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Yersinia pestis virulence regulator TyrR and applications thereof

A technology of Yersinia pestis and virulence regulator, applied in the field of genetic engineering, can solve the problem of incomplete understanding of the pathogenicity of Yersinia pestis

Inactive Publication Date: 2015-07-22
NANHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In conclusion, as a typical regulator, TyrR is affected by ATP, type and concentration of TyrR protein, composition and position of TyrR box, and type of aromatic amino acid. Regulation, there are no reports of TyrR regulating virulence at home and abroad
[0006] The current understanding of the regulation of Y. pestis gene expression is limited to some virulence factors regulated by specific environments, and the understanding of the pathogenicity of Y. pestis is still not comprehensive. Therefore, finding new virulence regulators is helpful to understand the pathogenicity of Y. pestis. disease mechanism, providing a basis for the preparation of live attenuated Yersinia pestis vaccines and drug therapeutic targets

Method used

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  • Yersinia pestis virulence regulator TyrR and applications thereof
  • Yersinia pestis virulence regulator TyrR and applications thereof
  • Yersinia pestis virulence regulator TyrR and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Discovery and Identification of 47kb Virulent Large Fragment

[0040] RyhB is a small RNA related to iron metabolism. Yersinia pestis has 2 copies of small RNA (RyhB1 and RyhB2) with a length of about 100bp. In the construction of the Yersinia pestis RyhB mutant strain, the application of the traditional one-step gene mutation technique was unsuccessful. In this example, two-step PCR was used to successfully construct △ryhB1 and △ryhB2 single-deleted strains, and the △ryhB1-deleted strain was constructed on the basis of △ryhB1::ryhB2 double deletion strain LD 50 Up to 1.5×10 6 CFU (see Table 1), the virulence is about 100,000 times lower than that of the wild strain, while the RyhB1 and RyhB2 single mutant strain LD 50 <8CFU, does not cause a drop in toxicity.

[0041] 1. The method of two-step PCR knockout gene is as follows:

[0042] (1) PCR amplification of the upstream and downstream homology arms and kanaboxes of the target gene to be knocked out. Pri...

Embodiment 2

[0054] Example 2 Determination of F1 antibody and immune protection effect of Yersinia pestis 47kb deletion strain

[0055] 1. Determination of F1 antibody of Yersinia pestis 47kb deletion strain: F1 antigen is an important neutralizing antigen of Yersinia pestis, and the humoral immune response ability of the vaccine can be reflected by measuring F1 neutralizing antibody. The mean F1 antibody titers produced between the two dose groups of Yersinia pestis 47kb large fragment deletion strain (and EV76 live vaccine) were 761.3±1.53 and 656.3±1.77 respectively, and there was no statistically significant difference (t=0.44, P>0.05), It shows that the large fragment of Yersinia pestis produces better humoral immune response after deletion of the 47kb large fragment, and the effect is similar to that of the traditional EV76 live vaccine (see figure 2 ).

[0056] The specific method is as follows: Three groups of BALB / c mice (ten mice in each group) were subcutaneously immunized wi...

Embodiment 3

[0058] Example 3 Discovery of Yersinia pestis virulence regulator TyrR

[0059] From the results of Example 1, it can be seen that the gene knockout technology based on Red homologous recombination in the present invention deletes the 47kb large fragment of Yersinia pestis, and after the 47kb deletion, the LD 50 1.5×10 6 CFU, the virulence is about 100,000 times lower than that of the wild strain, and it can produce F1 antibody and has good immune protection effect, so it can be used in the preparation of vaccines.

[0060] In order to further determine the gene attenuated after the deletion of the 47kb large fragment, the present invention adopts segmental knockout and LD 50 Experiment with combining strategies. The stepwise gene knockout method in this embodiment adopts the one-step gene knockout method and the two-step gene knockout method respectively. The one-step gene knockout method is to directly use PCR to amplify the linear mutation cassette, use the λRed system t...

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Abstract

A Yersinia pestis virulence regulator TyrR and applications thereof are provided. It is found that virulence of TyrR-deleted Yersinia pestis strains is lowered about 10000 times than virulence of wild strains, lack of the TyrR has no influence on in-vitro growth of the Yersinia pestis, and the TyrR can achieve negative regulation of transcription of five aromatic amino acid metabolism genes comprising aroF-tyrA, aroP, aroL and tyrP, and achieve positive regulation of two acid response genes which are hdeB and hdeD, so that the TyrR is proved to be a Yersinia pestis virulence regulator. In an environment having a pH value of 5.0, the survival rate and a growth speed of a Yersinia pestis TyrR mutant strain are lower than that of a wild strain, thus proving that the acid resistance of a TyrR mutant strain is lowered and that the TyrR regulator is closely related to acid resistance of the Yersinia pestis. Applications of the Yersinia pestis virulence regulator TyrR in preparation of anti-plague vaccines, and applications of the virulence regulator TyrR in preparation of medicines adopting the TyrR as a target are provided.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the discovery of a Yersinia pestis virulence regulator, in particular to the Yersinia pestis virulence regulator TyrR and its application. Background technique [0002] Plague is a natural foci disease caused by Yersinia pestis (Yersinia pestis for short). It is mainly transmitted among rodents through fleas as the medium of transmission, and occasionally invades the human body through the bite of infected fleas or contact with sick animals, causing bubonic plague, pneumonic plague and septicemic plague. Plague has an acute onset, rapid spread and high fatality rate. There have been three worldwide plague pandemics in history, killing at least 160 million people. From 1980 to 1999, 473 cases of human plague occurred in 7 western provinces including Inner Mongolia, Yunnan, Tibet, Gansu, Qinghai, Xinjiang and Sichuan, and 104 died. Since the 1990s, the incidence of plague has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/24A61K39/02A61K48/00A61P31/04C07K16/12G01N33/577G01N33/569
CPCY02A50/30
Inventor 邓仲良杨瑞馥韩延平王效义让蔚清王仁霞
Owner NANHUA UNIV
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