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Recombinant brevibacillus brevis expressing pig growth hormone gene, construction method and application

A technology of Bacillus brevis and growth hormone, which is applied in the field of recombinant Bacillus brevis expressing porcine growth hormone gene and its construction and application, can solve the problems of short half-life of growth hormone and achieve the effect of short half-life

Pending Publication Date: 2017-09-08
江西嘉博生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a recombinant Bacillus brevis expressing porcine growth hormone gene and its construction method and application. The present invention effectively solves the problem of short half-life of growth hormone and achieves a long-acting effect

Method used

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  • Recombinant brevibacillus brevis expressing pig growth hormone gene, construction method and application
  • Recombinant brevibacillus brevis expressing pig growth hormone gene, construction method and application
  • Recombinant brevibacillus brevis expressing pig growth hormone gene, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Acquisition of porcine growth hormone gene

[0031] The porcine growth hormone sequence (AAA31045) published on NCBI is shown in sequence 1 in the sequence table, optimized according to the codon preference of Bacillus brevis, the nucleotide sequence of 6 histidines is added upstream, and the stop codon is added downstream XbaI and ClaI restriction sites and protective bases were added at both ends, and the sequence was artificially synthesized. See sequence 2 in the sequence table.

[0032] Sequence 1

[0033] FPAMPLSSLFANAVLRAQHLHQLAADTYKEFERAYIPEGQRYSIQNAQAAFCFSETIPAPTGKDEAQQRSDVELLRFSLLLIQSWLGPVQFLSRVFTNSLVFGTSDRVYEKLKDLEEGIQALMRELEDGSPRAGQILKQTYDKFDTYLRSDDALLKGLLSFKKDLHKAETNLRSDDALLKGLLSFKKDLHKAETNLRSDDALLKGLLSFKKDLHK

[0034] Sequence 2

[0035] GCTCTAGACATCATCATCATCATCATTTCCCGGCAATGCCGCTGAGCAGCCTGTTCGCGAACGCGGTGCTGCGCGCGCAACACCTGCACCAGCTGGCGGCAGATACGTATAAAGAATTCGAACGCGCGTACATCCCGGAAGGCCAACGCTATAGCATCCAGAACGCGCAAGCGGCGTTCTGCTTCAGCGAGACGATCCCGGCGCCGACGGGCAAAGATGAAGCGCAACAGC...

Embodiment 2

[0037] Construction of the recombinant plasmid pGH-pNCM02 of Bacillus brevis

[0038] 1. Double enzyme digestion reaction of pGH gene and pNCM02 plasmid

[0039] Use restriction enzymes XbaI and ClaI to double digest the gene and pNCM02 plasmid obtained in Example 1. The double digestion reaction system is as follows:

[0040] system Volume (μL) XbaI2 ClaI2 pGH gene / pNCM02 plasmid20 10×M buffer4 ddH2O12 total capacity40

[0041] After reacting in a 37°C incubator for 3 hours, the agarose gel recovered the double digested fragments.

[0042] 2. The ligation reaction between pGH gene and pNCM02 plasmid

[0043] T4 ligase ligation reaction system is as follows:

[0044] system Volume (μL) pGH8 pNCM028 T42 10×buffer2 total capacity20

[0045] React overnight in a refrigerator at 4°C.

[0046] 3. The ligation product transforms JM109 E. coli competent cells

[0047] 3.1. Take out the E. coli JM109 competent cells (100μL) frozen at -80°C, hold them in the palm of your hand for...

Embodiment 3

[0061] Construction of Recombinant Bacillus brevis

[0062] 1. Preparation of relevant media and reagents

[0063] T2 liquid medium: glucose 1.0%, peptone 1.0%, beef extract 0.5%, yeast extract 0.2%, pH 7.0; T2 solid medium is T2 liquid medium supplemented with 2% agar powder.

[0064] Competent preparation medium: T2 medium is supplemented with MnSO4 0.001% (w / v, the same below), FeSO40.0001%, ZnSO4 0.0001%

[0065] Competent washing solution 1: sucrose 10%, HEPES 16mM, CaCl2 1mM, glycerol 15%, pH 7.0.

[0066] Competent washing solution 2: PEG6000 15%, HEPES 1mM, pH 7.0.

[0067] Repair medium: add 20mM MgCl2 to T2 liquid medium

[0068] 2. Preparation of Bacillus brevis SP3 Competent

[0069] 2.1. Resuscitate Bacillus brevis SP3 by streaking on a T2 solid medium plate, and culture it upside down at 37°C18;

[0070] 2.2. Cultivation seed solution: Pick a single colony of Bacillus brevis SP3 and inoculate it in 5ml T2 liquid medium, and cultivate overnight at 30°C and 250rpm.

[0071] 2.3. ...

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Abstract

The invention discloses a construction method of recombinant brevibacillus brevis expressing a pig growth hormone (pGH) gene. The construction method comprises the steps of (1) optimizing a pGH sequence published by NCBI (National Center of Biotechnology Information) according to brevibacillus brevis codon preference to obtain the pGH gene and a pNCM02 plasmid, (2) performing double enzyme digestion on the pGH gene and the pNCM02 plasmid by restriction enzyme XbaI and ClaI, (3) recycling a double enzyme digestion product, performing a coupled reaction, transforming a coupled product into an escherichia coli JM109 competent cell, and performing resistance screening on a transformant by ampicillin to obtain a recombinant plasmid pGH-pNCM02, and (4) extracting the recombinant plasmid, performing electrotransformation into brevibacillus brevis SP3, and performing the resistance screening on a transformant by neomycin to obtain the recombinant brevibacillus brevis expressing pGH. The construction method effectively solves the problem of short half-life period of growth hormone; and a long-acting effect is achieved.

Description

Technical field [0001] The invention relates to a recombinant Bacillus brevis expressing a porcine growth hormone gene, and a construction method and application. Background technique [0002] Bacillus brevis is a safe, non-toxic probiotic, and has the characteristics of strong protein secretion ability and low extracellular protease activity, so it is particularly suitable as an extracellular secretion protein expression system. [0003] Porcine growth hormone is a protein-like single-chain hormone secreted by the eosinophils of the anterior lobe of the pig pituitary. It is composed of 191 amino acids, has a molecular weight of 22kDa, and an isoelectric point of 6. It is a single-copy gene in the pig genome without other similar sequences. . Porcine growth hormone can promote the body's protein synthesis and tissue utilization of amino acids in the circulatory system, increase muscle protein content, reduce fat content, promote bone development, and increase pig growth. At the sa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/18C12N15/74C12N15/66A61K38/27A61P5/06C12R1/01
CPCC07K14/61A61K9/0019A61K38/27C12N15/66C12N15/74C12N2800/22
Inventor 曹远东周志潘烘张燕程晓文向勇
Owner 江西嘉博生物工程有限公司
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