A protective antigen of streptococcus suis and use thereof

Abstract

The application relates to a protective antigen of streptococcus suis disease and application thereof, and belongs to the technical field of biological vaccines. The amino acid sequence shown in SEQ ID No. 1 provided in the application is used as a protective antigen of streptococcus suis disease. The protective antigen is obtained by codon optimization of the nucleotide sequence shown in SEQ ID No. 2, construction of an expression vector and prokaryotic expression, and the protective antigen is verified to be capable of providing good immune cross-protection for type 2 and type 7 streptococcus suis infection in different ways. The application scheme has important significance for research on a general vaccine candidate antigen of multiple serotypes of streptococcus suis and scientific prevention and control of the disease.

Description

TECHNICAL FIELD

[0001] The application relates to a protective antigen of streptococcus suis and application thereof, and belongs to the technical field of biological vaccines. BACKGROUND

[0002] Streptococcus suis is an important zoonotic pathogenic bacteria, which can infect through respiratory tract, wound, etc., and pigs show symptoms of meningitis, myocarditis, arthritis and septicemia after the disease. In recent years, Streptococcus suis 2 and 7 are the two types of Streptococcus suis with higher prevalence and incidence in China, which have caused serious harm to the pig breeding industry. At present, the prevention and treatment of streptococcus suis disease in China still mainly relies on antibiotics, but the phenomenon of antibiotic abuse is very serious in recent years, which leads to the continuous emergence of drug-resistant strains, and the disease cannot be effectively prevented and treated, seriously endangering human and animal health. Vaccines play an important role in the prevention and control of streptococcus suis disease. Since there are many serotypes of streptococcus suis, there is currently no universal vaccine that can provide cross-protection against different serotypes of streptococcus suis pathogens, so it is of great significance to carry out research on the candidate antigens of the universal vaccine for multiple serotypes of streptococcus suis for the scientific prevention and control of the disease. SUMMARY

[0003] In order to solve the above problems, a protective antigen of streptococcus suis and application thereof are provided, the amino acid sequence shown in SEQ ID No. 1 in the application is used as a protective antigen of streptococcus suis, the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3 is amplified and expressed to obtain the protective antigen, which can provide good immune protection against type 2 and type 7 streptococcus suis infection, and is named as 5371 protein. The application scheme has great significance for carrying out research on the candidate antigens of the universal vaccine for multiple serotypes of streptococcus suis, and for the scientific prevention and control of the disease.

[0004] According to one aspect of the application, a protective antigen of streptococcus suis is provided, and the amino acid sequence of the protective antigen is shown in SEQ ID No. 1.

[0005] According to another aspect of the application, a gene sequence encoding a protective antigen of streptococcus suis is provided, and the nucleotide sequence is shown in SEQ ID No. 2 or SEQ ID No. 3.

[0006] According to another aspect of the application, a streptococcus suis vaccine is provided, and the antigen protein in the vaccine comprises the amino acid sequence shown in SEQ ID No. 1.

[0007] Optionally, the vaccine further comprises an adjuvant.

[0008] Optionally, the adjuvant is selected from aluminum phosphate, aluminum sulfate, aluminum hydroxide, sucrose ester, AEO-5 or mineral oil.

[0009] Optionally, the adjuvant is aluminum hydroxide.

[0010] It should be noted that the skilled in the art know that the addition of adjuvant can enhance the immune response of the vaccine and improve the immune effect, although the adjuvant used in the present technical solution is one of the aluminum adjuvants, i.e. aluminum hydroxide, and the skilled in the art know that other adjuvants commonly used in the art can also have the same effect.

[0011] According to another aspect of the present application, there is provided the use of any of the above-mentioned vaccines for providing immune protection against serotype 2, 7 Streptococcus suis infection.

[0012] Optionally, there is provided the use of any of the above-mentioned vaccines for providing immune protection against serotype 2 and / or 7 Streptococcus suis infection.

[0013] Optionally, the Streptococcus suis protective antigen protein can significantly increase the expression level of host animal antibodies.

[0014] Optionally, the Streptococcus suis protective antigen protein can significantly increase the expression level of cytokines IL-17A, and / or IL-10, and / or IFN-γ.

[0015] According to another aspect of the present application, there is provided a biological material, which is any of a) to c):

[0016] a) an expression cassette expressing the above protective antigen;

[0017] b) an expression vector comprising the expression cassette of a);

[0018] c) a host bacterium comprising the expression vector of b).

[0019] The beneficial effects of the present application include but are not limited to:

[0020] 1. The Streptococcus suis protective antigen according to the present application, when the antigen protein is mixed with aluminum adjuvant to immunize New Zealand white rabbits, the antibody level shows a significant upward trend after the second immunization, indicating that the antigen protein can induce the body to produce good humoral immune effect.

[0021] 2. The Streptococcus suis protective antigen according to the present application, the whole blood bactericidal effect on Streptococcus suis 2, 7 is obvious, which is consistent with the results of the New Zealand white rabbit challenge protection effect.

[0022] 3. The protective antigen of Streptococcus suis disease according to the present application, the stimulation of the antigen protein has obvious effect on IL-17A, indicating that the protein can stimulate the body to produce immune response to Streptococcus suis infection, and the stimulation of the antigen protein can obviously increase the levels of IFN-γ and IL-10, so that the pro-inflammatory factors and anti-inflammatory factors are balanced, and the two complement each other in the immune response system.

[0023] 4. The protective antigen of Streptococcus suis disease according to the present application, in the cross-protection evaluation test of the protein, the incidence and death of the rabbits in the 5371 protein immune group are reduced, the final protection rate against Streptococcus suis type 2 can reach 50%, and the protection rate against Streptococcus suis type 7 can reach 70%. BRIEF DESCRIPTION OF DRAWINGS

[0024] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings:

[0025] Figure 1 The figure for the results of the cytokine content involved in Test Example 4 of the present application Figure 1 Each subgraph in the figure is on the left side for PBS control and on the right side for 5371 antigen.

[0026] Figure 2 The figure for the results of the Streptococcus suis type 2 and type 7 challenge protection test involved in Test Example 5 of the present application. DETAILED DESCRIPTION

[0027] The present application will be described in detail below in combination with examples, but the present application is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present application are all purchased through commercial channels.

[0028] Example 1

[0029] According to the description of Streptococcus suis protein in the Protein database, the protein sequence is downloaded from the NCBI database, the transmembrane condition of the protein is screened through TMHMM, the protein vaccine characteristics are predicted through VAXIGN, and the proteins with adhesion parameters above 0.5 are reserved; then the signal peptide is predicted, and the possible signal peptide sequence is deleted, the proteins of different serotypes are compared through BLAST, and the protein sequences commonly or partially commonly shared by common pathogenic serotypes are reserved. This process is a reverse vaccine analysis process, which is mainly carried out by using computers through online and offline databases.

[0030] The final screening obtained the antigen protein with the amino acid sequence shown in SEQ ID No. 1, and the number 5371 protein, wherein the nucleotide sequence of the Streptococcus suis in the NCBI database is shown in SEQ ID No. 2, and the subsequent step uses E. coli for expression, and the optimized nucleotide sequence of the expression plasmid after optimization is shown in SEQ ID No. 3. It should be noted that those skilled in the art can perform various optimizations based on the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3 according to conventional means to obtain the 5371 antigen protein expressing the amino acid sequence shown in SEQ ID No. 1. Those skilled in the art know that the nucleotide sequence is different, which does not affect the antigen protein with the amino acid sequence shown in SEQ ID No. 1 to play the same function and effect as in the scheme of the present application.

[0031] Example 2 Antigen protein expression and purification

[0032] 1) The recombinant plasmid containing the nucleotide sequence shown in SEQ ID No. 3 is synthesized by General Biotech (Anhui) Co., Ltd. for subsequent tests. It should be noted that those skilled in the art can also use general means to insert the sequence into the same plasmid or other expression vectors for expression of the 5371 protein.

[0033] 2) Recombinant plasmid into expression bacteria: The recombinant plasmid is transformed into the genetically engineered expression strain BL21 (DE3) by heat shock.

[0034] 3) Protein expression: LB medium containing kanamycin, 37°C, 180rpm, growth to OD value of 0.6, addition of IPTG with final concentration of 1mmol / L, induction for 5h, centrifugation, and collection of bacterial cells.

[0035] 4) SDS-PAGE electrophoresis verification of expressed protein: 10-fold concentrated collected bacterial cells, ultrasonic crushing, and taking 30μL of supernatant and precipitate respectively for SDS-PAGE electrophoresis.

[0036] 5) Purification of expressed protein: The target protein mainly exists in the form of inclusion body, which is dissolved with 8mol / L urea and then purified on a HIS tag purification column.

[0037] 6) BCA method for determining the concentration of purified protein: BCA method is used to determine the concentration of the purified protein, and the concentration of the 5371 protein is 918.26μg / mL.

[0038] Example 3 New Zealand white rabbit immunization

[0039] Twenty 1.5 kg New Zealand white rabbits were randomly divided into two groups, 10 in each group, 5371 group and control group, then the purified test protein was mixed with aluminum adjuvant, and each rabbit was immunized with 100 μg of protein. The control group was not immunized. Blood was collected at 0, 7 days, and serum was prepared for use.

[0040] Test Example 1 ELISA detection of antibody titer

[0041] 1) Coating: The purified test protein was diluted with carbonic acid coating solution (CBS) to a concentration of 10 μg / mL per well as coating antigen and added to the enzyme-labeled plate, 100 μL / well, coated at 4°C overnight; 2) Washing: The coating solution was poured out the next day, washed with PBST 5 times, 3 min each time, emptied, and dried with filter paper. 3) Blocking: Add 5% skim milk powder to block, 250 μL / well, block at 37°C for 2 h. 4) Washing: Wash with PBST 5 times, 3 min each time, empty, and dry with filter paper. 5) Add the antibody to be tested: Add 1:400 diluted three test sera as primary antibody, 100 μL / well, incubate at 37°C for 1 h. 6) Washing: Wash with PBST 5 times, 3 min each time, empty, and dry with filter paper. 7) Add enzyme-labeled antibody: Add 1:2000 diluted HRP-staphylococcal A protein as enzyme-labeled antibody, 100 μL / well, incubate at 37°C for 1 h. 8) Washing: Wash with PBST 5 times, 3 min each time, empty, and dry with filter paper. 9) Color development: Add TMB color developing solution, 100 μL / well, develop color at room temperature for 15 min. 10) Stop: Stop the reaction by adding 2M H2SO4 stop solution, 50 μL / well. 11) Measure OD value: Read the absorbance value at 450 nm wavelength on the enzyme-labeled instrument.

[0042] The 5371 protein was subcutaneously injected into New Zealand white rabbits, and the serum of 4 New Zealand white rabbits was randomly collected after immunization. The antibody level change in the immune serum was evaluated by ELISA method, as shown in Table 1.

[0043] Table 1 Change of antibody titer after immunization with antigen protein

[0044] Animal number 7 days after immunization 14 days after immunization 21 days after immunization 1 0.3265 1.5972 1.6213 2 0.2548 1.3564 1.3546 3 0.2168 1.3264 1.4568 4 0.1689 0.9531 1.4862

[0045] The results show that after one-time booster immunization (second immunization), the antibody titer shows a significant increase (P<0.001).

[0046] Test Example 2 Whole blood bactericidal test

[0047] Streptococcus suis types 2, 7, and 9 were cultured in TSB medium (with 4% horse serum) to OD 630 0.6, then diluted to 10 -52) 50 μL 5371 positive serum and negative serum (1:4 dilution) were heat inactivated and mixed with equal amount of bacteria solution, incubated at 25°C for 20 min. 3) 400 μL healthy rabbit blood was added to the mixed bacteria solution, incubated at 37°C, 30 rpm for 3 h. 4) The mixed bacteria solution after incubation was diluted, 100 μL was added to blood plate (containing 4% horse serum), grown at 37°C overnight, and the colonies were counted. The antiserum of New Zealand white rabbits was reacted with Streptococcus suis type 2, 7 and 9 in fresh healthy rabbit blood for 3 h, and the colony growth results are shown in Table 2.

[0048] Table 2 Colony count results of whole blood bactericidal test

[0049]

[0050] It was found by calculating the whole blood bactericidal rate that the bactericidal effect of whole blood mediated by antiserum on Streptococcus suis type 2 was obvious, which could reach 37.2%; the bactericidal effect on Streptococcus suis type 7 was obvious, which could reach a maximum of 86.9%; the bactericidal effect of whole blood mediated by 5371 antiserum on Streptococcus suis type 9 was invalid.

[0051] Test Example 3 Cell Adhesion Inhibition Test

[0052] 1) PK15, Vero cells were plated in two 24-well plates at 2 x 10 6 cells per well, and the growth density of the cells was observed after 24 h, until the bottom of the plate was covered with a single layer of cells. 2) When Streptococcus suis strains grew to an OD 630 of about 0.5, the bacteria were collected and diluted 10 times with DMEM medium, and the number of bacteria in 40 μL Streptococcus suis solution was 2 x 10 5 cells. 3) 40 μL Streptococcus suis dilution solution was mixed with 10 μL 5371, negative rabbit serum (1:250 dilution of PBS), and 2.5 mL of DMEM medium was added, and incubated at 4°C for 1 h. 4) 400 μL of the mixture was added to the 24-well plate at an infection ratio of 1:10 (Streptococcus suis type 2 / PK15 cells or Vero cells), and incubated at 37°C in a 5% CO2 incubator for 2 h. The infected monolayer cells were washed 3 times with PBS to remove non-adherent Streptococcus suis type 2. 5) Then 0.2 mL of 0.25% trypsin-EDTA was added to the first two rows of one 24-well plate, and incubated at 37°C in a 5% CO2 incubator until the monolayer cells were completely detached. Then 0.8 mL of ice-cold 0.025% Triton X-100 was added to destroy the monolayer, and the mixture was aspirated and diluted to 10 -2The monolayer cells in the last two rows of the 24-well plate were treated with 20 μg / mL of ampicillin (with complete DMEM medium) for 10 min to kill the adhered S. suis cells, and then the monolayer cells were washed with PBS for 5 times, followed by adding 0.2 mL of 0.25% trypsin-EDTA, and incubating in a 37°C 5% CO2 incubator until the monolayer cells were completely detached, and then adding 0.8 mL of ice 0.025% Triton X-100 to destroy the monolayer, and aspirating the mixture, and taking 100 μL to be coated on a 5% sheep blood agar plate, and incubating at 37°C overnight, and counting the CFU of S. suis, which is the CFU of invasion. The adhered S. suis CFU = total CFU (adhesion and invasion) - invasion CFU. Each test was repeated 3 times, and the number of CFU on each plate was determined. The adhesion inhibition rate = [1 - CFU of positive serum / CFU of negative serum] x 100%. The experimental results are shown in Tables 3, 4 and 5.

[0053] Table 3 Results of S. suis 2 cell adhesion inhibition test

[0054]

[0055] Table 4 Results of S. suis 7 cell adhesion inhibition test

[0056]

[0057] Table 5 Results of S. suis 9 cell adhesion inhibition test

[0058]

[0059] From the results of the cell adhesion inhibition test, it can be concluded that the 5371 antiserum has different degrees of adhesion inhibition effect on S. suis types 2, 7 and 9, and the adhesion inhibition rate on S. suis type 2 is 60.1%, the adhesion inhibition rate on S. suis type 7 is 92.5%, and the adhesion inhibition rate on S. suis type 9 is 64.3%.

[0060] Test Example 4 Determination of cytokines

[0061] Ten six-month-old BALB / c mice were grouped, 5 in each group, into two groups, 5371 group and control group. The mice were immunized with the protein mixed with aluminum adjuvant, 100 μg of protein per mouse, once every seven days, for a total of three times. Mouse spleen primary cells were prepared and stimulated with 10 μg of PBS containing protein antigen or PBS alone, with the stimulation of beans protein A as a positive control. After 72 h of culture, the supernatant was collected and stored at -80°C. The content of cytokines after 72 h of antigen restimulation of spleen cells was detected by ELISA method, and the results are shown in Figure 1 Compared with the PBS control group, the stimulation of 5371 protein showed a significant increase in the level of cytokine IL-17A (P<0.01; P<0.001); the effect on cytokines IL-2 and IL-4 was not obvious; the stimulation of 5371 protein increased the level of cytokine IFN-γ by about 2-4 times (P<0.01; P<0.001), and increased the level of cytokine IL-10 by 2.5-4 times (P<0.001).

[0062] Test Example 5 Immunization of New Zealand White Rabbits and Protection Against Challenge

[0063] Thirty New Zealand white rabbits were randomly divided into three groups, 10 in each group, namely 5371 group type 2 challenge group, 5371 group type 7 challenge group, and control group. Then the tested protein after expression and purification was mixed with aluminum adjuvant, and the rabbits were immunized with 100 μg of protein per rabbit. The control group was not immunized. The immunization was boosted every seven days. After the end of immunization, the rabbits were challenged with the minimum lethal dose (2 type: 5 x 10 3 CFU / mL and 7 type: 5 x 10 7 CFU / mL) in the marginal ear vein after one week. The diet, mental state, and morbidity and mortality of the rabbits in each group were recorded within one week. The protective effect of the protein was evaluated according to the mortality and morbidity. The protection rate was calculated as follows: (number of survivors / total number) x 100%.

[0064] The 5371 protein immunized New Zealand white rabbits were challenged with Streptococcus suis type 2 and type 7, and the results are shown in Figure 2 Compared with the blank control group, the tested protein had a protective effect on Streptococcus suis type 2 and type 7, with a protection rate of 50% and 70% for Streptococcus suis type 2 and type 7, respectively.

[0065] The above is only an embodiment of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A Streptococcus suis disease protective antigen, characterized in that, The nucleotide sequence of the protective antigen is shown in SEQ ID No. 2 or SEQ ID No.

3.

2. A Streptococcus suis vaccine, characterized in that, The nucleotide sequence of the antigen protein in the vaccine is shown in SEQ ID No. 2 or SEQ ID No.

3.

3. The vaccine of claim 2, characterized in that, The vaccine further comprises an adjuvant.

4. The vaccine of claim 3, characterized in that, The adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate, aluminum hydroxide, sucrose ester, AEO-5 or mineral oil.

5. The vaccine of claim 4, characterized in that, The adjuvant is aluminum hydroxide.

6. Biomaterials characterized in that, The biological material is any one of a) ~ c): a) an expression cassette comprising the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3; b) an expression vector comprising the expression cassette of a); c) an engineered bacteria comprising the expression vector of b).

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