Method for soluble expression of recombinant protein of human brain natriuretic peptide and application

A technology of human brain natriuretic peptide and recombinant protein, which is applied to the preparation method of peptides, peptide/protein components, animal/human proteins, etc., can solve the problems of bacterial cell rupture, and achieve the effect of easy repeatability and simple operation

Active Publication Date: 2014-07-16
SHIJIAZHUANG WOTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But adding triton will cause the bacterium to rupture, and the contradiction between the amount of triton added and the improvement of protein yield cannot be resolved

Method used

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  • Method for soluble expression of recombinant protein of human brain natriuretic peptide and application
  • Method for soluble expression of recombinant protein of human brain natriuretic peptide and application
  • Method for soluble expression of recombinant protein of human brain natriuretic peptide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 BNP codon optimization

[0086] Synthetic DsbA mut -wtBNP (SEQ ID No11) and DsbA mut -optBNP (SEQ ID No3) gene sequence (synthesized by Invitrogen), and the His-Tag histidine tag sequence and the thrombin cleavage site sequence were introduced into the gene fragment respectively, and introduced at the 5' end of the artificially synthesized gene fragment Nco Ⅰ Endonuclease site, introduced at the 3' end Hind III endonuclease site. The synthetic fragment and the pET28a(+) vector were subjected to Nco Ⅰ and Hind Ⅲ Double enzyme digestion, T4 DNA ligase to connect gene fragments and vector fragments, routinely transform DH5α competent cells, use kanamycin resistance to screen to obtain positive clones, extract plasmids and carry out gene sequencing, and retain the correct clones and plasmids.

[0087] The wtBNP gene sequence is (SEQ ID No 1):

[0088] 5’-AGC CCC AAG ATG GTG CAA GGG TCT GGC TGC TTT GGG AGG AAG ATG GAC CGG ATC AGC TCC TCC AGT GGC CTG GGC...

Embodiment 2

[0094] Embodiment 2 Recombinant Vector and Engineering Bacteria Construction

[0095] Commissioned Invitrogen to synthesize His-DsbA mut -Thrombin recognition site-BNP sequence (SEQ ID No 3), and introduced at the 5' end of the gene fragment Nco Ⅰ Endonuclease site, introduced at the 3' end Hind III endonuclease site. The synthetic gene fragment and pET28a (+) vector ( figure 1 ) respectively via Nco Ⅰ and Hind Ⅲ double enzyme digestion, T4 DNA ligase to connect the gene fragment and the vector fragment, routinely transform DH5α competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.), screen positive clones according to kanamycin resistance, and extract plasmids. Recombinant plasmid via Nco Ⅰ and Hind Ⅲ Double enzyme digestion and agarose gel electrophoresis identification, and at the same time entrust Invitrogen to sequence the recombinant plasmid, use BioEdit software to analyze the sequencing results, the results are the same as the designed sequ...

Embodiment 3

[0097] Embodiment three engineering bacteria fermentation

[0098] 1. Take pET-28a-DsbA mut -BNP / BL21(DE3) seed solution, inoculated in LB liquid medium (containing 50 μg / mL kanamycin) at 1:500 at 37°C, 150 rpm for overnight culture;

[0099] 2. Measure the OD of the overnight culture solution 600 for 4 o'clock. It was inoculated into a fermenter containing 30 L of TB medium at a ratio of 1:20. The dissolved oxygen was controlled at 30%, the pH was controlled at 7.0, and cultured at 37°C. The stirring speed of the fermenter was automatically associated with the dissolved oxygen. Regular sampling, measure OD 600 value;

[0100] 3. When the culture solution OD 600 When it reaches 12, add IPTG (final concentration 0.5 mM) to the fermenter, control the dissolved oxygen to 30%, control the pH to 7.0, culture and induce at 37°C, take samples every hour to measure OD 600 value;

[0101] 4. After the engineered bacteria were induced for 4 hours, the fermentation culture was s...

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Abstract

The invention provides a method for soluble expression of recombinant protein of a human brain natriuretic peptide (Human Brain Natriuretic Peptide, hBNP). The method comprises the following steps: 1) obtaining a recombinant gene, namely obtaining the recombinant gene His-DsbAmut-BNP of expressing the recombinant protein of the human brain natriuretic peptide, wherein the sequence of the recombinant gene comprises a purified tag gene sequence, a molecular chaperone protein gene sequence, a protease recognition site sequence and a human brain natriuretic peptide sequence, which are sequentially arranged along the direction from 5' to 3'; 2) obtaining recombinant plasmids, namely inserting the recombinant gene obtained in the previous step into a carrier vector, so as to obtain the recombinant plasmids containing the recombinant gene; 3) obtaining genetically engineered bacterium, transferring the recombinant plasmids obtained in the previous step into host cells to obtain the genetically engineered bacterium; 4) expressing exogenous genes, fermenting the genetically engineered bacterium, and expressing fusion protein containing the human brain natriuretic peptide; 5) purifying target protein, splitting thallus, collecting fusion protein, removing molecular chaperone and carrying out chromatographic purification by protease digestion, so as to obtain the recombinant protein of the human brain natriuretic peptide.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method and application for soluble expression of human brain natriuretic peptide recombinant protein. The invention also relates to a recombinant gene for soluble expression of human brain natriuretic peptide recombinant protein. Background technique [0002] With the acceleration of the aging process of the population and the increase in the incidence of common cardiovascular diseases such as hypertension and coronary heart disease, the prevalence of heart failure is increasing year by year. According to the statistics of the United States, there are currently 4.9 million heart failure patients in the United States, and there are 400,000 to 700,000 new patients every year. The prevalence of heart failure in the population is about 1.5%-2.0%, and the population over 65 years old can reach 6%. -10%. The number of deaths due to heart failure has increased six-fold in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C07K14/575C07K1/22C07K1/20C07K1/18A61K38/22A61P9/04
Inventor 邹卫张世光周晓雷宋浩雷延志芬
Owner SHIJIAZHUANG WOTAI BIOTECH
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