Pasteurella multocida waaF gene and application thereof in attenuated live vaccine
By screening and constructing a Pasteurella multocida waaF gene deletion strain and creating a ΔwaaF mutant strain, the shortcomings of existing vaccines have been addressed, resulting in a genetically engineered attenuated live vaccine with high safety and immunoprotective efficacy.
Patent Information
- Application Number
- CN202511421558.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-12-30
AI Technical Summary
Existing Pasteurella multocida oil emulsion inactivated vaccines have shortcomings such as large immunization doses, significant side effects, and weak cross-protection, necessitating the development of safer and more efficient genetically engineered attenuated live vaccines.
By screening for the waaF gene in Pasteurella multocida, a ΔwaaF mutant strain was constructed. Then, a Pasteurella multocida strain lacking the waaF gene was constructed using the suicide plasmid-mediated homologous recombination method to form an attenuated live vaccine. The ΔwaaF mutant strain was used to conduct an immunization protection test on duck flocks.
The ΔwaaF mutant strain is highly attenuated in ducks, providing 81.25% oral immunoprotection and 75% intramuscular immunoprotection, significantly reducing bacterial pathogenicity and exhibiting high safety and immunoprotective effects.
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Figure CN121227751A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, specifically to the waaF gene of Pasteurella multocida and its application in live attenuated vaccines. Background Technology
[0002] Pasteurella multocida is an important pathogenic bacterium in livestock and poultry, causing highly pathogenic diseases such as fowl cholera. Currently, the prevention and control of fowl cholera mainly relies on oil-emulsion inactivated vaccines, but these vaccines have drawbacks such as high immunization doses, significant side effects, and weak cross-protection. Therefore, developing genetically engineered attenuated live vaccines has become an ideal alternative strategy, offering advantages such as a clearly defined genetic background, oral or nasal administration, induction of balanced humoral and mucosal immune responses, and strong cross-protection.
[0003] Screening and discovering more virulence-related genes associated with attenuated *Pasteurella multocida* is crucial for constructing live attenuated vaccines with diverse gene combinations and mutations. First, targeting the deletion of two or more virulence genes can effectively reduce the probability of virulence reversion in vaccine strains, improving vaccine safety. Second, identifying multiple virulence genes in *Pasteurella multocida* and constructing corresponding deletion mutants can help screen vaccines with superior immunoprotective efficacy and provide a rich genetic basis and selection for developing different types of live attenuated vaccines. Currently reported attenuation target genes include arcA, gatA, and grcA. Deletion of these genes can significantly weaken bacterial virulence and, to some extent, stimulate a protective immune response in the host. For example, a *Pasteurella multocida* double-gene mutant strain lacking arcA and gatA showed high attenuation in ducks while maintaining a certain degree of in vivo colonization ability and immunoprotective effect, although its protective effect was limited. Similarly, a mutant strain lacking the grcA gene showed at least a 10% reduction in virulence. 5 The oral immunization with this strain provided 87.5% protection against a lethal dose of Pasteurella multocida. These successful cases have laid the foundation for constructing an ideal attenuated Pasteurella multocida vaccine. However, most existing attenuated strains still have some limitations in vaccine application. For example, further improvements are needed to enhance the immunoprotective rate, and to construct a gene combination deletion vaccine with better safety, or multiple gene deletions may be required to achieve ideal safety and efficacy. Therefore, continuously discovering new virulence genes and verifying their attenuation effects is of great significance for enriching the research and development pathways of live attenuated vaccines. Summary of the Invention
[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a *Pasteurella multocida* waaF gene and its application in a live attenuated vaccine. The *Pasteurella multocida* waaF gene was screened, and its virulence was confirmed by constructing a mutant strain. Immunoprotection experiments on duck flocks confirmed the effectiveness of the *Pasteurella multocida* gene-deleted strain Δ. waa F has high safety and can be used to prevent Pasteurella multocida infection in ducks.
[0005] The objective of this invention is achieved through the following technical solution: the waaF gene of Pasteurella multocida, the sequence of which is shown in SEQ ID NO: 1.
[0006] The sequence of the waaF gene is as follows:
[0007]
[0008] The present invention also provides an application of the above-mentioned Pasteurella multocida waaF gene for attenuating Pasteurella multocida.
[0009] Furthermore, the method of application includes: constructing a multi-kill Pasteurella multocida lacking the waaF gene by suicide plasmid-mediated homologous recombination.
[0010] Furthermore, the method of application specifically includes:
[0011] S1. Construct a suicide plasmid that lacks the waaF gene;
[0012] S2. The suicide plasmid constructed in step S1 is transferred into competent E. coli and then conjugated with Pasteurella multocida for transfer, and positive colonies are screened.
[0013] S3. Pasteurella multocida lacking the waaF gene was obtained by PCR screening.
[0014] Furthermore, in step S1, the method for constructing a suicide plasmid lacking the waaF gene includes:
[0015] 1) Based on the sequence information of Pasteurella multocida and plasmid, primers were designed to amplify the upstream and downstream homologous arms of the waaF gene, as well as the kanamycin resistance gene fragment between the homologous arms. The resistance gene fragment and the upstream and downstream homologous arm fragments of the waaF gene each have a repeat sequence of 15±2bp.
[0016] 2) Using the whole genome of Pasteurella multocida as a template, amplify the upstream and downstream homologous arms of the waaF gene, and use plasmid as a template to amplify the kanamycin resistance gene;
[0017] 3) The product obtained in step 2) is amplified by PCR using primers and high-fidelity enzyme to obtain the fusion fragment;
[0018] 4) After digesting the plasmid with enzymes, ligate it with the fusion fragment, transform it into competent E. coli cells, and perform PCR identification to obtain a suicide plasmid with a positive deletion of the waaF gene.
[0019] Furthermore, the primers involved in the suicide plasmid-mediated homologous recombination method are shown in SEQ ID NO: 2 to SEQ ID NO: 11.
[0020] The present invention also provides a multi-kill Pasteurella multocida strain lacking the waaF gene prepared by the above-mentioned application.
[0021] The present invention also provides an application of the above-mentioned Pasteurella multocida lacking the waaF gene as a live attenuated vaccine for poultry.
[0022] The principle of this invention is as follows:
[0023] Among numerous virulence factors, the integrity of the inner core of bacterial lipopolysaccharide (LPS) is a key factor determining the outer membrane barrier function and complement resistance of *Pasteurella multocida*, and is closely related to the virulence and spread of the bacteria within the host. This invention focuses on the key gene waaF in the LPS inner core synthesis pathway. waaF encodes heptosyltransferase II, a secondary hexosyltransferase for the LPS inner core oligosaccharide. Its deletion leads to the synthesis of truncated "deep-rough" LPS, preventing the outer core region from extending. This significantly increases bacterial outer membrane permeability, making it highly sensitive to serum complement, thereby greatly reducing the bacteria's in vivo pathogenicity; simultaneously, more antigenic epitopes are exposed on the outer membrane, which is beneficial for inducing a protective immune response in the host. Based on the above mechanism, this invention uses the highly pathogenic locally isolated duck-derived *Pasteurella multocida* strain PM0818 as a parent and constructs a Δ... waa The F mutant strain was tested in ducklings via oral and intramuscular immunization to assess the degree of virulence reduction and immunoprotective potential. Results showed that Δ waa The F mutant strain was highly attenuated in ducks (virulence reduced by at least 10%). 6 (times), and, against lethal challenge of Pasteurella multocida, its oral and intramuscular immunization can provide 81.25% and 75% immune protection against lethal challenge of Pasteurella multocida, respectively.
[0024] The beneficial effects of this invention are:
[0025] Δ waa The F gene mutant strain is highly attenuated in ducks and exhibits a certain level of in vivo colonization, demonstrating its potential as a live attenuated vaccine. Immunization with this mutant strain via oral and intramuscular administration not only does not affect the production performance of duck flocks but also provides 81.25% and 75% immune protection against Pasteurella multocida challenge doses, respectively. Compared with the attenuated ΔgrcA strain reported in existing technologies (with approximately 10% reduction in virulence), this mutant is significantly more effective. 5 Compared to (duplicate, with an immunization protection rate of 87.5%), Δ waa The F mutant strain exhibited a greater reduction in virulence, at the cost of only a slightly lower rate of immunoprotection, demonstrating superior safety and a balanced immune response. This finding highlights that the waaF gene deletion can serve as an innovative target for developing a live attenuated vaccine against Pasteurella multocida, providing a novel strategy and theoretical basis for fowl cholera control that differs from existing technologies. Attached Figure Description
[0026] Figure 1For the identification of waaF gene deletion strains; lane M is the DL5000 marker. Lanes 1-5 are for waaF gene amplification, lanes 6-10 are for amplification with PM0818 specific primers, and lanes 11-15 are for amplification of upstream and downstream homologous arms of the waaF gene.
[0027] Figure 2 For monitoring the duck's body temperature;
[0028] Figure 3 For monitoring the weight of ducks;
[0029] Figure 4 The survival rate of the ΔwaaF vaccine strain after challenge to the parent strain;
[0030] Figure 5 The bacterial load of the ΔwaaF vaccine strain in the challenge parent strain. Detailed Implementation
[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings, but the scope of protection of the present invention is not limited to the following description.
[0032] Example 1: Pasteurella multocida Δ waa Construction of F deletion strains
[0033] 1. Primer design
[0034] According to the genome accession number of PM0818 (P. multocida strain DY120818) published by NCBI...
[0035] Based on the genome sequence of LUCZ01000000 and the plasmid pCZ4 sequence, two primer pairs, waaF-up-F / R and waaF-down-F / R, were designed to amplify the upstream and downstream homologous arms of the waaF gene, respectively. The upstream and downstream homologous arm fragments of the waaF gene each have approximately 15 bp of repetitive sequences with the resistance gene fragment. Furthermore, the kanamycin resistance gene fragment between the homologous arms was amplified using waaF-kan-F / R. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific primer names and sequences are shown in Table 1 below.
[0036] Table 1 Primers for constructing the ΔwaaF mutant strain
[0037]
[0038] 2. Amplification and fusion of upstream and downstream homologous arms of the waaF gene and the kanamycin resistance gene.
[0039] Using the PM0818 whole genome as a template, the upstream and downstream homologous arms of waaF were amplified, and the kanamycin resistance gene was amplified using plasmid pCZ4 as a template. The amplification system and procedure were as follows: 1 μL template DNA (bacterial genome), 30 μL 2×PrimeSTARMax (Takara Bio Inc.), 2 μL upstream primer, 2 μL downstream primer, and 25 μL ddH2O. The amplification conditions were: denaturation at 98℃ for 2 min, followed by cycling with the parameters 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 10 s. After 30 cycles, an extension at 72℃ for 5 min was performed. The amplified PCR products were analyzed by 1% agarose gel electrophoresis and then purified and recovered using a kit. The purified and recovered upstream and downstream homologous arms of the waaF gene and the kanamycin resistance gene fragment were concentrated using a Nanodrop 2000. Based on the fragment length and concentration, they were fused at a 1:1:1 ratio. PCR amplification was performed using 2 μL of the fusion fragment as a template, followed by PCR amplification using primers waaF-up-F / waaF-down-R and LATaq polymerase. The amplification conditions were: denaturation at 94℃ for 5 min, followed by cycling with the parameters 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 1 min. After 30 cycles, extension was performed at 72℃ for 5 min. The amplified fragments were analyzed by agarose gel electrophoresis and recovered using a kit.
[0040] 3. Ligation of the fusion fragment with the suicide plasmid pRE112
[0041] 1) Restriction digestion of suicide plasmid pRE112: in LB(Cm + Resuscitate *E. coli* DH5αλpir containing the suicide plasmid pRE112 on agar plates, and collect a single colony in 200 mL LB (Cm + The plasmid was cultured in liquid medium at 37°C in a shaker for 12 hours. Plasmids were extracted using a plasmid mini-extraction kit, and the concentration was determined using Nanodrop2000 before enzyme digestion.
[0042] 2) Ligation of plasmids and fragments: After the enzyme digestion system was placed in a 37°C metal bath for 3 hours, it was recovered using a DNA purification and recovery kit. The concentration was determined using Nanodrop 2000. After determining the concentration and size of the plasmid and fragment, they were placed in a 16°C metal bath for 18 hours according to the ratio. The ligation system consisted of 8 μL of enzyme digestion product + fusion fragment, 1 μL of T4 ligase, and 1 μL of T4 buffer.
[0043] 4. Preparation of competent Escherichia coli cells
[0044] In LB(Cm) + Resuscitate Escherichia coli DH5α and DH5αλpir on agar plates and pick single colonies to inoculate into 5 mL LB (Cm +After incubating in liquid medium at 37°C on a shaker for 12 hours, the culture was inoculated into 100 mL of LB liquid medium at a ratio of 1:100 and incubated on a shaker at 37°C until OD reached. 600 =0.6~0.8; Aliquot the bacterial culture into 50mL centrifuge tubes and incubate on ice for 10min; Centrifuge at 5000r / min for 10min at 4℃, and discard the supernatant; Add 20mL of CaCl2 (0.1mol / L) solution to each centrifuge tube to resuspend the bacterial cells, and incubate on ice for 10min; Centrifuge at 5000r / min for 10min at 4℃, and discard the supernatant; Add 800μL of CaCl2 (0.1mol / L) solution to each centrifuge tube to resuspend the bacterial cells, and incubate overnight at 4℃; The next day, add 15% sterile glycerol, mix well, and aliquot into 100μL tubes, and store at -80℃.
[0045] 5. Transformation and identification of the connection system
[0046] Remove DH5αλpir competent cells from a -80℃ freezer, add 10 μL of ligation product, and incubate on ice for 20 min; heat shock in a 42℃ metal bath for 90 s, then incubate on ice for 2 min; add 200 μL of LB liquid medium and incubate at 37℃ in a shaker for 2–3 h; spread 100 μL of the bacterial culture onto LB (Cm + The colonies on the resistant plate (Kan: 50 μg / mL) were incubated at 37℃ for 24 h. After expanding the colonies on the resistant plate, PCR amplification and identification were performed using the amplification primers waaF-up-F and waaF-down-R.
[0047] After agarose gel electrophoresis analysis, plasmids from positive colonies were extracted using a plasmid extraction kit. Amplification was performed using primers waaF-up-F and waaF-down-R, and the amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The correctly sequenced plasmids were transformed into E. coli SM10λpir competent cells, and the cultured colonies were then identified by PCR amplification using primers waaF-up-F and waaF-down-R. Colonies with positive identification results were expanded and named pRE112-waaF-kan.
[0048] 6. Identification of conjugation transfer and ΔwaaF gene deletion strains
[0049] 1) Conjugation transfer: Resuscitate recipient bacteria PM0818 and donor bacteria pRE112-waaF-kan, pick single colonies and culture them in 5 mL of BHI or LB (DAP: 50 μg / mL) liquid medium at 37°C with shaking for 12 h, then expand the culture at a 1:100 ratio to BHI or LB (DAP: 50 μg / mL) liquid medium and culture at 37°C with shaking until OD. 600=0.6~0.8. Mix the recipient bacteria and donor bacteria according to the specified ratio, with a total volume of 5 mL. Add the mixed bacterial solution and 5 mL of MgSO4 (0.1 mol / L) to a sterilized 50 mL centrifuge tube, filter using a sterile syringe and a sterile filter (including a filter membrane), and then plate the filtered membrane onto a TSA (DAP: 50 μg / mL) plate and incubate at 37°C for 12 h. After washing the filter membrane with 10 mL of MgSO4 (0.1 mol / L), aspirate 100 μL of the bacterial solution and spread it onto a BHI (Kan: 50 μg / mL) plate and incubate at 37°C for 24 h.
[0050] 2) Identification of waaF gene deletion strains: Single colonies from the above plates were picked and cultured amplified for PCR amplification and identification. The primers used for identification were waaF-F / R, 0818-F / R, and waaF-up-F / waaF-down-R. The identification results are as follows: Figure 1 .
[0051] Example 2: Virulence (LD50) of the ΔwaaF deletion strain 50 ) Measurement
[0052] Using Sichuan Muscovy ducks as an animal model, the LD50 of PM0818 and PM0818ΔwaaF in seven-day-old ducks was determined via oral and intramuscular injection. 50 , through LD 50 The virulence of the gene-deleted strain was compared with that of the wild-type strain. The results showed that the LD50 of PM0818ΔwaaF after infection via the muscle route was... 50 About 10 higher than PM0818 7 Times; LD50 of PM0818ΔwaaF after oral infection 50 About 10 cm taller than wild plants 6 Times (Table 2, Table 3).
[0053] In summary, the deletion of the waaF gene reduces the virulence of PM0818 in ducks by at least 10%. 6 The result of the multiple is that waaF is the virulence gene of Pasteurella multocida, therefore, the vaccine strain we constructed can be used for evaluation of immune protection.
[0054] Table 2 PM0818ΔwaaF LD50 after intramuscular injection in ducklings 50 Measurement
[0055]
[0056] Table 3 shows the LD50 of PM0818ΔwaaF after oral challenge in ducklings. 50 Measurement
[0057]
[0058] Example 3: Safety testing of Pasteurella multocida vaccine strain
[0059] 10 were administered orally and intramuscularly, respectively. 9 CFU, 10 6 Seven-day-old ducks were immunized with CFU PM0818ΔwaaF, and their body temperature and weight were measured for 14 consecutive days. A PBS control group was set up to record the health status of these ducks.
[0060] 1. Changes in duck body temperature
[0061] Body temperature monitoring showed that after PM0818ΔwaaF infection, the body temperature of the immunized group rose slightly on days 1-2, and the duck joints swelled slightly. After day 2, the body temperature of both the immunized and PBS groups stabilized with no significant difference. Figure 2 ).
[0062] 2. Changes in duck weight
[0063] Weight monitoring showed that the weight of ducks immunized with PM0818ΔwaaF increased from 0.2 kg to 0.7 kg within 14 days, similar to the PBS control group. Figure 3 In summary, the results of body temperature and weight monitoring indicate that PM0818ΔwaaF has a higher immunogenicity compared to the PBS control group.
[0064] Example 4: Determination of the immunoprotective effect of the ΔwaaF vaccine strain
[0065] 1) Immunoprotective efficacy determination: 120 one-day-old ducks purchased from the hatchery were randomly divided into three groups: an oral immunization group, an intramuscular immunization group, and a control group, with 40 ducks in each group. The ducks were allowed to acclimatize to the environment for one week. The oral immunization group received 500 μL of a solution containing 10... 9 CFU ΔwaaF PM0818 was administered orally to ducklings, with a booster immunization at the same dose and route 14 days later; the intramuscular immunization group received 100uL of a solution containing 10... 7 CFU ΔwaaF PM0818 was administered to ducklings via intramuscular injection, followed by a booster immunization 14 days later using the same dose and route. Two weeks after the second immunization, ducklings were treated with a 100-fold LD50 solution. 50 Ducklings were challenged with PM0818, and mortality rates were recorded after challenge. Results showed that oral immunization with PM0818ΔwaaF cleared PM0818 from ducks and provided 81.25% immune protection; intramuscular immunization with PM0818ΔwaaF cleared PM0818 from ducks and provided 75% immune protection. Figure 4 This indicates that the attenuated strain PM0818ΔwaaF that we constructed can be used to prevent infection with Pasteurella multocida in birds.
[0066] 2) Bacterial clearance effect test: Following the immunization procedure in 1), 12 hours after challenge, ducklings were randomly selected from each group and euthanized according to regulations. Heart blood, liver, spleen, and lungs were collected sequentially. These tissues were placed in disposable sterile sampling bags, and an appropriate amount of PBS was added for grinding. The grinding solution was diluted 10-fold and 100-fold and then dropped onto plates. The plates were incubated overnight at 37°C. The next day, the number of colonies on the plates was counted, the number of bacteria per gram of tissue was calculated, and the data were processed using GraphPadPrism5 software. The results showed that compared with the PBS control group, the bacterial clearance effect of the ΔwaaF immunization group was significantly higher. Figure 5 This is consistent with the results of the immune protection test.
[0067] In summary, our immunization protection experiments on ducklings have demonstrated that the ΔwaaF mutant strain we constructed can be used safely to prevent infection with avian Pasteurella multocida. Furthermore, the study confirms that the deletion of virulence factor-related genes is an ideal strategy for constructing Pasteurella multocida vaccine strains, laying the foundation for the development of novel Pasteurella vaccines.
[0068] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.
Claims
1. A Pasteurella multocida waaF gene characterized in that, The sequence of the waaF gene of Pasteurella multocida is shown as SEQ ID NO:
1.
2. Use of the waaF gene of Pasteurella multocida according to claim 1 for attenuating Pasteurella multocida.
3. Use according to claim 2, characterized in that, The method of the use comprises: constructing the Pasteurella multocida with the deleted waaF gene by the method of homologous recombination mediated by the suicide plasmid.
4. Use according to claim 3, characterized in that, The method of the use specifically comprises: S1, constructing the suicide plasmid with the deleted waaF gene; S2, transforming the suicide plasmid constructed in step S1 into the competent Escherichia coli and then performing conjugation transfer with the Pasteurella multocida to screen the positive colonies; S3, screening the Pasteurella multocida with the deleted waaF gene by PCR.
5. Use according to claim 4, characterized in that, In step S1, the method of constructing the suicide plasmid with the deleted waaF gene comprises: 1) according to the sequence information of the Pasteurella multocida and the plasmid, designing primers to respectively amplify the upstream and downstream homologous arms of the waaF gene and the kanamycin resistance gene fragment between the homologous arms, and the resistance gene fragment and the upstream and downstream homologous arm fragments of the waaF gene have 15±2 bp of repeated sequences; 2) using the whole genome of the Pasteurella multocida as a template to amplify the upstream and downstream homologous arms of the waaF gene, and using the plasmid as a template to amplify the kanamycin resistance gene; 3) using primers and high-fidelity enzymes to perform PCR amplification on the products obtained in step 2) to obtain a fusion fragment; 4) performing enzyme digestion on the plasmid, then performing ligation with the fusion fragment, transforming the competent Escherichia coli cells, and performing PCR identification to obtain the positive suicide plasmid with the deleted waaF gene.
6. Use according to claim 5, characterized in that, The primers involved in the method of homologous recombination mediated by the suicide plasmid are shown as SEQ ID NO: 2-SEQ ID NO:
11.
7. The Pasteurella multocida with the deleted waaF gene prepared by the use according to any one of claims 3-6.
8. Use of the Pasteurella multocida with the deleted waaF gene according to claim 7 as an attenuated live vaccine for poultry.