CN112680391A - APEC double-gene rfaH and hfq deletion strain and attenuated vaccine - Google Patents

Abstract

The invention belongs to the field of microorganisms, and discloses an APEC double-gene rfaH and hfq deletion strain and an attenuated vaccine, wherein the construction method of the APEC double-gene deletion strain is a Red homologous recombination method, and the double genes of rfaH and hfq are knocked out to obtain avian pathogenic escherichia coli FY26 delta rfaH/hfq which is preserved in China Center for Type Culture Collection (CCTCC) M2020612. The deletion strain has good biological safety and immunogenicity and weak toxicity, and can be used for preparing avian pathogenic escherichia coli attenuated vaccines and preventing avian pathogenic escherichia coli infection. The attenuated vaccine prepared by the avian pathogenic escherichia coli FY26 delta rfaH/hfq has immune protection power against avian pathogenic escherichia coli infection and good virus attacking immune protection effect.

Description

APEC double-gene rfaH and hfq deletion strain and attenuated vaccine

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an APEC double-gene rfaH and hfq deletion strain and an attenuated vaccine prepared from the APEC double-gene rfaH and hfq deletion strain.

Background

Avian Pathogenic Escherichia Coli (APEC) is an important bacterial pathogen that compromises the poultry industry, and belongs to the enteropathogenic escherichia coli (ExPEC). Avian pathogenic escherichia coli mainly infects poultry through respiratory tract, causes multi-system mixed infection of the poultry, even causes acute death of the poultry, causes serious loss to the poultry industry, and poses a threat to food safety. The enteropathogenic escherichia coli has a zoonosis potential, and pathogenic subtypes of the enteropathogenic escherichia coli are named, namely Urethra Pathogenic Escherichia Coli (UPEC), septicemia escherichia coli (SEPEC), Neonatal Meningitis Escherichia Coli (NMEC) and avian pathogenic escherichia coli. The evolution relation of the avian pathogenic escherichia coli and the human source enteropathogenic escherichia coli clinical isolate is relatively close, the avian pathogenic escherichia coli clinical isolate has very similar genome and virulence gene characteristics, and the conventional molecular epidemiology detection method cannot obviously distinguish the enteropathogenic escherichia coli isolate from different host sources. The avian pathogenic escherichia coli has the potential of infecting human beings through avian meat products, and researches show that the avian pathogenic escherichia coli carried by avian food can be an important source of the human pathogenic escherichia coli outside intestinal tracts, so that the pathogenicity, drug resistance and transmission mechanism of the avian pathogenic escherichia coli are deeply explored, and the avian pathogenic escherichia coli is very important to public health safety.

In recent years, the use of antibiotics in large quantities has led to a gradual increase in the resistance of avian pathogenic E.coli to antibiotics, and the strains of avian pathogenic E.coli of the dominant ST class exhibit a trend toward both high pathogenicity and broad resistance, these ST types including ST95, ST131, ST48, ST117, ST162, ST501, ST648 and ST2085, among others. The development of avian pathogenic Escherichia coli vaccine provides a new means for preventing and controlling avian Escherichia coli diseases. Although the traditional inactivated vaccine and subunit vaccine are continuously used, the immune effect is poor, and the immune path is inconvenient and gradually replaced by the attenuated live vaccine. A key factor for restricting the development of attenuated vaccines is the lack of vaccine strains with good immunogenicity and low toxicity. Therefore, screening suitable virulence genes, weakening the virulence of the strain and attenuating the virulence of the strain are the techniques for obtaining vaccine strains and are of great interest.

Disclosure of Invention

In view of the above, the present invention aims to provide an Avian Pathogenic Escherichia Coli (APEC) double-gene rfaH and hfq deletion strain with good immunogenicity and high safety and an attenuated vaccine prepared from the same, so as to achieve the effect of safely preventing avian colibacillosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an APEC double-gene rfaH and hfq deletion strain, wherein the APEC double-gene rfaH and hfq deletion strain is avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq which is preserved in China Center for Type Culture Collection (CCTCC) M2020612. The Escherichia coli is classified and named as Escherichia coli, the preservation address is Wuhan university in Wuhan, China, the postal code 430072, and the preservation date is 2020, 10 months and 20 days.

Further, the construction method of the APEC double-gene rfaH and hfq deletion strain comprises the following steps: and knocking out the rfaH and hfq genes in the avian pathogenic escherichia coli FY26 one by adopting a Red homologous recombination method to obtain the avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq.

Furthermore, the invention also provides application of the APEC double-gene rfaH and hfq deletion strain in preparing avian pathogenic escherichia coli attenuated vaccines for preventing and treating avian colibacillosis.

Furthermore, the invention also provides an avian pathogenic escherichia coli attenuated vaccine prepared from the APEC double-gene rfaH and hfq deletion strain.

Compared with the prior art, the APEC double-gene rfaH and hfq deletion strain constructed by the invention can obviously reduce the pathogenicity of avian pathogenic escherichia coli, has good immunoprotection and biological safety, does not contain resistance markers, and has wide market application prospect when being used for preparing avian pathogenic escherichia coli attenuated vaccines for preventing and treating avian colibacillosis.

Drawings

FIG. 1 is a graph comparing the adhesion of APEC double-gene rfaH and hfq-deleted strain FY26 Δ rfaH/hfq, APEC wild strain FY26 and single-gene-deleted strain FY26 Δ rfaH to chicken embryo fibroblasts (DF-1 cells);

FIG. 2 is a graph comparing the colonization ability of APEC double-gene rfaH and hfq-deleted strain FY26 delta rfaH/hfq, APEC wild strain FY26 and single-gene-deleted strain FY26 delta rfaH to the lung of chicks;

FIG. 3 shows the colonization ability of APEC double-gene rfaH and hfq-deleted strain FY26 delta rfaH/hfq, APEC wild strain FY26 and single-gene-deleted strain FY26 delta rfaH on the lungs of young ducks;

FIG. 4 is a graph showing the result of antibody titer determination after immunization of APEC double-gene rfaH and hfq-deleted strain FY26 Δ rfaH/hfq.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only illustrative of the present invention and should not be taken as limiting the scope of the claims. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Example 1 construction of APEC double Gene rfaH and hfq deleted Strain (avian pathogenic Escherichia coli rfaH and hfq double Gene deleted Strain)

The avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain is derived from a wild avian pathogenic escherichia coli virulent strain which is named FY26 and is separated from chickens suffering from the colibacillosis. (Zhuge X, Sun Y, Jiang M, Wang J, Tang F, Xue F, Ren J, Zhu W, Dai J. acetate metabolism retrieval of avian pathogenic Escherichia coli intracellular promotion with molecular cloning. Veterimental research.2019Dec 1; 50(1):31.) the invention mainly adopts Red homologous recombination method to knock out the rfaH and hfq double genes in the avian pathogenic Escherichia coli one by one, so as to construct a rfaH and hfq double-gene deletion avian pathogenic Escherichia coli.

The sequence of the deleted rfaH gene is shown in SEQ ID NO. 1.

The sequence of the deleted hfq gene is shown in SEQ ID NO. 2.

1. Main culture medium, reagent and instrument

The bacterial culture medium comprises LB culture medium, SOC culture medium and PBS buffer solution.

The reagents required include: DNA Gel Purification Kit (Takara), 2 XPCR PreMix (Biotech, Nanjing Novowed), inducer IPTG (Invitrogen), and antibiotic (Invitrogen).

The related apparatus comprises: an electric rotary instrument (MicroPulser, Bio-Rad) microplate reader (multiskanGO, BIO-RAD), a spectrophotometer (SmartSpec Plus, Thermo Scientific), and a gel imaging system (ChemiDoc MP, BIO-RAD).

2. Gene deletion procedure

Firstly, constructing a rfaH gene deletion strain of a strain FY26 by a Red homologous recombination method, preparing linear targeting DNA, amplifying DNA targeting fragments (shown in table 1) by using a pKD4 plasmid as a template through a primer rfaH-P1 (the gene sequence is shown in SEQ ID NO. 3) and a rfaH-P2 (the gene sequence is shown in SEQ ID NO. 4) with a homology arm of the rfaH gene, and recovering a target fragment according to a nucleic acid gel purification step.

Secondly, the linear targeting fragment is electrically transformed into competent cells, the method comprises the following steps: culturing FY26 strain containing pKD46 plasmid in LB culture medium, culturing to early stage of logarithm, adding L-arabinose into the culture medium, the final concentration is 1mM/L, inducing the expression of Red recombinase, collecting the cultured FY26 thallus, washing three times with 10% glycerol, preparing into electrically transformed FY26 competent bacteria by conventional method. The linear DNA targeting fragment was added to the competent bacteria and the rfaH gene targeting fragment was electrically transformed into FY26 competent bacteria under shock conditions of 2300V, 200. omega. and 25. mu.F. Then adding the competent bacteria into SOC culture medium, placing in a shaker at 37 ℃ for recovery culture for 2h, centrifugally collecting the bacteria, coating the bacteria on an LB agar plate containing kanamycin, and culturing overnight at 37 ℃. Selecting a single colony for pure culture, identifying a deletion strain by using front and back primers rfaH-up-F (the gene sequence is shown as SEQ ID NO. 5) and rfaH-down-R (the gene sequence is shown as SEQ ID NO. 6) PCR of the rfaH gene, electrically converting a PCP20 plasmid to identify a correct strain, and selecting a strain successfully removing a kanamycin resistance gene, wherein the strain is the rfaH gene deletion strain FY26 delta rfaH.

Then, a double-gene deletion strain of rfaH and hfq of FY26 is constructed by a Red homologous recombination method, linear targeting DNA is prepared, primers hfq-P1 (the gene sequence is shown as SEQ ID NO. 7) and hfq-P2 (the gene sequence is shown as SEQ ID NO. 8) with a hfq gene homology arm are used for amplifying a DNA targeting fragment by taking a pKD4 plasmid as a template, and a target fragment is recovered according to a nucleic acid gel purification step. The hfq gene linear targeting fragment was then electroporated into a strain with the single deletion of the rfaH gene. Firstly, the pKD46 plasmid is electrically transferred into a strain with FY26 single deletion of rfaH gene, the strain is cultured in LB culture medium, the culture medium is cultured to the early stage of logarithm, L-arabinose is added into the culture medium, the final concentration is 1mM/L, the expression of Red recombinase is induced, then the cultured thalli is collected, washed for three times by 10% glycerol, and the strain competent bacteria with single deletion of rfaH gene are prepared by the conventional method. The hfq gene linear targeting fragment was electrically transformed into single deletion competent bacteria under shock conditions of 2300V, 200. omega. and 25. mu.F. Then adding the competent bacteria into SOC culture medium, placing in a shaker at 37 ℃ for recovery culture for 2h, centrifugally collecting the bacteria, coating the bacteria on LB agar plate containing kanamycin, and culturing overnight at 37 ℃. Selecting a single colony for pure culture, identifying a deletion strain by PCR (polymerase chain reaction) of primers hfq-up-F (the gene sequence is shown as SEQ ID NO. 9) and hfq-down-R (the gene sequence is shown as SEQ ID NO. 10) before and after hfq genes, electrically transforming a PCP20 plasmid to identify a correct strain, and selecting a strain successfully removing a kanamycin resistance gene to serve as a rfaH and hfq double-gene deletion strain of FY26 (FY26 delta rfaH/hfq).

TABLE 1 deletion Strain construction and identification primers

The avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq constructed above is preserved in China center for type culture Collection, and the address is at Wuhan university, Wuhan, China, zip code 430072, and the preservation number is: CCTCC M2020612, date of deposit: 10 and 20 days in 2020.

Example 2: influence of deletion of rfaH and hfq genes on pathogenicity of avian pathogenic escherichia coli

In order to verify the attenuation effect of the deletion strain on avian pathogenic escherichia coli infection, the influence of the wild strain and the deletion strain on the chicken lethal dose is determined through various animal infection tests including a chicken embryo infection test, a chick infection test, a duckling infection test and the like. The effect of wild and deletion strains on the ability to infect DF-1 cells was tested, as well as the detection of the level of bacterial colonization in the individual viscera upon infection of chicks. The result shows that the deletion of the rfaH and hfq genes obviously reduces the pathogenicity of avian pathogenic escherichia coli, and the deletion strain FY26 delta rfaH/hfq is a low virulent strain.

1 chick embryo lethality test

Chick embryo lethality test was used to determine the virulence of wild type FY26 and deletion strain FY26 Δ rfaH/hfq, the strains were grown to mid-log late, washed twice with PBS, and 12-day-old SPF chick embryos (Jinan Sese poultry technology Co., Ltd.) were selected and injected via allantoic cavity with 50 μ L of about 500CFU bacteria, 20 chick embryos per strain, and the PBS-injected chick embryos served as a negative control. After challenge, the embryos were continuously observed for 4 days, and the death of the embryos in each group was counted.

And (3) detection results: the pathogenicity of the deletion of rfaH and hfq genes to avian pathogenic escherichia coli to avian hosts is identified through an embryo chick death test (ELA), as shown in Table 2, the embryo chick mortality of a wild type FY26 challenge group is obviously higher than that of a negative control group (strain MG1655 challenge group) (P is less than 0.05), and the embryo chick mortality of a deletion strain FY26 delta rfaH/hfq is obviously lower than that of the wild type FY 26. The result shows that the deletion strain FY26 delta rfaH/hfq obviously reduces the infectivity of avian pathogenic escherichia coli to chick embryos, and the deletion strain FY26 delta rfaH/hfq is a low virulent strain.

TABLE 2 lethality of Gene-deleted strains to chick embryos

^aData represent number of dead chick embryos/total number of chick embryos tested

^bNegative control avirulent strain

2 chick lethal test

The ability of wild type FY26 and deletion strain FY26 Δ rfaH/hfq to cause Avian Colibacillosis (Avian Colibacillosis) was evaluated by a chick (commercial broiler) lethality test, and 10 chicks of 1 day old in each group were injected with 0.1mL of a bacterial solution (5.0X 10) through the trachea (see above)⁶CFU), group injected with the non-strain MG1655 as a negative control group. Continuously observing for 7 days after the challenge, and counting the death and survival conditions of the chicks. All chicks were dissected after challenge and death or seven days after observation and scored for the presence of air sacculitis (score 1), pericarditis (score 2) and perihepatitis (score 3). Inoculating ring, selecting the tissue (air sac, intracardiac blood and brain tissue) of the virus attacking chick, inoculating the tissue into a Mackanka culture medium, culturing overnight at 37 ℃, and observing the growth of sterile colonies.

As shown in Table 3, the mortality, the organ separation rate and the lesion score of the chickens of each challenge group and the control group are evaluated, similar to the ELA test result, the mortality and other parameters of the wild type FY26 are obviously higher than those of the MG1655(P is less than 0.05) of the negative control group, while the Escherichia coli disease causing capability of the deletion strain FY26 delta rfaH/hfq is obviously weaker, and the deletion strain FY26 delta rfaH/hfq is proved to be a weak strain.

TABLE 3 pathogenicity of Gene-deleted Strain to 1 day old chick

3 duckling lethality test

7 days old ducklings (cherry valley duck) are used for determining pathogenicity of wild strain FY26 and deletion strain, the strain is cultured to logarithmic phase, PBS is used for washing for 2 times, multiple dilution is carried out after thalli are resuspended, 10 ducklings in each group are injected with 0.2mL of bacterial liquid, the toxicity dose is 1.0 multiplied by 10⁷And CFU/duck, continuously observing for seven days after the toxin is attacked, and recording the death condition of the ducklings.

The results are shown in Table 4, and are 5.0X 10 for intraperitoneal injection⁶The mortality rate of the wild type FY26 of the CFU to the ducklings is 100 percent, while the mortality rate of the ducklings of the deletion strain FY26 delta rfaH/hfq challenge group is only 20 percent under the same dosage, and the result shows that the deletion of rfaH and the deletion of rfaHhfq gene obviously reduces the ability of avian pathogenic colibacillosis causing duckling colibacillosis, and confirms that the deletion strain FY26 delta rfaH/hfq is a low virulent strain.

TABLE 4 pathogenicity of Gene-deleted Strain to 7-day-old ducklings

4 determination of the adhesion Capacity of the Strain to DF-1 cells

4.1 DF-1 cell culture

Digesting DF-1 chicken embryo fibroblasts cultured in a cell bottle by using pancreatin, transferring and culturing to a 24-hole cell culture plate, randomly selecting two holes of cells after the cells grow full, carrying out cell counting after the cells are digested by using the pancreatin, wherein the average value represents the average number of the cells in the 24-hole plate. And the cell wells were washed 3 times with a serum-free DMEM medium for use.

4.2 bacterial treatment and adhesion test

Wild type FY26 and deletion strain FY26 delta rfaH/hfq are respectively cultured in a conical flask to logarithmic phase, thalli are collected after ice bath, washed 3 times by PBS and re-suspended by DMEM culture medium, the bacterial number of each bacterial liquid is 100 times of the cell number in a hole (infection ratio is 1:100), each bacterial liquid is added into a cell culture hole, sterile DMEM is used as negative control, DF-1 cells are infected for 2 hours, the infected cells are washed by sterile PBS, the cells are lysed by 0.1% Triton X-100 after being washed three times by PBS, the lysate is diluted by 10 times of PBS, LB agar plates are coated, and bacterial counting and counting are carried out after overnight culture at 37 ℃.

4.3 Effect of deletion of rfaH and hfq genes on the adhesion Capacity of FY26

The adhesion capacity of wild type FY26 and deletion strain FY26 delta rfaH/hfq to DF-1 chick embryo fibroblasts is measured, as shown in figure 1, compared with the adhesion capacity of FY26, the adhesion capacity of deletion strain FY26 delta rfaH/hfq is obviously reduced to 23 percent (P is less than 0.01), and the results show that the deletion of genes rfaH and hfq obviously reduces the adhesion capacity of FY26, and the deletion strain FY26 delta rfaH/hfq has relatively weaker adhesion capacity.

5 determining the colonization ability of bacteria in lungs of chicks and ducks

Wild type FY26 and deletion strain FY26 delta rfaH/hfq were cultured to log phase, washed 2 times with PBS, resuspended and diluted to 1.0X 10⁸And CFU/mL, performing detoxification on chicks through an air pipe, injecting 0.2mL into each duckling, killing the chicks in each detoxification group by at least 10 chicks in each detoxification group, performing detoxification for 24 hours, dissecting and separating tissues such as brain, lung and the like under an aseptic condition, weighing, adding PBS (phosphate buffer solution) according to the specific gravity of 1mL/g, performing tissue homogenization, diluting by 10 times, coating an LB (Langmuir-Blodgett) plate, culturing at 37 ℃ overnight, performing bacterial counting, and counting the results.

The detection result is shown in figure 2, and the result of the chick lung colonization test shows that when the chick is infected with the early 24hpi strain, the colonization ability of the deletion strain FY26 delta rfaH/hfq in the chick lung is obviously lower than that of the wild type FY 26.

In vivo colonization test of duckling, wild type FY26 and deletion strain FY26 delta rfaH/hfq are cultured to logarithmic phase respectively, washed with PBS for 2 times, and resuspended in APEC thallus at 2.0 × 10⁶And (3) performing toxicity counteracting by using CFU/duck dosage, performing toxicity counteracting by using a trachea for 10 ducklings in each toxicity counteracting group, injecting 0.2mL of each ducklings, neutralizing the ducklings after 24h of toxicity counteracting, dissecting the ducklings under aseptic conditions, separating tissues such as brain, lung and the like, weighing internal organs, adding PBS (phosphate buffer solution) according to the proportion of 1mL/g, performing tissue homogenate, diluting by 10 times, coating an LB (Langmuir-Blodgett) flat plate, culturing overnight at 37 ℃, performing bacterial counting and counting results.

As shown in fig. 3, deletion of rfaH and hfq genes significantly reduced the ability of FY26 to colonize the lungs of ducklings by a ducklings infection model. The above results indicate that the deletion strain FY26 Δ rfaH/hfq has relatively weak in vivo colonization ability, and the above results fully indicate that the deletion strain FY26 Δ rfaH/hfq is a low virulent strain.

Example 3: avian pathogenic Escherichia coli attenuated vaccine (avian pathogenic Escherichia coli rfaH and hfq double gene deletion vaccine)

The obtained avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq is identified, each generation is inoculated on an LB agar-containing culture medium to observe the colony color, and the genes of the avian pathogenic escherichia coli are used for detection to identify the genetic stability of the deletion bacteria. FY26 Δ rfaH/hfq maintainer found after a second passageBecause of the deleted phenotypic characteristics, the identified gene is still deleted, and the heredity is stable. Culturing the rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq on a solid culture medium, and selecting a single colony to be cultured in a liquid culture medium until the concentration of viable bacteria reaches 5.0 multiplied by 10⁹CFU/mL. The gelatin protectant is prepared by adding sucrose 40g and gelatin 9g into deionized water 100mL, melting completely, sterilizing at 121 deg.C for 30min, and storing. Adding the gelatin protective agent according to the ratio of the bacterial liquid to the gelatin protective agent (volume: volume) of 7: 1. Subpackaging in sterilized lyophilized bottle at 2.0 mL/bottle, lyophilizing in a lyophilizer at-50 deg.C for 36h, capping, dissolving with 10% physiological salt of aluminum gel, counting viable bacteria (CFU), determining no contamination, and storing at-20 deg.C for use as attenuated vaccine strain.

Example 4: safety evaluation of avian pathogenic escherichia coli gene deletion vaccine FY26 delta rfaH/hfq

The safety evaluation of the FY26 delta rfaH/hfq attenuated vaccine on chicks selects 40 chicks with 7-day age and escherichia coli negativity, and the chicks are divided into 2 groups, and each group comprises 20 chicks. The FY26 delta rfaH/hfq gene deletion vaccine prepared by the invention is 0.1mL (containing 1.0 multiplied by 10) per chicken⁷CFU viable count) is injected into chicks through leg muscles, the chicks do not die after inoculation, the chicks injected with the gene deletion vaccine prepared by the invention have normal spirit and appetite and do not change abnormally, and escherichia coli antibodies can be detected in the day after inoculation. According to the same method, a second group of chicks are inoculated with the wild type FY26 according to the same dose, the inoculated chicks have clinical symptoms of lassitude, anorexia or abominability, rough hair and the like, death begins in the next day of inoculation, all death occurs within 7 days, and pathological anatomical examination on the group of chicks shows that the chicks all have typical pathological changes of acute colibacillosis. The attenuated vaccine prepared by the gene deletion strain FY26 delta rfaH/hfq is safe.

Example 5: immunoprotection evaluation of Gene-deleted Strain FY 26. delta. rfaH/hfq in chicks

1 immunization procedure of mice

The 7-day-old ducklings are divided into 2 groups, and each group of ducklings are sequentially immunized with attenuated vaccine FY26 delta rfaH (5.0 multiplied by 10)⁶CFU live count vaccine) and PBS (control group), secondary immunization was performed after 14 days, chick blood was collected one week after secondary immunization, and serum was separated and stored at-20 ℃.

Using FY26 mycoprotein as an Escherichia coli detection antigen, detecting the antibody titer in the serum of the immunized chicken by an ELISA method, coating a 96-well plate with 0.5 mu g/well FY26 mycoprotein in each well at 4 ℃ overnight, adding diluted chicken serum in each well in a multiple proportion, incubating for 2h, washing the plate by PBST, using HRP-labeled anti-chicken IgG as a secondary antibody (1:4000 dilution), incubating for 1h by the secondary antibody, washing the plate by PBST, adding 100 mu l of TMB color development solution (Solarbio) in each well, reacting for a certain time, adding concentrated sulfuric acid to terminate the reaction, determining the absorbance of each well by selecting a wavelength of 450nm, and calculating the last gradient well with the numerical ratio of the final gradient well to the negative control well being more than 2.1 times so as to calculate the antibody titer. Ten days after the secondary immunization (the titer of the antibody of the chick is obviously improved), the chick is injected with the wild strain FY26(5.0 multiplied by 10) through a trachea⁸CFU/one), continuously observing for one week, counting survival rate of chicks in each group, and selecting 10 chicks from each experimental group for FY26 lung colonization quantity determination after 24 hours of virus challenge.

Protective test of FY26 Δ rfaH/hfq Gene-deleted vaccine immunized chicks

The chick is immunized with FY26 delta rfaH/hfq gene deletion vaccine, the immunized chick is subjected to challenge test, whether the chick is immune-protected by the vaccination is researched, and as shown in figure 4, the titer of the anti-escherichia coli antibody in the chick serum after the chick is immunized for the second time basically reaches 10⁴The result shows that the FY26 delta rfaH/hfq gene deletion vaccine can stimulate the organism to generate IgG antibody with high titer. The challenge test is carried out on the immunized chicks, and the survival rate of the immunized chicks is 100 percent compared with that of a negative control group (the death rate is 100 percent). The in vivo colonization experiment result of the chicks shows that the immunization of the chicks with the FY26 delta rfaH/hfq gene deletion vaccine can reduce the colonization capacity of the FY26 in the lungs of the infected chicks. Relevant experiment results prove that the FY26 delta rfaH/hfq gene deletion vaccine has immunogenicity, and the vaccination has immune protection on chicks.

According to the results of the above examples, the knockout of the gene of the present inventionThe avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain has excellent immune protection effect and can be used as an attenuated vaccine, and the concentration of the avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain in the attenuated vaccine can be 5.0 x 10⁶CFU/0.1mL。

Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which should be covered by the claims of the present invention.

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Claims (4)

1. An APEC double-gene rfaH and hfq deletion strain, wherein the APEC double-gene rfaH and hfq deletion strain is avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq, and the preservation number of the APEC double-gene rfaH and hfq deletion strain is CCTCC NO. M2020612.

2. The APEC double-gene rfaH and hfq-deleted strain as claimed in claim 1, wherein the APEC double-gene rfaH and hfq-deleted strain is constructed by the following steps: and knocking out the rfaH and hfq genes in the avian pathogenic escherichia coli FY26 one by adopting a Red homologous recombination method to obtain the avian pathogenic escherichia coli rfaH and hfq double-gene deletion strain FY26 delta rfaH/hfq.

3. The use of the APEC double-gene rfaH and hfq deleted strain according to claim 1 in the preparation of avian pathogenic Escherichia coli attenuated vaccine for preventing and treating avian colibacillosis.

4. The avian pathogenic escherichia coli attenuated vaccine prepared by the APEC double-gene rfaH and hfq deletion strain of claim 1.

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