [0002] Homocysteine ​​is a metabolic intermediate in the methionine cycle. Studies in recent years have confirmed that hyperhomocysteinemia is closely related to the occurrence of various cardiovascular diseases such as atherosclerosis, hypertension, and myocardial infarction, which are the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/43C12N15/52C12N15/70C12N9/00A61P3/10A61P9/00
Inventor 钱令嘉冷雪赵云杲修杰
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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