CN104306964B - The subunit vaccine for recombinating haemophilus parasuis immune protective antigen HbpA2 purposes and being prepared with it - Google Patents

Abstract

The invention discloses the use of the recombinant HbpA2 protein with the amino acid sequence shown in SEQ ID NO: 2 in the preparation of medicines for preventing Haemophilus parasuis disease, and the recombinant HbpA2 protein with the amino acid sequence shown in SEQ ID NO: 2 and Subunit vaccine prepared from pharmaceutically acceptable auxiliary material or carrier and use thereof. The recombinant HbpA2 protein of the invention has good immunogenicity and protective properties, the level of HbpA2 specific antibody produced after immunization is high, the protection rate of the prepared subunit vaccine is high, and the clinical application prospect is good.

Description

Use and preparation of recombinant Haemophilus parasuis immunoprotective antigen HbpA2 subunit vaccine

technical field

The invention relates to the field of genetic engineering, in particular to the use of a recombinant Haemophilus parasuis immunoprotective antigen HbpA2 and a subunit vaccine prepared therefrom.

Background technique

Haemophilus parasuis (Hps) belongs to the genus Haemophilus (Pasteurellaceae) and is a small gram-negative bacillus with various forms. It colonizes the upper respiratory tract of healthy pigs and belongs to the normal flora , Under certain conditions such as immunosuppression, it will cause Glasser's dis-ease, which is mainly characterized by fibrinous polyserositis, meningitis, and arthritis. In recent years, due to the rapid development of large-scale breeding in pig farms , especially in the intensive farming of factories, the swine gerbil disease has become popular and broke out in the world, showing sudden mortality, high morbidity and fatality rate, seriously endangering the development of pig industry, and causing huge economic losses.

In recent years, my country has gradually begun to pay attention to the research on Haemophilus parasuis disease. There are many serotypes of Haemophilus parasuis, fifteen serotypes have been identified, and 15%-41% unidentified serotypes, of which 1, 5, 10, 12, 13 and 14 are strong strains, while The prevalent serotypes in my country are 4 and 5. The virulence of different serotypes of Haemophilus parasuis varies greatly. We know little about its virulence factors and pathogenic mechanism. So far, there is no effective general commercial vaccine and sensitive diagnostic method. The disease has not been effectively controlled. Bring huge economic losses to the global pig industry.

There is no mature vaccine in China to target Haemophilus parasuis disease like the attenuated vaccine of classical swine fever rabbitization and the gene deletion vaccine of pseudorabies. At present, most of them are traditional inactivated vaccines and attenuated vaccines, which lack good protection and lead to clinical Cases are common. The inactivated vaccine can only protect the strains of the same serotype and lacks cross-protection. The diversity of serotypes of Haemophilus parasuis and a large proportion of strains that cannot be typed affect the development of vaccines with high cross-protection ability. Subunit vaccines have the advantages of high immune efficacy of live vaccines, strong cross-protection and good safety of inactivated vaccines, and have very broad application prospects. Most of the untypable strains of Haemophilus parasuis and their serotype diversity have affected the development of vaccines with high cross-protection.

Heme-binding protein (HbpA) is a globulin widely present in pathogenic bacteria. Studies have found that there are one or more heme-binding regions in the HbpA protein molecule, which have physiological functions such as binding, transporting and transmitting heme, and participating in body metabolism. Sex is inseparable. Studies on Haemophilus influenzae of the Pasteurella family Haemophilus genus have shown that HbpA protein is an important virulence factor of Haemophilus influenzae, and HbpA protein is related to the utilization of heme. At present, there are few research reports on the function of HbpA protein of Haemophilus parasuis. Two HbpA genes were found in Haemophilus parasuis SH0165 that has undergone genome sequencing, and the size of both genes is 1595bp. The patent application with the publication number CN103288934A discloses a method for recombinantly expressing the HbpA gene (Gene ID: 7276898) by means of genetic engineering. Poor protection, its immune protection is only 50%.

Contents of the invention

In order to solve the above problems, the present invention provides a new subunit vaccine prepared from recombinant HbpA eggs.

The present invention firstly provides the use of the recombinant HbpA2 protein whose amino acid sequence is shown in SEQ ID NO: 2 in the preparation of a medicament for preventing Haemophilus parasuis disease.

Preferably, the medicament contains recombinant HbpA2 protein in an immunogenic effective amount.

The subunit vaccine of the present invention is prepared from the recombinant HbpA2 protein whose amino acid sequence is shown in SEQ ID NO: 2 and pharmaceutically acceptable auxiliary materials or carriers.

Preferably, the vaccine comprises recombinant HbpA2 protein whose amino acid sequence is shown in SEQ ID NO: 2, and pharmaceutically acceptable adjuvant or carrier in an immunogenic effective amount.

Preferably, the effective immunogenic dose is not less than 0.1 μg/ml.

Preferably, the adjuvant or carrier is an immune adjuvant and/or a preservative.

The adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant.

The preservative is thimerosal.

The present invention also provides the application of the aforementioned vaccine in the preparation of medicine for Haemophilus parasuis disease. Wherein, the medicament for preventing Haemophilus parasuis disease is administered by injection.

The recombinant protein HbpA2 obtained by recombinant expression in the method of genetic engineering in the present invention has good immunogenicity and protective properties, and the HbpA2 specific antibody level produced after immunization is high, and after being made into a vaccine, it can significantly protect mice against parasigophilia The protection rate of Haemophilus serotype 5 virulent strains is as high as 70%, which can effectively prevent Haemophilus parasuis disease, and has a good clinical application prospect.

The above-mentioned content of the present invention will be further described in detail through specific implementation in the form of examples below. However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 is a diagram of PCR amplification results of HbpA2 gene. M: DNA maker; 1, 2, 3: PCR amplification products;

Figure 2 Induction time optimization. M: protein maker; 1: pET-39b(BL21); 2-9: induction time: 0, 1h, 2h, 3h, 4h, 5h, 6h, 7h;

Figure 3 IPTG induction concentration optimization. M: protein maker; 1: pET-39b(BL21); 2~9: IPTG: final concentration: 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol /L, 1.2mmol/L, 1.5mmol/L;

Figure 4 Induction temperature optimization. M: protein maker; 1, 3, 5, 7: before pET-39b-HbpA2(BL21) induction; 2: induction temperature is 25°C; 4: induction temperature is 30°C; 6: induction temperature is 37°C; 7: The induction temperature is 42°C;

Fig. 5 Detection of HbpA2 protein expressed in pET-39b-HbpA2 (BL21). M: protein maker; 1: pET-39b(BL21); 2: pET-39b-HbpA2(BL21) after sonication; 3: supernatant; 4: precipitation;

Figure 6 SDS-PAGE of recombinant HbpA2 expression. M: protein maker; 1: before pET-39b(BL21) induction; 2: after pET-39b(BL21) induction; 3: before pET-39b-HbpA2(BL21) induction; 4: pET-39b-HbpA2(BL21) After induction; 5: After purification of HbpA2 protein (100mmol/L imidazole elution);

Figure 7 Western blotting of recombinant protein;

Figure 8 The relationship between the survival rate of mice and time.

detailed description

Experimental materials and reagents:

Haemophilus parasuis CVCC3361 was obtained from the National Veterinary Microbiological Culture Collection Center, strain number: CVCC3361. Escherichia coli DH5a, BL21 (DE3) competent cells were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Restriction enzymes, Ex-Taq DNA polymerase, DNA ligase, DNA gel recovery kit, plasmid extraction kit, DNAMarker, pMD19-T simple vector, etc. were purchased from Treasure Bio (Dalian) Engineering Co., Ltd. Bacterial Genome Extraction Kit is a product of Omega Company. The pre-stained protein Ladder is a product of NEB Company. Mice were purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

The formula of LB medium is as follows: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L, adjust the pH of the medium with NaOH to reach 7.2 -7.4, high pressure steam sterilization for 30min.

Sample buffer formulation: 50mM NaH2PO4, 300mM NaCl, 5mM imidazole, pH8.0.

100mM imidazole eluent: 50mM NaH2PO4, 300mM NaCl, 100mM imidazole, pH 8.0.

Embodiment 1 Preparation of Haemophilus parasuis immunoprotective antigen HbpA2 of the present invention

1. Recombinant expression

1.1 Cloning of HbpA2 gene and construction of expression vector

1.1.1 Primer design and synthesis

According to the HbpA2 gene sequence of Haemophilus parasuis (Gene ID: 7278927), a pair of primers were designed with biological software PremierPrimer 5.0 to amplify the HbpA2 gene.

Upstream primer: 5'-AGTACTCCGACAAATACATTGGTCAACTGT-3';

Downstream primer: 5'-CTCGAGTTAAGGCTTCAGACTTACGCCATA-3';

1.1.2 PCR amplification of HbpA2 gene

Using the genomic DNA of Haemophilus parasuis CVCC3361 as a template, PCR amplification was carried out according to the conventional method, the amplification system was 25 μL, and 4 replicates were performed. Amplification system:

Material volume genome template 1μL Primer (upstream, downstream) 2μL EX-Taq MasterMix 12.5μL ddH2O 9.5μL total capacity 25 μL

Cycle parameters: pre-denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 100s, 30 cycles; extension at 72°C for 5min, storage at 4°C. The PCR products were detected by 1% agarose gel electrophoresis, and the PCR fragments after electrophoresis were gel-recovered and purified with a DNA gel recovery kit (operated according to the instructions of the Omega gel recovery kit), and the purified PCR fragments were sequenced.

Nucleotide sequence (SEQ ID NO:1) of the purified target gene fragment:

It is also possible to directly synthesize the target gene fragment by means of synthesis.

1.1.3 Construction of expression vector

Cloning the target and gene fragments (recovered products from PCR) into the T vector to obtain pMD19-T-HbpA2, T vector cloning connection system:

Material Reagent volume PCR recovery product 3μL pMD19-T vector 1μL T4DNA Ligase 0.5μL 10×Buffer 1μL ATP 1μL ddH ₂ O 3.5 μL total capacity 10 μL

Ligate overnight at 16°C, quickly identify the ligated products by PCR, and transform the ligated products into competent cells Escherichia coli DH5a using the CaCl ₂ method. Dissolve 50 μL on ice, add 10 μL of pMD19-T-HbpA2 plasmid and mix well, ice-bath at -20°C for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and quickly ice-bath at -20°C for 5 minutes. (2) Add 600 μL of preheated LB medium to the sample treated above, and incubate at 37° C. at 180 rpm for 1.5 h. (3) Take out 100 μL of the bacterial liquid and spread it on the LB solid medium containing Amp resistance, and cultivate it in a 37°C incubator for 12-24h. Positive transformants were screened, suspected colonies were picked for PCR, recombinants containing pMD19-T-HbpA2 were identified and expanded for culture, and plasmids were extracted for sequencing identification.

The correctly sequenced pMD19-T-HbpA2 was directional cloned into the prokaryotic expression vector pET-39b with XhoI and ScaI to construct the recombinant expression vector pET-39b-HbpA2. The plasmid and the pET-39b plasmid were subjected to double enzyme digestion, and digested at 37°C for 3h. (2) After performing 1% agarose gel electrophoresis on the digested product, use a DNA gel recovery kit to recover and purify the target fragment after electrophoresis (operate according to the instructions of the Omega gel recovery kit), and store it at -20°C for future use. (3) Ligate and transform the digested target fragments to obtain the recombinant expression vector pET-39b-HbpA2 (the method of ligation and transformation is the same as T vector cloning), and carry out PCR identification, enzyme digestion identification, and sequencing identification.

1.1.4 Construction of recombinant bacteria

Finally, the constructed recombinant expression vector pET-39b-HbpA2 was transformed into the expression host strain BL21(DE3) to obtain the recombinant strain.

1.2 Induced expression of recombinant Escherichia coli

1.2.1 Initial expression and identification of recombinant protein

Inoculate Escherichia coli BL21(DE3) containing the recombinant plasmid pET-39b-HbpA2 into LB medium containing 50 μg/ml Kan, culture with shaking at 37°C for 3-4 hours, and add IPTG until the OD value is 0.5-0.6. Concentration 1mmoL/mL, continue shaking culture for 3~4h, centrifuge to remove supernatant, add SDS-PAGE loading buffer to the precipitate, boil for 5min, and carry out SDS-PAGE. The gel was stained with Coomassie Brilliant Blue and then decolorized, and analyzed by using a gel imaging system.

1.2.2 Optimization of recombinant protein expression conditions

1.2.2.1 Optimization of induction time

The recombinant bacteria were inoculated in LB liquid medium test tubes containing Kan at a ratio of 1:100. When the OD value was 0.5-0.6, IPTG was added to a final concentration of 1mmoL/mL, and the expression was induced at 0h, 1h, and 2h respectively. , After 3h, 4h, 5h, 6h, 7h, take 1ml of bacterial liquid, process the sample, and perform SDS-PAGE electrophoresis.

1.2.2.2 Optimization of IPTG concentration

Recombinant bacteria were inoculated in LB liquid medium test tubes containing 50 μg/ml Kan (kalamycin) n at a ratio of 1:100, cultured with shaking at 37°C until the OD value was 0.5-0.6, and then IPTG was added to a final concentration of : 0mmol/L, 0.2mmol/L, 0.4mmol/L, 0.6mmol/L, 0.8mmol/L, 1.0mmol/L, 1.2mmol/L, 1.5mmol/L, the best induction obtained from the above experiments Time, 1 mL of the bacterial solution was taken for each sample, and the samples were post-processed and subjected to SDS-PAGE electrophoresis.

1.2.2.3 Optimization of induction temperature

The recombinant bacteria were inoculated in LB liquid medium containing 50 μg/ml Kan at a ratio of 1:100, and when the OD value was 0.5 to 0.6, IPTG was added to the final concentration of the optimal IPTG concentration obtained from the above test, respectively. Induce culture at 25°C, 30°C, 37°C, and 42°C for the best induction time obtained from the above experiments, take 1 mL of each bacterial solution, post-process the samples, and perform SDS-PAGE electrophoresis.

1.2.3 Mass expression of recombinant protein

The recombinant bacteria were inoculated in LB liquid medium containing 50 μg/ml Kan at a ratio of 1:100, cultured until the OD value was 0.5-0.6, and IPTG was added to the bacterial solution until the final concentration was the optimal concentration for inducing expression, and then Grow at the optimum temperature for the optimum time.

1.3 Separation and purification

(1) Centrifuge the induced bacterial solution at 12,000 g/min for 5 minutes to collect the bacterial cells, add 80 mL of bacterial cell lysate containing 1 mg/mL lysozyme, and suspend the precipitate. Placed at 4°C for overnight reaction.

(2) Freeze and thaw the suspension three times between -20°C and room temperature.

(3) After melting, the suspension was placed on ice and crushed for about 10 minutes using an ultrasonic cell disruptor at 40% intensity. Centrifuge at 12000g/min for 10min, collect the supernatant, adjust the pH to 8.0 with 1mmol/L NaOH, and filter the sample with a 0.45μm filter membrane for subsequent purification.

(4) Pack the Ni ion packing into the column overnight, and wait for the packing to be compacted for later use, that is, the nickel ion chelating affinity chromatography packing chromatography column.

(5) Elute the ethanol with ultrapure water at a flow rate of 2ml/min, and equilibrate to the chromatography column. Equilibrate the chromatographic column with loading buffer at a flow rate of 1 ml/min for about 30 min. Then load the sample at a flow rate of 0.5ml/min. After the sample is loaded, equilibrate the chromatographic column with loading buffer to equilibrium. Use the elution buffer containing 30mmol/L, 50mmol/L, 80mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L imidazole in order to elute the protein at a flow rate of 1ml/min. The eluate was collected from the rising peak to the end of the falling peak, and after equilibration, the elution buffer was changed to the next concentration of imidazole to continue elution. The eluate that was eluted and purified with an elution buffer containing 100 mmol/L imidazole was collected, that is, a solution containing the recombinant protein HbpA2 of the present invention, stored at -80°C for future use, and analyzed by SDS-PAGE for the purity of the collected protein.

2. Determination

2.1Western blotting

After the purified HbpA2 protein was subjected to SDS-PAGE electrophoresis on a 12% gel, Western blotting was performed. The specific steps are as follows:

(1) Properly cut the gel block after SDS-PAGE electrophoresis, and cut six pieces of filter paper and one piece of NC membrane at the same time as the size of the gel; soak it in the transfer buffer and place it for 1 hour;

(2) Open the transfer box, stack the filter paper, gel and NC membrane soaked in the transfer buffer on the negative electrode of the transfer box according to "filter paper--gel--NC membrane--filter paper", and use a clean Roll the small test tube gently to eliminate air bubbles, close the transfer box and insert into the transfer cell;

(3) Turn on the power supply, transfer 25V for 30min;

(4) After the transfer, take out the NC membrane, add an appropriate amount of blocking buffer to block for about 1 hour; pour off the blocking solution, and wash the membrane 3 times with TBST, 10 min each time;

Put the NC membrane into a sealed small plastic bag, add the rabbit anti-CVCC3361 primary antibody diluted 1:1000, react at 37°C for 1 hour, discard the primary antibody, wash the membrane 3 times with TBST, 10min each time;

(5) Put the NC membrane into a sealed small plastic bag, add 1:1000 diluted goat anti-rabbit IgG secondary antibody, and react at 37°C for 1 hour; wash the membrane 3 times with TBST, 10 minutes each time;

(6) Add an appropriate amount of color developing solution and develop color for 20-30 minutes. After the target band is clearly visible, wash the membrane with deionized water to stop the color development. Absorb the water on the membrane with absorbent paper and dry it in the dark.

(7) The gel image processing system takes pictures and analyzes them.

3. Measurement results

As shown in Figure 1, using the genomic DNA of strain CVCC3361 as a template, a band with the same size as the expected fragment (159bp) appeared after PCR amplification (Figure 1). Sequencing results showed that the homology of the amplified fragment with the HbpA2 gene sequence published on Gen-Bank was 99.9%. The constructed recombinant expression plasmid pET-39b-HbpA2 was digested with XhoI and ScaI to reveal 2 bands, the size of which was consistent with the expected band size. It shows that the HbpA2 gene has been successfully inserted into the pET39b vector.

As shown in Figures 2-4, when inducing expression, the optimal concentration of IPTG is 1.5mmol/L, the optimal induction temperature is 30°C, and the optimal induction time is 5h.

As shown in Figures 5 to 6, after the expression of the Escherichia coli containing the recombinant plasmid pET-39b-HbpA2 of the present invention was induced, an obviously thickened band appeared at 70kd in SDS-PAGE analysis, and its size was the same as the theoretical molecular weight of the expected fusion protein. The same size indicates that the recombinant protein HbpA2 is obtained, and after separation and purification according to the method of the present invention, a pure protein is obtained.

The amino acid sequence (SEQ ID NO:2) of recombinant protein-HbpA2 is:

As shown in Figure 7, after the recombinant protein HbpA2 of the present invention fully reacts with the immune serum, there are specific color bands at the position of the recombinant protein, which proves that the protein has good reactogenicity.

Experimental results show that the present invention obtains pure recombinant protein HbpA2 through recombinant expression through genetic engineering.

The preparation of embodiment 2 subunit vaccine of the present invention

Take 50 μg of pure recombinant protein HbpA2 prepared in Example 1, make 135 μl of HbpA2 protein solution, add 115 μl of normal saline, and emulsify with complete Freund’s adjuvant at a volume ratio of 1:1 to obtain the product.

Embodiment 3 Preparation of subunit vaccine of the present invention

Take 50 μg of the pure recombinant protein HbpA2 prepared in Example 1, make 135 μl of HbpA2 protein solution, add 115 μl of normal saline, and emulsify with incomplete Freund’s adjuvant at a volume ratio of 1:1 to obtain the product.

Embodiment 4 Preparation of subunit vaccine of the present invention

Take 50 μg of the pure recombinant protein HbpA2 prepared in Example 1, make 135 μl of HbpA2 protein solution, add 115 μl of normal saline, emulsify with incomplete Freund’s adjuvant at a volume ratio of 1:1, and add an appropriate amount of thimerosal as a preservative.

The beneficial effect of the present invention is illustrated in the mode of experimental example below:

Experimental example 1 subunit vaccine of the present invention and the in vivo protective effect of subunit vaccine

1. Experimental method

1.1 Determination of the median lethal dose (LD50) of Haemophilus parasuis CVCC3361

1.1.1 Determination of LD0 and LD100

10 mice were used as a group, and each mouse was intraperitoneally injected with 1×10 ⁹ CFU of live bacteria (LD0 estimated in the preliminary experiment), and the death of the mice was counted after 7 days. If two or more died, the challenge The toxic dose was reduced by 0.1×10 ⁹ CFU, and if all survived, the challenge dose was increased by 0.1×10 ⁹ CFU until only one mouse died in a group, and the adjacent low dose was LD0. Obtain LD100 by the same method.

1.1.2 Determination of the challenge dose of the experimental group

The mice were randomly and equally divided into 7 groups, with 10 mice in each group, of which 7 groups were control groups, and groups 1-6 were test groups. Calculate the r value according to the following formula, Among them, G is the number of groups, and Dm/Dn is the ratio of LD100 to LD0. The challenge doses of the test groups were D1=Dn=LD0, D2=D1×r, D3=D2×r, D4=D3×r, D5=D4×r, D6=D5×r. After inoculation according to the above doses, the death of the mice was observed and recorded.

1.1.3 Calculation of LD50

Calculate LD50 by Bliss-LD50 software, enter the challenge dose, number of animals, and death number of each group in the program, without entering the data of the control group, click "Calculate" to display the LD50 parameters of CVCC3361 on mice.

1.2 Mice challenge protection test

40 mice were randomly divided into 4 groups, 10 in each group. Group 1 back subcutaneous multi-point immunization with 50 micrograms of purified HbpA2+ adjuvant (freund's complete adjuvant for the first immunization, incomplete Freund's adjuvant for the second immunization, preparation method: take 135 μl of HbpA2 protein solution, add 115 μl of normal saline, parasuis CVCC3361 inactivated vaccine + adjuvant in group 2 as positive control group; group 3 with normal saline as negative control group; group 4 with adjuvant as negative control Group. On the 14th day after the first immunization, booster immunization was given once, and the dose and method of immunization were the same as the first immunization. On the 14th day after the second immunization, all mice were intraperitoneally challenged with Haemophilus parasuis CVCC3361. Before the challenge, the mouse serum was collected to detect the HbpA2-specific antibody titer. The clinical manifestations and death of the mice were observed and recorded every day after challenge until the fifth day. After 5 days, the mice were euthanized, and bacteria were isolated and identified from all the dead mice to confirm the infection of Haemophilus parasuis.

1.3 Detection of specific antibodies

Serum used to detect HbpA2-specific antibodies was collected before challenge. Blood was collected from mice by docking their tails, and the collected serum was stored at -20°C for future use. Antibody detection was performed by indirect ELISA, and the specific method was as follows: After HbpA2 was purified, it was diluted with carbonate pH 9.6, 100 μL was added to each well of a 96-well ELISA plate, incubated at 37°C for 1 hour, and coated overnight at 4°C. The next day, after washing 3 times with PBST, block with 5% skimmed milk at 37°C for 1.5h. Discard the blocking solution and wash 3 times with PBST. After diluting the serum to be tested from 1:100 to 1:12500, add 100 μL to each well and incubate at 37°C for 1 hour. After washing 3 times, dilute goat anti-mouse IgG with PBST, add 100 μL to each well, and react at 37°C for 30 minutes. Wash 3 times, add 100 μL TMB solution to each well and react in the dark for 15 min at room temperature, add 50 μL 2.0 mol/L sulfuric acid solution to terminate the reaction, and read at 450 nm with a microplate reader. Wells with a value greater than 0.2 were considered to have HbpA2-specific antibodies present.

2. Experimental results

1. Mouse serum antibody detection

The results are shown in Table 1:

Table 1 The results of antibody detection in mouse serum

The antibody titer in the HbpA2 immunized group was significantly higher than that in the negative control group and the positive control group, indicating that the expressed HbpA2 and its prepared subunit vaccine had good immunogenicity.

2. Antivirus protection test

In order to evaluate the protective efficiency of HbpA2 immunization, mice were used as animal models in this study. After 2 times of immunization, they were challenged with 6x10 ⁹ CFU (5×LD50) virulent strain CVCC3361, and the clinical symptoms and death of mice were observed and recorded. , the experimental results are shown in Table 2 and Figure 8:

Table 2 The results of the virus attack protection test

It can be seen from Table 2 and Figure 8 that the mice in the negative control group all died within 1 day after the challenge, and multiple organ lesions were found after dissection, and the strains used for the challenge could be isolated from the lesioned organs; the HbpA2 immune group challenged the virus Afterwards, 3 mice died within 2 days, and the remaining mice were still alive after 5 days of observation. Two mice in the positive control group died within 2 days after being challenged.

The results show that the HbpA2 immunity of the present invention can significantly protect mice against the attack of the virulent strain of Haemophilus parasuis, and the immune protection of the subunit vaccine prepared by it is better, the protection rate is as high as 70%, and the protection effect is close to that of the whole bacteria inactivated vaccine .

To sum up, the present invention successfully constructs genetically engineered bacteria by means of genetic engineering, and expresses recombinant protein HbpA2. The recombinant protein HbpA2 has good immunogenicity and protective properties, and the HbpA2-specific antibody level produced after immunization with the subunit vaccine prepared by it can significantly protect mice against the virulent strain of Haemophilus parasuis serotype 5 The challenge shows that the recombinant protein HbpA2 is a protective antigen of Haemophilus parasuis, which can be prepared as a vaccine and has a good prospect for clinical application.

Claims (10)

1. Use of the recombinant HbpA2 protein whose amino acid sequence is shown in SEQ ID NO: 2 in the preparation of medicines for preventing Haemophilus parasuis disease; the protein is encoded by the nucleotide sequence shown in SEQ ID NO: 1. 2. The use according to claim 1, characterized in that: the medicine comprises recombinant HbpA2 protein in an immunogenic effective amount. 3. A subunit vaccine, characterized in that it is prepared from recombinant HbpA2 protein with amino acid sequence as shown in SEQ ID NO: 2 and pharmaceutically acceptable adjuvant or carrier. 4. The vaccine according to claim 3, characterized in that: said vaccine comprises recombinant HbpA2 protein and a pharmaceutically acceptable adjuvant or carrier with an amino acid sequence of an immunogenic effective amount as shown in SEQ ID NO: 2; The protein is encoded by the nucleotide sequence shown in SEQ ID NO:1. 5. The vaccine according to claim 4, characterized in that: said immunogenic effective dose is not less than 0.1 μg/ml. 6. The vaccine according to claim 4, characterized in that: said adjuvant or carrier is an immune adjuvant and/or a preservative. 7. The vaccine according to claim 6, characterized in that: the adjuvant is complete Freund's adjuvant or incomplete Freund's adjuvant. 8. The vaccine according to claim 6, characterized in that: the preservative is thimerosal. 9. Use of the vaccine according to any one of claims 3-8 in the preparation of a medicament for preventing Haemophilus parasuis disease. 10. The use according to claim 9, characterized in that: the medicament for preventing Haemophilus parasuis disease is administered by injection.