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Method for preparing targeted lidamycin production strain by utilizing gene disruption strain and application of lidamycin production strain

A technology for production of lidamycin and strains, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of complexity, low rate of gene homologous double exchange, difficulty in producing strains of daxamycin, etc.

Inactive Publication Date: 2011-05-04
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, due to the complex operation of gene replacement technology, the low rate of homologous double exchange of genes, and the heavy workload of screening recombinant strains, it takes a long time to obtain strains with correct gene replacement, and it is difficult to quickly obtain strains with multiple different targets. Lidamycin-producing strains

Method used

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  • Method for preparing targeted lidamycin production strain by utilizing gene disruption strain and application of lidamycin production strain
  • Method for preparing targeted lidamycin production strain by utilizing gene disruption strain and application of lidamycin production strain
  • Method for preparing targeted lidamycin production strain by utilizing gene disruption strain and application of lidamycin production strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Construction of lidamycin prosthetic protein gene blocking strain S.globisporus AKO

[0087] 1. Construction of cagA gene blocking plasmid

[0088] According to the known sequence of cagA gene, design PCR primers, use Pfu DNA polymerase, carry out PCR reaction with the total DNA of S. globisporus C-1027 as the template, and amplify the forearm Aup and hind arm Adown of the target fragment. The primers of the amplified forearm are AupF and AupR (see Table 3 for the primer sequence, SEQ ID No.1 and SEQ ID No.2), and the primers for the back arm are AdownF and AdownR (see Table 3 for the primer sequence, SEQ ID No.3 and SEQ ID No. 4).

[0089] After gel recovery and purification, the PCR products of the forearm and hind arm were ligated with pGEM-T (Promage, USA) using Taq DNA polymerase to add dATP to the tail of the product, and the ligated products were respectively transformed into E.coli DH5α competent cells (Dalian Bao Biological Company). Restriction si...

Embodiment 2

[0114] Example 2 Reply of lidamycin prosthetic protein gene blocking strain S.globisporus AKO

[0115] 1. Construction of cagA gene reversion plasmid

[0116] The inventors used a variety of different plasmids to perform recovery experiments on S. globisporus AKO. Plasmid pKC1139 is a multi-copy plasmid. Plasmid pSET152 is an integrative plasmid that does not contain Streptomyces replicons and contains ΦC31 attP site, which can be integrated into Streptomyces attB site. Plasmid pL646 was derived from pSET152 into which the strong promoter ermE*p and the SD sequence of the tuf1 gene were introduced upstream of the multiple cloning site.

[0117] Amplify the cagA gene coding region and its upstream promoter region and a fragment of about 900bp in the downstream terminator region by PCR (see Table 3 for the sequence, SEQ ID No.7 and SEQ ID No.8), and add enzymes to the two ends of the fragment respectively Fragment A9001 was amplified by cutting site EcoRI+BamHI; fragment A900...

Embodiment 3

[0135] Embodiment 3 is targeted at the construction of the lidamycin production bacterial strain of IV collagenase

[0136] 1. Construction of fusion protein expression plasmid

[0137] single domain antibody V H The nucleotide sequence and amino acid sequence are referenced [Tang Yong, Zhen Yongsu, Expression of Anti-IV Type Collagenase Single-Chain Antibody and Its Inhibitory Effect on Tumor Cell Invasion, Cancer, 2001, 20: 801-805], without change Under the principle of amino acid sequence, the codons of the original gene were replaced with codons preferred by Streptomyces.

[0138] In order to facilitate the cloning of fragments, XhoI and BglII restriction sites were introduced at both ends of the DNA sequence encoding the single-domain antibody gene; and when designing the upstream primer, the base sequence of the GGGGS flexible pentapeptide was added for connection Lidamycin prosthetic protein gene and anti-IV collagenase single domain antibody gene, so that lidamycin ...

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Abstract

The invention relates to a method for preparing a targeted lidamycin production strain by utilizing a gene disruption strain. The method comprises a step of introducing an expression plasmid of a fusion gene, which contains lidamycin apoprotein gene and a fusion gene consisting of different tumor targeted molecular coding sequences, into a lidamycin apoprotein gene disruption strain to obtain the lidamycin production strain with the target corresponding to the target of the tumor targeted molecule, wherein the gene interruption strain is a Streptomyces globisporus C-1027 lidamycin apoprotein gene (cagA) interruption strain, namely S.globisporus AKO; and the tumor targeted molecule includes, but not limited to tumor targeted antibody, tumor targeted peptide, cell factor and the like. The invention also relates to application of the production strain prepared by the method in producing targeted lidamycin.

Description

field of invention [0001] The invention relates to a method for preparing targeted lidamycin-producing strains by using gene blocking strains. Specifically, the method includes: introducing an expression plasmid comprising a fusion gene consisting of a lidamycin prosthetic protein gene and a coding sequence of a different tumor-targeting molecule into a lidamycin prosthetic protein gene-blocking strain to obtain Targeted lidamycin producing strain corresponding to the tumor targeting molecule, wherein the gene blocking strain is Streptomyces globisporus C-1027 (Streptomyces globisporus C-1027) lidamycin prosthetic protein gene (cagA) blocking strain Streptomyces globisporus AKO (S. globisporus AKO), the tumor targeting molecules include but not limited to, for example, tumor targeting antibodies, tumor targeting peptides, cytokines and the like. The present invention also relates to the use of the production strain obtained by the method to produce targeted lidamycin. Backg...

Claims

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Application Information

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IPC IPC(8): C12N15/76C12N1/21C12R1/465
Inventor 洪斌邵荣光王丽非崔智慧李保卫朱元军余腾斐
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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