CN112695005A

CN112695005A - APEC double sRNA gene deletion strain and attenuated vaccine prepared from same

Info

Publication number: CN112695005A
Application number: CN202011597050.4A
Authority: CN
Inventors: 诸葛祥凯; 戴建君; 姜敏; 周洲; 王忠星
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-04-23

Abstract

The invention discloses an APEC double sRNA gene deletion strain and an attenuated vaccine prepared by the same, wherein the construction method of the APEC double sRNA gene deletion strain is to knock out RyhA and GcvB double sRNA genes by a Red homologous recombination method to obtain avian pathogenic escherichia coli FY26 delta ryhA/gcvB, and the avian pathogenic escherichia coli FY26 delta ryhA/gcvB is preserved in China center for type culture collection with the preservation number of CCTCC M2020611. 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 ryhA/gcvB has immune protection power against avian pathogenic escherichia coli infection and good virus attacking immune protection effect.

Description

APEC double sRNA gene deletion strain and attenuated vaccine prepared from same

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to an APEC double sRNA gene deletion strain and an attenuated vaccine prepared from the APEC double sRNA gene deletion strain, in particular to an avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain and an attenuated vaccine prepared from the avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain.

Background

Avian Pathogenic Escherichia Coli (APEC) mainly infects poultry through respiratory tract, APEC pathogen with strong pathogenicity can break through the immune defense of lung and enter blood to cause septicemia, thereby causing multi-system mixed infection of poultry and even acute death of poultry. With the development of poultry industry in China, avian colibacillosis becomes one of the most serious bacterial infectious diseases harming the poultry industry, the morbidity of the disease is 11% -69%, the mortality is 3.8% -72.9%, huge loss is caused to the poultry industry, and great threat is caused to food safety. At present, the pathogenic mechanism of the APEC is not clarified, no effective vaccine is available for preventing and treating avian colibacillosis, and the avian colibacillosis is controlled only by drugs such as antibiotics, so that the drug resistance of APEC isolates is continuously increased and the risk of drug residues in avian products exists.

In recent years, it has been found that the expression Regulation of virulence factors of pathogenic bacteria also occurs at the mRNA translation level, and this Regulation is called Post-transcriptional Regulation (Post-transcriptional Regulation), while the elements that manipulate the Post-transcriptional Regulation are Small Non-coding Regulatory RNAs (sRNA). sRNA is commonly found in prokaryotic organisms as a post-transcriptional regulator, and different species of sRNA have various base lengths, but the length range is generally between 50bp and 500 bp. srnas are often directly associated with bacterial virulence, and some newly discovered srnas are involved in the stress response of pathogenic bacteria under infection conditions. Regulatory sRNA is closely related to expression of a series of pathogenic metabolic genes of pathogenic bacteria. Research indicates that sRNA RyhA can regulate the amino acid metabolism of Escherichia coli after transcription, and can regulate the expression of genes related to aromatic amino acid synthesis. sRNA GcvB can also regulate E.coli amino acid metabolism and transport. 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 technical means for obtaining the vaccine strain and are concerned.

Disclosure of Invention

In view of the above, the invention aims to provide an avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain with good immunogenicity and strong safety and an attenuated vaccine prepared by 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:

an APEC double sRNA gene deletion strain is avian pathogenic Escherichia coli RyhA and GcvB double sRNA gene deletion strain FY26 delta ryhA/gcvB, is preserved in China center for type culture collection (Wuhan university, Wuhan, China), has a preservation number of CCTCC M2020611, has a preservation date of 2020, 10 months and 20 days, and is classified and named as Escherichia coli.

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

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

Furthermore, the invention also provides the avian pathogenic escherichia coli attenuated vaccine prepared by the APEC double sRNA gene deletion strain.

Compared with the prior art, the avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain constructed and provided 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 ability of avian pathogenic E.coli RyhA and GcvB double sRNA gene-deleted strain FY26 delta ryhA/gcvB, avian pathogenic E.coli wild strain FY26 and single gene-deleted strain FY26 delta ryhA to chicken embryo fibroblast (DF-1 cell);

FIG. 2 is a diagram comparing the colonization ability of avian pathogenic E.coli RyhA and GcvB double sRNA gene deletion strain FY26 delta ryhA/gcvB, avian pathogenic E.coli wild strain FY26 and single gene deletion strain FY26 delta ryhA to the lung of chicks;

FIG. 3 shows the colonization ability of pathogenic avian Escherichia coli RyhA and GcvB double sRNA gene-deleted strain FY26 delta ryhA/gcvB, pathogenic avian Escherichia coli wild strain FY26 and single gene-deleted strain FY26 delta ryhA to the lungs of young ducks;

FIG. 4 is a graph showing the results of measurement of the antibody titer of the avian pathogenic E.coli RyhA and GcvB double sRNA gene-deleted strain FY 26. delta. ryhA/gcvB after immunization.

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 avian pathogenic Escherichia coli RyhA and GcvB double sRNA Gene-deleted Strain FY 26. delta. ryhA/gcvB

The avian pathogenic escherichia coli RyhA and GcvB double sRNA 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 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 bacteria genes intercellulary promotion with protein polymorphism. gateway research.2019Dec 1; 50(1):31.) the invention mainly adopts Red homologous recombination method to knock out RyhA and GcvB double sRNA genes in the avian pathogenic Escherichia coli one by one, so as to construct a RyhA and GcvB double sRNA gene deletion avian pathogenic Escherichia coli.

The sequence of the deleted RyhA gene is shown as SEQ ID NO.1, and the sequence of the deleted GcvB gene is shown as 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

First, a ryhA gene-deleted strain of the strain FY26 was constructed by Red homologous recombination to prepare a linear targeting DNA, and then a DNA targeting fragment (Table 1) was amplified using pKD4 plasmid as a template by using a primer ryhA-P1 (the gene sequence is shown in SEQ ID NO. 3) and ryhA-P2 (the gene sequence is shown in SEQ ID NO. 4) having a homology arm of the rfaH gene, and the desired fragment was recovered by 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 competent bacteria, and the ryhA gene targeting fragment was electrically transformed into FY26 competent bacteria under the 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 ℃. A single colony was selected and cultured, and the deletion strain was identified by PCR using primers ryhA-up-F (gene sequence shown in SEQ ID NO. 5) and ryhA-down-R (gene sequence shown in SEQ ID NO. 6) before and after the ryhA gene, and the strain from which the kanamycin-resistant gene was successfully removed was selected by electrotransformation of the PCP20 plasmid to identify the correct strain, which was the ryhA gene-deleted strain FY 26. delta. ryhA.

Then, a double sRNA gene deletion strain of RyhA and GcvB of FY26 is constructed by a Red homologous recombination method, linear targeting DNA is prepared, a DNA targeting fragment is amplified by using a pKD4 plasmid as a template through a primer gcvB-P1 (the gene sequence is shown as SEQ ID NO. 7) with a gcvB gene homology arm and a gcvB-P2 (the gene sequence is shown as SEQ ID NO. 8), and a target fragment is recovered according to a nucleic acid gel purification step. The linear targeted fragment of the gcvB gene was then electroporated into a strain with a single deletion of the ryhA gene. First, pKD46 plasmid was electrotransferred to FY26 single-deletion ryhA gene strain, cultured in LB medium to the logarithmic prophase, L-arabinose was added to the medium to a final concentration of 1mM/L to induce the expression of Red recombinase, and then the cultured cells were collected, washed three times with 10% glycerol, and prepared into electrotransferred single-deletion ryhA gene strain competent bacteria by a conventional method. Linear targeted fragments of the gcvB gene were electrically transformed into single deletion competent bacteria under shock conditions of 2300V, 200 Ω and 25 μ 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 ℃. A single colony is picked and purely cultured, a deletion strain is identified by PCR of a gcvB gene front primer and a gcvB primer, wherein the gene sequence is shown as SEQ ID NO.9, and a gcvB-down-R primer, wherein the gene sequence is shown as SEQ ID NO.10, the PCP20 plasmid is electrically transformed to identify a correct strain, and a strain which successfully removes a kanamycin resistance gene is selected to serve as a RyhA and GcvB double sRNA gene deletion strain (FY26 delta ryhA/gcvB) of FY 26.

TABLE 1 construction and identification primers for deletion strains

The avian pathogenic escherichia coli RyhA and GcvB gene deletion strain FY26 delta ryhA/gcvB constructed above is preserved in China typical culture collection center, the address is Wuhan university, Wuhan, China, zip code 430072, the preservation number is: CCTCC M2020611, date of deposit: 10 and 20 days in 2020.

Example 2 Effect of deletion of RyhA and GcvB Dual sRNA genes on virulence of avian pathogenic E.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 RyhA and GcvB double sRNA genes obviously reduces the pathogenicity of avian pathogenic escherichia coli, and the deletion strain FY26 delta ryhA/gcvB is an attenuated strain.

1 chick embryo lethality test

Chick embryo lethality test was used to determine the virulence of wild type FY26 and deletion strain FY 26. delta. ryhA/gcvB, the strains were grown to mid-log phase and washed twice with PBS, and 12 day old SPF chick embryos (Jinan Sese poultry technology Co., Ltd.) were selected and injected with 50. mu.L of about 500CFU of bacteria via the allantoic cavity, 20 chick embryos per strain, and PBS injected chick embryos served as a negative control. After challenge, the embryos were observed for 4 days and the death of the embryos in each group was counted.

And (3) detection results: the pathogenicity of the RyhA and GcvB deletion double sRNA genes to avian pathogenic Escherichia coli to avian hosts is identified by an avian embryo lethal test (ELA), and as shown in Table 2, the avian embryo lethality rate 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), while the avian embryo lethality rate of a deletion strain FY26 delta ryhA/gcvB is obviously lower than that of the wild type FY 26. The result shows that the deletion strain FY26 delta ryhA/gcvB obviously reduces the infectivity of avian pathogenic Escherichia coli to chicken embryos, and the deletion strain FY26 delta ryhA/gcvB 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 the deletion strain FY 26. delta. ryhA/gcvB to cause Avian Colibacillosis (Avian colibacilosis) 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 isolation rate and the lesion score of the chickens of each challenge group and the control group were evaluated, and similarly to the ELA test results, the mortality and other parameters of the wild-type FY26 were significantly higher than those of the negative control group MG1655(P < 0.05), while the E.coli disease-causing ability of the deletion strain FY 26. delta. ryhA/gcvB was significantly weaker, confirming that the deletion strain FY 26. delta. ryhA/gcvB is a low virulent 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 of the wild type FY26 of the CFU to ducklings is 100%, while the mortality of the ducklings of the deleted strain FY26 delta ryhA/gcvB virus attacking group is only 20% under the same dosage, and the result shows that the deletion of the RyhA and the GcvB double sRNA genes obviously reduces the capability of avian pathogenic escherichia coli caused ducklings escherichia coli, and the deletion strain FY26 delta ryhA/gcvB is proved to be an attenuated 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 ryhA/gcvB are respectively cultured in a conical flask to logarithmic phase, thalli are collected after ice bath, washed 3 times by PBS, and resuspended 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, after DF-1 cells are infected for 2 hours, the infected cells are washed by sterile PBS, after PBS is washed for three times, the cells are lysed by 0.1% Triton X-100, the lysate is diluted by 10 times of PBS, an LB agar plate is coated, and after being cultured overnight at 37 ℃, the bacterial count is carried out, and the result is counted.

4.3 Effect of deletion of RyhA and GcvB Dual sRNA genes on the adhesion Capacity of FY26

The adhesion ability of wild type FY26 and deletion strain FY26 delta ryhA/gcvB to DF-1 chick embryo fibroblasts was determined, as shown in FIG. 1, the adhesion ability of deletion strain FY26 delta ryhA/gcvB was significantly reduced to 23% (P < 0.01) compared with the adhesion ability of FY26, and the above results show that the deletion of the RyhA and GcvB double sRNA genes significantly reduces the adhesion ability of FY26, and the deletion strain FY26 delta ryhA/gcvB has relatively weak adhesion ability.

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

Wild type FY26 and deletion strain FY 26. delta. ryhA/gcvB 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.

As shown in FIG. 2, the result of the in-lung colonization test of the chicks shows that the colonization ability of the deletion strain FY26 delta ryhA/gcvB in the lungs of the chicks is obviously lower than that of the wild type FY26 when the early 24hpi chicks are infected.

In vivo colonization test of duckling, wild type FY26 and deletion strain FY26 delta ryhA/gcvB are cultured to logarithmic phase respectively, washed 2 times with PBS, and APEC thallus is resuspended 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 RyhA and GcvB dual sRNA 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 FY 26. delta. ryhA/gcvB has a relatively weak in vivo colonization ability, and the above results sufficiently indicate that the deletion strain FY 26. delta. ryhA/gcvB is a low virulent strain.

Example 3 avian pathogenic E.coli RyhA and GcvB double sRNA Gene deletion vaccine (attenuated vaccine)

Pathogenic disease of the obtained fowlThe sexual escherichia coli rfaH gene deletion strain FY26 delta ryhA/gcvB is identified, each generation is inoculated on an LB agar-containing culture medium to observe colony color, and the gene of avian pathogenic escherichia coli is used for detection to identify the genetic stability of the deletion bacteria. After the generation, the FY26 delta ryhA/gcvB is found to maintain the phenotype characteristic of gene deletion, and the identified gene is still deleted and has stable heredity. The RyhA and GcvB double sRNA gene-deleted strain FY26 delta ryhA/gcvB was cultured on a solid medium, and a single colony was selected and cultured in a liquid medium until the viable cell concentration reached 5.0X 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. The gelatin protective agent is added according to the ratio of 7:1 of the bacterial liquid to the gelatin protective agent (volume: volume). 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 evaluation of safety of avian pathogenic E.coli Gene deletion vaccine FY 26. delta. ryhA/gcvB

The safety evaluation of FY26 delta ryhA/gcvB attenuated vaccine on chicks selects 40 chicks with 7-day age and escherichia coli negative, and the chicks are divided into 2 groups, and each group comprises 20 chicks. The FY26 delta ryhA/gcvB gene deletion vaccine prepared by the invention is 0.1mL (containing 1.0X 10) per chicken⁷CFU viable bacteria 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 have no abnormal change, and escherichia coli antibodies can be detected all 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 ryhA/gcvB is safe.

Example 5 immunoprotection evaluation of the Gene-deleted Strain FY 26. delta. ryhA/gcvB of the present invention on 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 assay for FY 26. delta. ryhA/gcvB Gene-deleted vaccines in immunized chicks

The chick is immunized with FY26 delta ryhA/gcvB gene deletion vaccine, the challenge test is carried out on the immunized chick, whether the vaccination has immune protection to the chick 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 ryhA/gcvB gene deletion vaccine can stimulate the organism to produce high-titer IgG antibodies. 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 test result of the chicks shows that the chicks are immunized with FY26 delta ryhA/gcvB gene deletion vaccineCan reduce the colonization ability of FY26 in the lung of infected chicks. The results of related experiments prove that the FY26 delta ryhA/gcvB gene deletion vaccine has immunogenicity, and the vaccination has immunoprotection to chicks.

As a result of the verification of the above examples, the knock-out avian pathogenic Escherichia coli FY26 Δ ryhA/gcvB of the present invention has an excellent immunoprotection effect and can be used as an attenuated vaccine, and the concentration of the avian pathogenic Escherichia coli FY26 Δ ryhA/gcvB in the attenuated vaccine can be 5.0X 10⁶ CFU/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.

Sequence listing

<110> university of southeast Tong

<120> APEC double sRNA gene deletion strain and attenuated vaccine prepared from same

<130> 20201225

<141> 2020-12-27

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 121

<212> DNA

<213> Escherichia coli

<400> 1

gtgcggcctg aaaaacagtg ctgtgccctt gtaactcatc ataataattt acggcgcagc 60

caagatttcc ctggtgttgg cgcagtattc gcgcaccccg gtctagccgg ggtcattttt 120

t 121

<210> 2

<211> 205

<212> DNA

<213> Escherichia coli

<400> 2

acttcctgag ccggaacgaa aagttttatc ggaatgcgtg ttctggtgaa cttttggctt 60

acggttgtga tgttgtgttg ttgtgtttgc aattggtctg cgattcagac catggtagca 120

aagctacctt ttttcacttc ctgtacattt accctgtctg tccatagtga ttaatgtagc 180

accgcctaat tgcggtgctt ttttt 205

<210> 3

<211> 70

<212> DNA

<213> Unknown (Unknown)

<400> 3

aaaggggtcc gcatccgcga gccgcaatat acgcagggct gcaagaagat gtgtaggctg 60

gagctgcttc 70

<210> 4

<211> 70

<212> DNA

<213> Unknown (Unknown)

<400> 4

gcgaaggtac aaaaaattaa cgttttagca atagctatat aatatagcct catatgaata 60

tcctccttag 70

<210> 5

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 5

gagtgcgtga agagtggaaa ggggt 25

<210> 6

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 6

gacgactcat tgtgcttcct catta 25

<210> 7

<211> 70

<212> DNA

<213> Unknown (Unknown)

<400> 7

attataaatt gtccgttgag cttctaccag caaataccta tagtggcggc gtgtaggctg 60

gagctgcttc 70

<210> 8

<211> 70

<212> DNA

<213> Unknown (Unknown)

<400> 8

gacgtgaaag agatggtcga actggatcag taattcgcga tcgcaaggta catatgaata 60

tcctccttag 70

<210> 9

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 9

gagccattat aaattgtccg ttgag 25

<210> 10

<211> 25

<212> DNA

<213> Unknown (Unknown)

<400> 10

acgctaatgg caacaaacag cagat 25

Claims

1. An APEC double sRNA gene deletion strain is avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain FY26 delta ryhA/gcvB, and the preservation number is CCTCC NO. M2020611.

2. The APEC double sRNA gene deleted strain of claim 1, which is constructed by the following method: and knocking out the RyhA and GcvB double sRNA genes in the avian pathogenic escherichia coli FY26 one by adopting a Red homologous recombination method to obtain the avian pathogenic escherichia coli RyhA and GcvB double sRNA gene deletion strain FY26 delta ryhA/gcvB.

3. The use of the APEC double sRNA gene deletion strain of claim 1 in the preparation of an avian pathogenic Escherichia coli attenuated vaccine for the prevention and treatment of avian colibacillosis.

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