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Recombinant lysed Salmonella enterica and construction method and application thereof

A technology of Salmonella swine cholerae and a construction method, which is applied in the field of animal bacterial genetic engineering, can solve problems such as bacterial programmed lysis, and achieve the effect of improving biological safety and ensuring safety.

Active Publication Date: 2019-10-25
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the contrary, when the expression of the antitoxin MazE is controlled, the accumulated and synthesized MazF will enzymatically cleave the mRNA containing the ACA sequence in the bacterial chromosome, eventually leading to the programmed lysis of the bacterium

Method used

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  • Recombinant lysed Salmonella enterica and construction method and application thereof
  • Recombinant lysed Salmonella enterica and construction method and application thereof
  • Recombinant lysed Salmonella enterica and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] In this example, the arabinose-regulated Salmonella choleraesuis rSC0118 containing the MazEF cleavage system was constructed.

[0084] Its specific steps include:

[0085] 1.1 Construction of arabinose araC P BAD Activate the promoter-regulated mazE suicide vector (ΔendA::araCP BAD mazE TT)

[0086] According to the complete gene sequence of Salmonella choleraesuis released in Genbank, find the endA gene sequence (Genbank accession number is AE017220) and its upstream and downstream sequences, design primers according to the upstream and downstream genes of the gene, and the fragment of about 500bp of the upstream and downstream genes is Homology arm, knock out the ΔendA gene, a total of 1700bp, insert the cleavage system antitoxin gene mazE and araC P BAD The activated promoter sequence totaled 1592bp ( figure 1 );

[0087] Specific steps are as follows:

[0088] Using Salmonella choleraesuis C78-3 as a template, primers P1 and P2 were amplified by PCR to obtain...

Embodiment 2

[0141] In this implementation, recombinant prokaryotic expression vectors pET28a-mazE and pET28a-mazF were constructed and polyantibody serum against MazE and MazF proteins were prepared to detect the expression of MazE and MazF proteins in Salmonella choleraesuis lysate strain rSC0118.

[0142] The specific steps are:

[0143] 2.1 Construction of prokaryotic expression bacteria BL21(pET28a-mazE) and BL21(pET28a-mazF)

[0144] Using mazE, mazF gene sequences in Escherichia coli and pET-28a vector sequences to design homologous primers, namely mazE-F, mazE-R, mazF-F, mazF-R, amplify mazE using Escherichia coli K-12 as a template , mazF gene ( Figure 28 ); pET-28a was digested with XhoI and EcoRI, mazE, mazF gene and pET-28a were connected with homologous recombinase, and a single colony was taken for PCR and enzyme digestion ( Figure 29 ) and sequencing identification, the identified correct strains were stored at -70°C for later use.

[0145] Table 4: Amplification primer...

Embodiment 3

[0154] In this example, the relevant characteristics of the rSC0118 lysate regulated by arabinose (such as in vitro passage growth characteristics, dynamic changes in in vitro MazE and MazF protein expression, LD 50 , colonization ability) to evaluate and provide new ideas for the construction of biosafety Salmonella choleraesuis vaccine vectors.

[0155] 3.1 Identification of the growth phenotype of rSC0118 passaged under the regulation of arabinose

[0156] In order to detect the regulation of arabinose on the cleavage ability of rSC0118 in different media, C78-3 and rSC0117 were subcultured in LB and NB media respectively, and rSC0118 was cultured in culture media containing 0.2%, 0.1%, and 0.02% (mass fraction) arabinose, respectively. Passage in the LB, NB medium, detect OD 600 values ​​and plate counts.

[0157] 3.1.1 rSC0118 was passaged in LB and NB medium containing 0.02% arabinose

[0158] The specific steps are as follows: take C78-3 and rSC0117 and subculture in...

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Abstract

The invention discloses recombinant lysed Salmonella enterica and a construction method and application thereof. The elements introduced to genome of the recombinant lysed Salmonella enterica include:a mazE expression element including antitoxin gene mazE and its upstream promoter PBAD, and positive-negative regulator gene araC that encodes the promoter PBAD; an lacI expression element includinga gene lacI and its upstream promoter PBAD, and a positive-negative regulator gene araC that encodes the promoter PBAD; and an mazF expression element including toxin gene mazF and its upstream promoter Plac. The invention also discloses a construction method and application of the recombinant lysed Salmonella enterica. A Salmonella vector is modified through the combination of an arabinose promoter regulatory gene technology and an MazEF system so that programmed death occurs to the Salmonella vector due to immunostimulation; safety is good; allowing Salmonella to serve as a biosafe controllable polyvalent vaccine vector to deliver an exogenous antigen is guaranteed.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering of animal bacteria, and in particular relates to a recombinant lysed Salmonella choleraesuis and its construction method and application. Background technique [0002] With the development of molecular biology, there have been many reports on the genetic modification of pathogenic bacteria as their own preventive vaccines or the vaccines that deliver exogenous antigens at the same time. Among them, Salmonella has strong invasiveness to the host, and the heterologous protein expressed by the plasmid carried by it can continuously stimulate the host, and can better cause mucosal, cellular and humoral immune responses. Therefore, attenuated Salmonella has incomparable superiority as a vaccine carrier, and it has been widely used in the development of dual genetically engineered attenuated vaccines for viruses, bacteria, and parasites at home and abroad. However, Salmonella is a live reco...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12N1/06A61K39/112A61P31/04C12R1/42
CPCC12N15/74C07K14/245C12N9/22C12N1/06A61K39/0275A61P31/04C12N2800/60A61K2039/552A61K2039/522
Inventor 石火英陈芸芸
Owner YANGZHOU UNIV
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