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Human relaxin 2 precursor and preparation method thereof

A technology of human relaxin and relaxin, which is applied in the direction of relaxin, botany equipment and methods, biochemical equipment and methods, etc. It can solve the problems of low secretion expression and expensive C-peptide excision, and achieve low cost of culture medium , Reduce the cost of C-peptide excision, and increase the secretion expression

Inactive Publication Date: 2012-07-25
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0006] One object of the present invention is to provide a precursor of human relaxin 2, which is used to solve the problem of low secretion and expression of human relaxin 2 precursor in Pichia pastoris and its C-peptide The problem of expensive excision; another object of the present invention is to provide the preparation method of this human relaxin 2 precursor

Method used

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  • Human relaxin 2 precursor and preparation method thereof
  • Human relaxin 2 precursor and preparation method thereof
  • Human relaxin 2 precursor and preparation method thereof

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Embodiment 1

[0043] Expression of human relaxin 2 precursor (rhR2) and its C-peptide excision using the GAP1 promoter in Pichia pastoris.

[0044] 1. Synthesis of rhR2 gene and construction of expression vector pGAPZα-rhR2.

[0045] In order to facilitate the construction of yeast expression vectors, we added XhoI, XbaI restriction enzyme sites, stop codons to the rhR2 gene, and added a Kex2 cleavage site after XhoI, and then artificially synthesized figure 2 The gene sequence shown, was connected to the pUC-57 cloning vector, transformed into Escherichia coli DH5a competent, and screened by 100μg / mL Amp+resistant LB plate to obtain positive clones; positive clones were treated with 100μg / mL Amp+resistant LB Cultivate overnight in liquid medium, and then use a plasmid DNA mini-extraction kit to extract its recombinant plasmid; the recombinant plasmid and pGAPZαA plasmid are digested with XhoI and XbaI respectively, and then the relaxin precursor gene fragment and pGAPZα are recovered us...

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Abstract

The invention relates to a human relaxin 2 precursor and a preparation method thereof. According to the human relaxin 2 precursor, a B chain and an A chain of a natural human relaxin 2 are connected through an AsnGlyPheAsnGly artificial C peptide in a B-C-A order, so as to form a structure of a non-natural human relaxin 2 precursor. An amino acid sequence of the human relaxin 2 precursor is shown by a sequence 1 in a sequence list and is named as rhR2. According to the human relaxin 2 precursor and an optimized gene thereof, provided by the invention, a secreted expression amount of the human relaxin 2 precursor in pichia pastoris is greatly improved. The C peptide of the human relaxin 2 precursor is removed by using cheap hydroxyl amine rather than expensive TPCK (Tosyl-Phenylalanine Chloromethyl-Ketone)-trypsinase, so that the cost of removing the C peptide is reduced.

Description

[0001] 1. Technical field: [0002] The invention relates to the genetic engineering preparation of a human relaxin 2 precursor with a new structure, in particular to a human relaxin 2 precursor and a preparation method thereof. 2. Background technology: [0003] Relaxin belongs to the insulin family. It is a short peptide hormone composed of two short peptide chains connected by disulfide bonds. It has the functions of softening reproductive tract tissue, separating phalanx joints to promote childbirth, inhibiting uterine contraction, stimulating mammary gland growth and differentiation, and regulating follicles. Physiological effects such as development, dilation of blood vessels, anti-inflammation and promotion of wound healing, influence on stress response and appetite. Relaxin 2 can promote the increase of cAMP content in THP-1 cells. In recent years, it has been found that human relaxin also has the effect of treating heart failure clinically, which has attracted furt...

Claims

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Application Information

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IPC IPC(8): C07K14/64C12N15/16C12N15/81C12N1/19C12P21/02C12R1/84
Inventor 郭德军王淑慧厉保秋韩佳汕
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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