CN111040025B

CN111040025B - Application of Yp _2058 protein in regulation of toxicity of Yersinia pestis

Info

Publication number: CN111040025B
Application number: CN201911343786.6A
Authority: CN
Inventors: 赵腾; 曹诗洋; 游旸; 陈红艳; 焦扬; 杜宗敏
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-09-11
Anticipated expiration: 2039-12-24
Also published as: CN111040025A

Abstract

The invention discloses application of Yp _2058 protein in regulation and control of toxicity of Yersinia pestis. The amino acid sequence of Yp _2058 protein is shown in SEQ ID NO: 2, respectively. Injecting 100CFU of a rat plague bacterium delta yp _2058, a rat plague bacterium delta yp _2058 replenisher strain or a rat plague bacterium 201 strain into the inguinal, tail vein or nasal drip of a Balb/c mouse, and then normally feeding for 14 d; during which time the survival of the mice was observed. The result shows that compared with the plague bacteria 201 strain, the plague bacteria delta yp _2058 have obviously reduced virulence; the strain for replenishing the plague delta yp _2058 can completely replenish virulence. Therefore, the Yp _2058 protein has an important influence on the virulence of Yersinia pestis. The invention has great application value.

Description

Application of Yp _2058 protein in regulation of toxicity of Yersinia pestis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of Yp _2058 protein in regulation of toxicity of Yersinia pestis.

Background

Plague is a Chinese-legislated class A infectious disease, the pathogen of which is Yersinia pestis (Yersinia pestis). Plague is a natural epidemic disease, and the plague source of China is mainly distributed in 17 provinces and autonomous regions, and the total area is as much as 60 to ten thousand square kilometers. Yersinia pestis is mainly transmitted to host rodents by arthropod fleas through bites and possibly transmitted to human beings, which causes bubonic plague, pneumonic plague, septicemia and the like, and causes huge life and property loss to the human beings. The world health organization has listed plague as a newly emerging infectious disease and, therefore, must pay high attention to it.

Plague is a gram-negative bacillus pumilus that is spread by fleas, which are considered to be room temperature animals with an in vivo temperature of 26 c, and enter a 37 c environment after biting animals or humans. Therefore, plague has a set of mechanisms to cope with this temperature shift stimulus. The plague bacteria have important significance for temperature conversion and pathogenicity development by secreting virulence proteins.

Disclosure of Invention

The invention aims to reduce the toxicity of Yersinia pestis.

The invention firstly protects the application of Yp _2058 protein in regulation and control of Yersinia pestis virulence and/or prevention of plague.

In the above application, the Yp _2058 protein can be a1) or a2) or a3) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2, the N end or/and the C end of the protein shown in the figure is connected with a label to obtain a fusion protein;

a3) the protein related to the toxicity of the Yersinia pestis is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein shown in a1) or a 2).

Wherein, SEQ ID NO: 2 consists of 199 amino acid residues.

The invention also protects the application of the nucleic acid molecule for encoding the Yp _2058 protein in regulation of toxicity of Yersinia pestis and/or prevention of plague.

The nucleic acid molecule encoding the Yp _2058 protein can be a DNA molecule shown as b1) or b2) or b3) or b4) as follows:

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1;

b3) a DNA molecule derived from yersinia pestis and encoding said Yp _2058 protein, having 75% or more identity to the nucleotide sequence defined by b1) or b 2);

b4) a DNA molecule which is derived from Yersinia pestis and codes the Yp _2058 protein and hybridizes with the nucleotide sequence limited by b1) or b2) under strict conditions.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, SEQ ID NO: 1 consists of 600 nucleotides, SEQ ID NO: 1 encodes the DNA molecule shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In any of the above applications, the yersinia pestis strain may be yersinia pestis strain 201.

The invention also provides a recombinant Yersinia pestis, which is prepared by the following steps: inhibiting the expression quantity and/or activity of the Yp _2058 protein in the yersinia pestis, and obtaining a recombinant bacterium, namely the recombinant yersinia pestis; the recombinant yersinia pestis has reduced virulence compared to yersinia pestis.

In the above method, the "inhibition of the expression level and/or activity of the Yp _2058 protein in yersinia pestis" may be achieved by a method known in the art, such as RNA interference, homologous recombination, gene knockout, or gene site-directed editing, to inhibit the expression level and/or activity of the Yp _2058 protein.

In the above method, the "inhibition of the expression amount and/or activity of Yp _2058 protein in yersinia pestis" may be achieved by introducing a substance that inhibits the expression amount and/or activity of Yp _2058 protein into yersinia pestis.

In any of the above-mentioned methods, the "substance inhibiting the expression level and/or activity of Yp _2058 protein" may specifically be a PCR amplification product obtained by performing PCR amplification using a primer pair consisting of 2058-K-F and 2058-K-R of Table 1 in examples, using plasmid pKD4 as a template. The PCR amplification product has a kana mutation box with a homologous arm.

In any of the methods described above, the yersinia pestis can be yersinia pestis strain 201. In one embodiment of the present invention, when the yersinia pestis is yersinia pestis strain 201, the recombinant yersinia pestis may be specifically yersinia pestis Δ yp _ 2058.

The invention also discloses a recombinant Yersinia pestis B, and the preparation method comprises the following steps: improving the expression quantity and/or activity of the Yp _2058 protein in the yersinia pestis, wherein the obtained recombinant bacterium is the recombinant yersinia pestis B; the recombinant Yersinia pestis B has enhanced virulence compared to Yersinia pestis.

In the above method, the "increasing the expression level and/or activity of the Yp _2058 protein in yersinia pestis" may be achieved by a method known in the art, such as multiple copies, alteration of a promoter, a regulatory factor, and a transgene, to increase the expression level and/or activity of the Yp _2058 protein.

In the above method, the "increasing the expression level and/or activity of the Yp _2058 protein in yersinia pestis" may be performed by introducing a substance that increases the expression level and/or activity of the Yp _2058 protein into yersinia pestis. The "substance which increases the expression level and/or activity of Yp _2058 protein" may be a nucleic acid molecule encoding the Yp _2058 protein. The introduction of the substance that increases the expression level and/or activity of the Yp _2058 protein into Yersinia pestis can be specifically achieved by introducing a recombinant vector containing a nucleic acid molecule encoding the Yp _2058 protein into Yersinia pestis. The recombinant vector containing the nucleic acid molecule for encoding the Yp _2058 protein can be specifically prepared by replacing a small fragment between recognition sequences of restriction enzymes NheI and HindIII of a pBAD24 plasmid with a fragment of SEQ ID NO: 1 to obtain a recombinant plasmid.

In the above method, the yersinia pestis may be recombinant yersinia pestis or yersinia pestis strain 201. In one embodiment of the present invention, when Yersinia pestis is Yersinia pestis Δ yp _2058, the recombinant Yersinia pestis B can be Yersinia pestis Δ yp _2058 anaplerotic strain.

The invention also provides any substance which can inhibit the expression quantity and/or activity of Yp _2058 protein.

The invention also protects the application of any substance for inhibiting the expression quantity and/or activity of Yp _2058 protein in regulating the toxicity of Yersinia pestis and/or preventing plague.

In the above application, the yersinia pestis can be yersinia pestis strain 201.

In any of the above applications, the regulation of the toxicity of yersinia pestis may be a reduction in the toxicity of yersinia pestis or an increase in the toxicity of yersinia pestis.

In the embodiment of the invention, 100CFU of plague bacteria delta yp _2058 (equivalent to recombinant Yersinia pestis), plague bacteria delta yp _2058 anaplerotic strain or plague bacteria 201 strain are injected subcutaneously, in tail veins or in nasal drops into the groin of Balb/c mice, and then the mice are fed normally for 14 d; during which time the survival of the mice was observed. The result shows that compared with the plague bacteria 201 strain, the plague bacteria delta yp _2058 have obviously reduced virulence; the strain of plague bacillus delta yp _2058 can completely complement virulence. Therefore, the Yp _2058 protein has an important influence on the virulence of Yersinia pestis. The invention has great application value.

Drawings

FIG. 1 shows the results of the acid resistance test of example 3.

FIG. 2 is a growth curve prepared in example 4.

FIG. 3 is a graph of survival curves for groups of mice in the mice virulence experiment, step one of example 5.

FIG. 4 is a survival curve for groups of mice in the virulence experiment in step two mice in example 5.

FIG. 5 is a survival curve for groups of mice in the three-step mouse virulence experiment of example 5.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the quantitative experiments in the following examples, three replicates were set up and the results averaged.

The names and nucleotide sequences of the primers involved in the following examples are shown in Table 1.

TABLE 1

Note: the recognition sequence for the restriction enzyme Nhe I is single underlined, and the recognition sequence for the restriction enzyme HindIII is double underlined.

Yersinia pestis strain 201 is described in the following documents: song Y, Tong Z, Wang J, Wang L, Guo Z, Han Y, Zhang J, Pei D, Zhou D, Qin H et al complete genome sequence of Yersiniapestis strain 91001, an isocyanate average to humans DNA research: international journal for rapid publication of reports on genes and hedgermes 2004, 11(3): 179. about.197. Yersinia pestis strain 201 is hereinafter referred to as Yersinia pestis strain 201.

The plasmid miniprep kit is a product of Qiagen corporation.

Example 1 discovery of Yp _2058 protein

Yersinia pestis undergoes a transition from ambient temperature 26 ℃ to 37 ℃ from the transmission vector to the mammalian host, and many virulence genes are initiated at 37 ℃ and begin to be expressed in large numbers, which greatly increases the virulence of Yersinia pestis on the host. The existing research shows that the mycoprotein of the plague bacteria 201 strain is subjected to mass spectrometry by three different methods, namely Shotgun-LC-MS-MS, 1D-LC-MS-MS and 2D-MS, and 1193 proteins are identified in total and account for 28.7 percent of the CDS predicted by the genome. The proteomics of the plague 201 in vitro flea simulation condition is analyzed by an LTQ-FT method, 1926 proteins are identified in total, and the proportion of the proteins in the CDS predicted by the genome is 46.50%. The inventor identifies the difference of proteomes under simulation conditions of plague bacillus and fleas and human body simulation conditions by TMT (TandemMass tag) mass spectrometry technology, and after redundancy is removed, 2141 proteins are detected in total and account for 52.2% of CDS predicted by genome (the coverage rate is highest in reported literature). Then, by analysis, Yp _2058 protein which is possibly related to the virulence of Yersinia pestis is screened out. The method comprises the following specific steps:

1. a secreted protein sample 1 of the plague bacterium 201 strain at 26 ℃ (simulating flea internal environment), a secreted protein sample 2 of the plague bacterium 201 strain at 37 ℃ (simulating mouse internal environment), a mycoprotein sample 3 at 26 ℃ and a mycoprotein sample 4 at 37 ℃ are prepared.

2. After the step 1 is completed, quantitative analysis is respectively carried out on the secreted protein sample 1, the secreted protein sample 2, the mycoprotein sample 3 and the mycoprotein sample 4 by adopting a same quantity of different sequence chemical Tags (TMT) method, so that a plague bacterium 201 strain protein analysis platform is established.

3. And (3) after the step 2 is completed, analyzing the plague bacteria 201 strain protein analysis platform, and screening Yp _2058 protein from the secretory protein with the expression quantity remarkably increased in the secretory protein sample 2 by combining the protein expression quantity and the omics analysis result.

The amino acid sequence of Yp _2058 protein is shown in SEQ ID NO: 2, respectively. The gene coding the Yp _2058 protein (hereinafter referred to as Yp _2058 gene) is shown as SEQ ID NO: 1 is shown.

Example 2 construction of yp _2058 Gene knockout and complementation Strain

Construction of yp _2058 gene knockout strain (i.e. plague bacillus delta yp _2058)

Constructing gene mutant strains is one of the most important methods for studying gene functions. The inventor of the invention knocks out the yp _2058 gene by using a one-step mutation technology. The method is based on a lambda-Red recombination system of escherichia coli, and can ensure that yp _2058 gene and resistance gene (kana mutant box) with a homologous arm are subjected to homologous recombination and are replaced by the resistance gene, so that the yp _2058 gene fails to function. In the method, firstly, pKD46 plasmid of a coding lambda-Red recombination system is introduced into a plague bacterium 201 strain, and the plasmid can induce and express three proteins of Exo, Beta and Gam through arabinose (the Exo protein is lambda exonuclease and can cut double-stranded DNA; the Beta protein can mediate single-stranded DNA complementary renaturation and annealing reaction with the Beta protein; the Gam protein can inhibit the activity of exonuclease on the DNA); then transferring into a resistance gene (namely kana mutant box) with a homologous arm, and carrying out homologous recombination with the yp _2058 gene on the chromosome of the plague bacteria 201 strain under the action of recombinase so as to inactivate the yp _2058 gene; finally, the culture temperature was increased to eliminate the temperature-sensitive plasmid pKD 46.

1. Extraction of plasmid pKD46

(1) Escherichia coli DH 5. alpha. containing pKD46 plasmid was inoculated into 5mL LB liquid medium, and cultured at 37 ℃ with shaking at 200rpm until logarithmic phase to obtain a culture broth.

(2) And (2) taking the culture solution obtained in the step (1), and extracting the plasmid by using a plasmid miniprep kit to obtain a plasmid pKD46 (about 177 bp).

2. Preparation of competent cell of plague bacterium strain 201

(1) The plague bacterium 201 strain is inoculated in an LB liquid culture medium, and is subjected to shaking culture at 26 ℃ and 200rpm until the logarithmic phase, so that bacterium liquid 1 is obtained.

(2) Taking the bacterial liquid 1, inoculating to LB liquid culture medium (inoculation ratio is 1:50), shaking culturing at 26 deg.C and 200rpm to obtain bacterial liquid 2 with OD620nm value of 0.5-0.6.

(3) Adding arabinose into the bacterial liquid 2 to obtain an induction system; in the induction system, the concentration of arabinose was 10 mmol/L.

(4) Taking an induction system, carrying out shaking culture at 26 ℃ and 200rpm for 2h to obtain a bacterial liquid 3.

(5) Centrifuging the bacterial liquid at 4000rpm for 5min at 4 ℃ for 3 min, and collecting the thalli.

(6) And (3) taking the thallus collected in the step (5), washing twice by using a sterile 10% (v/v) glycerol aqueous solution, and then re-suspending by using 100 mu L of 10% (v/v) glycerol aqueous solution to obtain the plague bacteria 201 strain competent cell.

The competent cells of the strain of plague bacterium 201 are placed in an ice bath for standby.

3. Electric conversion

(1) Taking the competent cells of the plague bacillus 201 strain, adding 200 mu g of plasmid pKD46, uniformly mixing, adding into a precooled electric shock cup, carrying out ice bath for 10min, and then placing into an electric transfer apparatus for electric shock (the electric shock parameters are 25 mu F, 200 omega and 2.5 KV).

(2) After the step (1) is finished, adding 1mL of LB liquid culture medium into the electric shock cup, uniformly mixing, sucking into an EP tube (specification is 1.5mL), and carrying out shake culture at 26 ℃ and 200rpm for 2 h; and finally, uniformly coating 100 mu L of bacterial liquid on an LB solid plate (Amp resistance), and carrying out inverted culture at 26 ℃ for 36h to obtain a plurality of monoclonals.

(3) And (3) respectively plating each monoclonal for enrichment (Amp resistance, LB plate), taking a small amount of thallus in sterile water, treating for 10min at 99 ℃, centrifuging, and collecting the supernatant.

(4) Respectively taking the supernatant as a template, and taking pkd 46-F: 5'-GGAGCGCATGGCAGAACAC-3' and pkd 46-R: 5'-CAGAGCGGCAATAAGTCG-3' is used as a primer for PCR amplification. If the PCR amplification product contains 893bp DNA fragment, the corresponding monoclonal is positive. The positive single clone is the plague bacteria 201 strain containing KD46 plasmid.

Each positive monoclonal was glycerol-preserved at-20 ℃.

4. kana mutant cassette amplification

And carrying out PCR amplification by using the plasmid pKD4 as a template and a primer pair consisting of 2058-K-F and 2058-K-R to obtain a PCR amplification product of about 1500bp, wherein the PCR amplification product is the kana mutation box with a homology arm.

5. Preparation of plague bacterium 201 strain competent cell containing KD46 plasmid

Replacing the plague bacteria 201 strain with the positive monoclonal (namely the plague bacteria 201 strain containing the KD46 plasmid) obtained in the step 3 according to the method of the step 2, and obtaining the plague bacteria 201 strain competent cell containing the KD46 plasmid by the other steps.

6. Electric conversion

(1) Taking the competent cell of the plague bacteria 201 strain containing the KD46 plasmid, adding 1-2 mug of PCR amplification product obtained in the step 4, fully mixing uniformly, carrying out ice bath for 10min, and then adding a precooled electric shock cup (the diameter is 1 mm).

(2) After the step 1 is finished, the electric shock cup is placed in an electrotransfer instrument for electric shock (the electric shock parameters are 25 muF, 200 omega and 2.5 KV).

(3) After the step (2) is finished, adding 1mL of LB liquid culture medium into the electric shock cup, uniformly mixing, sucking into an EP tube (specification is 1.5mL), and carrying out shake culture at 26 ℃ and 200rpm for 2 h; finally, 100 mul of bacterial liquid is evenly coated on an LB solid plate (Amp resistance and kana resistance), and inverted culture is carried out for 36h at 26 ℃, so as to obtain a plurality of monoclonals.

(4) Each single clone was plated to increase the number of cells (Amp-resistant and kana-resistant, LB plates), a small amount of cells were taken in sterile water, treated at 99 ℃ for 10min, centrifuged, and the supernatant was collected.

(5) Respectively taking the supernatant as a template, and carrying out PCR amplification by taking a primer pair A (consisting of 2058-I-F and Kana-I-R and used for carrying out upper bridging PCR identification), a primer pair B (consisting of Kana-I-F and 2058-I-R and used for carrying out lower bridging PCR identification) or a primer pair C (consisting of 2058-N-F and 2058-N-R and used for carrying out internal gene PCR identification) as primers. If a certain monoclonal antibody adopts a primer pair A to amplify to obtain a PCR amplification product containing about 500bp DNA fragments, adopts a primer pair B to amplify to obtain a PCR amplification product containing about 500bp DNA fragments, and adopts a primer pair C to amplify to obtain a PCR amplification product without bands, the monoclonal antibody is a positive monoclonal antibody.

The positive monoclonal glycerol was stored at-80 ℃.

7. pKD46 plasmid elimination

(1) And (4) respectively taking the positive monoclonals obtained in the step (6), inoculating the positive monoclonals in an LB liquid culture medium, and carrying out shaking culture at 37 ℃ and 200rpm for 48 hours to obtain bacterial liquid.

(2) After the completion of step (1), 100. mu.L of the bacterial solution was diluted 1000-fold with LB liquid medium and spread evenly on LB solid plates (two types of LB solid plates, one type of Kana-resistant and the other type of Amp-resistant), and cultured by inversion at 26 ℃ for 36 hours.

If a positive monoclonal shows a clone on LB solid plate (kana resistance) and no clone on LB solid plate (Amp resistance), the pKD46 plasmid of the positive monoclonal is eliminated. A single clone (i.e., P. murraya. delta. yp-2058) on an LB solid plate (kana-resistant) was picked, glycerol was used for preservation, and the mixture was stored at-80 ℃.

Second, construction of yp _2058 gene complementation strain (i.e. plague bacterium delta yp _2058 complementation strain)

1. Extraction of pBAD24 plasmid

(1) Escherichia coli DH 5. alpha. containing pBAD24 plasmid was inoculated into 5mL LB liquid medium, and cultured at 37 ℃ with shaking at 200rpm until logarithmic phase to obtain a culture broth.

(2) And (2) taking the culture solution obtained in the step (1), and extracting plasmids by using a plasmid miniprep kit to obtain a pBAD24 plasmid (about 4542 bp).

2. Construction of recombinant plasmid pBAD24-yp _2058

(1) And (3) performing PCR amplification by using genome DNA of the plague bacteria 201 strain as a template and adopting a primer pair consisting of 2058-F and 2058-R to obtain a PCR amplification product.

(2) Taking the PCR amplification product obtained in the step (1), and recovering a DNA fragment of about 600 bp.

(3) And (3) taking the DNA fragment recovered in the step (2), carrying out enzyme digestion by using restriction enzymes Nhe I and HindIII, and recovering the enzyme digestion fragment.

(4) The plasmid pBAD24 was digested with restriction enzymes Nhe I and HindIII, and the vector backbone of about 4Kb was recovered.

(5) And (4) connecting the enzyme digestion fragment recovered in the step (3) with the vector skeleton recovered in the step (4) to obtain a recombinant plasmid pBAD24-yp _ 2058.

The recombinant plasmid pBAD24-yp _2058 was sequenced. According to the sequencing result, the recombinant plasmid pBAD24-yp _2058 is described as follows: the small fragment between the recognition sequences for the restriction enzymes Nhe I and HindIII of the pBAD24 plasmid was replaced with SEQ ID NO: 1.

3. Preparation of plague bacillus delta yp _2058 competent cells

Replacing the plague bacteria 201 strain with the plague bacteria delta yp _2058 according to the method in the step one (2), and obtaining the plague bacteria delta yp _2058 competent cells without changing other steps.

4. Electric conversion

(1) Taking plague bacillus delta yp _2058 competent cells, adding 200 mu g of recombinant plasmid pBAD24-yp _2058, uniformly mixing, adding a precooled electric shock cup, carrying out ice bath for 10min, and then placing in an electrotransformation machine for electric shock (the electric shock parameters are 25 mu F, 200 omega and 2.5 KV).

(2) After the step (1) is finished, adding 1mL of LB liquid culture medium into the electric shock cup, uniformly mixing, sucking into an EP tube (specification is 1.5mL), and carrying out shake culture at 26 ℃ and 200rpm for 2 h; finally, 100 mul of bacterial liquid is evenly coated on an LB solid plate (Amp resistance and kana resistance), and inverted culture is carried out for 36h at 26 ℃, so as to obtain a plurality of monoclonals.

(4) Respectively taking the supernatants as templates and taking 2058-F and 2058-R as primers to carry out PCR amplification. If the PCR amplification product contains DNA fragment of about 600bp, the corresponding monoclonal is positive. The positive monoclonals (i.e. the. DELTA. yp-2058 retransflement strain) were kept in glycerol at-80 ℃.

And detecting the expression conditions of Yp _2058 protein of bacteria to be detected (the plague bacteria 201 strain, the plague bacteria delta Yp _2058 or the plague bacteria delta Yp _2058 anaplerotic strain) in the bacterial liquid supernatant and the bacterial liquid sediment by an immunoblotting method. The result shows that the Yp _2058 protein is expressed in both the bacterial liquid supernatant and the bacterial liquid sediment of the plague bacteria 201 strain, the Yp _2058 protein is not expressed in both the bacterial liquid supernatant and the bacterial liquid sediment of the plague bacteria delta Yp _2058 strain, and the Yp _2058 protein is expressed in both the bacterial liquid supernatant and the bacterial liquid sediment of the plague bacteria delta Yp _2058 anaplerotic strain and has higher expression level than that of the plague bacteria 201 strain.

Example 3 acid resistance test

Minimal media are described in the following documents: f sebbank, CO Jarrett, JR Linkenhoker, BJHINnebubisch. evaluation of the roll of consistent isocitrate lyase activity in Yersinia pestis infection of the flow vector and a consistent host. infection & Immunity, 2004, 72 (12): 7334-7.

The percentage of the decrease in the test bacteria was defined as the number of bacteria in the minimum medium (ph3.5) containing 20mM glucose/the number of bacteria in the minimum medium (ph6.5) containing 20mM glucose × 100%.

One, experiment one

(1) And (3) taking bacteria to be detected (the plague bacteria delta yp _2058 or the plague bacteria 201 strain), inoculating the bacteria to be detected in an LB liquid culture medium, and carrying out shake culture at 26 ℃ and 200rpm until logarithmic phase to obtain a bacteria liquid 1.

(2) The bacterial liquid 1 was inoculated into LB liquid medium (inoculation ratio 1:50), and cultured with shaking at 26 ℃ and 200rpm to obtain bacterial liquid 2 with OD620nm of 1.0.

(3) The bacterial liquid 2 was diluted 100-fold with a minimal medium (pH6.5) containing 20mM glucose or a minimal medium (pH3.5) containing 20mM glucose to obtain a bacterial liquid dilution.

(4) And (3) taking the bacterial liquid diluent, standing for 30min, then diluting in a multiple ratio, coating plates, counting, and calculating the percentage of the bacteria to be detected which are reduced when standing for 30 min.

Second, experiment two

(1) Taking a reseeding strain of the plague bacillus delta yp _2058, inoculating the reseeding strain in an LB liquid culture medium, and carrying out shaking culture at 26 ℃ and 200rpm until the log phase to obtain a bacterium liquid 1.

(2) The bacterial liquid 1 was inoculated into LB liquid medium (inoculation ratio 1:50), and cultured with shaking at 26 ℃ and 200rpm to obtain bacterial liquid 2 with OD620nm of 0.8.

(3) Adding arabinose into the bacterial liquid 2 to obtain an induction system; in the induction system, the concentration of arabinose was 0.2% (m/v).

(4) And taking the induction system, carrying out shaking culture at 26 ℃ and 200rpm for 2h to obtain a bacterial liquid 3.

(5) The bacterial liquid 3 was diluted 100-fold with a minimal medium (pH6.5) containing 20mM glucose or a minimal medium (pH3.5) containing 20mM glucose to obtain a bacterial liquid diluent.

(6) And (3) taking the diluted bacteria liquid, standing for 30min, then diluting in a multiple ratio, coating plates, counting, and calculating the percentage of the reduction of the phytophthora parasitica delta yp _2058 anaplerotic strains when standing for 30 min.

The experimental results are shown in figure 1(1 is the strain 201 of the plague bacteria, 2 is the strain delta yp _2058 of the plague bacteria, and 3 is the replete strain delta yp _2058 of the plague bacteria). The result shows that the acid resistance of the plague bacteria delta yp _2058 is obviously reduced compared with the plague bacteria 201 strain; the acid resistance of the reseeding strain delta yp _2058 of the plague bacillus can be recovered by 90 percent.

Example 4 growth curves

1. Taking a monoclonal of a reseeding strain of the plague bacillus delta yp _2058, inoculating the monoclonal in an LB liquid culture medium, and carrying out shaking culture at 26 ℃ and 200rpm to obtain a bacterial liquid 1 with an OD620nm value of 0.8.

2. Adding arabinose into the bacterial liquid 1 to obtain an induction system; in the induction system, the concentration of arabinose was 0.2% (m/v).

3. And taking the induction system, carrying out shaking culture at 26 ℃ and 200rpm for 2h to obtain a bacterial liquid 2.

4. The bacterial liquid 2 was inoculated into LB liquid medium (inoculation ratio 1:50), cultured with shaking at 26 ℃ and 200rpm, and the OD620nm values of the bacterial liquid were measured at different culture times. The cultivation time is used as the abscissa, and the corresponding bacterial liquid OD620nm value is used as the ordinate, and a growth curve is drawn.

Taking a monoclonal of a bacterium to be detected (the plague bacteria delta yp _2058 or the plague bacteria 201 strain), inoculating the monoclonal to an LB liquid culture medium, carrying out shaking culture at 26 ℃ and 200rpm, and measuring the OD620nm value of a bacterium solution at different culture time. The cultivation time is used as the abscissa, and the corresponding bacterial liquid OD620nm value is used as the ordinate, and a growth curve is drawn.

The results of the experiment are shown in FIG. 2. The result shows that compared with the plague bacteria 201 strain, the growth speed of the plague bacteria delta yp _2058 is obviously reduced, the time for reaching the platform stage is obviously delayed, and the growth state is obviously inhibited; after the yp _2058 gene is complemented back (namely the plague bacillus delta yp _2058 complemented back strain), the growth state is recovered to be normal.

Example 5 mouse virulence experiments

First, inguinal subcutaneous route

30 Bal b/c mice were randomly divided into three groups (10 mice each) of mutant, anaplerotic and control groups. The following treatments were carried out:

mutation group: in the experiment 1d, 100CFU plague bacteria delta yp _2058 are injected subcutaneously in the groin of each mouse; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

and (3) a anaplerosis group: in the experiment 1d, 100CFU plague bacteria delta yp _2058 anaplerotic strains are injected subcutaneously in the groin of each mouse; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

control group: experiment 1d, 100CFU plague bacteria strain 201 was injected subcutaneously in the groin of each mouse; then feeding the chicken normally for 14 d; during which time the survival of the mice was observed.

The results are shown in Table 2. The results showed that mice in the anaplerotic and control groups started to die at 5d and all died at 5-7 d; the mutant group began to die at 7d and died at all 7-12 d. Therefore, compared with the plague bacteria 201 strain, the plague bacteria delta yp _2058 have obviously reduced virulence; the strain of plague bacillus delta yp _2058 can completely complement virulence and eliminate polar mutation.

TABLE 2-1

Tables 2 to 2

Survival curves for each group of mice were plotted using GraphPad Prism software (website: https:// www.graphpad.com/scientific-software/Prism /), and the results are shown in FIG. 3.

The difference between the groups was analyzed for statistical significance using GraphPad Prism software's own chi-square test method Logrank and Gehan-Breslow-Wilcoxon. The statistical difference between the control group and the mutant group is shown in Table 3, and the statistical difference between the control group and the anaplerotic group is shown in Table 4. The result shows that the p value in the difference statistical result of the control group and the mutation group is less than 0.0001, and the difference is significant; the statistical difference between the control group and the anaplerotic group was 0.4930, with no significant difference.

TABLE 3

TABLE 4

Survival curve comparison
		Timing verification
Square card	0.4930
		Degree of freedom	1
P value	0.4826
		Summary of P values	Without significant difference
Whether the survival curves have significant difference	Whether or not
		Gehan-Breslow-Wilcoxon test
Square card	0.6420
		Degree of freedom	1
P value	0.4230
		Summary of P values	Without significant difference
Whether the survival curves have significant difference	Whether or not

The second, tail vein route

mutation group: experiment 1d, 100CFU plague bacteria delta yp _2058 were injected intravenously at the tail of each mouse; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

and (3) a anaplerosis group: in the experiment 1d, 100CFU of the rat plague delta yp _2058 anaplerotic strain is injected into the tail of each mouse intravenously; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

control group: experiment 1d, 100CFU of plague bacillus strain 201 was injected intravenously per mouse; then feeding the chicken normally for 14 d; during which time the survival of the mice was observed.

The results are shown in Table 5. The results showed that mice in the control group began to die at 3d and all 3-4d died; mice in the anaplerotic group began to die at 3d and all died 3-5 d; mice in the mutant group began to die at 5d and all died 5-8 d. Therefore, compared with the plague bacteria 201 strain, the plague bacteria delta yp _2058 have obviously reduced virulence; the strain of plague bacillus delta yp _2058 can completely complement virulence and eliminate polar mutation.

TABLE 5-1

TABLE 5-2

Survival curves for each group of mice were plotted using GraphPad Prism software, and the results are shown in fig. 4.

The difference between the groups was analyzed for statistical significance using GraphPad Prism software's own chi-square test method Logrank and Gehan-Breslow-Wilcoxon. The statistical difference between the control group and the mutant group is shown in Table 6, and the statistical difference between the control group and the anaplerotic group is shown in Table 7. The result shows that the p value in the difference statistical result of the control group and the mutation group is less than 0.0001, and the difference is significant; the difference between the control group and the anaplerotic group was 1.339, with no significant difference.

TABLE 6

Survival curve comparison
		Timing verification
Square card	21.19
		Degree of freedom	1
P value	＜0.0001
		Summary of P values	***
Whether the survival curves have significant difference	Is that
		Gehan-Breslow-Wilcoxon test
Square card	18.46
		Degree of freedom	1
P value	＜0.0001
		Summary of P values	***
Whether the survival curves have significant difference	Is that

TABLE 7

Third, nasal drip infection route

mutation group: in the experiment 1d, 100CFU of plague bacillus delta yp _2058 is injected into each mouse by nasal drip; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

and (3) a anaplerosis group: in the experiment 1d, 100CFU plague bacteria delta yp _2058 replenisher strains are injected into each mouse by nasal drip; then feeding the chicken normally for 14 d; during which the survival of the mice was observed;

control group: experiment 1d, 100CFU of plague bacterium 201 strain was injected into each mouse by nasal drip; then feeding the chicken normally for 14 d; during which time the survival of the mice was observed.

The results are shown in Table 8. The results showed that mice in the control group began to die at 3d and all 3-5d died; mice in the anaplerotic group began to die at 3d and all died 3-6 d; mice in the mutant group began to die at 5d and all died 5-12 d. Therefore, compared with the plague bacteria 201 strain, the plague bacteria delta yp _2058 have obviously reduced virulence; the strain of plague bacillus delta yp _2058 can completely complement virulence and eliminate polar mutation.

TABLE 8-1

TABLE 8-2

Survival curves for each group of mice were plotted using GraphPad Prism software, and the results are shown in fig. 5.

The difference between the groups was analyzed for statistical significance using GraphPad Prism software's own chi-square test method Logrank and Gehan-Breslow-Wilcoxon. The statistical difference between the control group and the mutant group is shown in Table 9, and the statistical difference between the control group and the anaplerotic group is shown in Table 10. The result shows that the p value in the difference statistical result of the control group and the mutation group is less than 0.0001, and the difference is significant; the difference between the control group and the anaplerotic group was 2.19 in p-value, and there was no significant difference.

TABLE 9

Watch 10

Survival curve comparison
		Timing verification
Square card	2.190
		Degree of freedom	1
P value	0.1389
		Summary of P values	Without significant difference
Whether the survival curves have significant difference	Whether or not
		Gehan-Breslow-Wilcoxon test
Square card	1.436
		Degree of freedom	1
P value	0.2308
		Summary of P values	Without significant difference
Whether the survival curves have significant difference	Whether or not

The above results indicate that the Yp _2058 protein has an important effect on the virulence of Yersinia pestis.

<110> military medical research institute of military science institute of people's liberation force of China

Application of <120> Yp _2058 protein in regulation of toxicity of Yersinia pestis

<160>2

<170>PatentIn version 3.5

<210>1

<211>600

<212>DNA

<213> Yersinia pestis (Yersinia pestis)

<400>1

atgatggaga taggtacttt ttcattatct aacgtagata ctgatcattt gattgctgac 60

ccaactggga ttaaatcgat taaaacggta tgcgatcttc atatcgtcga actggctcaa 120

gtcgataatg aaaaaaaact gcatatagca gaaatagata aaggtattag tgctgtactg 180

ccgggcattg attcgtgcat tgggataatt ataaaaacag acttagggaa aattatcgcc 240

tcccatgttg ggatatatga ttgcggtgtg gataaaggat ttgatgccaa caaatttgat 300

gagagcgttt acgcaaacat tgagtcagaa aacatgcgca gttcgatagc gcttattact 360

gatgatctaa aaaagatgat aggtaaaaat aaaatatctg aaatcatgat catcggacaa 420

gatgcgggta gtgaatggcc tcatcaacag ttaaatgatt tggcgaaaga actgggcttt 480

tctggcgagt taataatttg tgggcataaa gaaggggtaa atacaacatg ctctgtgatt 540

atagacagcg ctggggatat atctatattg aatcgagatg gaaagagaat agagatttaa 600

<210>2

<211>199

<212>PRT

<213> Yersinia pestis (Yersinia pestis)

<400>2

Met Met Glu Ile Gly Thr Phe Ser Leu Ser Asn Val Asp Thr Asp His

1 5 10 15

Leu Ile Ala Asp Pro Thr Gly Ile Lys Ser Ile Lys Thr Val Cys Asp

20 25 30

Leu His Ile Val Glu Leu Ala Gln Val Asp Asn Glu Lys Lys Leu His

35 40 45

Ile Ala Glu Ile Asp Lys Gly Ile Ser Ala Val Leu Pro Gly Ile Asp

50 55 60

Ser Cys Ile Gly Ile Ile Ile Lys Thr Asp Leu Gly Lys Ile Ile Ala

65 70 75 80

Ser His Val Gly Ile Tyr Asp Cys Gly Val Asp Lys Gly Phe Asp Ala

85 90 95

Asn Lys Phe Asp Glu Ser Val Tyr Ala Asn Ile Glu Ser Glu Asn Met

100 105 110

Arg Ser Ser Ile Ala Leu Ile Thr Asp Asp Leu Lys Lys Met Ile Gly

115 120 125

Lys Asn Lys Ile Ser Glu Ile Met Ile Ile Gly Gln Asp Ala Gly Ser

130 135 140

Glu Trp Pro His Gln Gln Leu Asn Asp Leu Ala Lys Glu Leu Gly Phe

145 150 155 160

Ser Gly Glu Leu Ile Ile Cys Gly His Lys Glu Gly Val Asn Thr Thr

165 170 175

Cys Ser Val Ile Ile Asp Ser Ala Gly Asp Ile Ser Ile Leu Asn Arg

180 185 190

Asp Gly Lys Arg Ile Glu Ile

195

Claims

The application of Yp _2058 protein in regulation and control of Yersinia pestis virulence;

the Yp _2058 protein is a1) or a2) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.
2. The use of claim 1, wherein: the Yersinia pestis is Yersinia pestis strain 201.
3. The use of a nucleic acid molecule encoding a Yp _2058 protein for modulating the virulence of yersinia pestis;

the Yp _2058 protein is a1) or a2) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.
4. Use according to claim 3, characterized in that: the nucleic acid molecule encoding the Yp _2058 protein is a DNA molecule shown as b1) or b 2):

b1) the coding region is SEQ ID NO: 1;

b2) the nucleotide sequence is SEQ ID NO: 1.
5. The use of claim 3 or 4, wherein: the Yersinia pestis is Yersinia pestis strain 201.
6. A recombinant Yersinia pestis is prepared by the following steps: inhibiting the expression quantity and/or activity of Yp _2058 protein in Yersinia pestis, and obtaining recombinant bacteria, namely Yersinia pestis; reduced virulence of recombinant yersinia pestis compared to yersinia pestis;

the Yp _2058 protein is a1) or a2) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.
7. The recombinant Yersinia pestis bacterium of claim 6, wherein: the inhibition of the expression amount and/or activity of the Yp _2058 protein in the Yersinia pestis is realized by introducing a substance for inhibiting the expression amount and/or activity of the Yp _2058 protein into the Yersinia pestis.
8. The recombinant Yersinia pestis bacterium of claim 6 or 7, wherein: the Yersinia pestis is Yersinia pestis strain 201.
9. A recombinant Yersinia pestis B is prepared by the following steps: improving the expression quantity and/or activity of Yp _2058 protein in the yersinia pestis, wherein the obtained recombinant bacterium is the recombinant yersinia pestis B; enhanced virulence of recombinant yersinia pestis compared to yersinia pestis;

the Yp _2058 protein is a1) or a2) as follows:

a1) the amino acid sequence is SEQ ID NO: 2;

a2) in SEQ ID NO: 2 and the N end or/and the C end of the protein shown in the figure is connected with a label to obtain the fusion protein.
10. The recombinant yersinia pestis strain b of claim 9, wherein: the Yersinia pestis is Yersinia pestis strain 201.
11. The use of the substance capable of inhibiting the expression and/or activity of Yp _2058 protein as claimed in claim 7 for regulating the virulence of Yersinia pestis.
12. The use of claim 11, wherein: the Yersinia pestis is Yersinia pestis strain 201.