A method for constructing a gene regulation delayed attenuation and improved expression of exogenous antigen Salmonella choleraesuis vector

A Salmonella and exogenous antigen technology, which is applied in the construction field where the virulence gene of wild-type Salmonella choleraesuis is gradually deleted, and achieves the effect of significant economic benefits and large market applicability

Active Publication Date: 2017-11-17
YANGZHOU UNIV
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  • Application Information

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Problems solved by technology

But all of the above are studies on Salmonella typhimurium or human, studies using Salmonella choleraesuis as a carrier, especially the deletion of manA (the gene is equivalent to the pmi gene of typhoid fever) and the araC P BAD The application of activated promoter technology to construct attenuated Salmonella choleraesuis has not been reported at home and abroad

Method used

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  • A method for constructing a gene regulation delayed attenuation and improved expression of exogenous antigen Salmonella choleraesuis vector
  • A method for constructing a gene regulation delayed attenuation and improved expression of exogenous antigen Salmonella choleraesuis vector
  • A method for constructing a gene regulation delayed attenuation and improved expression of exogenous antigen Salmonella choleraesuis vector

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Embodiment 1

[0039] Construction of suicide vectors lacking manA, crp, relA and asd genes

[0040] 1. Construction of a suicide vector that deletes the crp gene

[0041] Search the whole gene sequence of Salmonella choleraesuis in Genbank, find the crp gene and its upstream sequence of Salmonella choleraesuis, design homology arm sequence primers in the crp gene and upstream gene, and delete 95 nucleotides in the promoter region of the crp gene , and then insert the transcription terminator (TT) and araC P BAD The activated promoter sequence is 1335bp, and its deletion and insertion system is yhfA partial sequence upstream of the crp gene, deletion of 95 nucleotides in the promoter region of the crp gene, transcription terminator (TT) of 1335bp and araC P BAD The crp sequence after activating the promoter sequence and deleting the 95bp crp gene promoter, the full name is ΔP crp ::TT araC P BAD crp (P means promoter, TT means transcription terminator) ( figure 1 ); The specific steps a...

Embodiment 2

[0073] Construction and identification of attenuated strain x0011 of Salmonella choleraesuis

[0074] 1. C78-3(ΔP crp ::TT araC P BAD crp) mutant strain construction and identification

[0075] 1.1C78-3(ΔP crp ::TT araC P BAD crp) mutant strain construction

[0076] Add recipient bacteria C78-3 (virulent wild-type Salmonella choleraesuis) to 1 mL of LB medium and culture overnight at 37°C; , DAP), 25mg / ml of chloramphenicol (chloramphenicol, Cm) in 1mLLB culture fluid, add donor bacteria χ7213 (pYAs001) (ΔP crp ::TTaraC P BAD crp suicide vector), cultivated overnight at 37°C to combine the recipient bacteria with the donor bacteria.

[0077] 1.2ΔP crp ::TT araC P BAD Screening of crp deletion strains

[0078] Streak a single colony of the combined colony on the LB solid culture plate containing chloramphenicol; take the grown single colony and culture it in 1mL of LB culture medium, and culture it with shaking at 37°C until the concentration of the bacterial solu...

Embodiment 3

[0110] Construction of Balanced-Lethal Bacteria-Vector System of Attenuated Salmonella Choleraesuis Strain χ0011

[0111] In order to avoid the use of drug-resistant or resistant gene-containing plasmid vectors and ensure the stability of the plasmid vectors in attenuated Salmonella in the host, we tested the attenuated Salmonella choleraesuis strain C78-3(ΔmanAΔP crp ::TT araC P BAD crpΔrelA::araC P BAD The asd gene was deleted on the chromosome of lacI TT), and this mutant strain was named χ0011 according to the naming sequence of our laboratory. When the asd gene-deleted strain is cultured in vitro, diaminopimelic acid (DAP) can be added to make up for the defect that the bacteria cannot grow due to the asd gene deletion. However, since there is no diaminopimelic acid in the animal body, Salmonella lacking the asd gene cannot survive in the animal body. Therefore, Dr. Curiss III's laboratory invented the addition of the asd gene of wild-type bacteria to the plasmid vect...

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Abstract

The invention provides a method for constructing a gene regulation delayed attenuation and improved expression of an exogenous antigen Salmonella choleraesuis vector, in which the recipient bacterium C78-3 is combined with the suicide vector Escherichia coli donor bacterium containing manA, crp, relA and asd deletions In combination, the mutations ΔmanA, ΔPcrp::TT araC PBAD crp, ΔrelA::araC PBAD lacI TT and ΔasdA were introduced into the wild-type Salmonella choleraesuis C78‑3 respectively, and the mutated Salmonella choleraesuis was called χ0011; and the four Phenotypic identification of mutations. The present invention can make wild-type Salmonella choleraesuis a safe and effective vaccine carrier for many exogenous antigens; provide attenuated vectors for various pig diseases, especially many bacterial diseases of pigs, which can improve the expression of exogenous antigens Salmonella choleraesuis by gene regulation , has great market applicability.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a construction method for gradually deleting the virulence gene of wild-type Salmonella choleraesuis. Background technique [0002] As an invasive intracellular bacterium, Salmonella has preferential invasion ability after attenuation, grows slowly in the body, and can effectively and continuously stimulate the body to produce strong cellular immunity, humoral immunity and local mucosal immunity. In addition, the Salmonella carrier has the function of immune adjuvant, and its LPS acts as an internal adjuvant, which can stimulate the host cells to release various cytokines. Therefore, attenuated Salmonella are more effective than inactivated or subunit vaccines in inducing humoral and cellular immune responses. Salmonella typhimurium weakened by genetic engineering has been used as a gene carrier to express foreign genes widely, and it is used in the treatment of tumors, viral diseas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/42
CPCC07K14/255C12N15/74
Inventor 石火英吉贞颖尚竞李玉安
Owner YANGZHOU UNIV
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