CN116492454A - Improved rabbit pasteurellosis inactivated vaccine, preparation and application thereof - Google Patents

Abstract

The invention belongs to the technical field of veterinary biological products, and provides an improved inactivated vaccine for pasteurellosis in rabbits and its preparation and application. The invention improves the inactivated vaccine (single vaccine or combined vaccine) of pasteurellosis in rabbits. The improved pasteurellosis inactivated vaccine for rabbits contains Pasteurella bacterium and recombinant Pasteurella protein in single or combined vaccines, and the immune protection effect has been improved, which is specifically manifested in the attack on homologous and heterologous strains. The immune protection efficacy of the virus has been improved, thus providing a good tool for better prevention and control of pasteurellosis in rabbits.

Description

An improved inactivated vaccine against pasteurellosis in rabbits and its preparation and application

technical field

The invention belongs to the technical field of veterinary biological products, and in particular relates to an improved rabbit pasteurella disease inactivated vaccine and its preparation and application in the prevention and control of rabbit pasteurella.

Background technique

Pasteurellosis in rabbits is a major bacterial disease caused by Pasteurella multocida, which seriously endangers the rabbit industry. Rabbits of various ages and breeds are susceptible to Pasteurella multocida, especially rabbits aged 2-6 months are susceptible to infection and disease, which is one of the main diseases that cause death of rabbits aged 9 weeks to 6 months one. With the widespread use of rabbit plague vaccine, the incidence and harm of rabbit plague (rabbit viral hemorrhagic disease) has decreased nationwide, and pasteurellosis has begun to rise as the main epidemic disease that endangers the rabbit industry.

At present, inactivated vaccines have been used for the prevention and control of pasteurellosis in rabbits, but because the preparation process is not stable enough, the antigen immunogenicity varies greatly between batches, and the challenge protection rate in the efficacy test is low. Moreover, the inactivated vaccine for pasteurellosis in rabbits has the inherent disadvantage of inactivated vaccines—poor immune protection against heterologous strains, so the clinical effect of pasteurellosis vaccine for rabbits is not completely satisfactory.

The purpose of the present invention is to provide an improved pasteurellosis inactivated vaccine for rabbits, which has stronger immune protection efficacy against homologous Pasteurella strains and heterologous Pasteurella strains.

Contents of the invention

The technical problem solved by the present invention is: obtain a kind of rabbit Pasteurella vaccine (single vaccine or combination vaccine) by screening and testing, this vaccine has stronger immune protective effect to the challenge virus of homologous bacterial strain, and has stronger immune protection effect to heterologous bacterial strain The challenge virus also has immune protection effect.

In order to solve the above technical problems, the present invention provides a combination of Pasteurella heavy antigens, which is used to prepare Pasteurella rabbit vaccines.

The vaccine comprises Pasteurella inactivated bacterium and recombinant Pasteurella protein, wherein the Pasteurella is C51-17 strain, and the Pasteurella protein is PlpE and PtfA recombinant protein.

The improved pasteurellosis inactivated vaccine for rabbits is characterized in that the Pasteurella C51-17 strain has a preservation number of: CCTCC M 2018401, a preservation date of June 26, 2018, and a preservation address It is the Chinese Type Culture Collection Center.

The amino acid sequence of the PlpE recombinant protein is shown in SEQ ID NO:1, and the amino acid sequence of the PtfA recombinant protein is shown in SEQ ID NO:2.

The nucleotide sequence of the gene encoding the PlpE recombinant protein is SEQ ID NO: 3, and the nucleotide sequence of the gene encoding the PtfA recombinant protein is SEQ ID NO: 4.

The vaccine also contains rabbit hemorrhagic disease virus VP60 antigen protein, and an improved rabbit pasteurellosis inactivated vaccine combination vaccine is obtained.

The preparation method of the improved pasteurellosis inactivated vaccine single vaccine for rabbits is as follows: after overnight culturing of Pasteurella C51-17 strain, adding PlpE recombinant protein and PtfA recombinant protein to the vaccine before inactivation and diluting the bacterial solution, so that The final concentration of the two recombinant proteins in the vaccine is 0.5mg/ml, the final concentration of Pasteurella is 7.5×10 ⁹ CFU/ml, and then add 0.2% formaldehyde solution of the total bacterial volume to inactivate for 24 hours, after being tested for sterility Add 1/10 volume of sterile aluminum gel adjuvant to make improved inactivated rabbit pasteurellosis vaccine.

The specific steps of the preparation method of the improved pasteurellosis inactivated vaccine combination vaccine for rabbits are as follows: After the Pasteurella C51-17 strain is cultivated overnight, the bacterial solution is diluted before inactivation, and PlpE recombinant protein and PtfA recombinant protein are added to the vaccine. Protein and rabbit hemorrhagic disease virus VP60 protein, so that the final concentration of Pasteurella in the vaccine is 7.5×10 ⁹ CFU/ml, the final concentration of the two recombinant proteins is 0.5mg/ml, and the hemagglutination value of VP60 protein is not lower than 1:256 , then add 0.2% formaldehyde solution of the total bacterial liquid volume to inactivate for 24 hours, and add 1/10 volume of sterile aluminum gel adjuvant to make the improved rabbit pasteurellosis inactivated vaccine combined vaccine after being tested for sterility.

The application of the improved rabbit pasteurellosis inactivated vaccine in the prevention and control of rabbit pasteurellosis and rabbit viral hemorrhagic disease.

The screening process of the combination of antigens that can be used to prepare rabbit pasteurellosis inactivated vaccine according to the present invention is as follows:

(1) Collect clinical isolates of Pasteurella from domestic rabbit farms in recent years, type them by MLST molecular typing method, and select representative strains of different groups for subsequent experiments.

(2) Comparing the immune protection effect of Pasteurella C51-17 strain inactivated vaccine on different strains.

(3) Screen the recombinant Pasteurella protein effective for the challenge of Pasteurella C51-17 strain, and determine the PlpE recombinant protein.

(4) Analyze the immune protection of recombinant PlpE protein against Pasteurella challenged from other rabbits, and determine the heterologous strains JY-1 and SD-7 that PlpE cannot provide effective immune protection.

(5) Prepare the recombinant protein of JY-1 strain bacterial protein, and screen the recombinant Pasteurella protein with immune protection against JY-1 strain challenge, and confirm that the PtfA recombinant protein meets the requirements.

(6) Prepare the culture solution of Pasteurella C51-17 strain, add recombinant PlpE protein and PtfA (further add rabbit haemorrhagic disease virus VP60 anti-principle to make joint vaccine), inactivate and add adjuvant to obtain improved rabbit Pasteurella The single vaccine of inactivated bacillosis vaccine has a better immune protection against homologous strains and heterologous strains.

The beneficial effect of the present invention is: the inactivated rabbit pasteurellosis vaccine has the problem that the immune protection against homologous bacterial strains is not strong enough, and the immune effect against heterologous bacterial strains is poor. ways to improve. The present invention screens the protective antigens of different types of rabbit Pasteurella on the basis of analyzing the group composition of the rabbit-derived Pasteurella, and finally screens out the Pasteurella antigen combinations that have immune protection effects on different bacterial strains. After the combination of antigens is prepared into an inactivated vaccine against pasteurellosis in rabbits, it can effectively protect against homologous strains and heterologous strains.

In a word, the present invention obtains an antigen combination by screening different types of Pasteurella antigens, which can effectively cope with the challenge of homologous strains and heterologous strains after being prepared into an inactivated vaccine, so as to provide a more effective vaccine for the later production of Pasteurella disease in rabbits. Inactivated vaccine single vaccine and combined vaccine and the prevention and control of pasteurellosis in rabbits provide the basis.

Pasteurella multocida C51-17 strain used in the present invention has a classification name of Pasteurella multocida; it is a sporeless, non-moving, facultatively anaerobic, Gram-negative small bacillus that often stains heavily at both ends of the thalline. The deposit number is: CCTCCM 2018401. It was preserved in the China Center for Type Culture Collection on June 26, 2018.

Description of drawings

Figure 1 is the RIDIC MLST typing results and population structure of Pasteurella from rabbits determined in the present invention.

Fig. 2 is the recombinant Pasteurella protein derived from Pasteurella C51-17 strain obtained in the present invention. All proteins have been Ni affinity purified.

Figure 3 shows the detection results of antibody levels against the immunized antigens in the mice of each immunized group 21 days after immunization. All sera were diluted 1/100. The PBS immunized group was used as the control (Control).

Fig. 4 is the recombinant Pasteurella protein derived from Pasteurella JY-1 strain obtained in the present invention. All proteins have been Ni affinity purified.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

Example 1: Analysis of Pasteurella population structure from rabbits

From 2011 to 2020, the present invention collected 30 strains of rabbit-derived Pasteurella (including rabbit Pasteurella disease vaccine strain C51-17). These strains were isolated from the main rabbit production areas in my country. The isolated rabbit farms are different and have no correlation. The isolated disease materials include nasal mucus from rabbits with rhinitis, lungs from rabbits with pneumonia, and liver from rabbits that died of sepsis. . All strains were MLST typed according to the RIRDC MLST molecular typing protocol: first, the seven housekeeping enzyme genes (adk, est, pmi, zwf, mdh, gdh, and pgi) of these Pasteurella bacteria were cloned and sequenced; The 7-locus sequence of each strain was searched in the MLST database (https://pubmlst.org/P.multocidaultocida/) and ST type or CC group was determined.

The results of population structure analysis of Pasteurella from rabbits are shown in Figure 1. The present invention shows that rabbit-derived Pasteurella has 8 ST molecular types: ST3 (n=1), ST9 (n=2), ST74 (n=8), ST129 (n=3), ST204 (n=7), ST302 (n=7), ST345 (n=1) and ST348 (n=1). Based on genetic relatedness, these ST types were further divided into three CC groups (clonal complex): CC9 (ST9, ST204 and ST345, 10/30) and CC74 (ST74, ST302 and ST347, 16/30), CC129 (ST129 ,3/30). That is, Pasteurella from rabbits mainly includes 3 populations, CC9, CC74 and CC129, and a representative strain (SD-7, JY-1, and C51-17) was selected for subsequent research.

Embodiment 2: the immunoprotective efficacy of Pasteurella C51-17 strain inactivated vaccine

Pasteurella C51-17 strain was cultured overnight and then the bacterial solution was diluted so that the vaccine finally contained 7.5×10 ⁹ CFU/ml of Pasteurella. Then add 0.2% formaldehyde solution of the total bacterial liquid volume to inactivate for 24 hours, and add 1/10 volume of sterile aluminum gel adjuvant to prepare the vaccine after being tested for sterility.

90 SPF grade ICR female mice (4-6 weeks old) were selected and randomly divided into 9 groups, 6 groups as the experimental group and 3 groups as the control group, with 10 mice in each group. Each mouse was immunized with Pasteurella C51-17 inactivated vaccine twice at intervals of 3 weeks, each immunization dose was 100 μl/mouse, and inoculated by subcutaneous injection. 10 days after the second inoculation, Pasteurella C51-17 strain, JY-1 strain, and SD-7 strain were used to attack the virus respectively, and each strain was detected under low dose (10×LD50) and high dose (100×LD50). In the challenge survival rate, the control group only detected the survival rate under the low challenge dose.

The experimental results (Table 1) show that after the immunization of the C51-17 strain, the animals can resist the challenge of low doses of homologous strains, and the immune protection effect of high doses of homologous strains is not good; animals are immune to some heterologous strains The protective effect is not good, especially there is no immune protective effect under high-dose challenge.

Table 1 The immune protection effect (survival rate, survival number/total number) of vaccine strain C51-17 to different bacterial strains

Example 3: Recombinant expression and screening of Pasteurella C51-17 cell surface protein

(1) Using the genome sequence (NZ_MAPQ01000003.1) of the rabbit-derived Pasteurella inactivated vaccine strain C51-17 as a reference sequence, find the ORFs of the following surface proteins: LspB, OapA, OmpW, OmpH, OmpA, ComE, OmpLA, VacJ, PlpE, PlpB. The primers were further designed as shown in Table 2 (lowercase letters are the same homology arms as the vector). The genome of Pasteurella inactivated vaccine strain C51-17 from rabbits was extracted using a bacterial genome extraction kit. According to the annealing temperature of the respective primers and the length of the corresponding fragments, PCR parameters were set for PCR amplification. After the amplified product was purified, the PET28a(+) plasmid was double-digested with BamHI and HindIII according to the instructions (Takara), purified by the gel recovery method (Meiji), and the above-mentioned homologous recombination cloning kit (Novazyme) was used to clone the plasmid. The PCR amplified fragment was integrated into the plasmid, and then the positive recombinant plasmid was transferred into BL21(DE3) competent cells. Pick positive clones for PCR identification and send them to the company for sequencing to determine whether the cloned genes are complete and correctly connected to the carrier. Next, the HB-PET self-induction medium was used to induce the expression of the recombinant protein overnight at 16°C and was broken with an ultrasonic breaker. The supernatant was collected by centrifugation and subjected to Ni affinity purification using a protein purifier (GE). For proteins expressed in inclusion bodies, SDS-PAGE and Coomassie Brilliant Blue staining were performed to detect the expression and purification effects.

Through these works, we have successfully completed the recombinant expression of these surface proteins, and obtained purified soluble recombinant proteins: LspB (~60kDa), OapA (~45kDa), OmpW (~30kDa), OmpH (~45kDa), OmpA ( ～40kDa), ComE(～67kDa), VacJ(～33kDa), OmpLA(～40kDa), PlpE(～43kDa), PlpB(～55kDa) (Figure 2).

Table 2 Primers for Gene Cloning of Recombinant Proteins

(2) Screening of proteins with strong immune protection efficiency SPF grade ICR female mice (4-6 weeks old) were randomly divided into 11 groups, 10 mice in each group, each group corresponding to a recombinant protein, and the last group was used as the immune control group. After adjusting the antigen concentration, the recombinant protein and aluminum hydroxide adjuvant were mixed evenly at a volume ratio of 9:1, so that the antigen content reached 1000 μg/ml (mixing PBS and aluminum hydroxide adjuvant was used as a control). Two immunizations were made at intervals of 3 weeks, each immunization dose was 100 μl/bird, and inoculated by subcutaneous injection. 10 days after the booster immunization, blood was collected to prepare serum, and the antibody level was determined. After confirming that the antibody level was high, the rabbit-derived virulent Pasteurella multocida virulent strain C51-17 was used to challenge the virus by subcutaneous injection, 10×LD50/mouse, and the incidence and death of mice were observed and recorded for one week.

The experimental results showed that 10 days after the booster immunization, the mice produced high levels of antibodies against each antigenic protein, as shown in Figure 3. After challenge, the PlpE immune group showed the best immune protection effect, and no death was found until the end of the observation period, and the protection rate was 100%. The immune protection effects of other proteins were not more than 50%, weaker than PlpE (Table 3). This result shows that PlpE is a protective antigen with high immune protection efficacy and can be used in subsequent experiments.

Table 3 Comparison of the immune protection effect of recombinant bacterium protein of Pasteurella C51-17 strain

(3) Detection of PlpE's immune protection against heterologous strains SPF grade ICR female mice (4-6 weeks old) were randomly divided into 4 groups, 10 mice in each group, two groups were immunized with PlpE recombinant protein, and the other two groups were immunized with the control group PBS. The immune method is the same as (2). Two weeks after the booster immunization, Pasteurella SD-7 strains (CC9 group, 10×LD50) and JY-1 (CC74 group, about 10×LD50) were used to challenge the virus subcutaneously, and the incidence and death of mice were observed and recorded for one week.

The experimental results (Table 4) show that after the challenge, all mice in the control group died, and PlpE immunity did not show immune protection against the challenge of SD-7 and JY-1 strains, and the protection rate was close to 0%. This shows that only the Pasteurella PlpE recombinant protein is not enough to improve the efficacy of inactivated vaccines, and other Pasteurella recombinant proteins need to be added.

Table 4 PlpE immune protection test

Example 4: Recombinant expression and screening of Pasteurella JY-1 strain bacterial protein

Using the genome sequence of Pasteurella multocida 3480 strain (NC_017764.1) as a reference sequence, the ORFs of some bacterial proteins were found and primers were designed for amplification. The bacterial proteins and primer information are shown in Table 5 (lowercase letters are same homology arms as the vector). The genome of the JY-1 strain was extracted using a bacterial genome extraction kit. According to the annealing temperature of the respective primers and the length of the corresponding fragments, PCR parameters were set for PCR amplification. After purification, the amplified product was connected to the linearized pET28a(+) or pET32a plasmid vector through a homologous recombination cloning kit, and then the positive recombinant plasmid was transferred into BL21(DE3) competent cells. Pick positive clones for PCR identification and send them to the company for sequencing to determine whether the cloned genes are complete and correctly connected to the carrier. Next, the HB-PET self-induction medium was used to induce the expression of the recombinant protein overnight at 26°C and was broken with an ultrasonic breaker. The supernatant was collected by centrifugation and subjected to Ni or GST affinity purification using a protein purifier (GE). For proteins expressed in the form of inclusion bodies, they should be renatured before use after purification. Finally, SDS-PAGE was performed, and Coomassie Brilliant Blue staining was performed to detect expression and purification effects.

Table 5 JY-1 bacterial protein involved in the present invention and its cloning primer

Through these works, we successfully completed the recombinant expression of these surface proteins, and obtained the purified Pasteurella recombinant proteins: TbpA (~35kDa), Pm1809 (~68kDa), Pm0663 (~43kDa), Pm1069 (~45kDa), PtfA (-22 kDa), Pm1428 (-70 kDa), Pm0336 (-35 kDa), Pm0592 (-70 kDa) (Figure 4).

(2) Protein screening for immune protection efficiency SPF grade ICR female mice (4-6 weeks old) were randomly divided into 9 groups, 10 mice in each group, each group corresponding to a recombinant protein, and the last group was used as the immune control group. After adjusting the antigen concentration, the recombinant protein and aluminum hydroxide adjuvant were mixed evenly at a volume ratio of 9:1, so that the antigen content reached 1000 μg/ml (mixing PBS and aluminum hydroxide adjuvant was used as a control). Two immunizations were performed at intervals of 3 weeks, and the dose of each immunization was 100 μl. Two weeks after the booster immunization, Pasteurella JY-1 strain was used to challenge the virus by subcutaneous injection, 10×LD50/mouse, and the incidence and death of mice were observed and recorded for one week.

The experimental results showed that after the challenge, the PtfA immune group showed the best immune protection effect, and the protection rate was 80% by the end of the observation period. The immune protection effects of other proteins were not more than 30% (Table 6). This result shows that PtfA is a protective antigen with high immune protection efficacy against JY-1 strain challenge, which can be used in subsequent experiments.

Table 6 Comparison of the immune protection effect of Pasteurella JY-1 strain recombinant bacterium protein

The preparation of embodiment 5 improved rabbit pasteurellosis inactivated vaccines

(1) Preparation of the original inactivated vaccine: Pasteurella C51-17 strain was cultured overnight and the bacterial solution was diluted so that the vaccine finally contained 7.5×10 ⁹ CFU/ml of Pasteurella. Then add 0.2% formaldehyde solution of the total bacterial liquid volume to inactivate for 24 hours, and add 1/10 volume of sterile aluminum gel adjuvant to make traditional rabbit pasteurellosis inactivated vaccine after being tested for sterility.

(2) Subunit vaccine preparation: Dilute the purified PlpE recombinant protein and PtfA recombinant protein so that the final concentration of the two recombinant proteins in the vaccine is 0.5 mg/ml, and finally add 1/10 volume of sterile aluminum gel adjuvant Make a vaccine.

(3) Preparation of improved inactivated vaccine single vaccine: Pasteurella C51-17 strain was cultured overnight and added PlpE recombinant protein and PtfA recombinant protein to the vaccine before inactivation and diluted the bacterial solution to make the two recombinant proteins in the vaccine The final protein concentration was 0.5mg/ml, and the final concentration of Pasteurella was 7.5×10 ⁹ CFU/ml. Then add 0.2% formaldehyde solution of the total bacterial liquid volume to inactivate for 24 hours, and add 1/10 volume of sterile aluminum gel adjuvant after the sterility test to prepare a single vaccine of the improved rabbit pasteurellosis inactivated vaccine.

(4) Preparation of improved inactivated vaccine combination vaccine: Pasteurella C51-17 strain was cultured overnight and diluted the bacterial solution before inactivation and added PlpE recombinant protein, PtfA recombinant protein and rabbit hemorrhagic disease virus VP60 protein to the vaccine, The final concentration of Pasteurella in the vaccine is 7.5×10 ⁹ CFU/ml, the final concentration of the two recombinant proteins is 0.5 mg/ml, and the hemagglutination value of VP60 protein is not lower than 1:256. Then add 0.2% formaldehyde solution of the total bacterial liquid volume to inactivate for 24 hours, and add 1/10 volume of sterile aluminum gel adjuvant to prepare the improved rabbit pasteurellosis inactivated vaccine combined vaccine after being tested for sterility.

Embodiment 6 Improved rabbit pasteurellosis inactivated vaccine protection analysis

(1) Immune protection against homologous strains Rabbits (35-42 days old) were randomly divided into 5 groups, 10 in each group, half male and half female, and the four groups were the experimental groups immunized with original inactivated vaccine and subunit vaccine respectively. , improved inactivated vaccine single vaccine, improved inactivated vaccine combined vaccine, and the last group was used as the immunization control group. Select the part with loose skin on the neck of the rabbit, sterilize the injection site with 75% alcohol cotton ball, and inject 1.0ml of the vaccine into each rabbit. The control group was immunized with the same volume of PBS (containing 10% aluminum gel adjuvant). Twenty-one days after the immunization, Pasteurella C51-17 was used to challenge the virus by subcutaneous injection, 100 CFU/rat (about 50×MLD), and the incidence and death of rabbits were observed and recorded for one week. The experimental results are shown in Table 7. The original inactivated vaccine could not provide effective immune protection against the challenge of homologous or heterologous strains, while the improved inactivated vaccine and the improved combined vaccine immunization group showed good immune protection efficacy, that is, improved Type inactivated vaccine has better immune protection against homologous strain challenge.

Table 7 The immune protection efficacy of Pasteurella inactivated vaccine to homologous strain challenge before and after improvement

(2) Immune protection against heterologous strains. 100 rabbits (35-42 days old) were randomly divided into 10 groups, 10 in each group, half male and half female. Two groups were immunized with original inactivated vaccine, and two groups were immunized with subunit Vaccines, two groups of immune-improved inactivated vaccine single vaccine, two groups of immune-improved inactivated vaccine combined vaccine, and the remaining two groups immunized with PBS (containing 10% aluminum glue adjuvant) as a control group. Select the part with loose skin on the neck of the rabbit, sterilize the injection site with 75% alcohol cotton ball, and inject 1.0ml of the vaccine into each rabbit. After 21 days, the SD-7 strain of Pasteurella or JY-1 were injected subcutaneously to attack the virus respectively, and the attack dose was 1×MLD, and the incidence and death of the rabbits were observed and recorded for seven consecutive days. The experimental results are shown in Table 8. The original inactivated vaccine or subunit vaccine cannot provide complete immune protection against all heterologous strains, while the improved inactivated single vaccine group and the improved combined vaccine group have no effect on different heterologous strains. All of them showed good immune protection efficacy.

Based on the experimental results of (1) and (2), it can be seen that the single or combined vaccine of the improved inactivated vaccine has better immune protection against the challenge of Pasteurella homologous strains or heterologous strains.

The protective power of Pasteurella inactivated vaccine after table 8 heterologous strain challenge

(3) Select 20 healthy susceptible rabbits aged 2-3 months, and randomly divide them into two groups, one group is control and the other is experimental group, 10 rabbits in each group. The experimental group was immunized with improved combination vaccine, 1ml each; the control group was immunized with PBS, using the same adjuvant as that of the experimental group. After 3 weeks of immunization, rabbit haemorrhagic disease virus Wanfu strain was used to challenge the virus, 100×LD50 per rabbit, and the survival of rabbits was observed for 7 consecutive days. All the rabbits in the experimental group survived after challenge with rabbit hemorrhagic disease virus (survival rate 100%), and all rabbits in the control group died (survival rate 0/10). These experimental results show that the improvement work will not affect the rabbit hemorrhagic disease virus antigen in the combined vaccine.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible, which fall within the scope of the claimed invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims (10)

1. an improved pasteurellosis inactivated vaccine for rabbits is characterized in that, comprising Pasteurella inactivated thalline and recombinant Pasteurella protein in the vaccine, and the Pasteurella is Pasteurella multocida C51-17, the Pasteurella protein is PlpE and PtfA recombinant protein. 2. A kind of improved inactivated vaccine for rabbit pasteurellosis according to claim 1, characterized in that, the Pasteurella multocida C51-17 has a preservation number of: CCTCC M 2018401, and the preservation date is 2018 On June 26, 2010, the deposit address was China Center for Type Culture Collection. 3. a kind of improved rabbit pasteurellosis inactivated vaccine according to claim 1, is characterized in that, described PlpE recombinant protein amino acid sequence is as shown in SEQ ID NO:1, and PtfA recombinant protein amino acid sequence is as SEQ ID NO :2 shown. 4. a kind of improved rabbit pasteurellosis inactivated vaccine according to claim 2, is characterized in that, the gene nucleotide sequence of coding PlpE recombinant protein is SEQ ID NO: 3, the gene nucleoside of coding PtfA recombinant protein The acid sequence is SEQ ID NO:4. 5. A kind of improved rabbit pasteurellosis inactivated vaccine according to claim 1, is characterized in that, described vaccine also comprises rabbit hemorrhagic disease virus VP60 antigenic protein, obtains improved rabbit pasteurellosis inactivated vaccine combination Seedling. 6. The preparation method of the improved pasteurellosis inactivated vaccine for rabbits described in claim 1 is: adding PlpE and PtfA recombinant protein in Pasteurella multocida C51-17 culture fluid, inactivating and adding adjuvant, obtaining improved Pasteurella rabbit type inactivated vaccine. 7. according to the preparation method of the described improved rabbit pasteurellosis inactivated vaccine of claim 6, its concrete steps are: after overnight cultivation of Pasteurella multocida C51-17 strain, add PlpE in the vaccine before inactivation Recombinant protein and PtfA recombinant protein and dilute the bacterial liquid, so that the final concentration of the two recombinant proteins in the vaccine is 0.5mg/ml, and the final concentration of Pasteurella multocida C51-17 is 7.5×10 ⁹ CFU/ml, and then add 0.2% formaldehyde solution of the total bacterial liquid volume was inactivated for 24 hours, and after the sterility was tested, 1/10 volume of sterile aluminum gel adjuvant was added to make an improved rabbit pasteurellosis inactivated vaccine. 8. The method for preparing the improved pasteurellosis inactivated vaccine for rabbits described in claim 5, its specific steps are: after the Pasteurella multocida C51-17 strain is cultivated overnight, dilute the bacterium solution before inactivation and apply it to the vaccine PlpE recombinant protein, PtfA recombinant protein and rabbit hemorrhagic disease virus VP60 protein were added to the vaccine, so that the final concentration of Pasteurella in the vaccine was 7.5×10 ⁹ CFU/ml, the final concentration of the two recombinant proteins was 0.5 mg/ml, and the VP60 protein blood The coagulation price is not lower than 1:256, and then add 0.2% formaldehyde solution of the total bacterial volume to inactivate for 24 hours. After the sterility is tested, add 1/10 volume of sterile aluminum gel adjuvant to make improved rabbit pasteurellosis Inactivated vaccine combination vaccine. 9. The application of a kind of improved rabbit pasteurellosis inactivated vaccine described in claim 1 in the prevention and control of rabbit pasteurellosis. 10. The application of the improved pasteurellosis inactivated vaccine for rabbits described in claim 5 in the prevention and control of rabbit viral hemorrhagic disease.