CN105039203A - Riemerella anatipestifer gene deletion strain and application thereof - Google Patents

Abstract

The invention discloses a riemerella anatipestifer gene deletion strain having deletion of a double-component regulator control system 06537/o6542 gene, and an application of the riemerella anatipestifer gene deletion strain, belonging to the technical field of preparation of animinal genetic engineering vaccines. The riemerella anatipestifer gene deletion strain is preserved in the China Center for Type Culture Collection with the preservation number of CCTCC M 2015306; due to the deletion of the 798bp of the double-component regulator control system 06537/o6542 gene, the riemerella anatipestifer gene deletion strain has obviously lowered virulence, and the lost capacity of liquefying gelatin; the sequence of the deleted gene is shown as SEQ ID NO:2. After the riemerella anatipestifer gene deletion strain is inoculated to ducklings, the ducklings can have the protective immunity reaction resisting the serum 1 type riemerella anatipestifer virulent strain, so that the infection from the riemerella anatipestife is prevented.

Description

A gene-deleted strain of R. anatipestifer and its application

technical field

The invention belongs to the technical field of animal bacterial genetic engineering, and relates to related fields such as animal molecular biology and immunology. Specifically, it relates to a 06537/06542 gene deletion strain of the two-component regulatory system of R. anatipestifer and its application.

Background technique

Riemerella anatipestifer infection is an acute, contact, septic infectious disease of ducklings caused by Riemerella anatipestifer (RA), due to the high incidence, high mortality and growth retardation of the disease , has become a relatively serious problem in the process of large-scale breeding of meat ducks in my country. At present, 21 serotypes have been reported abroad, and 4 new serotypes have been discovered in China. Currently, only serotype 1 has been found among the RA strains isolated and identified from our province. At present, little is known about the virulence-related genes of R. anatipestifer. The virulence factors that have been identified include outer membrane protein A (OmpA) and dipeptide peptidase Ⅳ (DPPⅣ). In addition, there are some factors that are speculated to be related to virulence. Although some progress has been made in the research of RA, its molecular pathogenic mechanism is still unclear. Therefore, it is of great practical significance to strengthen the research on the molecular biology and pathogenic mechanism of RA to fundamentally prevent the occurrence of the disease.

Drug treatment is the main method to treat and prevent R. anatipestifer infection in poultry. At present, antibiotics and sulfa drugs are generally used to prevent and treat this disease, but the widespread use of antibiotics easily leads to the emergence of drug-resistant strains. Due to the complexity of RA serotypes, strains in different regions have different effects on the use of different drugs. Therefore, before using antibiotics, relevant drug susceptibility tests should be carried out on the strains, and the best drug regimen can be determined through comparison. In terms of vaccine immunity, because there are many serotypes of RA, and there is basically no or only very low cross-immunity protection between different serotypes, scholars from all over the world are committed to the research of genetically engineered vaccines for R. anatipestifer. Strive to obtain a safer and more efficient new vaccine.

Bacterial genetically engineered vaccines are one of the development directions of modern vaccines. There are many advantages to using a genetically engineered live vaccine with high safety and weak toxicity as a vaccine: first, it can be inoculated through nasal drops or sprays, which can stimulate the body to produce effective mucosal immunity, and at the same time stimulate the body's systemic immunity; , it is inoculated through nasal drops or sprays, and the operation is simple and easy; third, it has good immunogenicity and can produce good protection; fourth, the genetically engineered live vaccine can produce different serotypes of R. anatipestifer Protective power; fifth, in terms of production, it is fast and economical, suitable for large-scale promotion at the grassroots level; sixth, it can carry large foreign gene fragments, which is easy to construct multivalent genetic engineering vaccines, so as to achieve the purpose of preventing multiple diseases with one vaccine. With the in-depth research on the molecular biology and molecular pathogenic mechanism of R. anatipestifer, the use of modern molecular biology techniques and genetic engineering methods, through the inactivation of factors related to metabolism, to construct low-virulence strains , the development of a genetically engineered attenuated live vaccine for R. anatipestifer is of great significance to the prevention and control of R. anatipestifer disease.

Scholars at home and abroad have begun to study R. anatipestifer as a live attenuated vaccine, but so far no relatively mature product has come out. In the early stage of the present invention, a pair of new genes, namely 06537/06542, induced and expressed by RA Yunmeng strain (RA-YM) in infected ducks were found through in vivo antigen induction technology and bioinformatics analysis. It is speculated that the genes may be related to virulence. Therefore, we first cloned the 06537/06542 gene and its upstream and downstream homologous arm genes, constructed recombinant suicide plasmids, and screened the 06537/06542 gene deletion strains by using spectinomycin resistance and PCR, and evaluated the genetic stability, toxicity Liji studied the pathogenicity and immunogenicity of ducklings. Using self-constructed genetic engineering strains to develop genetic engineering vectors with my country's intellectual property rights and apply them to production will be a powerful measure for my country to cope with the challenges faced by the animal husbandry industry today and improve its competitiveness.

references

1. Chen Fenfen. Screening and Application of Induced Antigen Genes of R. anatipestifer in vivo. CNKI. Wuhan, Hubei: Huazhong Agricultural University, 2008

2. Li Jixiang. Research on pathogenicity-related factors of Riemerella anatipestifer. [Ph.D. Dissertation]. Chongqing: Southwest University, 2009

3. Xie Miaomiao. Construction of ECFssigma gene deletion mutant strain of R. anatipestifer and research on its biological characteristics. [Master's Dissertation]. Wuhan, Hubei: Huazhong Agricultural University, 2014

4. Xiong Tao. Research on Riemerella anatipestifer in Hubei Province. [Master's Dissertation]. Wuhan, Hubei: Huazhong Agricultural University, 2001

Contents of the invention

The purpose of the present invention is to provide a gene deletion strain of R. anatipestifer. Due to the deletion of a part of the 06537/06542 gene of the two-component regulatory system, the strain has reduced virulence. The invention also provides the application of the bacterial strain in the preparation of the attenuated R. anatipestifer vaccine.

The overall technical roadmap of the present invention is shown in FIG. 1 , and the construction route of the gene-deleted strain of R. anatipestifer is shown in FIG. 2 .

The present invention is achieved like this:

In order to realize the present invention, the applicant first consulted the annotation of the whole genome sequence of RA-YM strain. The results showed that there were 3 classic TCSs. In this experiment, Chen Fenfen (2008) selected the two-component regulatory system Ive-3, namely 06537/06542, which was screened by in vivo antigen induction technology, as the research object. The position of its coding gene on RA-YM genome is 52760bp-53989bp. Among them, the coding gene Ive-3a of histidine kinase HK is located on RAYM_06537, consists of 524 nucleotides, and encodes a total of 174 amino acids. The coding gene Ive-3b of the response regulator RR is located on RAYM_06542, which consists of 688 nucleotides and encodes a total of 228 amino acids. It can be seen from Figure 3 that the two coding genes are adjacent to each other on the genome, with the same transcription direction and only 18 bp between them, which is consistent with the structural characteristics of the general two-component regulatory system.

Using the RA-YM strain genome reported in the literature as a template, using LeftarmL1 and LeftarmL2, RightarmR1 and RightarmR2 as primers, the extracted RA-YM strain genome was used as a template for PCR amplification, and the amplified band was displayed by electrophoresis, and the size was about 500bp Left and right, consistent with the expected target fragments 454bp and 456bp, the amplified product was recovered. Since ^RA -YM is sensitive to spectinomycin, primers Spc RS1 and Spc RS2 were used to amplify the spectinomycin resistance gene from the ^pIC333 plasmid as a screening marker. The amplified product was shown by electrophoresis, and the band was about 1000bp Left and right, the size is consistent with the expected target fragment 1100bp, and the amplified product is recovered. The recovered Leftarm, Rightarm and Spc ^R fragments were ligated by OverlapPCR, the amplified products were recovered and ligated with the pMD18-T vector, and the positive clones transformed by the ligated products were identified by Spc ^R resistance gene PCR and double enzyme digestion. The positive clones identified by enzyme digestion were sent to Beijing Qingke for sequencing, and the homology of BLASTn comparison was 99%. The suicide plasmid pRE112 and the constructed pT-LSR plasmid containing the target fragment were double-digested with KpnI and SacI, respectively. The target fragment was recovered, ligated overnight in a 16°C water bath, and the ligated product was transformed into E.coliX7213. The positive clones were expanded and cultured, the plasmids were extracted, and identified by double enzyme digestion with KpnⅠ and SacⅠ. The double enzyme digestion electrophoresis pattern showed two bands, one band was about 2000bp in size and the other was about 5000bp in size, which was consistent with the expected 2010bp target fragment and pRE112 plasmid. At the same time, PCR identification of Spc ^R gene also indicated that the recombinant suicide plasmid pRE112-LSR had been successfully constructed. The E.coliX7213 strain transformed with the recombinant suicide plasmid pRE-LSR was conjugatively transferred to the RA-YM strain, and the homologous arms were exchanged, and the recombinant suicide plasmid pRE-LSR was integrated into the chromosome of the RA-YM strain, resulting in The phenotype of the positive zygote combined with transfer is Spc ^R , which can grow on the TSA plate containing Spc (50 μg/mL), pick a single colony on the resistant plate and put it in the TSB medium containing Spc (50 μg/mL) overnight nourish. Using C-Spc ^R -CS1 and C-Spc ^R -CS2 as primers, the spectinomycin resistance gene was amplified, and a Spc ^R gene fragment of about 1100 bp was obtained. The amplification results showed that the Spc ^R fragment was successfully inserted into the genome. Design two pairs of primers Deta-D1, Deta-D2 and Check-C1, Check-C2 to amplify the fragment and full length of the planned deletion respectively. The overnight culture solution of the positive zygote is used as a template for PCR amplification reaction, and the former cannot be amplified. , the latter gets fragments around 1968bp. Using the RA-YM strain as a control, the PCR amplification reaction obtained fragments of about 330bp and 1708bp. The electropherogram of the PCR product showed that the 798bp fragment in the 06537/06542 gene had been successfully deleted. The screened RA-YM06537/06542 gene deletion strains were blindly passed in TSB medium containing Spc, and primers C-Spc ^R -CS1, C-Spc ^R -CS2 and Deta-D1, Deta-D2 were used for each generation. for identification. The identification results showed that the missing 06537/06542 gene fragment was not amplified after 5 consecutive generations.

The principle of the invention is the homologous recombination of genes, through the recombination of homologous sequences between exogenous DNA and chromosomal DNA, thereby changing its genetic characteristics. We usually use electroporation transformation, combined with transfer to achieve homologous recombination. The electric shock transformation method is to make the bacteria into a competent state, and use high-voltage electric pulses to produce micropores in the cell membrane of the bacteria, and the foreign DNA fragments enter the nucleus of the bacteria through the micropores, recombine with the genome, the plasma membrane is subsequently repaired, and the bacteria resume growth . According to the different types of cells, the electric shock transformation method can select and optimize a series of parameters such as the electric field strength and DNA concentration during the electric shock transformation process to obtain a relatively high DNA transformation rate. Combination transfer is the process of plasmid transfer, the donor bacteria and the recipient bacteria are in close contact through binding, the plasmid is transferred from the donor bacteria to the recipient bacteria, and the plasmid is replicated at the same time. The transfer plasmid contains the tra gene, which can completely encode the transferase. The plasmid can be transferred from one cell to another cell autonomously. If the plasmid does not contain the tra gene, the transfer plasmid should contain the plasmid transfer initiation site oriT, although it cannot Translocates autonomously, but can be induced by other plasmids containing the complete tra gene.

Compared with the prior art, the present invention has the following outstanding advantages:

1. The material used in the present invention is the wild pathogenic strain RA-YM of R. anatipestifer, which is a serotype 1 bacterial strain isolated from a duck farm in Yunmeng, Hubei, and serotype 1 is currently popular in my country. Therefore, the 06537/06542 gene-deleted strain RA-YMΔ06537/06542 constructed with this pathogenic bacterium has broad application prospects in Hubei and even the whole country.

2. The present invention found that the 06537/06542 gene in RA-YM is a new combination of two-component regulatory system, which provides a basis for in-depth research on the molecular mechanism of R. anatipestifer.

3. After the deletion of the two-component regulatory system 06537/06542 of R. anatipestifer, the virulence of the 06537/06542 deletion strain obtained was significantly reduced, and the ability to liquefy gelatin was lost.

4. The 06537/06542 gene deletion strain can be vaccinated by various methods such as injection or intranasal drops, and the protection against virus is not much different from that of traditional inactivated vaccines.

Description of drawings

Figure 1: Overall technical roadmap of the present invention.

Fig. 2: A technical roadmap for constructing a gene-deleted strain of R. anatipestifer of the present invention.

Fig. 3: Gene structure diagram of 06537/06542 in the present invention.

Fig. 4: Identification of transfer plasmid in the present invention: wherein:

Figure 4A: The result of digestion and identification of the recombinant suicide plasmid pRE-LSR, figure 1: the figure of pRE112-LSR digested with KpnI and SacI, M: DL15000DNAMarker;

Figure 4B: PCR identification of the recombinant suicide plasmid pRE-LSR, Figure 1: PCR product of Spc ^R gene, M: DL15000DNAMarker.

Figure 5: PCR identification of the 06537/06542 gene deletion strain of R. anatipestifer in the present invention, in the figure 1: the full-length PCR amplification product of the 06537/06542 gene in RAΔ06537/06542, 2: 06537/06542 in RA-YM Full-length PCR product of the gene, 3: PCR product of the target deletion gene fragment in RAΔ06537/06542, 4: PCR product of the target deletion gene fragment in RA-YM, 5: PCR product of the Spc ^R gene fragment in RAΔ06537/06542, 6: RA-YM No Spc ^R gene fragment in middle, 7: 16sRNA gene PCR product in RAΔ06537/06542, 8: 16sRNA gene PCR product in RA-YM, M: DL2000DNAMarker.

Figure 6: The growth curve of the 06537/06542 gene-deleted strain of R. anatipestifer.

Fig. 7: The immunization program of the R. anatipestifer 06537/06542 gene-deleted strain to ducklings.

Figure 8: Antibody titer level after immunizing ducklings with the gene deletion strain of R. anatipestifer 06537/06542.

Detailed ways

The present invention will be described in detail below in conjunction with specific embodiments.

1. Cloning of left and right arms of RA-YM06537/06542 gene

According to the gene sequence of RA serotype 1 YM strain (RA-YM) 06537/06542 screened by Chen Fenfen (2008), two pairs of primers were designed using Premier 5.0 (all primers were synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.) and amplified It can be used for the left and right arms of OverlapPCR. A KpnI restriction site was added to the upstream primer L15'-end of Leftarm, and a SacI restriction site was introduced at the downstream primer R25'-end of Rightarm. The primer sequences are as follows:

L1: 5'-C GGTACC TTTTAACTATTGTATTTTATTTAC-3' (KpnI)

L2: 5'-CTTTTAAAACTACTGTCCAGTAGATTGATAAGC-3'

Primers L1 and L2 amplify the upstream homology arm 454bp

R1: 5'-GTTTCGTTCCACTGTTTTGAAGTTGTTTT-3'

R2: 5'-TGAGCTCGGAAGGAAGCGTCTTGCAATATT-3'(SacI)

Primers R1 and R2 amplify the upstream homology arm 456bp

Weigh 4.0 g of improved TSA (i.e. tryptic soy agar medium, purchased from Difco Company, with the medium as the basic component, adding 10% calf serum by volume), distilled water to 100 mL, and pressurize at 121 ° C. After sterilization, cool to 50°C and pour it into a petri dish. After cooling and solidification, the lyophilized RA-YM was placed in solid medium for overnight culture. The next day, pick a single colony and inoculate it in the modified TSB (i.e., tryptic soybean medium, purchased from Difco Company, using this medium as the basic component, adding 10% calf serum by volume) medium, 37 ° C, 200 r/min Cultivate for 5-6h.

Take the bacterial solution as a template, and the amplification reaction is in 25 μL. The reaction system is as follows: Hifi enzyme 0.5 μL, 10×PCRBuffer 2.5 μL, 10 mmol/LdNTPs 0.5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, bacterial solution 0.5 μL, ddH ₂ 020 μL up to 25 μL.

PCR amplification conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 sec, 55°C for 45 sec, and 72°C for 1.5 min; extension at 72°C for 10 min; storage at 12°C. The amplified band was analyzed by 0.8% agarose gel electrophoresis, and the size was about 500bp, which was consistent with the expected target fragment 454bp and 456bp. The amplified product was recovered and purified.

2. Amplification of Spectinomycin Resistance Gene (Spc ^R )

Spectinomycin was selected as a selection marker, referring to the spectinomycin resistance gene sequence on the PIC333 plasmid, primers that could be used for OverlapPCR were designed to amplify the spectinomycin resistance gene (including the promoter).

S1: 5'-TATCAATCTACTGGACAGTAGTTTTTAAAAG-3'

S2: 5'-AAAACAACTTCAAAACAGTGGAACGAAAAC-3'

Using the pIC333 plasmid as a template, the reaction system was as follows: Hifi enzyme 0.5 μL, 10×PCRBuffer 2.5 μL, 10 mmol/L dNTPs 0.5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, plasmid template 0.5 μL, ddH ₂ 020 μLupto 25 μL.

The amplification conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C for 1 min and 20 sec; extension at 72°C for 10 min; storage at 12°C.

After the PCR product was identified by 0.8% agarose gel electrophoresis, the band was about 1000 bp, which was consistent with the expected target fragment, and the target fragment was recovered and purified.

3. Overlap PCR

The purified PCR product was ligated by OverlapPCR, and the reaction system was as follows: Hifi enzyme 0.5 μL, 10×PCRBuffer 2.5 μL, 10 mmol/L dNTPs 0.5 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, template 0.5 μL, ddH ₂ 020 μLupto 25 μL.

The amplification conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C for 1 min and 20 sec; extension at 72°C for 10 min; storage at 12°C. After the PCR products were identified by 1% agarose gel electrophoresis, the target fragments were recovered and purified, and the purified PCR products were respectively connected to the cloning vector pMD18-T vector (purchased from Bao Company Engineering (Dalian) Co., Ltd.) to obtain the pT of the target fragments. -LSR plasmid. The positive clones identified by enzyme digestion were sent to Beijing Qingke for sequencing.

4. Construction of recombinant suicide plasmid pRE112-LSR

The suicide plasmid pRE112 (purchased from BiovectorNTCC Inc.) and the pT-LSR plasmid constructed to contain the target fragment were double-digested with KpnI and SacI, respectively. The target fragment was recovered, ligated overnight in a water bath at 16°C, and the ligated product was transformed into E.coliX7213 (purchased from BiovectorNTCC Inc.). The positive clones were expanded and cultured, the plasmids were extracted, and identified by double enzyme digestion with KpnⅠ and SacⅠ. The double enzyme digestion electrophoresis pattern showed two bands, one with a size of about 2000bp and the other with a size of about 5000bp, consistent with the expected 2010bp target fragment and the pRE112 plasmid (Fig. 4A). At the same time, PCR identification of the Spc ^R gene also indicated that the recombinant suicide plasmid pRE112-LSR had been successfully constructed ( FIG. 4B ).

5. Transformation of recombinant suicide plasmid into E.coliX7213

Place E.coliX7213 competent cells stored in a -80°C refrigerator on ice for 10min-15min to thaw, add 10μL of the ligated recombinant plasmid and mix well. After ice bathing for 30 minutes, heat shock at 42°C for 90 seconds, and immediately transfer to ice bath for 2 minutes. Add 600 μL of antibiotic-free LB medium, shake at 180 r/min at 37°C for 45min-1h to recover. Centrifuge the revived bacterial solution at 5000r/min for 3min, discard about 500μL supernatant, and spread the remaining 100μL resuspended pellet on an LB agar plate containing ampicillin (50μg/mL) resistance (if the transformation product is a plasmid, no centrifugation is required. ). Place it upright at 37°C for 30 minutes, then invert the plate and incubate at 37°C for 14h-16h until colonies appear.

6. Construction and identification of RA-YM06537/06542 gene deletion strain

The E.coliX7213 strain containing the plasmid to be transformed was used as the donor bacterium, and the R. anatipestifer serotype 1 Yunmeng strain (RA-YM) was used as the recipient bacterium, and the deletion strain was constructed by conjugative transfer. The donor bacterium and the recipient bacterium were respectively cultured overnight on a suitable agar plate, the recipient bacterium was inoculated in TSB medium, and cultured with shaking at 200 r/min at 37°C for 12h-16h. Donor bacteria were inoculated in LB liquid medium containing appropriate antibiotics, and cultured overnight at 37°C with shaking at 200r/min. The donor bacteria and the recipient bacteria were revived and cultured for 5h-6h, respectively, until the OD ₆₀₀ reached about 0.8. Centrifuge at 5000r/min for 3min to collect donor bacteria and recipient bacteria respectively. Resuspend with 1 mL of 10 mmol/L MgSO ₄ and repeat washing three times. Add 1 mL of 10 mmol/L MgSO ₄ to resuspend. Take 100 μL of each and mix them, stick a sterile nitrocellulose membrane on a TSA plate containing DAP, drop the mixed bacteria solution on the filter membrane, and culture overnight at 37°C CO ₂ . Wash the bacterial solution on the filter membrane, wash it twice with fresh TSB medium, spread it on a TSA plate containing spectinomycin and culture it overnight at 37°C CO ₂ . Pick a single colony and inoculate it into TSB medium containing spectinomycin, and observe the growth of bacterial clones.

The phenotype of the positive zygote that undergoes binding transfer is Spc ^R and can grow on the TSA plate containing Spc (50 μg/mL), pick a single colony on the resistant plate to the TSB medium containing Spc (50 μg/mL) Incubate overnight. Using C-Spc ^R -CS1 and C-Spc ^R -CS2 as primers, the spectinomycin resistance gene was amplified, and a Spc ^R gene fragment of about 1100 bp was obtained. The amplification results showed that the Spc ^R fragment was successfully inserted into the genome. Design two pairs of primers Deta-D1, Deta-D2 and Check-C1, Check-C2 to amplify the fragment and full length of the planned deletion respectively. The overnight culture solution of the positive zygote is used as a template for PCR amplification reaction, and the former cannot be amplified. , the latter gets fragments around 1968bp. Using the RA-YM strain as a control, the PCR amplification reaction obtained fragments of about 330bp and 1708bp. The electropherogram of the PCR product showed that the 798bp fragment in the 06537/06542 gene had been successfully deleted ( FIG. 5 ).

D1: 5'-AAAGGTATGGGAATGGAG-3'

D2: 5'-GCCGTCTGTAGCAAGGGT-3'

C1: 5'-ACATTATTTCTATCAATT-3'

C2: 5'-TTCTACAAGTTCTTGTAT-3'

CS1:5'-TAAGCTTCAGTGGAACGAAAACTCACGTT-3'

CS2: 5'-TGGATCCCAGTAGTTTTTAAAAGTAAGCACCTG-3'

7. Biological characteristics and identification of RA-YM06537/06542 gene deletion strain

(1) Genetic stability analysis of RA-YM06537/06542 gene deletion strains, the screened RA-YM06537/06542 gene deletion strains were blindly passed in TSB medium containing Spc, and primers C-Spc ^R -CS1, C- Spc ^R -CS2 and Deta-D1, Deta-D2 were identified for each generation. The identification results showed that the missing 06537/06542 gene fragment was not amplified after 5 consecutive generations.

(2) Determination of the growth curve of the RA-YM06537/06542 gene deletion strain, the overnight cultured RA-YM06537/06542 gene deletion strain and the wild strain RA-YM strain were transferred to 10mL of TSB medium at a ratio of 1:100, Shake culture at 200r/min at 37°C, dilute the bacterial solution taken out every hour by 10 times, count on a plate, and draw a growth curve based on the counting results (Figure 6). It was found that the growth rate of the deletion strain was slower than that of the wild strain.

(3) Biochemical identification of the RA-YM06537/06542 gene deletion strain, utilization of carbon and nitrogen sources by the RA-YM06537/06542 gene deletion strain and the wild strain RA-YM strain of R. anatipestifer. Comparing the biochemical characteristics of the 06537/06542 gene deletion strain with the wild strain RA-YM strain, the strain does not produce H ₂ S and does not liquefy gelatin. However, the three RA-YMECFs gene deletion mutants constructed by Xie Miaomiao (2014) were able to liquefy gelatin. Li Jixiang et al. (2009) compared the effects of the culture filtrates of natural strong and naturally attenuated strains on the morphology of duck embryo fibroblasts, and found that the culture filtrates of the strong strains that are virulent and capable of liquefying gelatin can all cause a reaction with gelatinase. The cell morphology changes with consistent effect, and the culture filtrate of the attenuated strain without virulence and non-liquefied gelatin does not show cytotoxicity, so it is speculated that liquefied gelatin is a hallmark biochemical characteristic of the virulent strain of R. anatipestifer. Therefore, compared with other constructed RA-YM gene deletion strains, the RA-YM06537/06542 gene deletion strain of the present invention has lost the ability to liquefy gelatin possessed by the virulent strain.

The RA06537/06542 gene deletion strain prepared by the present invention compares with the biochemical characteristics of RA-YM strain in table 1

-: Negative; +: Positive

The above experiments confirmed that the construction of the RA-YM06537/06542 gene deletion strain of the present invention was successful, and the strain was named Riemerella anatipestifer (Riemerella anatipestifer) YMΔ06537/06542, and was sent to Wuhan, Hubei Province on May 15, 2015. It is preserved in the China Center for Type Culture Collection (CCTCC) within Wuhan University, and the preservation number is: CCTCC NO: M2015306.

8. Determination of LD ₅₀ of RA-YM06537/06542 gene deletion strain

The RA-YM06537/06542 gene deletion strain of the present invention and the control parent strain RA-YM were cultured on TSA-modified medium, single colonies were cultured in TSB-modified medium at 37°C for 5 hours, and counted by plate method. Then, 10-fold equidistant dilution from 10 ⁵ -10 ¹¹ CFU was used to inoculate 12-day-old ducklings by flipper injection, and the control group was injected with the same amount of sterilized phosphate buffered saline (PBS) in the same way. Each inoculated with 0.5mL, generally within 24 hours after inoculation, the ducklings will develop symptoms. Taking 7 days after inoculation as an observation period, observe and record the incidence of the whole process, and count the death of ducklings in each group, and calculate according to the Korbor method. LD ₅₀ of R. anatipestifer on ducklings. The calculation formula is:

LogLD ₅₀ ＝Xm-d(ΣP＝0.5)

In the above formula: Xm is the logarithm of the maximum dose, d is the logarithm of the adjacent dose ratio, p is the mortality rate of each group (death rate is expressed as a decimal), and ΣP is the combination of mortality rates of each group.

The results are shown in Table 2. In this test, the LD ₅₀ of the RA-YM wild strain was 4×10 ⁷ CFU, and the LD ₅₀ of the RA-YM06537/06542 gene deletion strain was greater than 10 ¹⁰ CFU. The LD ₅₀ of the three RA-YMECFs gene deletion mutants constructed by Xie Miaomiao (2014) were 3.16×10 ⁶ CFU, 3.98×10 ⁶ CFU, and 5.01×10 ⁶ CFU, respectively. The virulence of the RA-YM06537/06542 gene deletion strain of the present invention is significantly lower than that of wild strains and RA-YMECFs gene deletion mutants.

RA06537/06542 gene deletion strain prepared by the present invention and RA-YM strain inoculated duckling death situation (number of deaths/total number) of table 2

9. Protection test of ducklings immunized with RA-YM06537/06542 gene deletion strain

(1) The immunization program of ducks (Fig. 7), select the R. anatipestifer antigen, 48 7-day-old ducklings with negative antibody, the test is divided into 6 groups, and the 1st-3rd group is the gene deletion vaccine injection group (5× 10 ⁹ CFU, 1×10 ⁹ CFU, 5×10 ⁸ CFU), the fourth group was gene deletion vaccine intranasal group (1×10 ⁹ CFU), the fifth group was R. anatipestifer-Escherichia coli dual propolis The inactivated vaccine (Huahong Biology, Veterinary Medicine (2012) 150102198) group, and the sixth group was a sterile phosphate buffer (PBS, pH=7.2) control group.

The R. anatipestifer 06537/06542 gene-deleted vaccine was used to immunize ducklings by subcutaneous injection (0.5mL/head) or intranasally (0.1mL/head) respectively. In the inactivated vaccine group, 0.5mL (0.5mL/ml The vaccine contains inactivated duck Escherichia coli≧3.4×10 ⁹ CFU, R. anatipestifer≧1×10 ¹⁰ CFU), and the PBS control group was subcutaneously injected with sterilized PBS (0.5mL/bird) on the back of the neck or 0.1mL intranasally (0.1mL/piece) (5 pieces for each). Blood was collected 14 days after immunization, and the antibody level of the ducklings against the whole bacteria of R. anatipestifer was detected by conventional ELISA method.

(2) ELISA antibody level detection against the whole bacteria of R. anatipestifer. Blood was collected from the medial metatarsal vein 14 days after immunization, and the serum was separated. The antibody against the whole bacteria of R. anatipestifer was detected by indirect ELISA method. Negative (Figure 8).

(3) In the test of the protection rate of immunized ducklings against viruses, 14 days after each group was immunized, 2.5×10 ⁶ CFU of wild-type R. anatipestifer YM strains were injected through the flippers. After the challenge, the ducklings were continuously observed for 7 days, and the clinical symptoms were observed. Finally, calculate the attack protection rate. The results showed that the protective power of the 06537/06542 gene deletion strain was not much different from that of the traditional inactivated vaccine.

Table 3 The result of the RA06537/06542 gene deletion strain prepared by the present invention to the challenge protection of ducklings (survival number/total number)

Claims (2)

1. A gene deletion strain of Riemerella anatipestifer, characterized in that the bacterial strain is Riemerella anatipestifer (Riemerella anatipestifer) YMΔ06537/06542, preserved in the China Center for Type Culture Collection, and its preservation number is CCTCCM2015306, and the bacterial strain has deleted The 798bp of the 06537/06542 gene of the two-component regulatory system leads to a significant decrease in the strain's virulence, and the deleted gene sequence is shown in SEQ ID NO:2. 2. the application of the R. anatipestifer gene deletion strain described in claim 1 in the preparation of the R. anatipestifer attenuated vaccine.