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Streptococcus suis efficient transposition mutation system and application thereof

A high-efficiency technology for Streptococcus suis, which is applied in the field of high-efficiency transposition mutation system for Streptococcus suis, and can solve the problems of not finding a high-efficiency random transposition mutation system

Pending Publication Date: 2022-07-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no similar high-efficiency random transposition mutation system found in Streptococcus suis

Method used

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  • Streptococcus suis efficient transposition mutation system and application thereof
  • Streptococcus suis efficient transposition mutation system and application thereof
  • Streptococcus suis efficient transposition mutation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The Streptococcus suis gene inducible expression system of the present invention comprises a Mariner transposase expression cassette composed of a gapdh gene promoter and a transposase gene Mariner, and an erythromycin resistance expression cassette (Erm R ) connection composition (as shown in SEQ ID NO.2), named as Tn fragment, the nucleotide sequence of gapdh gene promoter is as shown in SEQ ID NO.14, the nucleotide sequence of transposase gene Mariner is as shown in SEQ ID NO. .15, the erythromycin resistance expression cassette (Erm R ) nucleotide sequence shown in SEQ ID NO.18, erythromycin resistance expression cassette (Erm R ) including the erythromycin resistance gene, the transposase recognition sequence (shown in SEQ ID NO. 17) between the erythromycin resistance gene and the Mariner transposase expression cassette, and the transposase recognition sequence on the other side (as shown in SEQ ID NO. 16).

[0028] The Streptococcus suis high-efficiency transpo...

Embodiment 2

[0057] Example 2 Construction of Streptococcus suis transposition mutants using pSET4s-Tn plasmid

[0058] (1) Add about 100 ng of pSET4s-Tn plasmid to the competent Streptococcus suis, mix well on ice, and perform click transformation, then add 400 μl of TSB to culture at 28°C for 2 hours, and then spread the culture with the final concentration 5% bovine serum, 10 μg / mL erythromycin and 100 μg / mL spectinomycin on TSA culture plate, incubator at 28℃ for overnight culture;

[0059] (2) Using the primer pair shown in SEQ ID NO. 6 and SEQ ID NO. 7 and the primer pair shown in SEQ ID NO. 8 and SEQ ID NO. 9: Erm-F: 5'-GAAACCGATACCGTTTACG-3' Colony PCR with Erm-R: 5'-GACGATATTCTCGATTGACC-3' and Spc-F: 5'-GATCAGGAGTTGAGAGTG-3' and Spc-R: 5'-CTCTTCTCACATCAGAAAATGG-3' to confirm the successful transformation of the plasmid into S. suis;

[0060] (3) Inoculate a single colony of Streptococcus suis containing pSET4s-Tn plasmid into a TSB liquid medium containing a final concentration o...

Embodiment 3

[0063] Example 3 Identification of transposon insertion sites

[0064] (1) 10 Streptococcus suis transposon mutants were randomly selected, inoculated into TSB liquid medium containing a final concentration of 5% bovine serum and 5 μg / mL erythromycin, and cultured at 37°C for 12 hours, using genomic DNA Extraction kits extract bacterial genomes.

[0065] (2) Using the genomic DNA extracted above as a template, using primers arb-2: 5'-GGCCACGCGTCGACTAGTCANNNNNNNNNNGATCA-3' and erm5-4: 5'-GGTTGAGTACTTTTTCACTCG- 3' for the first round of PCR. The reaction system is: upstream and downstream primers 0.5nM each, genomic DNA 2μL, PCR Mix 10μL, ddH 2 O supplemented to 20 μL; PCR reaction conditions were 94°C, 2 min; 94°C, 30s; 42°C, 30s; 72°C, 3min, the above reaction was repeated for 6 cycles; 94°C, 30s; 65°C, 30s; 72°C, 3min , the above reaction was repeated for 25 cycles; 4°C, 10min;

[0066] (3) Using one round of PCR products as templates, primers arb-3 as shown in SEQ ID NO....

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Abstract

The invention discloses a streptococcus suis efficient transposon mutation system, namely a pSET4s-Tn plasmid, which comprises: (1) a streptococcus suis temperature-sensitive replication element Ts ori; (2) an escherichia coli replication element ColE1ori; (3) a Mariner transposase expression box; (4) a spectinomycin resistance expression cassette (SpcR); and (5) an erythromycin resistance expression cassette (ErmR) containing a transposase recognition sequence (IR) at each of the upstream and the downstream. By utilizing the transposon mutation system, efficient transposon mutation in streptococcus suis can be realized, more than 98% of streptococcus suis can be used as transposon mutants in a single experiment, the transposon is inserted into a TA base site in a streptococcus suis genome, and the transposon insertion site has no obvious preference.

Description

technical field [0001] The invention belongs to the technical field of gene mutation, in particular to a high-efficiency transposition mutation system of Streptococcus suis and its application. Background technique [0002] Streptococcus suis is an important zoonotic pathogen that can cause meningitis, arthritis and septicemia in pigs, causing huge economic losses to the pig industry, and can also infect humans, leading to toxic shock-like syndrome and death. high rate, endangering public health safety. The study of pathogenic biology and functional genomics of Streptococcus suis is of great significance for the identification of novel antibacterial drug targets and the discovery of vaccine candidate antigens. [0003] Efficient and rapid genetic manipulation of pathogenic bacteria is the key to study the growth, metabolism and pathogenic mechanism of pathogenic bacteria. Therefore, the development of genetic manipulation tools for Streptococcus suis is of great significanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/54C12N1/21C40B50/06C12R1/46
CPCC12N15/746C12N15/65C12N9/1241C40B50/06C12N2800/90
Inventor 黄琦张咏晴周锐邱德新黎璐
Owner HUAZHONG AGRI UNIV
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