CN115029291A

CN115029291A - Recombinant avian pasteurella multocida for expressing duck circovirus antigen Cap protein, preparation method and application thereof

Info

Publication number: CN115029291A
Application number: CN202210615032.7A
Authority: CN
Inventors: 刘成; 张安定; 张承凤; 李玉保; 司振书
Original assignee: Huazhong Agricultural University; Liaocheng University
Current assignee: Huazhong Agricultural University; Liaocheng University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2022-09-09

Abstract

The invention relates to the technical field of biology, in particular to recombinant avian pasteurella multocida for expressing duck circovirus antigen Cap protein, a preparation method and application thereof. The recombinant pasteurella multocida obtained by the invention has good genetic stability and safety, can induce immune ducks to respectively generate specific humoral immune response aiming at pasteurella multocida and duck circovirus, has good protection effect on weight gain and slow down and immune system injury caused by duck circovirus infection, and can be used as vaccine strains for preventing pasteurella multocida and duck circovirus at the same time.

Description

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

Technical Field

The invention relates to the technical field of biology, in particular to recombinant avian pasteurella multocida for expressing duck circovirus antigen Cap protein, a preparation method thereof and a vaccine.

Background

The duck circovirus disease and the avian pasteurella multocida disease are two important infectious diseases affecting the healthy development of the duck breeding industry.

The duck circovirus disease is an immunosuppressive disease caused by duck circovirus, and can cause clinical symptoms such as ill duck feather hair breeding, growth retardation and the like. The Cap protein is the only structural protein of the virus, consists of 218-273 amino acids and has the molecular weight of about 30 KD. The Cap protein plays a crucial role throughout the viral replication cycle, including virus attachment, invasion, genome uncoating, and packaging of newly formed virions. Cap protein is used as the only protein of the circovirus displayed on the surface of the particle, contains main antigenic determinant, and is the first choice for preparing diagnostic reagent and vaccine. In recent years, the duck circovirus is widely spread in duck groups, has the characteristic of recessive infection and is easy to ignore. But it affects the growth and development of the sick duck and seriously reduces the feed conversion rate. In addition, the immune system of the sick duck is damaged, so that the immune suppression of the sick duck is caused, and the secondary infection is easily caused. Due to the short research history, the research on the pathogenic mechanism of the duck circovirus is slow, the duck circovirus cannot be cultured in vitro at present, the research on the traditional vaccine is limited, and a prevention and treatment method is not available clinically.

The avian pasteurellosis has high contagious property, high morbidity and high mortality, is one of the most serious bacterial infectious diseases which endanger the duck breeding industry, and greatly threatens the development of the poultry breeding industry. Due to the large number of serotypes, current vaccines suffer from a number of deficiencies. The treatment of avian pasteurellosis is overly dependent on antibiotics, which however results in the emergence of resistant bacteria and the problem of drug residues that people are concerned about. With the implementation of national banning of resistance-limiting policies, prevention of disease is becoming more and more important. Therefore, effective prevention of the occurrence of avian pasteurellosis is the focus of research.

In view of this, the invention is particularly proposed.

Disclosure of Invention

In order to solve the technical problems, the invention provides a recombinant avian pasteurella multocida for expressing a duck circovirus antigen Cap protein, a preparation method thereof and a vaccine.

The invention provides a recombinant avian pasteurella multocida for expressing a duck circovirus antigen Cap protein, which is preserved in China general microbiological culture collection center with the preservation number of CGMCC 24875.

The invention provides a preparation method of recombinant poultry pasteurella multocida, which at least comprises the following steps:

s1, obtaining a target gene sequence for expressing a duck circovirus antigen Cap protein;

s2, constructing a recombinant plasmid containing a target gene:

s3, constructing a recombinant strain: and integrating the target gene sequence into the genome of the avian pasteurella multocida deleted strain delta aroA-GX-PM by using the constructed recombinant plasmid to obtain the recombinant avian pasteurella multocida with the preservation number of CGMCC 24875.

The invention provides a gene recombinant vector vaccine, which is a gene engineering vaccine for preventing poultry pasteurella multocida and duck circovirus at the same time, and is prepared from a poultry pasteurella multocida liquid with the preservation number of CGMCC 24875.

The invention provides application of the pasteurella multocida in preparation of a genetic engineering vaccine for simultaneously preventing pasteurella multocida and duck circovirus.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:

the invention successfully constructs the recombinant avian pasteurella multocida for expressing the Cap protein of the duck circovirus antigen, and the growth of the strain is not influenced by the expression of the Cap protein.

The recombinant pasteurella multocida obtained by the invention has good in vitro growth, morphological characteristics, genetic stability and safety, can induce ducks to respectively generate specific humoral immune response aiming at the pasteurella multocida and duck circovirus, has good protection effect on weight growth and slow down and immune system injury caused by duck circovirus infection, and can be used for preparing recombinant gene engineering vaccines for simultaneously preventing pasteurella multocida and duck circovirus.

Drawings

FIG. 1 shows the result of prediction of B-cell epitopes of Cap proteins;

FIG. 2 shows the results of PCR amplification of the DuCV Cap gene and vector in example 1: wherein (A) PCR amplification of the DuCV Cap gene: 1, Cap-PGEX-RH; m: DL2000 DNA Marker; (B) PCR amplification of the vector: 1: PGEX-GST; m: DL15000 DNAmarker;

FIG. 3 shows the results of PCR amplification of the asnA gene promoter, CTA1-DD-RH, optimized DuCV Cap gene, homology arms, wherein: 1: aroA-L-RH; 2: asnA-pro-RH; 3: CTA 1-DD-RH; 4: Cap-RH; 5: aroA-R-RH; m: DL2000 DNA marker;

FIG. 4 is the post-fusion results for the fragments in FIG. 3: wherein: in Panel A, 1: cap + aroA-R-RH; 2: aroA-L + asnA-pro-RH; m: DL2000 DNA marker; in panel B, 1: CTA1-DD + Cap + aroA-R-RH; m: DL5000 DNA marker; in panel C, 1: aroA-LR + asnA-pro + CTA1-DD + Cap; m: DL5000 DNA marker;

FIG. 5 is a diagram showing the results of PCR amplification of recombinant plasmid vectors: 1: pSHK5(TS) -CbAgo plasmid vector; m: DL15000 DNA marker;

FIG. 6 is a diagram showing the results of PCR identification of recombinant plasmids: 1-7: a single colony to be identified; 8: plasmid control; 9: h ₂ O；M：DL5000 DNA marker；

FIG. 7 shows the identification of recombinant plasmid constructsThe result chart of (2): 1-3: a single colony to be identified; 4: Δ aroA-GX-PM; 5: GX-PM; 6: h ₂ O；M：DL5000 DNA marker；

FIG. 8 is a graph showing the results of identifying plasmid elimination: 1-4: single bacterial colony to be identified; 5: aroA-L asnA-pro-CTA1-DD-Cap-Cbago-pSHK5(TS) plasmid control; 6: h ₂ O；M：DL5000 DNA Marker；

FIG. 9 is a diagram showing the result of Cap protein expression detection in recombinant strains, wherein, a diagram is protein detection in recombinant strains, and a diagram B is secreted protein detection of recombinant strains; 1: Δ aroA-GX-PM; 2: DuCV Cap- Δ aroA-GX-PM; m: PageRuler ^TM Prestained Protein Ladder；

FIG. 10 shows the measurement results of growth curves;

FIG. 11 is a graph showing the gram staining results of GX-PM, deletion mutants and recombinant strains: FIG. A is GX-PM; panel B is Δ aroA-GX-PM; panel C is DuCV Cap- Δ aroA-GX-PM;

fig. 12 is a graph showing the results of identifying the genetic stability of a recombinant strain, in which 1: generation 1 recombinant strain; 2: generation 10 recombinant strain; 3: generation 20 recombinant strain; 4: a 30 th generation recombinant strain; 5: Δ aroA-GX-PM; 6: GX-PM; 7: h ₂ O；M：DL5000 DNA Marker；

FIG. 13 is 10 ⁶ CFU、10 ⁸ A weight result graph of the duck 14 days after the CFU recombinant bacteria are infected;

FIG. 14 is a graph showing the results of antibody levels against avian Pasteurella multocida after the recombinant bacteria have immunized a duck for 14 days;

FIG. 15 is a graph showing the results of the tissue bacterial load after GX-PM challenge;

FIG. 16 is a graph showing the results of antibody levels against Cap protein 14 days after immunization with recombinant bacteria;

FIG. 17 is a graph of the results of weight monitoring after DuCV challenge;

FIG. 18 is the result chart of the immune organ index after the duck circovirus challenge.

Bacterial preservation description:

1. the recombinant avian pasteurella multocida for expressing the duck circovirus antigen Cap protein is preserved in China general microbiological culture collection center with the preservation number of CGMCC 24875, and the preservation date is 2022, 5 months and 9 days.

2. The pasteurella multocida deleted strain Δ aroA-GX-PM used in the present invention was published by the literature "construction of pasteurella multocida aroA, hexD and hexABC gene deletion mutants and its immune potency study [ D ]. university of agriculture in huazhong, 2020. king no.

3. The Pasteurella multocida type A strain GX-PM used in the embodiment of the invention is separated from liver tissues of dead chickens in a large-scale chicken farm in Guangxi in 2013, and is prepared by the following documents of Yu C, Sizhu S, Luo Q, Xu X, Fu L, Zhuang A.genome sequencing of a viral antigen Pasteurella multocida strain GX-Pm derivatives the candidate genes involved in the pathogenesis. 105:23-7.doi:10.1016/j. rvsc.2016.01.013.epub 2016Jan 18.PMID:27033902. "published, and stored in chatting university and Chinese agriculture university.

4. The duck circovirus histotoxicum used in the embodiment of the invention is preserved in chatting university.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, aspects of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

The embodiment of the invention provides a recombinant avian pasteurella multocida for expressing a duck circovirus antigen Cap protein, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC 24875. The recombinant strain can induce ducks to respectively generate specific liquid immune reactions aiming at the avian pasteurella multocida and the duck circovirus, can be used for preparing the genetic engineering vaccines of the avian pasteurella multocida and the duck circovirus, and is used for preventing and treating infectious diseases caused by the avian pasteurella multocida and the duck circovirus at the same time.

The embodiment of the invention provides a preparation method of the recombinant poultry pasteurella multocida, which at least comprises the following steps:

s2, constructing a recombinant plasmid containing a target gene:

s3, constructing a recombinant strain: and integrating a target gene sequence into the pasteurella multocida deleted strain delta aroA-GX-PM by using the constructed recombinant plasmid to obtain the recombinant pasteurella multocida with the preservation number of CGMCC 24875. As a preferred technical scheme of the embodiment of the invention, the target gene sequence at least comprises an asnA gene promoter sequence, an optimized Cap gene sequence, an upstream homology arm sequence, a downstream homology arm sequence and an adjuvant sequence. Wherein, a signal peptide sequence containing OmpH in the promoter sequence of the asnA gene enables secretory expression of the Cap protein. The upstream homology arm sequence and the downstream homology arm sequence enable the target gene and delta aroA-GX-PM to generate homologous recombination; it has been shown that the asnA promoter on the recombinant plasmid is effective in promoting expression of the VP60 gene in Pasteurella, and therefore the fusion asnA gene promoter was selected in the examples of aspects of the invention.

The Cap protein of the duck circovirus is difficult to be expressed in a soluble way in an escherichia coli prokaryotic expression system, so the Cap protein is optimized in the following specific mode: truncating an N-terminal arginine-enriched NLS sequence (comprising 1-54 amino acids), selecting a main epitope region of the Cap protein by predicting a B cell epitope of the Cap protein, preferably comprising 54-198 amino acids, and obtaining an optimized Cap gene sequence after codon optimization.

Adjuvants are of interest in vaccine research because they are critical for enhancing the immune response during the course of immunization. The effective and safe immunologic adjuvant has extremely important significance for vaccine immunization. CTA1-DD is one of the most promising mucosal adjuvants known to promote a wide range of specific immune responses and induce long-term immunological memory, enhance antibody levels and initiate CD4+ and CD8+ T cells to respond to various antigens.

As a preferred technical scheme of the embodiment of the invention, the target gene sequence is obtained by connecting an upstream homology arm sequence, an asnA gene promoter, an adjuvant sequence, an optimized Cap gene sequence and a downstream homology arm sequence in sequence; wherein a signal peptide sequence comprising OmpH is ligated to the 3' -end of the asnA gene promoter sequence.

As a preferred technical scheme of the embodiment of the invention, an adjuvant sequence is connected with an optimized Cap gene sequence through a flexible peptide, wherein the flexible peptide is a nucleotide sequence for coding 4-10 amino acids, preferably glycine and serine; the flexible peptide is preferably G4S linker, and the amino acid sequence is four glycines and one serine.

As a preferred technical scheme of the embodiment of the invention, the C end of the optimized Cap gene sequence is connected with a tag for assisting protein purification, and the tag is preferably 5-15 His tags.

As a preferred technical scheme of the embodiment of the invention, the site selected by the upstream homology arm sequence refers to the genome sequence of strain Pm70 (accession number: AE004439) published in GenBank, and the position 988186-988953 of the genome; the site selected by the downstream homology arm sequence is between 990277 th site and 991082 th site of the genome of the strain.

As a preferred technical scheme of the embodiment of the invention, the optimized Cap gene sequence is a gene sequence corresponding to 54-198 amino acids after 1-54 amino acids are removed.

As a preferred technical scheme of the embodiment of the invention, the nucleotide sequence of the asnA gene promoter sequence is shown as SEQ ID NO. 1; the asnA gene promoter (asnA-pro-RH) is amplified by a primer asnA-pro-RH-F/R1/R2 (the sequence is preferably SEQ ID NO: 12-14) by taking GX-PM genome DNA as a template, and the size is 275 bp.

As a preferred technical scheme of the embodiment of the invention, the nucleotide sequence of the optimized Cap gene sequence is shown as SEQ ID NO. 2; synthesized by biological companies, amplified by using primers Cap-CTA1-DD-RH-F/R (the sequence is preferably SEQ ID NO:17, 18) to obtain Cap-RH with the size of 543 bp.

As a preferred technical scheme of the embodiment of the invention, the nucleotide sequence of the upstream homology arm sequence is shown as 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 aroA gene takes GX-PM genome DNA as a template, primers aroA-L-RH-F/R (the sequence is preferably SEQ ID NO:10 and 11) and aroA-R-RH-F/R (the sequence is preferably SEQ ID NO:19 and 20) in the table 2 are respectively amplified by PrimeSTAR HS DNA Polymerase to obtain the upstream homology arm (aroA-L-RH) and the downstream homology arm (aroA-R-RH) of the aroA gene, and the sizes are 809bp and 847bp respectively.

As a preferred technical scheme of the embodiment of the invention, the nucleotide sequence of the adjuvant sequence is shown in SEQ ID NO:5, the adjuvant is synthesized by biological companies, and primers CTA1-DD-RH-F/R (the sequences are preferably SEQ ID NO:15 and SEQ ID NO: 16) are adopted for amplification to obtain CTA1-DD-RH with the size of 1032 bp.

As a preferred technical scheme of the embodiment of the invention, the nucleotide sequence of the target gene sequence of the embodiment of the invention is shown as SEQ ID NO. 6. The method for constructing the plasmid utilizes fusion PCR technology. The principle of the fusion PCR technology is that a primer with a complementary end is adopted to amplify a PCR product with an overlapped chain, and two different PCR product fragments with the overlapped chain are connected through the action of a kit fusion enzyme.

As a preferred technical scheme of the embodiment of the invention, the knocked-out gene in the avian pasteurella multocida deleted strain is aroA gene, preferably, the aroA gene at least comprises a sequence with at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 7, further preferably 95% homology, more preferably 99% homology, and most preferably the nucleotide sequence shown in SEQ ID NO. 7.

The embodiment of the invention also relates to the application of the pasteurella multocida in preparing the recombinant pasteurella multocida genetic engineering vaccine for preventing duck circovirus at the same time.

The embodiment of the invention also relates to a recombinant genetic engineering vaccine, which is prepared by using the pasteurella multocida liquid with the preservation number of CGMCC 24875 to simultaneously prevent pasteurella multocida and duck circovirus. The recombinant bacteria of the embodiment of the invention can induce ducks to respectively generate specific humoral immune reactions aiming at the avian pasteurella and the duck circovirus, so that the recombinant bacteria can be used for preparing a genetic engineering vaccine capable of simultaneously preventing the avian pasteurella multocida and the duck circovirus, and has a good protection effect.

The nucleotide sequences used in the examples of the present invention are specifically shown in table 1:

table 1:

the PCR primers used in the examples of the present invention are specifically shown in Table 2:

table 2: PCR primer

Some abbreviations in the examples are as follows:

Δ aroA-GX-PM: an aroA gene deletion mutant strain of avian pasteurella multocida GX-PM;

Cap-PGEX-RH: a Cap-PGEX fusion fragment obtained by PCR;

DuCV Cap-delta aroA-GX-PM recombinant strain: expressing a pasteurella multocida GX-PM mutant strain with aroA gene deletion of duck circovirus Cap protein;

asnA-pro-RH: the asnA gene promoter;

aroA-L-RH: aroA left homology arm;

aroA-R-RH: aroA Right homology arm.

The biomaterials used in the specific examples were:

coli DH 5. alpha. competent cells were purchased from Takara bioengineering (Dalian) Co., Ltd.;

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 for expressing Cap protein of duck circovirus.

After the pasteurella multocida GX-PM and the delta aroA-GX-PM are revived on a TSA plate added with 10% newborn bovine serum, a single colony is picked and inoculated in a TSB culture medium added with 10% newborn bovine serum, and shake culture is carried out in a shaker at 37 ℃. The Escherichia coli strain is cultured in LA or LB medium. Antibiotics were added to the medium as required for the experiment at the following concentrations: kanamycin is used for culturing escherichia coli, and the concentration of kanamycin is 50 mug/mL; the concentration of the culture medium for GX-PM strain is 100 mug/mL. Ampicillin was used in the E.coli culture at a concentration of 100. mu.g/mL.

The reagent consumables used in the examples are shown in table 3:

TABLE 3

Example 1

This example illustrates the construction of prokaryotic expression vectors for recombinant Cap proteins

PCR amplification of the gene and vector of interest: the N-terminal arginine-enriched NLS sequence (1-54 aa) of the Cap protein is truncated, B-cell epitope of the Cap protein (257aa) is predicted according to the website IEDB. org, Free epitope database and prediction resource, and the result of predicting the B-cell epitope of the Cap protein is shown in figure 1. A main antigen epitope region (54-198 aa) of the Cap protein is selected, synthesized after codon optimization, cloned to a PGEX-6P-1 plasmid vector, and the soluble expression of the Cap protein is promoted by GST tag protein.

The vector fragment PGEX-GST and the inserted target gene Cap-PGEX-RH are respectively amplified by 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 the table 2, the sizes of the vector fragment PGEX-GST and the inserted target gene Cap-PGEX-RH are 5025bp and 541bp respectively, and the experimental result is shown in the table 2. As can be seen from FIG. 2, the amplification results were in agreement with the expectations.

Example 2

This example illustrates the construction and identification of DuCV Cap- Δ aroA-GX-PM recombinant strains.

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

1.1 amplification of the Gene of interest

In order to stably express and secrete DuCV Cap protein in Pasteurella multocida and stimulate the body to generate correct immune response, a Cap protein expression cassette needs to be constructed, which comprises a strong promoter asnA promoter in Pasteurella multocida, a signal peptide sequence of Pasteurella multocida ompH, an adjuvant CTA1-DD, base sequences of Cap genes are optimized according to codon preference of Pasteurella multocida, a 10 XHis sequence is connected for detecting expression of Cap protein, and the optimized Cap gene is shown as SEQ ID NO. 2. In order to successfully construct a knock-in fragment into the Δ aroA-GX-PM genome, it was necessary to amplify the upstream and downstream homology arms of the aroA gene.

The steps of amplifying and connecting the gene segments with the added homologous sequences are as follows:

(1) the asnA gene promoter (asnA-pro-RH) was amplified from the primers SEQ ID NO:12 to 14(asnA-pro-RH-F/R1/R2) in Table 2, and a signal peptide sequence of 275bp in size and OmpH was ligated to the C-terminus of asnA-pro by the primers asnA-pro-RH-R1/R2.

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

(3) The optimized DuCV Cap gene is amplified by primers SEQ ID NO:17 and SEQ ID NO:18(Cap-CTA1-DD-RH-F/R) in Table 2 to obtain Cap-RH with the size of 543 bp.

(4) The homology arms were obtained by amplification with 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 809bp and 847bp, respectively.

The amplification results of the respective fragments are shown in FIG. 3, and the bands are suspected to be correct.

1.2 tandem connection of genes of interest

The amplified 5 fragments were concatenated using overlapping PCR:

(1) firstly, the aroA-L-RH and asnA-pro-RH segments are connected in series, and the size of the aroA-L + asnA-pro-RH segments after the aroA-L + asnA-pro-RH segments are 1043bp after the aroA-L + asnA-pro-RH segments are connected in series;

(2) the Cap-RH and the aroA-R-RH are connected in series to obtain Cap + aroA-R-RH, the size is 1348bp, and the PCR amplification result accords with the expectation.

(3) Fragments CTA1-DD-RH and Cap + aroA-R-RH are connected in series to obtain a fragment CTA1-DD + Cap + aroA-R-RH with the size of 2338bp, and the PCR amplification result is in line with expectation.

(4) The aroA-L + asnA-pro-RH and CTA1-DD + Cap + aroA-R-RH are connected in series, and the finally obtained insertion fragment aroA-LR + asnA-pro + CTA1-DD + Cap is 3363bp in size. The PCR amplification result is as expected.

The PCR amplification results are shown in FIG. 4.

1.3 amplification of plasmid vectors

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

1.4 fusion transformation and identification of recombinant plasmids

After fusion transformation of the plasmid vector pSHK5(TS) -CbAgo with the insert aroA-LR + asnA-pro + CTA1-DD + Cap-RH using the fusion PCR reaction system of Table 4 and the reaction conditions of the fusion PCR of Table 5, a single colony on a LA + Kan plate was picked up and identified. PCR amplification was performed with the primers SEQ ID NO:25 and SEQ ID NO:26(pSHK5(TS) -MCS-ID-F/R) in Table 2, and the amplification product size of the correctly fused transformant was 3819bp, as shown in FIG. 6.

As can be seen from FIG. 6, the sizes of the PCR product bands of the single colonies No. 1, 2, 3, 5 and 7 are suspected to be correct, and the sequencing results are compared to confirm that the plasmid vector and the insert are correctly fused without mutation, which indicates that the recombinant plasmid is correctly constructed, and the plasmid is extracted for subsequent experiments.

Table 4: fusion PCR reaction system

Table 5: fusion PCR reaction conditions

Time	Temperature of
		37℃	15min
50℃	8min
		52℃	8min
54℃	8min
		58℃	8min
60℃	8min
		25℃	1min

Screening and identification of 2DuCV Cap-delta aroA-GX-PM recombinant strain

2.1 screening of DuCV Cap- Δ aroA-GX-PM recombinant Strain

And (3) electrically transferring the correctly identified plasmid into a delta aroA-GX-PM strain, adding a nonresistant TSB culture medium, recovering for 2 hours in a shaker at 28 ℃, coating a TSA + Kan plate, and placing the plate in an incubator at 28 ℃ for growth for 36-48 hours. After the colonies grow out, selecting a single colony, coating a new TSA + Kan plate, placing the plate at room temperature for growing, after solid passage for a plurality of times, identifying whether the single colony is recombined or not by using primers SEQ ID NO:29 and SEQ ID NO:30(LR-aroA-LR-JD-F/R) in the table 2. The amplification product of the wild GX-PM is 3473bp, the amplification product of the delta aroA-GX-PM is 2156bp, and the recombinant strain with gene insertion is used for amplifying 3898bp product which is larger than the wild strain product. The results of the experiment are shown in FIG. 7.

As shown in FIG. 7, the band of the single colony to be identified as No. 3 is suspected to be correct, indicating that the gene recombination of the single colony No. 3 may have occurred.

2.2 purification of DuCV Cap- Δ aroA-GX-PM recombinant Strain

The single colony No. 3 is transferred to a non-resistant TSB culture medium and cultured in a shaker at 37 ℃ for several times of passage. When the strain is transferred to the 4 th generation, the strain liquid is diluted and coated on a non-resistant TSA plate, and grown single colonies are selected and subjected to dot-dash on the non-resistant TSA plate and the Kan-resistant TSA plate respectively. Single colonies that did not grow on the Kan-resistant plates and grew on the non-resistant plates were picked. The primers SEQ ID NO 27 and SEQ ID NO 28(CbAgo-inner-JD-F/R) identified on the recombinant plasmids in Table 2 were used for identification, and the results are shown in FIG. 8.

As shown in FIG. 8, none of the single colonies amplified any band, indicating that the recombinant plasmid had been successfully eliminated. And amplifying a single large band by using the primer LR-aroA-LR-JD-F/R again, sending the PCR identification product to a company for sequencing, wherein the sequencing result shows that the asnA-pro + CTA1-DD + Cap fragment is successfully inserted into the delta aroA-GX-PM genome and no mutation is generated. The above results show that DuCV Cap-delta aroA-GX-PM recombinant strains with inserted Cap gene expression cassettes without molecular markers are successfully constructed.

2.3 detection of Cap protein expression of DuCV Cap-delta aroA-GX-PM recombinant Strain

Selecting and identifying the correct DuCV Cap-delta aroA-GX-PM recombinant strain, and recovering 1: 1000 were transferred to 10mL serum-free TSB medium, while Δ aroA-GX-PM was transferred under the same conditions as control. The two kinds of bacteria are cultured for 12h at 37 ℃ by a shaking table and then are respectively collected. The cells were resuspended in 1mL PBS and sonicated to prepare samples. After TCA precipitation of the supernatant, 200uL of 2 XSDS loading buffer was boiled and dissolved. Western Blot analysis was performed on the mycoprotein and secreted protein samples. The results of the experiment are shown in FIG. 9.

As can be seen from FIG. 9, a band of the desired size was detected in the cells, indicating that the recombinant Cap protein was successfully expressed in the recombinant DuCV Cap- Δ aroA-GX-PM strain. In the culture supernatant, the presence of recombinant Cap protein was also detected, indicating that the recombinant Cap protein can be expressed in DuCV Cap- Δ aroA-GX-PM recombinant bacteria and secreted extracellularly. The results show that the DuCV Cap-delta aroA-GX-PM recombinant strain for recombinant expression of the Cap protein of the duck circovirus has been successfully constructed and can be used for subsequent experiments.

Experimental example 1

This example serves to illustrate the in vitro growth characteristics of the recombinant DuCV Cap- Δ aroA-GX-PM strain: in order to research whether the gene knockin and the expression of protein have influence on the growth of the strain, respectively selecting wild bacteria GX-PM, gene deletion attenuated bacteria delta aroA-GX-PM and DuCV Cap-delta aroA-GX-PM recombinant bacteria to resuscitate in a TSB culture medium containing 10% serum, and respectively 1: 1000 into fresh medium, add sample pan, 200 μ L per well, set for 3 replicates, and set TSB medium with 10% serum as a blank control, according to OD measured every half hour _600nm And drawing a bacterial growth curve. The results of the experiment are shown in FIG. 10.

As can be seen from FIG. 10, when the control is established, under the same conditions, the growth rate of the wild strain GX-PM is fastest, the growth rate of the wild strain is slowed down as that of the wild strain delta aroA-GX-PM, and the growth curves of the DuCV Cap-delta aroA-GX-PM recombinant strain and the original strain delta aroA-GX-PM are not obviously different, which indicates that knocking-in of the Cap gene expression cassette and expression of the Cap protein have no obvious influence on the growth of the strain.

Experimental example 2

The true bookThe examples serve to illustrate the in vitro morphological properties of the DuCV Cap- Δ aroA-GX-PM recombinant strain: respectively recovering wild strain GX-PM, gene deletion mutant strain delta aroA-GX-PM and recombinant strain DuCV Cap-delta aroA-GX-PM, transferring to fresh culture medium, and culturing at 37 deg.C to OD _600nm At a value of about 0.9, the smear was stained under a microscope. The results of the experiment are shown in FIG. 11.

As can be seen from FIG. 11, after gram staining, Pm is in a small spherical shape or a short rod shape under a mirror, and exists in a single or short chain shape, and the forms of GX-PM, delta aroA-GX-PM and recombinant bacteria DuCV Cap-delta aroA-GX-PM are not obviously different, which indicates that the knock-in of genes and the expression of exogenous Cap proteins have no obvious influence on the bacterial forms.

Experimental example 3

This example serves to illustrate the in vitro genetic stability of the DuCV Cap- Δ aroA-GX-PM recombinant strain: in order to verify whether the knocked-in gene can stably exist on the bacterial genome, the recombinant bacterium DuCV Cap-delta aroA-GX-PM is continuously passaged in a liquid culture medium and is identified once every 10 generations, and the experimental result is shown in FIG. 12.

As can be seen from FIG. 12, the expression cassette of the knocked-in Cap gene still exists stably after the recombinant strain is continuously passaged to 30 generations, which indicates that the DuCV Cap-delta aroA-GX-PM recombinant strain has good in vitro genetic stability.

Experimental example 4

This example serves to illustrate the safety of the DuCV Cap- Δ aroA-GX-PM recombinant strain:

the safety of the DuCV Cap- Δ aroA-GX-PM recombinant strain was evaluated by comparatively recording the body weight change, mental status, clinical symptoms and survival of the infected and PBS groups. The results of the experiment are shown in FIG. 13.

The results of the body weight of the duck 14 days after the infection with the recombinant bacteria shown in FIG. 13 indicate that 10 days after the infection with the recombinant bacteria ⁸ After CFU infection, the body weight of the PBS group is obviously higher than that of the infected group, which shows that the recombinant bacteria do not cause the clinical symptoms of the duck, but have influence on the body weight growth of the duck. To use 10 ⁶ After 14 days of infection with CFU recombinant bacteria, there was no difference between the final average body weight of the infected group and that of the PBS group, indicating that 10 is used ⁶ CFU infection dosage, the DuCV Cap-delta aroA-GX-PM recombinant bacteria can not cause diseases of the ducklings, can not generate negative influence on the growth of the ducklings,10 ⁶ CFU can be used as an immunization dose for the next experiment.

Experimental example 5

This example is used for the immunopotency of DuCV Cap- Δ aroA-GX-PM recombinant strain:

in order to verify whether the recombinant bacteria can generate immune protection effect on ducks, 40 healthy ducklings of 7 days old are divided into four groups, namely an immune + DuCV group, a PBS + DuCV group, an immune + GX-PM group and a PBS + GX-PM group, wherein the immune dose of the immune group is 10 ⁶ And (4) CFU. After 14 days of immunization, three ducks are taken from each group for blood collection, the immune + DuCV group and the PBS + DuCV group are detoxified by 100 mu L of duck circovirus tissue stock solution, and the immune + GX-PM group and the PBS + GX-PM group are detoxified by 10 ⁸ CFU GX-PM is used for counteracting toxic materials, changes of body weight are monitored, clinical symptoms are observed, a caesarean test is carried out 14 days after immunization, and 3 bacteria carrying amount of blood and liver are detected in each group.

1.1 immune protection efficacy research of recombinant bacteria on avian Pasteurella infection

After 14 days of immunization, three groups of the immunization + GX-PM and the PBS + GX-PM are respectively taken to collect blood, and serum is collected. According to the square matrix test, the optimal coating concentration of the antigen GX-PM is determined to be 2 multiplied by 10 ⁸ The optimal dilution factor of the serum is 1: 80. the GX-PM whole bacteria is used as an antigen, the collected duck serum is used as a primary antibody, a rabbit anti-duck IgG antibody is used as a secondary antibody, and the level of the antibody generated by the immune antibody aiming at the GX-PM is detected through indirect ELISA. The results of the experiment are shown in FIG. 14.

As can be seen from FIG. 14, the recombinant strain DuCV Cap- Δ aroA-GX-PM can induce the body to produce higher antibody level against the pasteurella avium after being immunized, and the difference is very significant compared with the control group (P < 0.01). The DuCV Cap-delta aroA-GX-PM recombinant bacteria can cause ducks to generate humoral immunity aiming at the fowl pasteurella multocida GX-PM after immunization, and the recombinant bacteria have better immunogenicity.

After 14 days of GX-PM challenge, the amount of bacteria carried in blood and liver was compared, and the experimental results are shown in FIG. 15.

As can be seen from FIG. 15, the tissue load of the immune + GX-PM group was significantly lower than that of the PBS + GX-PM group (P < 0.05).

1.2 immune protection efficacy research of recombinant bacteria on DuCV infection

The prepared recombinant Cap protein is coated, collected serum is used as a primary antibody, a rabbit anti-duck IgG antibody is used as a secondary antibody, and an indirect ELISA test is carried out to detect the antibody level 14 days after immunization, so that whether the immune response of an organism can be caused after the immunization of the recombinant bacteria is reflected. According to the matrix test, the optimal coating condition of the recombinant Cap protein is confirmed to be 3 mug/mL, and the optimal dilution condition of the serum is 1: 40. the results of the experiment are shown in FIG. 16.

As can be seen from fig. 16, the IgG antibody levels in the duck sera were higher and significantly different in the immunized group compared to the non-immunized group (P < 0.01). The DuCV Cap-delta aroA-GX-PM recombinant bacteria can induce ducks to generate humoral immunity aiming at Cap protein after immunization, and generate higher antibody level. Therefore, the recombinant bacteria have better immunogenicity.

Because the mortality rate of the duck circovirus is low when the duck circovirus is infected alone, but the diseased duck can cause the growth and development of the diseased duck to become slow, feather and hair are badly bred, disordered, immunosuppression and the like, the study mainly monitors the weight of two groups of ducks after the virus is attacked for 14 days, and observes the clinical symptoms, feeding conditions and the like of the ducks. After DuCV challenge, no obvious clinical signs were seen in either group, but food intake was reduced in the PBS group relative to the immunized group. The results of the experiment are shown in FIG. 17.

As can be seen from fig. 17, from the weight increase rate after challenge, the body weights of the two groups steadily increased after the DuCV challenge, but the body weight increase of the PBS group was significantly lower than that of the immune group, and after 14 days, the final average body weight of the PBS group was 1.413Kg, the final average body weight of the immune group was 1.566Kg, and the weight difference between the two groups was very significant. The DuCV Cap-delta aroA-GX-PM recombinant bacteria are proved to be capable of effectively protecting the appearance of the symptoms of growth retardation and weight growth limitation caused by DuCV infection after the DuCV infection after immunization, and the DuCV Cap-delta aroA-GX-PM recombinant bacteria have good immune protection effect.

The duck circovirus mainly attacks the immune system and necroses lymphocytes to damage immune organs, so that after 14 days of virus challenge, three immune groups and three PBS control groups are taken from each group, the main immune organs are respectively taken after weighing and weighed, and immune organ indexes are calculated, so that the immune level of the duck circovirus is indirectly reflected. The results of the experiment are shown in FIG. 18.

As can be seen from fig. 18, the immune organ index of the immune group is higher than that of the PBS control group in comparison with the results of the two groups, wherein the thymus index of the immune group is significantly higher than that of the control group (P < 0.05). The result shows that the immunity of the DuCV Cap-delta aroA-GX-PM recombinant bacteria has a certain immune protection effect on immune organs of ducks.

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> chat university

Huazhong Agricultural University

<120> recombinant avian pasteurella multocida for 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 (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 (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 (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 (Artificial Sequence)

<400> 4

cctacagata agtactcagc aaaaagcgaa gtattttata atacttcgct ttttgagtga 60

cgaatcgggt ctgagataat gcaagaaaac accttaagac tgaatccgaa aactcgccat 120

ataatttgcg ctgacatctt tattgagcca gaatttaccc gtcaatggat tgtaatgata 180

gcctaccata tctaaacagg ttaattgtgc ttcatcacac caatgaagca attccgctgg 240

cttaataaat ttgtcgtaat cgtgtgtccc tttcggcaac attttcaaca catattccgc 300

accaataatc accaatgccc aggctttaag ggttcgatta atggtggaga aaaaaatcac 360

cccgtttggt tttaataatt gcttacaaca ggcaataatc gaactcgggt caggcacatg 420

ctcaagcatt tccatgcaag taatcacatc aaacttttcg tcctcccctc tttcagcaaa 480

aagtgcggtc tgattttgca aaaattcctc aatcgtaatt tgttgataat caatgtgtag 540

cccgctttct aaagcatgtt ttcttgccac ttgtaatggg gcagaagaca tatcaatccc 600

cgtcacaatt gcgccttgct ttgccatgct ttctgacaaa atgccaccgc cacagcccac 660

atccagcact tttttccccg ttagcccgtt ggcttgctgt gcaatatagc ttaaacgtaa 720

cggattaagt tgatggatcg gtttgaaatc cccttgcgga tcccaccagc tttttgccat 780

tttctcaaac ttatcaagtt cttgtt 806

<210> 5

<211> 948

<212> DNA

<213> Artificial Sequence (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 (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 gagtagattc ctttacgtta ctcaaaaaac 300

actctaatct tgtgatttgt cgtacccttt ctaaagccta tggtttagcg ggtttgcgca 360

ttggttacgc ggtttcttct gccgagattg ctgacctctt taatcgcgtt cgccaaccgt 420

ttaactgtaa tagtctggca ttagccgctg ctaccgcggt gctgcatgat gatgcattta 480

ttgcgaaagt ggctgaaaac aaccgacaag ggttgaagtt attggaagac ttctttacag 540

ccaaaggttt gaactatatt ccttcaaaag gcaattttgt catgttagat gtcaatcaac 600

cagccttacc aatttatcag gcattattac aaaaaggcgt gattgtgcgt ccgattgcag 660

gttatggctt accaaatcat ttacggatta gcatcggttt accagaagaa aaccaacgtt 720

tcttactcgc attaaacgaa gtattagggc tttaagccaa acttatccag tgtataggaa 780

atataaatca aacacgcaat atttgctgat caagattatc aatattgtgc atagttgacc 840

ttttttagaa ttttttcaaa aataaatacg ttatattgta taaaattcaa agtatatcgc 900

ttatttcaaa aaaagtattg agttataggg cttttgtatt aaataatatt cagtgaattt 960

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

atgaaatttt aaatatgccg aatctgaatg aagcacaacg caatggcttt attcagagtt 1740

tgaaggatga tccgagtcaa agtaccaatg ttttgggcga agccaagaag ttgaatgaaa 1800

gtcaggcgcc gaaggcggat gcgcagcaaa ataattttaa taaagatcag cagtcggcgt 1860

tttatgaaat tttgaatatg ccgaatttga atgaagcgca gcgcaatggt tttattcagt 1920

cattgaaaga tgatccgtca cagagtacca atgttttagg cgaagccaaa aagttgaatg 1980

aatcgcaggc gccgaaacat catcatcatc atcatcatca tggtggcgga ggtagcggat 2040

ccatgggttc tggtacaggt ccaacagcag caggtaaatg gcaatcttta agtttagaag 2100

atggtgcaca atatacagat ccaccagcac atggtaataa tatttgtggt ttaaatatgc 2160

gttgggcaat gtttggtgat acaaatagtt atatgacagg tacaacacca aattatcatt 2220

atccatatga ttattatatt attaaaggtg tggcaattac attacgtcca gcatataata 2280

tttatgaaaa atctaaaaca caaggttcta cagttattga taaagatggt caaattgtga 2340

aaacaagtac tacaggttgg tctattgatc catatggttc aacatctagt cgtagaacag 2400

gtgatccatc tagagttcat cgtcgttatt ttgttccaaa accaattatt caaggtgcag 2460

gtgaaggtac agaatattct ggtggtggtg gttcacatca tcatcatcat catcatcatc 2520

atcattaaga attctaacct acagatagat actaagcaaa aagcgaagta ttttataata 2580

cttcgctttt tgagtgacga atcgggtctg agataatgca agaaaacacc ttaagactga 2640

atccgaaaac tcgccatata atttgcgctg acatctttat tgagccagaa tttacccgtc 2700

aatggattgt aatgatagcc taccatatct aaacaggtta attgtgcttc atcacaccaa 2760

tgaagcaatt ccgctggctt aataaatttg tcgtaatcgt gtgtcccttt cggcaacatt 2820

ttcaacacat attccgcacc aataatcacc aatgcccagg ctttaagggt tcgattaatg 2880

gtggagaaaa aaatcacccc gtttggtttt aataattgct tacaacaggc aataatcgaa 2940

ctcgggtcag gcacatgctc aagcatttcc atgcaagtaa tcacatcaaa cttttcgtcc 3000

tcccctcttt 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 (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

aatgaaatag tcttgactgg cgaacctcgt atgaaagaac gcccaataca acatttagtt 420

gatgcattat gtcaagctgg cgcagaaatt cagtatttag aacaagaagg ttacccacct 480

atcgccattc gaaataccgg actcaaaggc ggacgaatac aaattgatgg gtcagtttct 540

tctcaatttt tgaccgcact tttaatggct gccccgatgg cagaggcgga tacggaaatt 600

gaaatcatcg gtgagctggt ttccaaacct tacattgata tcacccttaa gatgatgcaa 660

acctttggcg ttgaagttga aaaccaagcc tatcaacgct ttttggtgaa aggtcatcag 720

caataccaat caccacacag gtttctagta gaaggcgatg cctcttctgc ttcttatttt 780

cttgccgcgg cagcaatcaa gggaaaagta aaagtcacag gcgtcggtaa aaatagcatt 840

caaggggatc gtctgtttgc ggatgtgcta gaaaaaatgg gggcgcatat cacttggggc 900

gacgatttta ttcaagtgga aaaaggcaac ctcaaaggca tcgatatgga tatgaaccat 960

attcccgatg cggcaatgac cattgccacc acagcgcttt ttgcagaagg tgaaacggtc 1020

attcgtaata tttataactg gcgcgtaaaa gaaactgatc gcttgaccgc gatggcgacc 1080

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> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agcttgatat cgaattcctg cag 23

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tatcgatacc gtcgacctcg ag 22

<210> 10

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcgaggtcga cggtatcgat aatccccttg ttacacaagc tatca 45

<210> 11

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcagcaaata ttgcgtgttt gatttatatt tcctatacac tggat 45

<210> 12

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agtgtatagg aaatataaat caaacacgca atatttgctg atcaaga 47

<210> 13

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgctaatgct acgattgtct ttttcatttt cgatacctta aaata 45

<210> 14

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgctgaagtt gctgctactg ctgcgactgc taatgctacg attgt 45

<210> 15

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtagcagcaa cttcagcaaa cgcaaatgat gataagctgt atcgc 45

<210> 16

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgtaccagaa cccatggatc cgctacctcc gccaccatga tg 42

<210> 17

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

catcatggtg gcggaggtag cggatccatg ggttctggta caggt 45

<210> 18

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

tgcttagtat ctatctgtag gttagaattc ttaatgatga tgatg 45

<210> 19

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

catcatcatt aagaattcta acctacagat agatactaag caaaa 45

<210> 20

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

caggaattcg atatcaagct aacaagaact tgataagttt gagaaa 46

<210> 21

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

catcatcatc attaagaatt cgaattcccg ggtcgactcg ag 42

<210> 22

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gtaccagaac ccatggatcc cggggatccc aggggcccct gg 42

<210> 23

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ccaggggccc ctgggatccc cgggatccat gggttctggt ac 42

<210> 24

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ctcgagtcga cccgggaatt cgaattctta atgatgatga tg 42

<210> 25

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ttttggtagt agggacgctt cgc 23

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ttatagtcct gtcgggtttc gcc 23

<210> 27

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tcctgtatag tctggacgaa ctg 23

<210> 28

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cccaggatgt tcagtgcgat a 21

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

taatctgacg cgctaccctg aca 23

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cgatacgagc ggatttacga taa 23

Claims

1. A recombinant avian pasteurella multocida for expressing duck circovirus antigen Cap protein is preserved in China general microbiological culture collection center with the preservation number of CGMCC 24875.

2. A preparation method of recombinant avian pasteurella multocida is characterized by comprising at least the following steps:

s2, constructing a recombinant plasmid containing the target gene:

3. The production method according to claim 2, wherein the target gene sequence includes at least 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 promoter sequence of the asnA gene comprises a signal peptide sequence of OmpH.

4. The preparation method according to claim 3, wherein 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 comprising OmpH was ligated to the 3' end of the asnA gene promoter sequence.

5. The method according to claim 3,

the adjuvant sequence and the optimized Cap gene sequence are connected through flexible peptide, the flexible peptide is a nucleotide sequence for encoding 4-10 amino acids, and the flexible peptide is preferably G4S linker;

the C end of the optimized Cap gene sequence is connected with a tag for assisting protein purification, and the tag is preferably 5-15 His tags.

6. The production method according to claim 3,

the position 988186-988953 of the genome of the Pm70 strain selected by the upstream homology arm sequence; the site selected by the downstream homology arm sequence is between 990277 site and 991082 site of the genome of the Pm70 strain;

the optimized Cap gene sequence is a gene sequence which removes an NLS sequence enriched by N-terminal arginine of the Cap gene and comprises a main antigen epitope region.

7. The production method according to claim 3,

the nucleotide sequence of the asnA gene promoter sequence is shown as SEQ ID NO. 1;

the nucleotide sequence of the optimized Cap gene sequence is shown as SEQ ID NO. 2;

the nucleotide sequence of the upstream homologous arm sequence is shown as SEQ ID NO. 3;

the nucleotide sequence of the downstream homologous arm sequence is shown as SEQ ID NO. 4;

the nucleotide sequence of the adjuvant sequence is shown as SEQ ID NO. 5;

the nucleotide sequence of the target gene sequence is shown as SEQ ID NO. 6;

the optimal selection of the primer for amplifying the sequence shown in SEQ ID NO. 1 is the nucleotide sequence shown in SEQ ID NO. 12-14;

the primer for amplifying the sequence shown in SEQ ID NO. 2 is preferably the nucleotide sequence shown in SEQ ID NO. 17 and SEQ ID NO. 18;

the primer for amplifying the sequence shown in SEQ ID NO. 3 is preferably the nucleotide sequence shown in SEQ ID NO. 10 and SEQ ID NO. 11;

the primer for amplifying the sequence shown in SEQ ID NO. 4 is preferably the nucleotide sequence shown in SEQ ID NO. 19 and SEQ ID NO. 20;

the primer for amplifying the sequence shown in SEQ ID NO. 5 is preferably the nucleotide sequence shown in SEQ ID NO. 15 and SEQ ID NO. 16.

8. The preparation method according to claim 1, wherein the knocked-out gene in the avian pasteurella multocida deleted strain is aroA gene, preferably, the aroA gene at least comprises a sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID NO. 7.

9. A gene recombinant vector vaccine is characterized in that a gene engineering vaccine for simultaneously preventing avian pasteurella multocida and duck circovirus is prepared from an avian pasteurella multocida liquid with the preservation number of CGMCC 24875.

10. Use of pasteurella multocida according to claim 1 for the preparation of a genetically engineered vaccine for the simultaneous prevention of pasteurella multocida and duck circovirus.