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spc-PcopA-yoeBVp gene cassette and preparation method and application thereof

A gene cassette and gene technology, applied in the field of genetic engineering, can solve the problems of unfavorable PCR amplification and subsequent identification, and achieve the effect of avoiding polarity effect and efficient traceless gene knockout

Active Publication Date: 2021-07-02
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the gene cassette is relatively large, and the left and right homology arms of the target gene are usually more than 4000 bp, which is not conducive to PCR amplification and subsequent identification; another disadvantage of this method is that some bacterial cells often have sacB The expression product of the gene exhibits resistance, limiting sacB Use as a negative selection marker

Method used

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  • spc-PcopA-yoeBVp gene cassette and preparation method and application thereof
  • spc-PcopA-yoeBVp gene cassette and preparation method and application thereof
  • spc-PcopA-yoeBVp gene cassette and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Example 1: spc -P copA - yoe B Vp Gene cassette construction

[0024] (1) PCR amplification separately spc, P copA, yoeB Vp DNA fragment

[0025] Using the Escherichia coli-Streptococcus suis shuttle plasmid pSET2 as a template, PCR amplification was performed with primers spc1 and spc2 to obtain a DNA fragment of 1130 bp, namely spc . Using the genome of Streptococcus suis type 2 SC19 strain as a template, PCR amplification was performed with primers PcopA1 and PcopA2 to obtain a 246 bp DNA fragment, which is P copA; using the genome of Vibrio parahaemolyticus RIMD 2210633 as a template, PCR amplification with primers yoeB1 and yoeB2 to obtain a 427 bp DNA fragment, namely yoe B Vp ; The PCR reaction system is: PrimeSTAR Max Premix 25 μL, F and R primers (10 μmol / L) each 2 μL, DNA template 1 μL, water 20 μL. The reaction conditions were: 98 °C for 3 min; 30 cycles of 98 °C for 10 s, 54 °C for 15 s, and 72 °C for 15 s; 72 °C for 1 min; 16 °C for ∞. The...

Embodiment 2

[0028] Embodiment 2: use spc -P copA - yoe B Vp Streptococcus suis pmtA knockout mutant

[0029] (1) Preparation of polypeptide (GNWGTWVEE);

[0030] The peptide (GNWGTWVEE) was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. with a purity of 90-95%. It was dissolved in ultrapure water to a final concentration of 5 mM and stored in a –80 °C refrigerator. Remove one tube per use and thaw on ice.

[0031] (2) build with spc -P copA - yoe B Vp gene cassette and pmtA Linear DNA fragments of the left and right homology arms of the gene;

[0032] Using Primer Premier 5 software to design and amplify Streptococcus suis pmtA Gene left and right homology arms and P copA - yoe B Vp The primers of the gene cassette, the sequence is as follows:

[0033] pmtA-LA-F:5'-tgtgtagccctcatttattgg-3';

[0034] pmtA-Fir-LA-R:5'-tattcacgaacctatcaacctcccactcgg-3';

[0035] pmtA-Fir-RA-F: 5'-tatattttacaagagcggtcgtgatgatgt-3';

[0036] pmtA-RA-R:5'-ataggtcttgctgtgct...

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Abstract

The invention discloses a spc-PcopA-yoeBVp gene cassette and a preparation method thereof, and belongs to the technical field of gene engineering, and the spc-PcopA-yoeBVp gene cassette comprises a positive selection marker spc and a negative selection marker PcopA-yoeBVp. The negative selection marker expresses YoeBVp toxin under the induction of copper sulfate and can inhibit the growth of an intermediate strain. By utilizing the gene box, the streptococcus suis traceless gene knockout can be efficiently realized. The gene knockout mutant strain does not contain any resistance marker, so that the polar effect caused by the resistance marker is avoided. In addition, because the gene knockout mutant strain does not contain a resistance marker, the gene knockout mutant strain can be used for subsequent vaccine research.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a negative selection marker for seamlessly knocking out the target gene of Streptococcus suis based on homologous recombination, in particular to a spc -P copA - yoe B Vp Gene cassette and its preparation method and application. Background technique [0002] Streptococcus suis ( Streptococcus suis , referred to as S. suis ) is a facultative anaerobic Gram-positive pathogen that can infect pigs and cause meningitis, sepsis, pneumonia, endocarditis and arthritis, etc., bringing huge economic losses to the global pig farming industry. In addition, Streptococcus suis can infect people through skin wounds and gastrointestinal tracts, leading to meningitis and streptococcal toxic shock syndrome, which poses a serious threat to public health. As of the end of 2013, more than 1,600 cases of human infection with S. suis had been reported worldwide, some of which were f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/64C12R1/46
CPCC12N15/74C12N15/65C12N15/64Y02A50/30
Inventor 郑成坤卫慢邱军
Owner YANGZHOU UNIV
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