A double gene deletion of a live attenuated vaccine strain of haemophilus parasuis serotype 5, construction method and application thereof

Abstract

This invention belongs to the field of genetic engineering technology, specifically relating to a double-gene-deleted serotype 5 Haemophilus parasuis attenuated vaccine strain, its construction method, and its application. This invention mainly utilizes modern genetic engineering principles and molecular biology techniques to delete the cpxA and cpxR genes, potential virulence factors of HPS, constructing the HN1570 double-gene-deleted strain HPS5△cpxAR. This double-gene-deleted strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 16, 2023, with accession number CGMCC NO: 45596. Compared with the parent strain, this double-gene-deleted strain exhibits significantly reduced virulence while maintaining the same immunogenicity. The attenuated live vaccine prepared from this strain as an antigen provides 100% protection against serotype 5 infection and good cross-protection against other serotypes and untyped Haemophilus parasuis infections. Furthermore, the vaccine preparation process eliminates the need for inactivation and inactivation testing, simplifying the preparation method and increasing production efficiency. This lays the foundation for further development of novel, highly effective genetically engineered live vaccines that provide high cross-protection efficacy.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a double-gene-deleted serum Haemophilus parasuis type 5 attenuated vaccine strain, its construction method, and its application. Background Technology

[0002] Haemophilus parasuis is a common bacterium in the upper respiratory tract of pigs and is the pathogen that causes Glazer's disease in pigs. Under certain stressful environmental conditions, it can invade the body of piglets and cause infectious diseases such as pleurisy, peritonitis, arthritis and meningitis in the host.

[0003] As the natural environment continues to change, many Gram-negative bacteria have evolved membrane stress systems that can both absorb sufficient nutrients and sensitively detect and adapt to changes in the external environment. The Cpx two-component system is one of the important membrane stress systems of bacteria, which can be induced by various stress conditions and, in turn, regulate various bacterial phenotypes.

[0004] The CPX system comprises a cpxA sensor (histidine kinase) and a cpxR response regulator. When cpxA is stimulated by different environmental signals (such as pH changes or low iron conditions), it is activated, transferring a phosphate group from cpxA to cpxR. The phosphorylated cpxR then binds to DNA, initiating gene expression regulation. cpxA, a histidine protein kinase, senses external environmental stimuli and transmits the signal to the response regulator cpxR via autophosphorylation. Phosphorylated cpxR regulates the transcription and expression of target genes, influencing bacterial physiological activity to cope with external stimuli, enabling bacteria to adapt to the environment and survive. After the external stimulus ends, cpxA degrades the phosphorylated cpxR, thus terminating the regulatory response. The CPX two-component system maintains a moderate bacterial response to specific environmental signals by controlling the phosphorylation level of the response regulator cpxR. Recent studies on the cpx two-component system and bacterial antibiotic resistance have revealed its association with resistance to various other antibiotics. As a bacterial cell membrane stress response system, the CPX two-component system's influence on antibiotic resistance is often achieved through alterations in cell membrane components. In addition, the study found that cpx affects the ability of bacteria to form biofilms.

[0005] The CPX system, as a cell membrane stress response system, plays a regulatory role in the infection process of many pathogens. The CPX system regulates the virulence of various pathogens, and in some bacteria, it influences the infection process by affecting the expression of virulence factors such as bacterial secretion systems or fimbriae. Due to the broad scope of action of the CPX two-component system, its mechanism of influence on bacterial virulence is highly complex. From initially affecting bacterial surface structure and thus virulence, to more recently regulating the expression of other regulatory factors and thus influencing bacterial virulence, as well as the interactions between the CPX two-component system and other regulatory systems, all indicate that the regulatory role of the CPX two-component system is very broad and complex.

[0006] With the transformation of farming models and the emergence of immunosuppressive diseases, Grazer's disease caused by Haemophilus parasuis has become one of the most common, prevalent, and serious respiratory diseases in pig farms. The overuse of clinical antibiotics has led to increasingly strong bacterial resistance, resulting in higher treatment costs. The CPX two-component system, as a signal transduction system for cell membrane stress response, plays an important role in the survival and pathogenicity of various pathogens. Since serotype 5 has the highest clinical isolation rate, is highly virulent, and poses the greatest threat, this invention constructs a CPXAR two-component gene-deleted strain of Haemophilus parasuis serotype 5, providing a scientific basis for the development of a novel, highly effective, and cross-protective attenuated vaccine against Haemophilus parasuis CPXAR and for the comprehensive prevention and control of this disease. Summary of the Invention

[0007] In response to the problem that the prevalent serotypes of Haemophilus parasuis vary across regions, and that multiple inactivated vaccines lack cross-protective attenuated live vaccines against serotype 5 Haemophilus parasuis, leading to increased bacterial resistance, this invention provides a cpxAR two-component gene deletion strain of Haemophilus parasuis serotype 5 and develops a novel, highly effective, cross-protective attenuated live vaccine against Haemophilus parasuis cpxAR two-component gene deletion.

[0008] The solution adopted by this invention to solve its technical problem is: a double-gene deletion strain of Haemophilus parasuis, wherein the Haemophilus parasuis is a double-gene deletion strain HPS5△cpxAR of clinical isolate HN1570 of Haemophilus parasuis serotype 5, with deletions of cpxA and cpxR genes, accession number CGMCC NO: 45596, accession date June 16, 2023, accession institution China General Microbiological Culture Collection Center, accession address No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.

[0009] This invention also provides a method for constructing a double-gene-deleted Haemophilus parasuis, characterized by comprising the following steps:

[0010] Step 1: Primer Design and Synthesis: Referring to the complete genome sequence No. CP001321 of Haemophilus parasuis serotype 5 strain SH0165 reported in GenBank, primers cpxARuF / cpxARuR and cpxARdF / cpxARdR, containing restriction enzyme sites and the DNA uptake signal sequence USS, were designed to amplify the upstream and downstream homologous arms of cpxAR. Primers KanF / KanR were designed based on the pSHK3-Kan sequence to amplify the Kan gene. The primer sequences used are as follows:

[0011] cpxARuF:CGGAATTCACCGCTTGTAAGTGGTATTTCTTAGCAAT

[0012] cpxARuR: TATTTTTATCTTGTGCAATGAGCTCTATTTCCCTATTCCAT

[0013] cpxARdF:TAATCAGAATTGGTTAATTGTATCAATATATTGTCAATATC

[0014] cpxARdR:CGGGATCCACAAGCGGTAGCCGCAAACATACTTTGAC

[0015] KanF:CATTGCACAAGATAAAAATATAT

[0016] KanR:TTATTCCACCCACAACGGCAA;

[0017] Step 2: Cloning and Recombinant Construction of Suicide Plasmid: Using the genome of Haemophilus parasuis serotype 5 clinical isolate HN1570 as a template, the upstream and downstream fragments of the cpxAR gene were amplified using primer pairs cpxARuF / cpxARuR and cpxARdF / cpxARdR, respectively. The kanamycin gene was amplified from the pSHK3 plasmid using primers KanF / KanR. Using the principle of overlap extension PCR, the upstream and downstream fragments of the cpxAR gene and the kanamycin gene were ligated by overlap extension PCR using primers cpxARuF / cpxARdR to form the overlap fragment UKD. The UKD fragment was digested with BamHI and EcoRI and ligated into pK18mobsacB to construct the suicide plasmid pKUKD.

[0018] Step 3: Construction of the double gene deletion strain: Following the natural transformation method, a large amount of recombinant plasmid pKUKD was extracted and its concentration was determined; transformants were selected, and their genomes were extracted. The UKD sequence, cpxAR gene, and Kan gene were amplified using primers cpxARuuF / cpxARddR, cpxARF / cpxARR, and KanF / KanR, respectively. Electrophoresis was used to detect the size and presence of the target fragment to identify whether the cpxAR deletion strain was successfully constructed, and sequencing was used for verification.

[0019] In the above-mentioned method for constructing Haemophilus parasuis with double gene deletion, step two uses the genome of Haemophilus parasuis strain HN1570 as a template to amplify the upstream 683bp and downstream 658bp fragments of the cpxAR gene using primer pairs cpxARuF / cpxARuR and cpxARdF / cpxARdR; and amplifies the 909bp kanamycin gene from the pSHK3 plasmid using primers KanF / KanR.

[0020] The above-mentioned method for constructing Haemophilus parasuis with double gene deletion involves the extraction of recombinant plasmid pKUKD via natural transformation in step three. Specifically, a single colony of HN1570 is picked and placed in 5 mL of TSB, and cultured at 37°C until OD500 is reached. 600 The value was 0.8. 20 μL of bacterial culture was added to 20 μL of 8 mmol / L cAMP, mixed well, and reacted for 10 min. Then, 2 μg of recombinant plasmid pKUKD was added, mixed well, and reacted for another 10 min. The mixture was then transferred to TSA, spread evenly, and incubated at 37°C for 5 h. The bacteria were washed off with TSB and transferred to TSA solid medium containing Kan, and incubated at 37°C for 24–48 h.

[0021] This invention also provides the application of double-gene-deleted Haemophilus parasuis in the preparation of Haemophilus parasuis attenuated live vaccine.

[0022] In the above application, the content of Haemophilus parasuis HPS5ΔcpxAR in the attenuated live vaccine is 1×10⁻⁶. 10 CFU / mL.

[0023] In the above application, the preparation method of the Haemophilus parasuis attenuated live vaccine is as follows: after the Haemophilus parasuis is successively proliferated and concentrated, the Haemophilus parasuis antigen solution is obtained, and an immune adjuvant is added to the antigen solution to obtain the vaccine.

[0024] In the above application, the immune adjuvant is Summit S550, an oil-in-water adjuvant for livestock and poultry.

[0025] The above-mentioned application, the preparation method of the Haemophilus parasuis attenuated live vaccine is as follows:

[0026] (1) Proliferation: The double gene deletion strain HPS5△cpxAR of Haemophilus parasuis was propagated and cultured to obtain 8 strains of Haemophilus parasuis bacterial suspension, and viable bacteria were counted separately.

[0027] (2) Concentration: The Haemophilus parasuis double-gene deletion strain HPS5△cpxAR was concentrated using an ultrafiltration device. Based on the viable cell count, the final concentration was adjusted to 1.0 × 10⁻⁶ with sterile physiological saline. 10 CFU / mL;

[0028] (3) Vaccine preparation: The HPS5△cpxAR antigen solution of Haemophilus parasuis double gene deletion strain obtained after proliferation and concentration is mixed with 30% of livestock and poultry water-in-oil-in-water adjuvant and attenuated live Haemophilus parasuis vaccine.

[0029] The beneficial effects of the present invention are as follows: Using the genome of Haemophilus parasuis strain HN1570, which was screened from clinical isolates, as a template, the present invention constructs a serum 5-type cpxAR double gene deletion strain HPS5△cpxAR. This double gene deletion strain has extremely weak pathogenicity to weaned piglets and cannot cause disease in weaned piglets.

[0030] Experiments showed that the attenuated live vaccine prepared using Haemophilus parasuis strain HPS5△cpxAR as the strain antigen had a 100% protection rate against type 5 infection and good cross-protection against other serotypes and untyped Haemophilus parasuis infections, indicating that the Haemophilus parasuis strain HPS5△cpxAR has good immunogenicity. Moreover, the vaccine preparation process of this invention is simple, has no toxic side effects, good safety, long-lasting immunity, and good immunization effect, making it suitable for industrial production. Attached Figure Description

[0031] Figure 1 The figures show the amplification results of the upstream and downstream fragments of the cpxAR gene, the kanamycin gene, and the overlapping extended upstream fragment of cpxAR + Kan + downstream fragment of cpxAR. In the figure, M1 is the DL5000 Marker, with lengths from top to bottom of 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; M2 is the DL2000 Marker, with lengths from top to bottom of 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp; lane 1 is the upstream fragment; lane 2 is the downstream fragment; lane 3 is Kan; and lane 4 is UKD.

[0032] Figure 2The results show the identification of HPS5△cpxAR deletion strains; in the figure, M1 is the DL5000 marker, from top to bottom: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp; M2 is the DL2000 marker, from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp; lane 1 is the primer cpxARuuF / cpxARddR for identifying HPS5△cpxAR; lane 2 is the primer cpxARuuF / cpxARddR for identifying HN1570; lane 3 is the primer cpxARuuF / cpxARddR for identifying the ddH2O control; lane 4 is the primer... cpxARF / cpxARR was used to identify HPS5△cpxAR; lane 5 was used with primer cpxARF / cpxARR to identify HN1570; lane 6 was used with primer cpxARF / cpxARR to identify ddH2O control; lane 7 was used with primer KanF / KanR to identify HPS5△cpxAR; lane 8 was used with primer KanF / KanR to identify HN1570; lane 9 was used with primer KanF / KanR to identify ddH2O control.

[0033] Figure 3 The colony morphology of Haemophilus parasuis HN1570 and HPS5△cpxAR is shown in this invention.

[0034] Figure 4 The images show the morphology of Haemophilus parasuis HN1570 and HPS5△cpxAR under an optical microscope at 1000x magnification.

[0035] Figure 5 The morphology of Haemophilus parasuis HN1570 and HPS5△cpxAR under a scanning electron microscope at 100,000x magnification is shown in this invention.

[0036] Figure 6 The results of the growth curves of Haemophilus parasuis HN1570 and HPS5 ΔcpxAR are presented in this invention. Detailed Implementation

[0037] To more clearly illustrate the technical solution of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0038] Example 1: Construction of the HPS5△cpxAR double-gene deletion strain of Haemophilus parasuis serotype 5 cpxAR

[0039] 1. Primer design and synthesis

[0040] Based on the complete genome sequence (No. CP001321) of Haemophilus parasuis serotype 5 strain SH0165 reported in GenBank, primers cpxARuF / ARuR and cpxARdF / cpxARdR, containing restriction enzyme sites and ACGCTTGTA (DNA uptake signal sequence, USS), were designed to amplify the upstream and downstream homologous arms of cpxAR. cpxARF / cpxARR was used to amplify the cpxAR gene. cpxARuuF / cpxARddR, with the restriction enzyme sites and USS sequences removed, was used to identify the parental and deletion strains of cpxAR. KanF / KanR was designed based on the pSHK3-Kan plasmid sequence to amplify the Kan gene. Primers used for constructing and identifying Haemophilus parasuis cpxAR gene deletion strains are shown in Table 1.

[0041] Table 1 Primers used for constructing and identifying cpxAR deletion strains

[0042]

[0043] 2. Construction and identification of the double-gene deletion strain HPS5△cpxAR of Haemophilus parasuis serotype 5 cpxAR

[0044] Using the genome of Haemophilus parasuis serotype 5 clinical isolate HN1570 as a template, primers cpxARuF / cpxARuR and cpxARdF / cpxARdR amplified 683 bp upstream and 658 bp downstream fragments of the cpxAR gene, respectively. Primers KanF / KanR amplified the 909 bp kanamycin gene from the pSHK3 plasmid. Utilizing the overlap extension PCR principle, primers cpxARuF / cpxARdR were used to ligate these three fragments via overlap extension PCR, forming an overlap fragment UKD (e.g., ...). Figure 1 The UKD fragment was digested with BamHI and EcoRI and ligated into pK18mobsacB. The recombinant suicide plasmid pKUKD was identified. The digested fragment size was correct and the sequencing was 100% correct, proving that the recombinant suicide plasmid was successfully constructed.

[0045] The recombinant plasmid pKUKD was extracted in large quantities using the natural transformation method reported in the reference (Jiang CS, Cheng YF, Cao H, et al. Effect of cAMP Receptor Protein Gene on Growth Characteristics and Stress Resistance of Haemophilus parasuis Serovar 5[J]. Frontiers in Cellular and Infection Microbiology, 2020.00019.), and its concentration was determined. A single colony of HN1570 was picked and cultured in 5 mL of TSB at 37°C until OD500 reached. 600 The value was 0.8. 20 μL of bacterial culture was added to 20 μL of 8 mmol / L cAMP, mixed well, and reacted for 10 min. Then, 2 μg of recombinant plasmid pKUKD was added, mixed well, and reacted for another 10 min. The mixture was then transferred to TSA, spread evenly, and incubated at 37°C for 5 h. The bacteria were washed off with TSB and transferred to TSA solid medium containing Kan, and cultured at 37°C for 24–48 h. Transformants were picked, and their genomes were extracted. The UKD sequence, cpxAR gene, and Kan gene were amplified using primers cpxARuuF / cpxARddR, cpxARF / cpxARR, and KanF / KanR. Electrophoresis was used to detect the size and presence of the target fragment to identify whether the cpxAR deletion strain was successfully constructed, and sequencing was performed for verification.

[0046] The successfully constructed recombinant suicide plasmid pKUKD was transformed into HN1570 using natural transformation. PCR was used to confirm the successful construction of the cpxAR deletion strain in HN1570. Primers cpxARuuF / cpxARddR identified a 2250 bp deletion fragment in the deletion strain, compared to 3513 bp in the parental strain. Primers cpxARF / cpxARR showed that the deletion strain could not amplify the 2172 bp cpxAR gene fragment, while the parental strain could. Primers KanF / KanR showed that the deletion strain amplified the 909 bp kanamycin gene, while the parental strain could not. PCR results are as follows: Figure 2 Sequencing and alignment of the positive amplified fragments showed 100% homology. This indicates that the cpxAR gene deletion strain of HN1570 was successfully constructed.

[0047] Example 2: Growth characteristics of Haemophilus parasuis serotype 5 cpxAR double gene deletion strain

[0048] Both the parental strain HN1570 and the deletion strain HPS5△cpxAR were simultaneously streaked onto TSA medium and incubated at 37°C. Colorless, transparent small colonies were observed in both strains. The HPS5△cpxAR colonies were similar to those of HN1570 (e.g., ...). Figure 3Gram staining and scanning electron microscopy revealed that all bacteria were Gram-negative short bacilli. The morphology of HPS5△cpxAR was similar to that of HN1570 (e.g., Figure 4 ,5).

[0049] The growth curves of strains HN1570 and HPS5△cpxAR were measured, and the results showed no significant difference in the growth characteristics of the two strains (e.g., Figure 6 This indicates that the cpxAR gene has no significant effect on the growth of HN1570.

[0050] Example 3: Virulence test of Haemophilus parasuis serotype 5 cpxAR double gene deletion strain

[0051] Forty-five clean-grade female guinea pigs, each weighing approximately 250g, were randomly divided into nine groups (four groups each of HN1570 and HPS5△cpxAR strains, and one control group), with five guinea pigs in each group. Single colonies of HN1570 and HPS5△cpxAR were inoculated with TSB and cultured overnight. Viable cell counts were performed, and the concentration was adjusted to 1.0 × 10⁻⁶. 10 The solution was diluted 10-fold to four titers, with each titer administered intraperitoneally to five guinea pigs (1 mL per pig). The control group received physiological saline. Simultaneously, the stock solution was diluted 10-fold for viable bacterial count. Guinea pig morbidity and mortality were observed for 14 consecutive days. Organ tissue smears from dead guinea pigs were collected for examination and inoculated onto TSA medium.

[0052] Observations revealed the following: the morbidity and mortality rates in the guinea pig infection experiment are shown in Table 2.

[0053] Table 2 Results of pathogenicity tests

[0054]

[0055] The results showed that HN1570 had a 1.0×10 7 Two mice in the dosage group developed the disease, with a mortality rate of 1.0 × 10⁻⁶. 8 1.0×10 9 1.0×10 10 All five birds in the dosage group developed symptoms, with obvious symptoms appearing 2 hours after infection, such as lethargy, rough coat, tremors, curling up, and huddling together. Death occurred 6 hours after infection, with a mortality rate of 1.0 × 10⁻⁶. 7 No deaths occurred in the dosage group (1.0 × 10⁻⁶). 8 Three mice in the dosage group died, with a mortality rate of 1.0 × 10⁻⁶. 9 1.0×10 10 All five guinea pigs in the dosage group died. Three days later, the surviving guinea pigs recovered from tolerance symptoms and returned to normal. HPS5ΔcpxAR was 1.0 × 10⁻⁶. 7 1.0×10 8 1.0×10 9No disease developed in any of the dosage groups (1.0×10). 10 Two animals in the dosage group developed symptoms, but no deaths occurred. The symptoms in the HPS5△cpxAR group were significantly milder than those in the HN1570 group, while the control group showed no symptoms. Short rod-shaped bacteria were observed in tissue smears from the liver, lungs, heart, blood, spleen, kidneys, and brain. After inoculation with TSA solid medium, bacteria consistent with the inoculated strains were isolated from the liver, lungs, heart, blood, spleen, kidneys, and brain.

[0056] Based on the combined virulence test results, the HPS5△cpxAR strain was determined to be a strain with extremely weak virulence, and the virulence of HPS5△cpxAR was significantly lower than that of HN1570.

[0057] Example 4: Preparation, safety, and efficacy testing of a live attenuated vaccine against Haemophilus parasuis serotype 5 cpxAR double gene deletion strain.

[0058] I. Vaccine Preparation

[0059] 1. Strains and strain propagation

[0060] Primary seed propagation

[0061] Freeze-dried strains of Haemophilus parasuis serotype 5 HN1570 and the cpxAR double gene deletion strain HPS5△cpxAR were inoculated onto TSA plates containing newborn calf serum (final concentration 5%) and NAD (final concentration 0.01%), respectively, and incubated at 37°C for 18–24 h. Typical colonies that met the requirements were selected, subcultured, and inoculated onto TSA plates containing fetal bovine serum (final concentration 5%) and NAD (final concentration 0.01%), respectively, and incubated at 37°C for 24 h. After passing the test, they were used as primary seed culture.

[0062] Secondary seed propagation

[0063] Each primary seedling was inoculated into TSB liquid medium containing newborn calf serum (final concentration 5%) and NAD (final concentration 0.01%), and cultured at 37°C in a shaker for 18–24 h. After passing the purity test, the seedlings were used as secondary seeds.

[0064] 2. Preparation of Antigen Bacterial Solution

[0065] Secondary seed cultures of strains HN1570 and HPS5△cpxAR were inoculated at 1% in TSB liquid medium containing newborn calf serum (final concentration 5%) and NAD (final concentration 0.01%), respectively, and cultured at 37°C with shaking for 18–24 h before harvesting the bacterial culture.

[0066] 3. Viable bacteria count

[0067] Viable cell counts were performed using TSA medium containing 5% newborn calf serum and 0.01% NAD, suitable for the growth of this bacterium, according to the appendix method of the current Chinese Veterinary Pharmacopoeia. Shake flask culture was used, and the viable cell concentration and viable cell content of the strain were both within 1.0 × 10⁻⁶. 9 CFU / mL or higher.

[0068] 4. Inactivation of bacterial solution

[0069] Add 0.2% formaldehyde solution to the culture medium of Haemophilus parasuis strain HN1570 and inactivate it at 37°C for 48 hours. Inoculate 0.2 mL of the inactivated HN1570 strain onto a suitable TSA plate (containing 5% newborn calf serum and 0.01% NAD) and incubate at 37°C for 48 hours. No bacterial growth indicates successful inactivation. HPS5ΔcpxAR does not require inactivation.

[0070] 5. Antigen Concentration

[0071] The qualified HPS5△cpxAR strain bacterial solutions were concentrated using an ultrafiltration system. Based on the viable cell count before concentration, the bacterial antigen concentration was adjusted to 1.0 × 10⁻⁶ using sterile physiological saline. 10 CFU / mL.

[0072] 6. Adjuvant preparation

[0073] The water-in-oil-in-water adjuvant Summit-S550 for livestock and poultry was bottled, sterilized at 121°C for 30 minutes, and stored at room temperature for later use.

[0074] 7. Vaccine preparation

[0075] Add 30% of Summit-S550 (a water-in-oil-in-water adjuvant for livestock and poultry) to the concentrated antigen solution, stir thoroughly, and then package to obtain a live attenuated vaccine against Haemophilus parasuis infection using the water-in-oil-in-water adjuvant for livestock and poultry. The final vaccine contains a total live bacterial count of 1.0 × 10⁻⁶ strains before inactivation. 9 CFU / mL.

[0076] II. Vaccine efficacy trials

[0077] Two hundred and forty clean-grade female guinea pigs weighing approximately 250g were selected and divided into three groups: an HN1570 inactivated vaccine group, an HPS5△cpxAR live vaccine group, and a control group, with 16 groups in each group (n=5 per group). Each vaccine group received 1mL of vaccine. Three weeks later, the vaccine groups received a second immunization with the same dose of vaccine, while the control group received saline. Eight days after the second immunization, the vaccine-immunized groups and the control group received 10 mL of vaccine. 9 CFU-containing Haemophilus parasuis strains 1-15 and untyped strains were challenged intraperitoneally. Clinical manifestations, morbidity, and mortality were observed in each group of guinea pigs for two weeks. Bacterial isolation and culture were performed on the lungs and other organs of the deceased pigs.

[0078] Observations revealed that both the HN1570 inactivated vaccine group and the HPS5△cpxAR live vaccine group showed good protective efficacy against different serotype standard strains, with no significant difference between the two vaccine groups. Guinea pigs in the control group showed varying degrees of morbidity and mortality rates. The results of the vaccine immunization challenge are shown in Table 3.

[0079] Table 3 Results of challenge tests on different serotype strains

[0080]

[0081] The results of the above immune challenge test show that two immunizations with a 1mL dose of the HPS5△cpxAR attenuated live vaccine provide varying degrees of protection against Haemophilus parasuis strains 1-15 and untyped strains, indicating that the attenuated live vaccine prepared by the strain of this invention has good cross-protection against all Haemophilus parasuis infections.

[0082] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of Haemophilus parasuis with double gene deletion, characterized by: The Haemophilus parasuis is Haemophilus parasuis ( Haemophilus parasuis The double-gene deletion strain HPS5△cpxAR, which contains the deletions of cpxA and cpxR genes of the clinical isolate HN1570 of serum type 5, has the accession number CGMCC NO: 45596, the deposit date is June 16, 2023, the depositary institution is China General Microbiological Culture Collection Center, the depositary address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A method for constructing a double-gene-deleted Haemophilus parasuis as described in claim 1, characterized in that: Includes the following steps: Step 1: Primer Design and Synthesis: Referring to the complete genome sequence No. CP001321 of Haemophilus parasuis serotype 5 strain SH0165 reported in GenBank, primers cpxARuF / cpxARuR and cpxARdF / cpxARdR, containing restriction enzyme sites and the DNA uptake signal sequence USS, were designed to amplify the upstream and downstream homologous arms of cpxAR. Primers KanF / KanR were designed based on the pSHK3-Kan sequence to amplify the Kan gene. The primer sequences used are as follows: cpxARuF:CGGAATTCACCGCTTGTAAGTGGTATTTCTTAGCAAT cpxARuR: TATTTTTATCTTGTGCAATGAGCTCTATTTCCCTATTCCAT cpxARdF: TAATCAGAATTGGTTAATTGTATCAATATATTGTCAATATC cpxARdR:CGGGATCCACAAGCGGTAGCCGCAAACATACTTTGAC KanF:CATTGCACAAGATAAAAATATAT KanR:TTATTCCACCCACAACGGCAA Step 2: Cloning and Recombination of Genes and Construction of Suicide Plasmids: Using the genome of Haemophilus parasuis serotype 5 clinical isolate HN1570 as a template, the upstream and downstream fragments of the cpxAR gene were amplified using primer pairs cpxARuF / cpxARuR and cpxARdF / cpxARdR, respectively; the kanamycin gene was amplified from the pSHK3 plasmid using primers KanF / KanR. Using the principle of overlap extension PCR amplification, the upstream and downstream fragments of the cpxAR gene and the kanamycin gene were ligated by overlap extension PCR with primers cpxARuF / cpxARdR to form the overlap fragment UKD; the UKD fragment was digested with BamHI and EcoRI and ligated into pK18mobsacB to construct the suicide plasmid pKUKD. Step 3: Construction of double gene deletion strains: Following the natural transformation method, a large amount of recombinant plasmid pKUKD was extracted and its concentration was determined; Transformants were selected, and their genomes were extracted. The UKD sequence, cpxAR gene, and Kan gene were amplified using primers cpxARuuF / cpxARddR, cpxARF / cpxARR, and KanF / KanR, respectively. Electrophoresis was used to detect the size and presence of the target fragment to identify whether the cpxAR deletion strain was successfully constructed, and sequencing was used for verification.

3. The method for constructing double-gene-deleted Haemophilus parasuis according to claim 2, characterized in that: In step two, using the genome of Haemophilus parasuis strain HN1570 as a template, the upstream 683bp and downstream 658bp fragments of the cpxAR gene were amplified using primer pairs cpxARuF / cpxARuR and cpxARdF / cpxARdR; and the 909bp kanamycin gene was amplified from the pSHK3 plasmid using primers KanF / KanR.

4. The method for constructing double-gene-deleted Haemophilus parasuis according to claim 2, characterized in that: In step three, the recombinant plasmid pKUKD was extracted using the natural transformation method. The specific steps were as follows: HN1570 single colonies were picked and placed in 5 mL of TSB and cultured at 37°C until the OD600 value was 0.

8. 20 μL of bacterial culture was taken and 20 μL of 8 mmol / L cAMP was added. The mixture was mixed and reacted for 10 min. 2 μg of recombinant plasmid pKUKD was added and mixed. After reacting for 10 min, the mixture was added to TSA, spread evenly, and incubated at 37°C for 5 h. The bacteria were washed off with TSB and transferred to TSA solid medium containing Kan. The medium was then cultured at 37°C for 24–48 h.

5. The application of the double-gene-deleted Haemophilus parasuis as described in claim 1 in the preparation of a live attenuated Haemophilus parasuis vaccine.

6. The application according to claim 5, characterized in that: The content of Haemophilus parasuis HPS5 ΔcpxAR in the attenuated live vaccine is 1×10⁻⁶. 10 CFU / mL.

7. The application according to claim 5, characterized in that: The preparation method of the Haemophilus parasuis attenuated live vaccine is as follows: after the Haemophilus parasuis is successively proliferated and concentrated, an Haemophilus parasuis antigen solution is obtained, and an immune adjuvant is added to the antigen solution to obtain the vaccine.

8. The application according to claim 7, characterized in that: The immune adjuvant is Summit S550, an oil-in-water adjuvant for livestock and poultry.

9. The application according to claim 5, characterized in that: The preparation method of the Haemophilus parasuis attenuated live vaccine is as follows: (1) Proliferation: The double gene deletion strain HPS5△cpxAR of Haemophilus parasuis was propagated and cultured to obtain 8 strains of Haemophilus parasuis bacterial solution, and viable bacteria were counted separately. (2) Concentration: The Haemophilus parasuis double gene deletion strain HPS5△cpxAR was concentrated using an ultrafiltration device. Based on the viable cell count, the final concentration was adjusted to 1.0 × 10⁻⁶ with sterile physiological saline. 10 CFU / mL; (3) Preparation of vaccine: The HPS5△cpxAR antigen solution of Haemophilus parasuis double gene deletion strain obtained after proliferation and concentration is mixed with 30% of the final volume of livestock and poultry water-in-oil-in-water adjuvant to prepare a live attenuated Haemophilus parasuis vaccine.

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