Recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, preparation method and application thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, and a preparation method and application thereof. The recombinant avian Pasteurella multocida obtained by the present invention has good genetic stability and safety, can induce immune ducks to produce humoral immune responses specific to avian Pasteurella multocida and duck circovirus, respectively, and has a good protective effect on slow weight growth and immune system damage caused by duck circovirus infection, and can be used as a vaccine strain for simultaneously preventing avian Pasteurella multocida and duck circovirus.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, and a preparation method and vaccine thereof. Background Art

[0002] Duck circovirus disease and avian Pasteurellosis multocida are two important infectious diseases that affect the healthy development of the duck industry.

[0003] Duck circovirus disease is an immunosuppressive disease caused by duck circovirus, which can cause clinical symptoms such as poor feather development and growth retardation in sick ducks. Cap protein is the only structural protein of the virus, consisting of 218 to 273 amino acids and a molecular weight of about 30KD. Cap protein plays a vital role in the entire viral replication cycle, including virus attachment, invasion, genome uncoating, and packaging of newly formed virus particles. As the only protein presented on the surface of circovirus particles, Cap protein contains the main antigenic determinants and is the first choice for preparing diagnostic reagents and vaccine development. Duck circovirus has been widely spread among ducks in recent years. It has the characteristics of latent infection and is easily overlooked. However, it affects the growth and development of sick ducks and seriously reduces feed conversion rate. In addition, it will damage the immune system of sick ducks, causing immunosuppression in sick ducks, which can easily lead to secondary infection. Due to its short research history, the research on its pathogenic mechanism has been slow. Duck circovirus cannot be cultured in vitro at present, which limits the research on its traditional vaccine and there is no clinical prevention and treatment method.

[0004] Avian Pasteurellosis multocida is highly contagious, has high morbidity and mortality, and is one of the most serious bacterial infectious diseases that endanger the duck industry, posing a great threat to the development of the poultry industry. Due to its numerous serotypes, current vaccines have many deficiencies. The treatment of avian Pasteurellosis multocida relies too much on antibiotics, but over-reliance on antibiotics leads to the emergence of drug-resistant bacteria and the problem of drug residues that people are worried about. With the implementation of the national policy of banning and limiting antibiotics, disease prevention is becoming increasingly important. Therefore, effective prevention of the occurrence of avian Pasteurellosis is the focus of research.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, a preparation method and a vaccine thereof.

[0007] The invention provides a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, which is deposited in China General Microbiological Culture Collection Center with the deposit number of CGMCC 24875.

[0008] The present invention provides a method for preparing a recombinant avian Pasteurella multocida, comprising at least the following steps:

[0009] S1. Obtaining the target gene sequence for expressing duck circovirus antigen Cap protein;

[0010] S2. Construction of recombinant plasmid containing target gene:

[0011] S3. Construction of recombinant strain: Using the constructed recombinant plasmid, the target gene sequence was integrated into the genome of the avian Pasteurella multocida deletion strain ΔaroA-GX-PM to obtain the recombinant avian Pasteurella multocida with the deposit number of CGMCC 24875.

[0012] The invention provides a gene recombination vector vaccine, which is prepared from avian Pasteurella multocida bacterial liquid with the deposit number of CGMCC 24875 to obtain a genetic engineering vaccine for simultaneously preventing avian Pasteurella multocida and duck circovirus.

[0013] The present invention provides the use of the avian Pasteurella multocida in preparing a genetic engineering vaccine for simultaneously preventing avian Pasteurella multocida and duck circovirus.

[0014] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0015] The invention successfully constructs a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, and the expression of Cap protein does not affect the growth of the strain.

[0016] The recombinant avian Pasteurella multocida obtained by the present invention has good in vitro growth, morphological characteristics, genetic stability and safety, can induce ducks to produce humoral immune responses specifically against avian Pasteurella multocida and duck circovirus, respectively, and has a good protective effect on the slowing of weight growth and immune system damage caused by duck circovirus infection, and can be used to prepare a recombinant genetic engineering vaccine for simultaneously preventing avian Pasteurella multocida and duck circovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the prediction result of B cell antigen epitope of Cap protein;

[0018] Figure 2The PCR amplification results of the DuCV Cap gene and vector in Example 1: (A) PCR amplification of the DuCV Cap gene: 1: Cap-PGEX-RH; M: DL 2000 DNA Marker; (B) PCR amplification of the vector: 1: PGEX-GST; M: DL15000 DNA Marker;

[0019] Figure 3 The PCR amplification results of asnA gene promoter, CTA1-DD-RH, optimized DuCV Cap gene, and homology arms, where: 1: aroA-L-RH; 2: asnA-pro-RH; 3: CTA1-DD-RH; 4: Cap-RH; 5: aroA-R-RH; M: DL2000 DNA marker;

[0020] Figure 4 for Figure 3 The fusion results of the fragments in: In which: In Figure A, 1: Cap + aroA-R-RH; 2: aroA-L + asnA-pro-RH; M: DL2000 DNA marker; In Figure B, 1: CTA1-DD + Cap + aroA-R-RH; M: DL5000 DNA marker; In Figure C, 1: aroA-LR + asnA-pro + CTA1-DD + Cap; M: DL5000 DNA marker;

[0021] Figure 5 Figure 1 shows the PCR amplification results of the recombinant plasmid vector: 1: pSHK5(TS)-CbAgo plasmid vector; M: DL15000 DNA marker;

[0022] Figure 6 Figure 1 shows the PCR identification results of the recombinant plasmid: 1-7: single colonies to be identified; 8: plasmid control; 9: H2O; M: DL5000 DNA marker;

[0023] Figure 7 Results for identifying the construction of recombinant plasmids: 1-3: single colonies to be identified; 4: ΔaroA-GX-PM; 5: GX-PM; 6: H2O; M: DL5000 DNA marker;

[0024] Figure 8 Figure 1 shows the results of identifying plasmid elimination: 1-4: single colonies to be identified; 5: aroA-L asnA-pro-CTA1-DD-Cap-CbAgo-pSHK5 (TS) plasmid control; 6: H2O; M: DL5000 DNA Marker;

[0025] Fig. 9 The results of Cap protein expression detection in recombinant strains are shown in Figure A, where Figure A is the protein detection in the recombinant bacteria, and Figure B is the secretory protein detection of the recombinant bacteria; 1: ΔaroA-GX-PM; 2: DuCV Cap-ΔaroA-GX-PM; M: PageRuler TM Prestained Protein Ladder;

[0026] Fig.10 This is the result of growth curve measurement;

[0027] Fig.11 The Gram staining results of GX-PM, deletion mutants and recombinant strains are shown in Figure A: GX-PM; Figure B: ΔaroA-GX-PM; Figure C: DuCV Cap-ΔaroA-GX-PM;

[0028] Fig.12 The results of identifying the genetic stability of the recombinant strains are shown in Figure 1, where 1: the first-generation recombinant strain; 2: the tenth-generation recombinant strain; 3: the twentieth-generation recombinant strain; 4: the thirtieth-generation recombinant strain; 5: ΔaroA-GX-PM; 6: GX-PM; 7: H2O; M: DL5000 DNAMarker;

[0029] Fig.13 For 10 6 CFU, 10 8 The weight results of ducks 14 days after infection with CFU recombinant bacteria;

[0030] Fig.14 This is the result of antibody level against avian Pasteurella multocida in ducks 14 days after immunization with recombinant bacteria;

[0031] Fig.15 This is the result of tissue bacterial load after GX-PM challenge;

[0032] Fig.16 This is the result of the antibody level against Cap protein 14 days after immunization with the recombinant bacteria;

[0033] Fig.17 This is the result of body weight monitoring after DuCV challenge;

[0034] Fig.18 This is the result graph of immune organ index after duck circovirus challenge.

[0035] Instructions for strain preservation:

[0036] 1. The recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein of the present invention is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC 24875 and the deposit date May 9, 2022.

[0037] 2. The avian Pasteurella multocida deletion strain ΔaroA-GX-PM used in the present invention is disclosed in the document "Construction of avian Pasteurella multocida aroA, hexD and hexABC gene deletion mutant strains and study of their immune efficacy [D]. Huazhong Agricultural University, 2020. Wang Shan." and is preserved in Liaocheng University and Huazhong Agricultural University.

[0038] 3. The avian Pasteurella multocida type A strain GX-PM used in the embodiments of the present invention was isolated from the liver tissue of dead chickens in a large-scale chicken farm in Guangxi in 2013, and was disclosed in the document "Yu C, Sizhu S, Luo Q, Xu X, Fu L, Zhang A. Genome sequencing of a virulent avian Pasteurella multocida strain GX-PM reveals the candidate genes involved in the pathogenesis. Res Vet Sci. 2016Apr;105:23-7.doi:10.1016 / j.rvsc.2016.01.013.Epub 2016Jan 18.PMID:27033902." and is preserved in Liaocheng University and Huazhong Agricultural University.

[0039] 4. The duck circovirus tissue virus used in the examples of the present invention was stored in Liaocheng University. DETAILED DESCRIPTION

[0040] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0041] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0042] The embodiment of the present invention provides a recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC 24875. The recombinant bacteria can induce ducks to produce humoral immune responses specific to avian Pasteurella multocida and duck circovirus, respectively, and can be used to prepare genetic engineering vaccines of avian Pasteurella multocida and duck circovirus, and is used to simultaneously prevent and treat infectious diseases caused by avian Pasteurella multocida and duck circovirus.

[0043] The present invention provides a method for preparing the recombinant avian Pasteurella multocida, which comprises at least the following steps:

[0044] S1. Obtaining the target gene sequence for expressing duck circovirus antigen Cap protein;

[0045] S2. Construction of recombinant plasmid containing target gene:

[0046] S3, constructing a recombinant strain: using the constructed recombinant plasmid, the target gene sequence is integrated into the avian Pasteurella multocida deletion strain ΔaroA-GX-PM to obtain a recombinant avian Pasteurella multocida with a deposit number of CGMCC 24875. As a preferred technical solution of an embodiment of the present invention, the target gene sequence at least includes an asnA gene promoter sequence, an optimized Cap gene sequence, an upstream homologous arm sequence, a downstream homologous arm sequence, and an adjuvant sequence. Among them, the signal peptide sequence of OmpH contained in the asnA gene promoter sequence enables the Cap protein to be secreted and expressed. The upstream homologous arm sequence and the downstream homologous arm sequence enable the target gene to undergo homologous recombination with ΔaroA-GX-PM; studies have shown that the asnA promoter on the recombinant plasmid can effectively promote the expression of the VP60 gene in Pasteurella, so the fusion asnA gene promoter is selected in the embodiment of the present invention.

[0047] Since duck circovirus Cap protein is difficult to express soluble in the Escherichia coli prokaryotic expression system, the Cap protein was optimized by truncating its N-terminal arginine-rich NLS sequence (including 1 to 54 amino acids), predicting the B cell antigen epitope of the Cap protein, selecting the main antigen epitope region of the Cap protein, preferably including 54 to 198 amino acids, and obtaining the optimized Cap gene sequence after codon optimization.

[0048] Adjuvants have attracted much attention in vaccine research because they are crucial for enhancing immune responses during the immunization process. Effective and safe immune adjuvants are extremely important for vaccine immunization. CTA1-DD can promote a wide range of specific immune responses and induce long-term immune memory, enhance antibody levels, and activate CD4+ and CD8+ T cells to respond to various antigens. It is one of the most promising mucosal adjuvants known.

[0049] As a preferred technical solution of an embodiment of the present invention, the target gene sequence is obtained by sequentially connecting the upstream homology arm sequence, the asnA gene promoter, the adjuvant sequence, the optimized Cap gene sequence, and the downstream homology arm sequence; wherein the signal peptide sequence containing OmpH is connected to the 3' end of the asnA gene promoter sequence.

[0050] As a preferred technical solution of an embodiment of the present invention, the adjuvant sequence and the optimized Cap gene sequence are connected by a flexible peptide, and the flexible peptide is a nucleotide sequence encoding 4 to 10 amino acids, preferably glycine and serine; the flexible peptide is preferably a G4S linker, and the amino acid sequence is four glycines and one serine.

[0051] As a preferred technical solution of the embodiment of the present invention, a tag for facilitating protein purification is connected to the C-terminus of the optimized Cap gene sequence, and the tag is preferably 5 to 15 His tags.

[0052] As a preferred technical solution of an embodiment of the present invention, the site selected by the upstream homology arm sequence refers to the genome sequence of the Pm70 strain (accession number: AE004439) published in GenBank, between positions 988186 and 988953 of its genome; the site selected by the downstream homology arm sequence is between positions 990277 and 991082 of the genome of the strain.

[0053] As a preferred technical solution of an embodiment of the present invention, the optimized Cap gene sequence is a gene sequence corresponding to 54 to 198 amino acids after removing amino acids 1 to 54.

[0054] As a preferred technical solution of an embodiment of the present invention, the nucleotide sequence of the asnA gene promoter sequence is shown in SEQ ID NO: 1; the asnA gene promoter (asnA-pro-RH) is amplified by primers asnA-pro-RH-F / R1 / R2 (the sequence is preferably SEQ ID NO: 12 to 14) using GX-PM genomic DNA as a template, with a size of 275 bp.

[0055] As a preferred technical solution of an embodiment of the present invention, the nucleotide sequence of the optimized Cap gene sequence is shown in SEQ ID NO: 2; it is synthesized by a biological company and amplified with primers Cap-CTA1-DD-RH-F / R (the sequence is preferably SEQ ID NO: 17, 18) to obtain a Cap-RH size of 543bp.

[0056] As a preferred technical solution of an embodiment of the present invention, the nucleotide sequence of the upstream homology arm sequence is shown in SEQ ID NO: 3; the nucleotide sequence of the downstream homology arm sequence is shown in SEQ ID NO: 4. The amplification of the left and right homology arms of the aroA gene is performed using GX-PM genomic DNA as a template, using the primers aroA-L-RH-F / R (preferably SEQ ID NOs: 10, 11) and aroA-R-RH-F / R (preferably SEQ ID NOs: 19, 20) in Table 2, and using PrimeSTAR HS DNA Polymerase to amplify the upstream homology arm (aroA-L-RH) and downstream homology arm (aroA-R-RH) of the aroA gene, with sizes of 809 bp and 847 bp, respectively.

[0057] As a preferred technical solution of an embodiment of the present invention, the nucleotide sequence of the adjuvant sequence is shown in SEQ ID NO: 5, which is synthesized by a biological company and amplified using primers CTA1-DD-RH-F / R (the sequence is preferably SEQ ID NO: 15, 16) to obtain CTA1-DD-RH with a size of 1032 bp.

[0058] As a preferred technical solution of the embodiment of the present invention, the nucleotide sequence of the target gene sequence of the embodiment of the present invention is shown in SEQ ID NO: 6. The methods for constructing the plasmid all use fusion PCR technology. The principle of fusion PCR technology is to use primers with complementary ends to amplify PCR products with overlapping chains, and connect two different PCR product fragments with overlapping chains through the action of the kit fusion enzyme.

[0059] As a preferred technical solution of an embodiment of the present invention, the gene knocked out in the avian Pasteurella multocida deletion strain is the aroA gene. Preferably, the aroA gene at least includes a sequence having at least 90% homology to the nucleotide sequence shown in SEQ ID NO:7, and further preferably has 95% homology, more preferably has 99% homology, and most preferably is the nucleotide sequence shown in SEQ ID NO:7.

[0060] The embodiment of the present invention also relates to the use of the above-mentioned avian Pasteurella multocida in the preparation of a recombinant avian Pasteurella multocida genetic engineering vaccine that can simultaneously prevent duck circovirus.

[0061] The embodiment of the present invention also relates to a recombinant genetic engineering vaccine, which is prepared from the avian Pasteurella multocida bacterial solution with the deposit number CGMCC 24875 to obtain a genetic engineering vaccine that simultaneously prevents avian Pasteurella multocida and duck circovirus. The recombinant bacteria of the embodiment of the present invention can induce ducks to produce humoral immune responses specific to avian Pasteurella multocida and duck circovirus, respectively, and can therefore be used to prepare a genetic engineering vaccine that can simultaneously prevent avian Pasteurella multocida and duck circovirus, and has a good protective effect.

[0062] The nucleotide sequences used in the examples of the present invention are specifically shown in Table 1:

[0063] Table 1:

[0064]

[0065]

[0066]

[0067] The PCR primers used in the embodiments of the present invention are specifically shown in Table 2:

[0068] Table 2: PCR primers

[0069]

[0070]

[0071] Some abbreviations in the embodiments are represented as follows:

[0072] ΔaroA-GX-PM: aroA gene deletion mutant of avian Pasteurella multocida GX-PM;

[0073] Cap-PGEX-RH: Cap-PGEX fusion fragment obtained by PCR;

[0074] DuCV Cap-ΔaroA-GX-PM recombinant strain: a mutant strain of avian Pasteurella multocida GX-PM with a deletion of the aroA gene that expresses the duck circovirus Cap protein;

[0075] asnA-pro-RH: asnA gene promoter;

[0076] aroA-L-RH: aroA left homology arm;

[0077] aroA-R-RH: aroA right homology arm.

[0078] The biological materials used in the specific embodiments are:

[0079] Escherichia coli DH5α competent cells were purchased from Takara Biotechnology (Dalian) Co., Ltd.;

[0080] The E. coli-Pasteurella multocida shuttle plasmid pSHK5 (TS) is a commercially available plasmid. The expression vector plasmid PGEX-6p-1 is commercially available and is used to express the Cap protein of duck circovirus.

[0081] After reviving avian Pasteurella multocida GX-PM and ΔaroA-GX-PM on TSA plates supplemented with 10% newborn calf serum, single colonies were picked and inoculated into TSB medium supplemented with 10% newborn calf serum and cultured in a shaker at 37°C. Escherichia coli strains were cultured in LA or LB medium. Antibiotics were added to the culture medium according to experimental needs, with the following concentrations: kanamycin was used for Escherichia coli culture at a concentration of 50 μg / mL; for GX-PM strain culture at a concentration of 100 μg / mL. Ampicillin was used for Escherichia coli culture at a concentration of 100 μg / mL.

[0082] The reagents and consumables used in the specific embodiments are shown in Table 3:

[0083] Table 3

[0084]

[0085] Example 1

[0086] This example is used to illustrate the construction of a prokaryotic expression vector for recombinant Cap protein

[0087] PCR amplification of target gene and vector: The NLS sequence (1-54 aa) rich in arginine at the N-terminus of Cap protein was truncated. The B cell antigen epitope of Cap protein (257 aa) was predicted according to the website IEDB.org: Free epitope database and prediction resource. The prediction results of B cell antigen epitope of Cap protein are as follows: Figure 1 The main antigenic epitope region (54-198aa) of the Cap protein was selected, synthesized after codon optimization, cloned into the PGEX-6P-1 plasmid vector, and the soluble expression of the Cap protein was promoted by GST tag protein.

[0088] The vector fragment PGEX-GST and the inserted target gene Cap-PGEX-RH were amplified using the primers PEGX-Cap-RH-F / R (SEQ ID NO: 21, SEQ ID NO: 22) and Cap-RH-F / R (SEQ ID NO: 23, SEQ ID NO: 24) in Table 2, with sizes of 5025 bp and 541 bp, respectively. The experimental results are shown in FIG. Figure 2As shown. Figure 2 It can be seen that the amplification results are consistent with expectations.

[0089] Example 2

[0090] This example is used to illustrate the construction and identification of the DuCV Cap-ΔaroA-GX-PM recombinant strain.

[0091] 1. Construction and identification of DuCV Cap-pSHK5(TS)-CbAgo recombinant plasmid

[0092] 1.1 Amplification of target gene

[0093] In order to stably express and secrete DuCV Cap protein in Pasteurella and stimulate the body to produce a correct immune response, it is necessary to construct a Cap protein expression cassette, including the strong promoter asnA promoter in Pasteurella, the signal peptide sequence of Pasteurella ompH, the adjuvant CTA1-DD, and optimize the base sequence of the Cap gene according to the codon preference of avian Pasteurella multocida, and connect the 10×His sequence for the detection of Cap protein expression. The optimized Cap gene is shown in SEQ ID NO: 2. In order to successfully construct the gene knock-in fragment into the ΔaroA-GX-PM genome, it is necessary to amplify the upstream and downstream homologous arms of the aroA gene.

[0094] The steps of amplifying and concatenating the gene fragments with added homologous sequences are as follows:

[0095] (1) The asnA gene promoter (asnA-pro-RH) was amplified with primers SEQ ID NO: 12 to 14 (asnA-pro-RH-F / R1 / R2) in Table 2. The size was 275 bp. The signal peptide sequence of OmpH was connected to the C-terminus of asnA-pro by primers asnA-pro-RH-R1 / R2.

[0096] (2) CTA1-DD-RH was amplified using primers SEQ ID NO: 15 and SEQ ID NO: 16 (CTA1-DD-RH-F / R) in Table 2, with a size of 1032 bp.

[0097] (3) The optimized DuCV Cap gene was amplified using primers SEQ ID NO: 17 and SEQ ID NO: 18 (Cap-CTA1-DD-RH-F / R) in Table 2 to obtain a Cap-RH with a size of 543 bp.

[0098] (4) The homology arms were amplified using primers SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 20 (aroA-L-RH, aroA-R-RH) in Table 2, and the sizes were 809 bp and 847 bp, respectively.

[0099] The amplification results of each fragment are as follows Figure 3 As shown, the bands appear to be correct.

[0100] 1.2 Tandem replication of target genes

[0101] The five amplified fragments were concatenated using overlapping PCR:

[0102] (1) First, the aroA-L-RH and asnA-pro-RH fragments were concatenated. After concatenation, the size of aroA-L+asnA-pro-RH was 1043 bp.

[0103] (2) Cap-RH and aroA-R-RH were concatenated to obtain Cap+aroA-R-RH, with a size of 1348 bp. The PCR amplification result was in line with expectations.

[0104] (3) The fragments CTA1-DD-RH and Cap+aroA-R-RH were concatenated to obtain a fragment CTA1-DD+Cap+aroA-R-RH with a size of 2338 bp. The PCR amplification result was in line with expectations.

[0105] (4) AroA-L+asnA-pro-RH was concatenated with CTA1-DD+Cap+aroA-R-RH to obtain an insert fragment of aroA-LR+asnA-pro+CTA1-DD+Cap with a size of 3363 bp. The PCR amplification results were in line with expectations.

[0106] PCR amplification results are as follows Figure 4 shown.

[0107] 1.3 Amplification of plasmid vector

[0108] Using pSHK5(TS)-CbAgo plasmid as template, the linearized fusion plasmid vector was amplified using primers SEQ ID NO:8 and SEQ ID NO:9 (pSHK5Ts-MCS-RH-F / R) in Table 2. The experimental results are shown in Figure 5 As shown, the size is 5036 bp.

[0109] 1.4 Fusion transformation and identification of recombinant plasmids

[0110] Using the fusion PCR reaction system in Table 4 and the reaction conditions of fusion PCR in Table 5, the plasmid vector pSHK5(TS)-CbAgo was fused with the insert fragment aroA-LR+asnA-pro+CTA1-DD+Cap-RH, and then a single colony on the LA+Kan plate was picked for identification. PCR amplification was performed using primers SEQ ID NO:25 and SEQ ID NO:26 (pSHK5(TS)-MCS-ID-F / R) in Table 2. The size of the amplified product of the correctly fused transformant should be 3819 bp. The experimental results are shown in Figure 2. Figure 6 shown.

[0111] Depend on Figure 6 It can be seen that the band sizes of the PCR products of single colonies 1, 2, 3, 5, and 7 are suspected to be correct. After comparison, the sequencing results confirmed that the plasmid vector and the inserted fragment were correctly fused and there was no mutation, indicating that the recombinant plasmid had been correctly constructed, and the plasmid was extracted for subsequent experiments.

[0112] Table 4: Fusion PCR reaction system

[0113]

[0114]

[0115] Table 5: Fusion PCR reaction conditions

[0116] time temperature 37℃ 15min 50℃ 8min 52℃ 8min 54℃ 8min 58℃ 8min 60℃ 8min 25℃ 1min

[0117] Screening and identification of 2DuCV Cap-ΔaroA-GX-PM recombinant strains

[0118] 2.1 Screening of DuCV Cap-ΔaroA-GX-PM recombinant strains

[0119] The correctly identified plasmid was electroporated into the ΔaroA-GX-PM strain, and the non-resistant TSB culture medium was added to resuscitate in a shaker at 28°C for 2 hours, then coated on a TSA+Kan plate and placed in a 28°C incubator for growth for 36 to 48 hours. After the colonies have grown, pick a single colony and coat it on a new TSA+Kan plate, place it at room temperature for growth, and after several solid passages, use the primers SEQ ID NO:29 and SEQ ID NO:30 (LR-aroA-LR-JD-F / R) in Table 2 to identify whether recombination has occurred in the single colony. The size of the amplified product of wild GX-PM is 3473bp, the size of the amplified product of ΔaroA-GX-PM is 2156bp, and the recombinant bacteria with gene insertion amplify a product of 3898bp that is larger than the wild strain. The experimental results are as follows Figure 7 shown.

[0120] like Figure 7As shown, the band of the single colony No. 3 to be identified is suspected to be correct, indicating that genetic recombination may have occurred in the single colony No. 3.

[0121] 2.2 Purification of DuCV Cap-ΔaroA-GX-PM recombinant strain

[0122] Transfer single colony No. 3 to TSB medium without resistance, culture in a shaking incubator at 37°C, and subculture several times. When it reaches the 4th generation, dilute the bacterial solution and spread it on a TSA plate without resistance. Pick out the grown single colonies and align them on the TSA plate without resistance and the TSA plate with Kan resistance. Pick out the single colonies that do not grow on the Kan-resistant plate but grow on the non-resistant plate. Use the identification primers SEQ ID NO:27 and SEQ ID NO:28 (CbAgo-inner-JD-F / R) on the recombinant plasmid in Table 2 for identification. The experimental results are shown in the figure. Figure 8 shown.

[0123] like Figure 8 As shown, no bands were amplified from the single colonies, indicating that the recombinant plasmid had been successfully eliminated. The primers LR-aroA-LR-JD-F / R were used to amplify a single large band again, and the PCR identification product was sent to the company for sequencing. The sequencing results showed that the asnA-pro+CTA1-DD+Cap fragment had been successfully inserted into the ΔaroA-GX-PM genome, and no mutations were generated. The above results indicate that the DuCV Cap-ΔaroA-GX-PM recombinant strain with the Cap gene expression frame inserted without molecular markers was successfully constructed.

[0124] 2.3 Detection of Cap protein expression in DuCV Cap-ΔaroA-GX-PM recombinant strain

[0125] The correctly identified DuCV Cap-ΔaroA-GX-PM recombinant strain was picked and transferred to 10mL serum-free TSB medium at a ratio of 1:1000 after recovery. At the same time, ΔaroA-GX-PM was transferred under the same conditions as a control. The two bacteria were cultured in a shaking incubator at 37°C for 12 hours and then collected separately. The bacteria were resuspended in 1mL PBS and ultrasonically disrupted for sample preparation. The supernatant of the culture medium was precipitated with TCA and then boiled and dissolved in 200uL 2×SDS loading buffer. The bacterial protein and secretory protein samples were subjected to Western Blot analysis. The experimental results are shown in the figure. Fig. 9 shown.

[0126] Depend on Fig. 9It can be seen that a band of the target size was detected in the bacteria, indicating that the recombinant Cap protein was successfully expressed in the DuCV Cap-ΔaroA-GX-PM recombinant bacteria. The presence of recombinant Cap protein was also detected in the culture supernatant, indicating that the recombinant Cap protein can be expressed in the DuCV Cap-ΔaroA-GX-PM recombinant bacteria and secreted into the extracellular space. The above results show that the DuCV Cap-ΔaroA-GX-PM recombinant bacteria that recombinantly express duck circovirus Cap protein have been successfully constructed and can be used for subsequent experiments.

[0127] Experimental Example 1

[0128] This experimental example is used to illustrate the in vitro growth characteristics of the DuCV Cap-ΔaroA-GX-PM recombinant strain: In order to study whether gene knock-in and protein expression have any effect on strain growth, wild strain GX-PM, gene-deficient attenuated strain ΔaroA-GX-PM and DuCV Cap-ΔaroA-GX-PM recombinant strain were picked up and revived in TSB medium containing 10% serum, and then transferred to fresh medium at a ratio of 1:1000. Sample plates were added, 200 μL per well, 3 replicates were set, and TSB medium with 10% serum was set as a blank control. According to the OD measured every half hour, 600nm , draw the bacterial growth curve. The experimental results are as follows Fig.10 shown.

[0129] Depend on Fig.10 It can be seen that when the control was established, under the same conditions, the wild bacteria GX-PM grew the fastest, and the growth rate of ΔaroA-GX-PM slowed down as previously proven, while the growth curve of the DuCV Cap-ΔaroA-GX-PM recombinant bacteria was not significantly different from that of the starting strain ΔaroA-GX-PM, indicating that the knock-in of the Cap gene expression frame and the expression of the Cap protein had no obvious effect on the growth of the strain.

[0130] Experimental Example 2

[0131] This example is used to illustrate the in vitro morphological characteristics of the DuCV Cap-ΔaroA-GX-PM recombinant strain: The wild-type GX-PM, the gene-deleted mutant ΔaroA-GX-PM and the recombinant DuCV Cap-ΔaroA-GX-PM were revived and transferred to fresh culture medium and cultured at 37°C until OD 600nm When the value is about 0.9, the smear is stained and examined under a microscope. Fig.11 shown.

[0132] Depend on Fig.11It can be seen that after Gram staining, Pm appeared as small spheres or short rods under the microscope, existing singly or in short chains. There was no significant difference in the morphology between GX-PM, ΔaroA-GX-PM and recombinant bacteria DuCV Cap-ΔaroA-GX-PM, indicating that gene knock-in and expression of exogenous Cap protein had no significant effect on bacterial morphology.

[0133] Experimental Example 3

[0134] This example is used to illustrate the in vitro genetic stability of the DuCV Cap-ΔaroA-GX-PM recombinant strain: In order to verify whether the knocked-in gene can be stably present in the bacterial genome, the recombinant bacteria DuCV Cap-ΔaroA-GX-PM were continuously passaged in liquid culture medium and identified every 10 generations. The experimental results are shown in Fig.12 shown.

[0135] Depend on Fig.12 It can be seen that the expression frame of the knocked-in Cap gene still exists stably after the recombinant strain was continuously passaged for 30 generations, indicating that the DuCV Cap-ΔaroA-GX-PM recombinant strain has good in vitro genetic stability.

[0136] Experimental Example 4

[0137] This experimental example is used to illustrate the safety of the DuCV Cap-ΔaroA-GX-PM recombinant strain:

[0138] The safety of the DuCV Cap-ΔaroA-GX-PM recombinant strain was evaluated by comparing the weight changes, mental state, clinical symptoms and survival of the infection group and the PBS group. Fig.13 shown.

[0139] Fig.13 The results of the weight of ducks 14 days after infection with the recombinant bacteria showed that 8 After CFU infection, the weight of the PBS group was significantly higher than that of the infected group, indicating that although the recombinant bacteria did not cause clinical symptoms in ducks, it did affect the weight gain of ducks. 6 After 14 days of infection with CFU recombinant bacteria, there was basically no difference in the final average body weight between the infection group and the PBS group, indicating that the use of 10 6 CFU infection dose, DuCV Cap-ΔaroA-GX-PM recombinant bacteria neither caused disease in ducklings nor had a negative impact on the growth of ducklings, 10 6 CFU can be used as the immune dose for the next experiment.

[0140] Experimental Example 5

[0141] This example is used for the immune efficacy of the DuCV Cap-ΔaroA-GX-PM recombinant strain:

[0142] In order to verify whether the recombinant bacteria can produce immune protection in ducks, 40 7-day-old healthy ducklings were divided into four groups, namely, the immunization + DuCV group, the PBS + DuCV group, the immunization + GX-PM group, and the PBS + GX-PM group. 6 After 14 days of immunization, blood was collected from three ducks in each group. The immunization + DuCV group and the PBS + DuCV group were challenged with 100 μL duck circovirus tissue stock solution, and the immunization + GX-PM group and the PBS + GX-PM group were challenged with 10 8 The mice were challenged with CFU GX-PM, and the changes in body weight and clinical symptoms were monitored. An autopsy was performed 14 days after immunization, and 3 mice from each group were selected to test the bacterial load in blood and liver.

[0143] 1.1. Study on the immune protection efficacy of recombinant bacteria against avian Pasteurella infection

[0144] 14 days after immunization, blood was collected from three rats in the immunization + GX-PM group and the PBS + GX-PM group, and serum was collected. According to the square array test, the optimal coating concentration of the antigen GX-PM was determined to be 2×10 8 The optimal dilution of serum was 1:80. The whole bacteria of GX-PM were used as antigen, the collected duck serum was used as primary antibody, and the rabbit anti-duck IgG antibody was used as secondary antibody. The antibody level against GX-PM after immunization was detected by indirect ELISA. The experimental results are shown in Fig.14 shown.

[0145] Depend on Fig.14 It can be seen that after immunization with the recombinant bacteria DuCV Cap-ΔaroA-GX-PM, the body can produce a higher level of antibodies against avian Pasteurella, and the difference is very significant compared with the control group (P<0.01). This shows that after immunization with the recombinant bacteria DuCV Cap-ΔaroA-GX-PM, ducks can produce humoral immunity against avian Pasteurella multocida GX-PM, indicating that the recombinant bacteria has good immunogenicity.

[0146] After 14 days of GX-PM infection, the bacterial load in blood and liver was compared. The experimental results are as follows Fig.15 shown.

[0147] Depend on Fig.15 It can be seen that the tissue bacterial load in the immunization+GX-PM group was significantly lower than that in the PBS+GX-PM group (P<0.05).

[0148] 1.2. Study on the immune protection efficacy of recombinant bacteria against DuCV infection

[0149] The prepared recombinant Cap protein was coated, and the collected serum was used as the primary antibody and the rabbit anti-duck IgG antibody was used as the secondary antibody to perform an indirect ELISA test to detect the antibody level 14 days after immunization, thereby reflecting whether the recombinant bacteria can induce the body's immune response after immunization. According to the square array test, it was confirmed that the optimal coating condition of the recombinant Cap protein was 3μg / mL and the optimal serum dilution condition was 1:40. The experimental results are as follows Fig.16 shown.

[0150] Depend on Fig.16 It can be seen that compared with the non-immunized group, the IgG antibody level in the duck serum of the immunized group was higher and the difference was very significant (P<0.01). This shows that after immunization with the DuCV Cap-ΔaroA-GX-PM recombinant bacteria, ducks can be induced to produce humoral immunity against the Cap protein and produce higher antibody levels. Therefore, the recombinant bacteria has good immunogenicity.

[0151] Since duck circovirus has a low mortality rate when infected alone, but it can cause slow growth and development of sick ducks, poor feather development, messy feathers, and immunosuppression, this study mainly monitored the weight of the two groups of ducks for 14 days after infection, and observed the clinical symptoms and feeding conditions of the ducks. After DuCV infection, neither group showed obvious clinical symptoms, but the food intake of the PBS group was reduced compared with the immunized group. The experimental results are as follows Fig.17 shown.

[0152] Depend on Fig.17 It can be seen that from the weight gain rate after the DuCV challenge, the weight of the two groups increased steadily after the DuCV challenge, but the weight gain of the PBS group was significantly lower than that of the immunized group. After 14 days, the final average weight of the PBS group was 1.413Kg, and the final average weight of the immunized group was 1.566Kg. The difference in weight between the two groups was very significant. It was proved that after immunization with the DuCV Cap-ΔaroA-GX-PM recombinant bacteria, ducks can effectively protect themselves from the symptoms of growth retardation and limited weight gain caused by infection with DuCV, indicating that the DuCVCap-ΔaroA-GX-PM recombinant bacteria has a good immune protection effect.

[0153] Duck circovirus mainly attacks the immune system, causing lymphocyte necrosis and thus damaging immune organs. Therefore, 14 days after infection, three ducks were taken from each of the immunization group and the PBS control group, and their main immune organs were taken and weighed respectively. The immune organ index was calculated to indirectly reflect their immune level. Fig.18 shown.

[0154] Depend on Fig.18It can be seen from the comparison of the results of the two groups that the immune organ indexes of the immunized group were higher than those of the PBS control group, and the thymus index of the immunized group was significantly higher than that of the control group (P<0.05). This shows that the immunization of DuCV Cap-ΔaroA-GX-PM recombinant bacteria has a certain immune protection effect on the immune organs of ducks.

[0155] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments described herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein. Sequence Listing <110> Liaocheng University Huazhong Agricultural University <120> Recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, preparation method and application thereof <160> 30 <170> SIPOSequenceListing 1.0 <210> 1 <211> 200 <212> DNA <213> Artificial Sequence <400> 1 aaacacgcaa tatttgctga tcaagattat caatattgtg catagttgac cttttttaga 60 attttttcaa aaataaatac gttatattgt ataaaattca aagtatatcg cttatttcaa 120 aaaaagtatt gagttatagg gcttttgtat taaataatat tcagtgaatt tgatgtagta 180 tatattttaa ggtatcgaaa 200 <210> 2 <211> 486 <212> DNA <213> Artificial Sequence <400> 2 atgggttctg gtacaggtcc aacagcagca ggtaaatggc aatctttaag tttagaagat 60 ggtgcacaat atacagatcc accagcacat ggtaataata tttgtggttt aaatatgcgt 120 tgggcaatgt ttggtgatac aaatagttat atgacaggta caacaccaaa ttatcattat 180 ccatatgatt attatattat taaaggtgtg gcaattacat tacgtccagc atataatatt 240 tatgaaaaat ctaaaacaca aggttctaca gttattgata aagatggtca aattgtgaaa 300 acaagtacta caggttggtc tattgatcca tatggttcaa catctagtcg tagaacaggt 360 gatccatcta gagttcatcg tcgttatttt gttccaaaac caattattca aggtgcaggt 420 gaaggtacag aatattctgg tggtggtggt tcacatcatc atcatcatca tcatcatcat 480 cattaa 486 <210> 3 <211> 768 <212> DNA <213> Artificial Sequence <400> 3 atccccttgt tacacaagct atcaatgcca aaaaagtgga aattccagcg aaaaattatg 60 gggctgattt agacggtttt ttacaggcaa taagtgataa aaccaaatta atttatctcg 120 cgaatcccaa taatccgaca ggcacttttt taagtgcggg tgaaatttcc caatttttaa 180 atcaagtgcc cgcgcatgtc atcgtggtat tagatgaggc ttatactgaa tttaccttgc 240 cggaagagcg agtagattcc tttacgttac tcaaaaaaca ctctaatctt gtgatttgtc 300 gtaccctttc taaagcctat ggtttagcgg gtttgcgcat tggttacgcg gtttcttctg 360 ccgagattgc tgacctcttt aatcgcgttc gccaaccgtt taactgtaat agtctggcat 420 tagccgctgc taccgcggtg ctgcatgatg atgcatttat tgcgaaagtg gctgaaaaca 480 accgacaagg gttgaagtta ttggaagact tctttacagc caaaggtttg aactatattc 540 cttcaaaagg caattttgtc atgttagatg tcaatcaacc agccttacca atttatcagg 600 cattattaca aaaaggcgtg attgtgcgtc cgattgcagg ttatggctta ccaaatcatt 660 tacggattag catcggttta ccagaagaaa accaacgttt cttactcgca ttaaacgaag 720 tattagggct ttaagccaaa cttatccagt gtataggaaa tataaatc 768 <210> 4 <211> 806 <212> DNA <213> Artificial Sequence <400> 4 cctacagata agtactcagc aaaaagcgaa gtattttata attacktcgct ttttgagtga cgaatcgggt ctgagataat gcaagaaaac accttaagac tgaatccgaa aactcgccat ataatttgcg ctgacatctt tattgagcca gaatttaccc gtcaatggat tgtaatgata gcctaccata tctaaacagg ttattgtgc ttcatcacac caatgaagca attccgctgg cttaataat ttgtcgtaat cgtgtgtccc tttcggcaac attttcaaca catattccgc accataatc accaatgccc aggctttaag ggttcgatta atggtggaga aaaaaatcac cccgtttggt tttaatt gcttacaaca ggcaataatc gaactcgggt caggcacatg 480. ctcaagcatt tccatgcaag taatcacatc aaacttttcg tcctcccctc tttcagcaaa aagtgcggtc tgattttgca aaaattcctc aatcgtaatt tgttgataat caatgtgtag cccgctttct aaagcatgtt ttcttgccac ttgtaatggg gcagaagaca tatcaatccc 600. cgtcacaatt gcgccttgct ttgccatgct ttctgacaaa atgccaccgc cacagcccac 660 atccagcact tttttccccg ttagcccgtt ggcttgctgt gcaatatagc ttaaacgtaa cggattaagt tgatggatcg gtttgaaatc cccttgcgga tcccaccagc tttttgccat 780 tttctcaaac ttatcaagtt cttgtt 806 <210> 5 <211> 948 <212> DNA <213> Artificial Sequence <400> 5 aatgatgata agctgtatcg cgcggatagt cggccgccgg atgaaattaa gcagagcggc 60 ggcttgatgc cgcggggcca aagtgaatat tttgatcggg gcacccagat gaatattaat 120 ttgtatgatc atgcgcgcgg tacccagacc ggctttgttc gccatgatga tggctatgtt 180 agtacctcaa tttcgttgcg gagtgctcat ttggttggtc agaccatttt gtcgggccat 240 tcgacctatt atatttatgt tattgcgact gcgccgaata tgtttaatgt taatgatgtt 300 ctgggcgcgt attctccgca tccggatgaa caggaagttt cggcgttggg cggtattccg 360 tatagtcaga tttatggttg gtatcgcgtt cattttggcg ttttggatga acagttgcat 420 cggaatcggg gctatcgcga tcgctattat agtaatttgg atattgcgcc ggccgccgat 480 ggctatggtt tggcgggttt tccgccggaa catcgggcgt ggcgcgaaga accgtggatt 540 catcatgcgc cgccgggctg tggcaatgcg ccgcgtagtt cggcagatgc gcagcagaat 600 aattttaata aggatcagca gagtgcgttt tatgaaattt taaatatgcc gaatctgaat 660 gaagcacaac gcaatggctt tattcagagt ttgaaggatg atccgagtca aagtaccaat 720 gttttgggcg aagccaagaa gttgaatgaa agtcaggcgc cgaaggcgga tgcgcagcaa 780 aataatttta ataaagatca gcagtcggcg ttttatgaaa ttttgaatat gccgaatttg 840 aatgaagcgc agcgcaatgg ttttattcag tcattgaaag atgatccgtc acagagtacc 900 aatgttttag gcgaagccaa aaagttgaat gaatcgcagg cgccgaaa 948 <210> 6 <211> 3363 <212> DNA <213> Duck circovirus <400> 6 tcgaggtcga cggtatcgat aatccccttg ttacacaagc tatcaatgcc aaaaaagtgg 60 aaattccagc gaaaaattat ggggctgatt tagacggttt tttacaggca ataagtgata 120 aaaccaaatt aatttatctc gcgaatccca ataatccgac aggcactttt ttaagtgcgg 180 gtgaaatttc ccaattttta aatcaagtgc ccgcgcatgt catcgtggta ttagatgagg 240 cttatactga atttaccttg ccggaagagc gagtagttc ctttacgtta ctcaaaaaac actctaatct tgtgatttgt cgtacccttt ctaaagccta tggtttagcg ggtttgcgca 360 ttggttacgc ggtttcttct gccgagattg ctgacctctt taatcgcgtt cgccaaccgt 420 ttaactgtaa tagtctggca ttagccgctg ctaccgcggt gctgcatgat gatgcattta 540. ttgcgaaagt ggctgaaaac aaccgacaag ggttgaagtt attggaagac ttctttacag ccaaggttt gaactatatt ccttcaaaag gcaattttgt catgttagat gtcaatcaac cagccttacc aatttatcag gcattattac aaaaaggcgt gattgtgcgt ccgattgcag gttatggctt accaatcat ttacggatta gcatcggttt accagaagaa aaccaacgtt tcttactcgc attack gtattagggc tttagcca acttatccag tgtatagga fathers fathers fathers fathers fathers fathers ttttttagaa ttttttcaaa aataatcg ttttttgta taaaattcaa agtatatcgc ttatttcaaa aaaagtattg agttataggg cttttgtatt aaataatt cagtgaattt gatgtagtat atattttaag gtatcgaaaa tgaaaaagac aatcgtagca ttagcagtcg 1020 cagcagtagc agcaacttca gcaaacgcaa atgatgataa gctgtatcgc gcggatagtc 1080 ggccgccgga tgaaattaag cagagcggcg gcttgatgcc gcggggccaa agtgaatatt 1140 ttgatcgggg cacccagatg aatattaatt tgtatgatca tgcgcgcggt acccagaccg 1200 gctttgttcg ccatgatgat ggctatgtta gtacctcaat ttcgttgcgg agtgctcatt 1260 tggttggtca gaccattttg tcgggccatt cgacctatta tatttatgtt attgcgactg 1320 cgccgaatat gtttaatgtt aatgatgttc tgggcgcgta ttctccgcat ccggatgaac 1380 aggaagtttc ggcgttgggc ggtattccgt atagtcagat ttatggttgg tatcgcgttc 1440 attttggcgt tttggatgaa cagttgcatc ggaatcgggg ctatcgcgat cgctattata 1500 gtaatttgga tattgcgccg gccgccgatg gctatggttt ggcgggtttt ccgccggaac 1560 atcgggcgtg gcgcgaagaa ccgtggattc atcatgcgcc gccgggctgt ggcaatgcgc 1620 cgcgtagttc ggcagatgcg cagcagaata attttaataa ggatcagcag agtgcgtttt 1680 atgaatttt aaatatgccg aatctgaatg aagcacaacg caatggcttt attcagagtt tgaaggatga tccgagtcaa agtaccaatg ttttgggcga agccaagaag ttgaatgaaa gtcaggcgcc gaaggcggat gcgcagcaaa ataattttaa taaagatcag cagtcggcgt tttatgaaat tttgaattg ccgaatttga atgaagcgca gcgcaatggt tttattcagt cattgaaaga tgatccgtca cagagtacca atgttttagg cgaagccaaa aagttgaatg aatcgcaggc gccgaaacat catcatcatc atcatcatca tggtggcgga ggtagcggat ccatgggttc tggtacaggt ccaacagcag caggtaaatg gcaatcttta agtttagaag atggtgcaca father ccaccagcac atggtaata tatttgtggt ttaaatatgc gttgggcaat gtttggtgat acaaatagtt atatgacagg tacaacacca aattatcatt atccatatga ttattatatt attack tggcaattac attack gcatataata tttatgaaaa atctaaaaca caaggttcta cagttattga taaagatggt caaattgtga 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400. 2400 gtgatccatc tagagttcat cgtcgttatt ttgttccaaa accaattatt caaggtgcag 2460 gtgaaggtac agaatattct ggtggtggtg gttcacatca tcatcatcat catcatcatc 2520 atcattaaga attctaacct acagatagat actaagcaaa aagcgaagta ttttataata 2580 cttcgcttt tgagtgacga atcgggtctg agataatgca agaaaacacc ttaagactga 2640 atccgaaaac tcgccatata atttgcgctg acatctttat tgagccagaa tttaccccgtc 2700 aatggattgt aatgatagcc taccatatct aaacaggtta attgtgcttc atcacaccaa 2760 tgaagcaatt ccgctggctt aataaatttg tcgtaatcgt gtgtcccttt cggcaacatt 2820 ttcaacacat attccgcacc aataatcacc aatgcccagg cttaagggt tcgattaatg 2880 gtggagaaaa aaatcacccc gtttggtttt aataattgct tacaacaggc aataatcgaa 2940 ctcgggtcag gcacatgctc aagcatttcc atgcaagtaa tcacatcaaa cttttcgtcc 3000 tcccctctt cagcaaaaag tgcggtctga ttttgcaaaa attcctcaat cgtaatttgt 3060 tgataatcaa tgtgtagccc gctttctaaa gcatgttttc ttgccacttg taatggggca 3120 gaagacatat caatccccgt cacaattgcg ccttgctttg ccatgctttc tgacaaaatg 3180 ccaccgccac agcccacatc cagcactttt ttccccgtta gcccgttggc ttgctgtgca 3240 atatagctta aacgtaacgg attaagttga tggatcggtt tgaaatcccc ttgcggatcc 3300 caccagcttt ttgccatttt ctcaaactta tcaagttctt gttagcttga tatcgaattc 3360 ctg 3363 <210> 7 <211> 1323 <212> DNA <213> avian Pasteurella multocida <400> 7 gtgataaaag atgcgaccgc tattactctc aatcccatca gctatattga aggcgaggtg 60 cgtttaccgg gctccaaaag cttatccaat cgcgcactct tactttccgc attagctaaa 120 ggaaaaacaa cattaaccaa tctgttagat agtgatgatg tgcgccatat gttaaatgcg 180 ttaaaagaac ttggcgtgac ttatcaactc tcagaagaca aatccgtctg tgaaattgaa 240 ggcttaggac gtgcttttga atggcaaagt ggcttagctt tatttttggg caatgcaggg 300 acggcgatgc gtcccttgac tgccgcgctt tgtttatcta caccgaacaa ggaaggcaaa 360 420. aatgaaatag tcttgactgg cgaacctcgt atgaaagaac gcccaataca acatttagtt 480. gtgcattat gtcaagctgg cgcagaaatt cagtatttag aacaagaagg ttacccacct atcgccattc gaataccgg actcaaaggc ggacgaatac aaattgatgg gtcagtttct tctcaatttt tgaccgcact tttaatggct gccccgatgg cagaggcgga tacggaatt gaaatcatcg gtgagctggt ttccaaacct tacattgata tcacccttaa gatgatgcaa acctttggcg ttgaagttga aaaccaagcc tatcaacgct ttttggtgaa aggtcatcag snow snow snow snow snow snow snow snow snow snow snow snow snow cover cttgccgcgg gggaaaagta aaagtcacag gcgtcggtaa aaatagcatt caaggggatc gtctgtttgc ggatgtgcta gaaaaaatgg gggcgcatat cacttggggc gacgatttta ttcaagtgga aaaaggcaac ctcaaaggca tcgatatgga tatgaaccat attcccgatg cggcaatgac cattgccacc acagcgcttt ttgcagaagg tgaaacggtc attcgtaata tttataactg gcgcgtaaaa gaactgatc gcttgaccgc gatggcgacc gaactgcgta aggtgggggc ggaagtggaa gaaggcgaag attttattcg tatccagcca 1140 ttgaatctgg cgcaatttca acatgctgaa attgaaacat acaatgatca ccgcatggcg 1200 atgtgctttg ctttaatcgc attgtcgcaa acgtcggtca cgattttaga cccgagctgt 1260 accgcaaaaa cgtttcctac gttttttgat acttttttac gcttaacaca cgcagaaagt 1320 tag 1323 <210> 8 <211> twenty three <212> DNA <213> Artificial Sequence <400> 8 agcttgatat cgaattcctg cag 23 <210> 9 <211> twenty two <212> DNA <213> Artificial Sequence <400> 9 tatcgatacc gtcgacctcg ag 22 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <400> 10 tcgaggtcga cggtatcgat aatccccttg ttacacaagc tatca 45 <210> 11 <211> 45 <212> DNA <213> Artificial Sequence <400> 11 tcagcaaata ttgcgtgttt gatttatatt tcctatacac tggat 45 <210> 12 <211> 47 <212> DNA <213> Artificial Sequence <400> 12 agtgtatagg aaatataaat caaacacgca atatttgctg atcaaga 47 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <400> 13 tgctaatgct acgattgtct ttttcatttt cgatacctta aaata 45 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <400> 14 tgctgaagtt gctgctactg ctgcgactgc taatgctacg attgt 45 <210> 15 <211> 45 <212> DNA <213> Artificial Sequence <400> 15 gtagcagcaa cttcagcaaa cgcaaatgat gataagctgt atcgc 45 <210> 16 <211> 42 <212> DNA <213> Artificial Sequence <400> 16 tgtaccagaa cccatggatc cgctacctcc gccaccatga tg 42 <210> 17 <211> 45 <212> DNA <213> Artificial Sequence <400> 17 catcatggtg gcggaggtag cggatccatg ggttctggta caggt 45 <210> 18 <211> 45 <212> DNA <213> Artificial Sequence <400> 18 tgcttagtat ctatctgtag gttagaattc ttaatgatga tgatg 45 <210> 19 <211> 45 <212> DNA <213> Artificial Sequence <400> 19 catcatcatt aagaattcta acctacagat agatactaag caaaa 45 <210> 20 <211> 46 <212> DNA <213> Artificial Sequence <400> 20 caggaattcg atatcaagct aacaagaact tgataagttt gagaaa 46 <210> twenty one <211> 42 <212> DNA <213> Artificial Sequence <400> twenty one catcatcatc attaagaatt cgaattcccg ggtcgactcg ag 42 <210> twenty two <211> 42 <212> DNA <213> Artificial Sequence <400> twenty two gtaccagaac ccatggatcc cggggatccc aggggcccct gg 42 <210> twenty three <211> 42 <212> DNA <213> Artificial Sequence <400> twenty three ccaggggccc ctgggatccc cgggatccat gggttctggt ac 42 <210> twenty four <211> 42 <212> DNA <213> Artificial Sequence <400> twenty four ctcgagtcga cccgggaatt cgaattctta atgatgatga tg 42 <210> 25 <211> twenty three <212> DNA <213> Artificial Sequence <400> 25 ttttggtagt agggacgctt cgc 23 <210> 26 <211> twenty three <212> DNA <213> Artificial Sequence <400> 26 ttatagtcct gtcgggtttc gcc 23 <210> 27 <211> twenty three <212> DNA <213> Artificial Sequence <400> 27 tcctgtatag tctggacgaa ctg 23 <210> 28 <211> twenty one <212> DNA <213> Artificial Sequence <400> 28 cccaggatgt tcagtgcgat a 21 <210> 29 <211> twenty three <212> DNA <213> Artificial Sequence <400> 29 taatctgacg cgctaccctg aca 23 <210> 30 <211> twenty three <212> DNA <213> Artificial Sequence <400> 30 cgatacgagc ggatttacga taa 23

Claims

1. A recombinant avian Pasteurella multocida expressing duck circovirus antigen Cap protein, which is deposited in China General Microbiological Culture Collection Center with the deposit number CGMCC 24875.

2. A method for preparing a recombinant avian Pasteurella multocida, characterized in that: At least the following steps: S1. Obtaining the target gene sequence for expressing duck circovirus antigen Cap protein; S2. Construction of a recombinant plasmid containing the target gene: S3. Constructing a recombinant strain: using the constructed recombinant plasmid, integrating the target gene sequence into the genome of the avian Pasteurella multocida deletion strain ΔaroA-GX-PM, and obtaining the recombinant avian Pasteurella multocida with the deposit number of CGMCC 24875.

3. The preparation method according to claim 2, characterized in that: The target gene sequence at least includes an asnA gene promoter sequence, an optimized Cap gene sequence, an upstream homology arm sequence, a downstream homology arm sequence and an adjuvant sequence; The asnA gene promoter sequence contains the signal peptide sequence of OmpH.

4. The preparation method according to claim 3, characterized in that: The target gene sequence is obtained by sequentially connecting an upstream homology arm sequence, an asnA gene promoter, an adjuvant sequence, an optimized Cap gene sequence, and a downstream homology arm sequence; The signal peptide sequence containing OmpH is connected to the 3' end of the asnA gene promoter sequence.

5. The preparation method according to claim 3, characterized in that: The adjuvant sequence and the optimized Cap gene sequence are connected via a flexible peptide, and the flexible peptide is G4Slinker; The C-terminus of the optimized Cap gene sequence is connected with a tag for facilitating protein purification, and the tag is 10 His tags.

6. The preparation method according to claim 3, characterized in that: The site selected by the upstream homology arm sequence is between positions 988186 and 988953 of the Pm70 genome; the site selected by the downstream homology arm sequence is between positions 990277 and 991082 of the Pm70 genome.

7. The preparation method according to claim 3, characterized in that: The nucleotide sequence of the asnA gene promoter sequence is shown in SEQ ID NO: 1; The nucleotide sequence of the optimized Cap gene sequence is shown in SEQ ID NO: 2; The nucleotide sequence of the upstream homology arm sequence is shown in SEQ ID NO: 3; The nucleotide sequence of the downstream homology arm sequence is shown in SEQ ID NO:4; The nucleotide sequence of the adjuvant sequence is shown in SEQ ID NO: 5; The nucleotide sequence of the target gene sequence is shown in SEQ ID NO:6; The primers used to amplify the sequence shown in SEQ ID NO: 1 have the nucleotide sequences shown in SEQ ID NO: 12 to 14; The primers used to amplify the sequence shown in SEQ ID NO:2 have the nucleotide sequences shown in SEQ ID NO:17 and SEQ ID NO:18; The primers used to amplify the sequence shown in SEQ ID NO:3 have the nucleotide sequences shown in SEQ ID NO:10 and SEQ ID NO:11; The primers used to amplify the sequence shown in SEQ ID NO:4 have the nucleotide sequences shown in SEQ ID NO:19 and SEQ ID NO:20; The primers used to amplify the sequence shown in SEQ ID NO:5 have the nucleotide sequences shown in SEQ ID NO:15 and SEQ ID NO:

16.

8. The preparation method according to claim 2, characterized in that: The gene knocked out in the avian Pasteurella multocida deletion strain is the aroA gene, and the aroA gene has a nucleotide sequence shown in SEQ ID NO:

7.

9. A genetically recombinant vector vaccine, characterized in that: The avian Pasteurella multocida bacterial liquid with the deposit number of CGMCC 24875 is used to prepare a genetic engineering vaccine for simultaneously preventing avian Pasteurella multocida and duck circovirus.

10. Use of the avian Pasteurella multocida according to claim 1 in the preparation of a genetic engineering vaccine for simultaneously preventing avian Pasteurella multocida and duck circovirus.

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