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A kind of constitutive lactic acid bacteria promoter, recombinant vector and its recombinant bacteria and application

A technology of recombinant vectors and promoters, applied in the field of genetic engineering, can solve the problem of low protein expression and achieve the effect of improving protein expression activity

Active Publication Date: 2021-05-28
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, in order to solve the problem of low protein expression in the current lactic acid bacteria system, the present invention provides an ultra-efficient constitutive lactic acid bacteria promoter

Method used

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  • A kind of constitutive lactic acid bacteria promoter, recombinant vector and its recombinant bacteria and application
  • A kind of constitutive lactic acid bacteria promoter, recombinant vector and its recombinant bacteria and application
  • A kind of constitutive lactic acid bacteria promoter, recombinant vector and its recombinant bacteria and application

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preparation example Construction

[0043] The preparation method of the drug delivery system: comprising the following steps:

[0044] 1) Synthesize the nucleotide sequence of the target polypeptide or protein;

[0045] 2) inserting the nucleotide sequence synthesized in step 1) into the vector containing the promoter to construct a recombinant expression vector;

[0046] 3) transforming the recombinant expression vector in step 2) into normal intestinal bacteria;

[0047] 4) Inoculate the recombinant bacteria obtained in step 3) in the culture medium for expression.

[0048] Alternatively, the method comprises the steps of:

[0049] 1) Synthetic promoter-the nucleotide sequence of the target polypeptide or protein;

[0050] 2) inserting and integrating the nucleotide sequence synthesized in step 1) into the chromosome of normal intestinal bacteria;

[0051] 3) Inoculate the recombinant bacteria obtained in step 2) in the culture medium for expression.

[0052] In the present invention, the pharmaceutical ...

Embodiment 1

[0055] P gapA Cloning of the promoter and containing P gapA Construction of promoter vector pGapA

[0056] Based on the food-grade protein expression vector pNZ8149 (MoBiTec, Germany), the food-grade expression vector pGapA was transformed and constructed. The food-grade expression vector pNZ8149 has a low protein expression level, which limits its application to a certain extent. In order to solve this problem, the strong promoter P gapA The inducible promoter pNisin on pNZ8149 was replaced, and a food-grade protein expression vector with a higher expression level was obtained, which was named pGapA.

[0057] First, artificially synthesized BstYI-P gapA -NcoI nucleotide sequence (SEQ ID No.4: ggatct aataaaaactccttaaacatttttagtaaaaaaaattgacactaaataaaaaaagtagtatcatttatattagagataagagatgtctccaataactataaattcatatttatattataccacagcagttgacctgaaacaatgtttcgtattacgaagagtgatatttgtagtagaaaaagagaaattctcatcatctaagagataaaactacataac ccatgg , where the underline represents the enzyme cle...

Embodiment 2

[0059] Design and construction of sPD-1 protein expression vectors pNZ8149-sPD-1 and PgapA-sPD-1

[0060] Taking human PD-1 extracellular domain (sPD-1) as an example, sPD-1 is used as a reporter gene to test the activity of the promoter of the present invention.

[0061] 首先,人工合成SphI-SPusp45-DDDDK-sPD-1-XbaI的核苷酸序列(SEQ ID No.5:gcatgctcatgaaaaaaaagattatctcagctattttaatgtctacagtgatactttctgctgcagccccgttgtcaggtgtttacgctgatactaattctgatttggaaatatcgtcgacttgtgatgctgacgatgacgataagccaggatggttcttagactccccagacaggccctggaacccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagccccagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggccaggactgccgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggcgcaatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccagttccaaaccctggtgtaatctaga)。

[0062]其中,所述sPD-1为PD-1的胞外域,其为天然的人源PD-1 / PD-L1信号通路抑制剂,核苷酸...

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Abstract

The invention provides a constitutive lactic acid bacteria promoter, a recombinant vector and its recombinant bacteria and application, belonging to the field of genetic engineering. The promoter P provided by the present invention gapA The nucleotide sequence of is shown in SEQ ID No.1. The promoter P gapA It can promote the high-efficiency expression of downstream genes in lactic acid bacteria, and the protein expression activity is significantly higher than that of traditional inducible P NisinA It is an ultra-efficient promoter. The protein expression cassette composed of the promoter PgapA and the target gene of the present invention, the expression vector and the host bacteria can be used for oral drug delivery of peptide and protein drugs, which not only has good application prospects in genetic engineering, but also can improve Patients' medication compliance is expected to generate considerable social and economic benefits.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a constitutive lactic acid bacteria promoter, a recombinant vector, recombinant bacteria and applications thereof. Background technique [0002] With the development of biotechnology, many active peptides and proteins have been developed into drugs and applied clinically. Drugs such as peptides, proteins, enzymes, hormones, vaccines, cell growth factors, and monoclonal antibodies used for treatment or diagnosis are constantly emerging. Nearly a hundred kinds of peptides and protein drugs represented by erythropoietin, interferon, and insulin are already in clinical practice. application. Compared with traditional chemically synthesized drugs, peptide and protein drugs have the advantages of specific targets, clear in vivo effects, strong drug effects, and less toxic and side effects, so they are increasingly used in the prevention and treatment of modern ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12R1/01C12R1/225C12R1/46
CPCC07K14/70521C07K14/71C07K2319/00C12N15/63
Inventor 金万洙
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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