CN109939225B

CN109939225B - Rough brucella abortus of recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof

Info

Publication number: CN109939225B
Application number: CN201910284652.5A
Authority: CN
Inventors: 丁家波; 范学政; 朱良全; 冯宇; 彭小薇; 秦玉明; 王芳; 许冠龙; 李秋辰; 蒋卉
Original assignee: China Institute of Veterinary Drug Control
Current assignee: China Institute of Veterinary Drug Control
Priority date: 2019-04-10
Filing date: 2019-04-10
Publication date: 2022-04-26
Anticipated expiration: 2039-04-10
Also published as: CN109939225A

Abstract

The invention relates to construction of rough brucella of a recombinant chlamydia psittaci (Cps) outer membrane protein MOMP gene and a vaccine thereof. The invention successfully constructs a recombinant Brucella RA343-MOMP strain capable of efficiently expressing the Roughtficia psittaci outer membrane protein MOMP gene by taking a rough type Brucella abortus attenuated strain RA343 as a parent strain and taking a sucrose suicide plasmid as a carrier and inserting the MOMP gene of the psittaci chlamydia outer membrane protein containing a specific promoter sequence into a Brucella genome tracelessly after codon optimization. The recombinant strain not only keeps the rough characteristic of the original parent strain RA343 and has good immune protection to brucellosis (brucellosis), but also can generate MOMP antibody to Chlamydia psittaci after the recombinant strain is used for immunizing animals, thereby realizing immune protection to Chlamydia psittaci. The vaccine prepared by the recombinant vaccine strain can realize double immune protection to the brucellosis and the chlamydia psittaci after immunizing animals.

Description

Rough brucella abortus of recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof

Technical Field

The invention relates to rough brucella of a recombinant chlamydia psittaci outer membrane protein MOMP gene and a vaccine production method thereof, belonging to the field of biological products for livestock.

Technical Field

Brucellosis (brucellosis) is a human and animal co-disease caused by Brucella and characterized by abortion and fever, seriously threatening the life health of humans and various animals. The disease not only has serious harm to the reproduction and production performance of animals, but also is difficult to cure after people are infected with brucella, thereby causing serious public health problems. Therefore, in countries where brucella is prevalent, eradication of brucella has always been one of the most important goals in public health programs. The main method for eliminating the disease worldwide is to combine killing and immunization at present, and the vaccine prevention becomes the main control means of the disease in China with relatively serious disease distribution due to high cost of killing.

In 2007-2012, 41 strains of bovine Brucella, 74 strains of ovine Brucella and 9 strains of canine Brucella are successively separated from the bodies of animals such as cattle, sheep, dogs and deer in different provinces and regions of China. In the research process, we preliminarily found a bovine brucella isolate with virulence between virulent and vaccine strains, and the patent applicant carried out comprehensive biochemical detection on the bovine brucella isolate and measured the virulence by using mice and guinea pigs respectively. As a result, the virulence of the brucella Abortus is found to be between strong virulence and weak virulence, and the brucella Abortus is determined to be moderate virulence and is named as Abortus 343. The invention induces and domesticates the separated strain, screens and obtains a stable rough brucella vaccine strain which is named as RA343 and obtains a related patent (ZL 201410240895.6). At present, the vaccine strain has completed all laboratory research and development and clinical tests, and enters a new veterinary drug registration link.

Chlamydia psittaci (Cps) is one of the main pathogenic bacteria causing local abortion of sheep, and the abortion rate of pregnant sheep and goats infected with Cps for the first time is as high as 30% and 70%, respectively, which causes great loss to animal husbandry in many countries. Vaccine research is an important means for preventing pathogenic infection, and genetic engineering subunit vaccines are increasingly favored due to their safety. The Main Outer Membrane Protein (MOMP) of the Cps is a species-specific antigen, and the r-Cps-MOMP expressed by genetic engineering has good immunogenicity and immune protection effect.

Disclosure of Invention

The invention aims to construct a recombinant Brucella RA343-MOMP strain capable of efficiently expressing a Brucella psittaci outer membrane protein MOMP gene by using a rough type Brucella attenuated strain RA343 as a parent strain and using a sucrose suicide plasmid as a vector, wherein the Brucella psittaci outer membrane protein MOMP gene containing a specific promoter sequence is inserted into a Brucella genome without trace after codon optimization, and the recombinant Brucella RA343-MOMP strain is constructed by a successful biotechnology. The recombinant strain not only keeps the rough characteristic of the original parent strain RA343 and has good immune protection to brucellosis (brucellosis), but also can generate antibodies aiming at Cps MOMP after the recombinant strain is inoculated to animals, thereby realizing immune protection to Chlamydia psittaci. The vaccine prepared by the recombinant vaccine can realize double immune protection to the brucellosis and the psittacosis chlamydia after immunizing animals.

Technical scheme of the invention

1. A recombinant live Brucella vaccine is characterized in that after the vaccine is used for immunizing animals, the vaccine can simultaneously realize double immune protection against Brucella disease and Chlamydia psittaci disease; the production strain is a recombinant brucella RA343-MOMP strain (the recombinant strain RA343-MOMP strain is shortened for the invention) which can efficiently express chlamydia psittaci outer membrane protein (Cps MOMP); the strain is prepared by adding P₃Carrying out codon optimization on the promoter and the MOMP sequence, artificially synthesizing a gene segment, and constructing a gene containing P₃-MOMP sequence and sucrose suicide plasmid vector pUC/P of upstream and downstream homology arms of Brucella₃-MOMP⁺The Chlamydia psittaci outer membrane protein MOMPPart of the gene expression frame is inserted into brucella genome without trace, positive clone is screened out through double screening of ampicillin and sucrose, subculture is carried out, stability of the brucella is identified through PCR and sequencing, a recombinant brucella (Bovine brucella) RA343-MOMP strain is obtained, and the strain is delivered to China general microbiological culture Collection center of China institute of microbiology, No. 3 of Ministry of China academy of sciences, North Dynasty, West Anthony, No.1 of North Dynasty, Beijing City, 03 and 14 days in 2019, and the preservation number is as follows: CGMCC No. 17330.

2. The recombinant brucella live vaccine is characterized in that the specificity of the recombinant brucella RA343-MOMP strain for production, which is different from other bacteria, is that a pair of specific PCR primers (sequence 21 and sequence 22) respectively aiming at brucella genome and Chlamydia psittaci MOMP gene are adopted to amplify a specific fragment (sequence 23) with the size of 1529 bp;

the MOMP gene expression verification is to verify the expression of MOMP gene transcription level by means of real-time fluorescent quantitative PCR (qPCR);

verifying the expression of the MOMP gene at the protein level by a protein imprinting method (Western Blot);

the recombinant bacteria are used for immunizing mice, and the expression condition of MOMP antibodies in the mice is monitored.

3. The recombinant live Brucella vaccine is characterized in that the production method of the vaccine is to use tryptone soybean broth as a culture medium of Brucella CGMCC No. 17330; after the culture medium is sterilized, brucella CGMCC No.17330 strain seed bacterial liquid is inoculated according to 1-2% of the amount of the culture medium, fermentation culture is carried out for 28-38 h at 37 ℃ according to a conventional method, a common freeze-drying protective agent for veterinary biological products is added, the mixture is fully and uniformly mixed, and the mixture is packaged in a vaccine bottle and then is frozen and dried in vacuum, so that the brucella RA343-MOMP strain live vaccine is obtained.

4. The invention relates to a recombinant live Brucella vaccine, which is characterized in that a freeze-drying protective agent in the vaccine comprises the following components in percentage by weight: 5-15 g of PVP, 5-15 g of BSA, 50-100 g of trehalose, 50g of mannitol, 10-15 g of thiourea, 2g of ascorbic acid, 2g of vitamin C, 2g of 199 culture, 1.25g of dipotassium hydrogen phosphate and 0.52g of potassium dihydrogen phosphate, and dissolving the mixture in 1000ml of water for injection.

5. The recombinant live Brucella vaccine is characterized in that the vaccine immunization effect is evaluated by respectively adopting Brucella and Chlamydia psittaci to carry out challenge protection tests after the vaccine is used for immunizing sheep.

The invention has the advantages of

(1) The brucellosis and the psittacosis are both important diseases causing the abortion of the sheep, the brucellosis RA343 strain is used as a carrier, a novel vaccine strain capable of successfully expressing the MOMP gene of the outer membrane protein of the psittacosis chlamydia (Cps) is constructed, the prevention and control problems of the brucellosis and the psittacosis chlamydia are skillfully solved, the concept of preventing two diseases by one injection is realized, and the immune cost and the labor cost in the production practice are reduced; (2) at present, the key technical problem which troubles the immune control of the cloth disease worldwide is that the antibody (smooth antibody) generated by the vaccine immunization interferes the clinical diagnosis of the cloth disease. The invention skillfully adopts the rough brucella RA343 strain as a carrier, and rough antibodies are generated after the vaccine is used for immunizing animals, so that the clinical diagnosis of the brucella is not interfered, and the weakness of the existing brucella vaccine is fundamentally improved; (3) the invention solves the dilemma that the psittacosis does not have vaccines. Because the Chlamydia psittaci is extremely difficult to culture, the pathogen has long growth time, high cost and is easy to be polluted and the like, no commercial vaccine for the Chlamydia psittaci exists so far. The Chlamydia psittaci outer membrane protein MOMP is the main immune protective antigen of Chlamydia psittaci, and the antibody produced after the animal is immunized by the antigen can provide good protection for Chlamydia psittaci disease. Based on the reasons, the invention skillfully uses the rough brucella (RA343 strain) as a carrier to express MOMP protein, thereby realizing the immune protection against the Chlamydia psittaci disease; (4) in order to ensure stable and efficient expression of MOMP, on one hand, the invention adopts a traceless insertion technology to replace plasmid mediation, directly inserts MOMP into the genome of Brucella RA343 strain, and verifies the stability through passage, thereby ensuring that exogenous genes are not lost in the production passage process. On the other hand, in the implementation process of the invention, the codons which are easy to translate by Brucella are used for replacing rare codons in MOMP genes, and the method for optimizing the codons promotes the high-efficiency expression of exogenous genes. (ii) a (5) The conventional live animal vaccine generally needs to be stored and transported at the temperature of 20 ℃ below zero, 4 groups of different heat-resistant protective agent formulas are designed, and one optimal formula is optimized to be used as a freeze-drying protective agent actually adopted, so that the live animal vaccine can be stored at the temperature of 4 ℃ for a long time, and the storage and transportation cost is reduced.

Information on microbial resources related to the present invention

The microbial resource related to the invention is a Brucella abortus RA343 strain, which is a rough type attenuated strain obtained by inducing and domesticating a Brucella abortus A343 strain separated from a cow body in a certain dairy farm in Shandong in 2009 in a laboratory, and the strain is delivered to the general microorganism preservation center of China Committee for microorganism preservation of China institute of academy of sciences No. 3, North Cheng West Lu No.1 of south china institute of sciences, in the Yangzhou, Beijing city, 05-20 days in 2014: CGMCC No. 8886; the brucella abortus 2308 strain (CVCC 788 strain) and brucella abortus BM28 strain (CVCC 920 strain) are preserved and supplied by the China veterinary microbial strain preservation management center, which is the China veterinary medical products institute, and is compiled by the China veterinary microbial strain preservation management center, China veterinary medical products catalog second edition, China agricultural science and technology publishing agency, 2002, p24 and p 29.

Drawings

FIG. 1 Green fluorescent plasmid GFP-pZL1790 plasmid map.

FIG. 2 shows PCR specificity results of recombinant Brucella RA343-MOMP strain, wherein 1 is marker, 2 is recombinant Brucella RA343-MOMP strain, 3 is Escherichia coli, 4 is Staphylococcus and 5 is Salmonella.

FIG. 3qPCR shows relative expression values on The ordinate of The relative expression of MOMP gene (The relative expression of MOMP gene) in The graph. 1 is RA343 strain, and 2 is RA343-MOMP strain.

FIG. 4 Western Blot identification of recombinant vaccine with absorbance at 450nm on the ordinate (absorbance at 450 nm); the abscissa represents the time after infection (day post infection). 1 is RA343-MOMP strain, 2 is RA343 vaccine strain, and 3 is marker.

FIG. 5MOMP gene induced specific antibody response results.

FIG. 6 is a schematic diagram of the lyophilization curve with temperature (. degree. C.) on the ordinate; the abscissa represents the lyophilization time.

Detailed description of the invention

1. Construction of recombinant Brucella RA343-MOMP strain

(1) Selection of promoter for foreign Gene expression by Brucella RA343 (for details, see example 1)

According to the analysis of the whole genome and gene function prediction of Brucella RA343(CGMCC No.8886, ZL201410240895.6), three molecular chaperone promoters P1, P2 and P3 which can be expressed in Brucella continuously at high level are screened out, and Brucella promoter P1, P2 and P3 are respectively inserted into the upstream of the green fluorescent group of green fluorescent plasmid GFP-pZL1790 (constructed in the laboratory)₁、P₂、P₃And common promoters tac, trc and T7 to construct six green fluorescent plasmids P carrying different promoters₁-GFP-pZL1790，P₂-GFP-pZL1790，P₃GFP-pZL1790, tac-GFP-pZL1790, trc-GFP-pZL1790, and T7-GFP-pZL 1790. Respectively transferring the genes into Brucella RA343 strains, and screening out proper strong promoter P for exogenous gene expression according to the relative expression quantity of green fluorescent protein₃。

(2) By adding P₃Carrying out codon optimization on the promoter and the MOMP sequence, artificially synthesizing a gene segment, and constructing a gene containing P₃-MOMP sequence and sucrose suicide plasmid vector pUC/P of upstream and downstream homology arms of Brucella₃-MOMP⁺Inserting part of an outer membrane protein MOMP gene expression frame of Chlamydia psittaci into a brucella genome without trace, screening out positive clones by ampicillin and sucrose double screening, carrying out subculture, and identifying the stability of the clones by PCR and sequencing to obtain a recombinant brucella RA343-MOMP strain.

2. General biological characteristics of recombinant Brucella RA343-MOMP Strain

(1) Morphological and biochemical Properties

The colony edge of the RA343-MOMP strain recombinant Brucella is neat, round and smooth, is in a drop shape, and is irradiated by oblique lightAnd observing micro-strip blue opalescence in a backlight mode. The staining morphology is coccobacillus, single scattered and does not form spores and capsules. The size is 0.3-0.6 μm. Gram staining was negative. The growth of the bacterium is independent of CO₂Capable of growing on medium containing thionine and basic rubine, H₂The S test is strong positive.

(2) The results of a thermal agglutination test, an acridine yellow test and a colony crystal violet test show that the RA343-MOMP strain bacteria of the recombinant brucella retain the rough RA343 characteristic.

(3) Specificity of

1) The serological specificity is that the recombinant brucella RA343-MOMP strain culture is prepared into antigen, and the antigen and the smooth brucella positive serum do not agglutinate and the rough serum do agglutinate.

2) The specificity of the PCR specificity recombination brucella RA343-MOMP strain different from other bacteria is that a pair of specificity PCR primers (sequence 21, sequence 22) aiming at brucella genome and Chlamydia psittaci MOMP gene are adopted to amplify a specificity fragment (sequence 23) with the size of 635 bp; the amplified product was identified by electrophoresis on 1.5% agarose gel, and 1 specific PCR band (FIG. 2) was observed, which was 1529bp in size. This method is distinguished from other bacterial identification methods.

(4) Virulence

The recombinant Brucella RA343-MOMP strain culture cultured for 48-72 h on a solid culture medium is washed down by normal saline, diluted into 10 hundred million suspension containing viable bacteria per milliliter, and 5 guinea pigs (Hartley strain) with the weight of 350-400 g are injected subcutaneously into groin, wherein each guinea pig is 1 ml. And (3) killing the cells after 14-15 days, taking spleens, mixing and weighing the spleens to prepare an emulsion, inoculating a TSA (TSA) plate, and calculating the bacteria content of the phagocytosed spleens according to the number of growing colonies, wherein the bacteria content of each 1g of the spleens is not more than 20 ten thousand.

(5) Safety of mice

Dividing Balb/C mice with the weight of 20-20 g into 5 mice/group, and diluting the recombinant Brucella RA343-MOMP strain into 1 × 10¹²CFU/ml to 1X 10⁸CFU/ml bacterial suspension, inguinal injection of 0.1 ml/one, and the control group. The health of the mice was observed and recorded within 1 week after inoculation, knotsFruit inoculation 10¹¹CFU/mice in the immunized group of only the following recombinant bacteria were all alive for 6 days. The result shows that the recombinant brucella has good safety when used for immunizing non-target animals at different doses.

3. Preparation of recombinant brucella (RA343-MOMP strain) live vaccine

The vaccine is produced with recombinant Brucella RA343-MOMP strain expressing MOMP gene as outer membrane protein of Chlamydia psittaci as the production strain, and the production process includes: tryptone soybean broth is used as a culture medium of Brucella CGMCC No. 17330; inoculating brucella CGMCC No.17330 strain seed bacterial liquid according to 1-2% of the culture medium after sterilizing the culture medium, fermenting and culturing for 28-38 h at 37 ℃ according to a conventional method, adding a freeze-drying protective agent commonly used by veterinary biological products, preferably a heat-resistant protective agent, fully mixing uniformly, subpackaging in a vaccine bottle, and then freezing and drying in vacuum to obtain the recombinant brucella (RA343-MOMP strain) live vaccine, wherein the vaccine can realize double immune protection on brucellosis and psittacosis after immunizing animals.

Examples

The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention.

Example 1 construction of recombinant Brucella RA343-MOMP Strain

1. Preparation of Brucella RA343 competent cells

Picking out a single colony of the Brucella RA343 strain, inoculating the colony and culturing the colony in 100ml of TSB culture medium until the bacteria grow to logarithmic phase, and cooling the colony in ice water. Centrifuging at 12000r/min for 10min, discarding the liquid culture medium, and repeatedly washing with sterile deionized water with different volumes for several times. Finally, the obtained thalli is resuspended in 1ml of 10% glycerol aqueous solution, and the prepared parent strain RA343 infected bacteria are stored at-80 ℃ for standby.

2. Promoter screening of Brucella RA343 expression exogenous gene

(1) Construction of Green fluorescent plasmid GFP-pZL1790

The pZL1790 plasmid is stored in the laboratory, the GFP green fluorescence gene is synthesized by Shanghai bio-engineering company, primers (sequence 28 and sequence 29) are designed to amplify the green fluorescence gene and add enzyme cutting sites, and the sequences of the primers are as follows:

sequence 28 GFP-F: tgagtcgaca tggtctcgaa gggcgaagaa 30

Sequence 29 GFP-R: ataccgcggt tacttataca gttca 25

Adding an upstream primer into a Sal I restriction site, adding a downstream primer into a Sac I restriction site, and carrying out PCR by using the primers and a GFP green fluorescent gene as a template, wherein the reaction system is as follows:

PCR reaction (50. mu.L):

max 2 XBuffer 25. mu.L, upstream and downstream primers (10. mu.M) 1. mu.L each, ddH₂O22. mu.L, template DNA 1. mu.L.

Reaction conditions are as follows: 5min at 95 ℃; 31 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 30 s; 10min at 72 ℃.

After fully mixing 100. mu.L of 6 XLoading Buffer with 20. mu.L of PCR amplification product, adding the mixture into a 0.8% agarose gel well, carrying out electrophoresis at 150V for about 25min, carrying out imaging analysis by using a gel imaging system, cutting an agarose gel block containing a target fragment, and carrying out recovery and purification of PCR products according to the instruction of a Takara gel recovery kit (Code No: 9762). After the green fluorescent gene recovery product and pZL1790 plasmid are quantified, double enzyme digestion is respectively carried out, and the reaction system is as follows:

sal I1. mu.L, Sac I1. mu.L, 10 XBuffer 5. mu.L, nucleic acid 1. mu.g, ddH₂The content of O is filled to 50 mu L.

After the enzyme digestion is carried out for 30min at 37 ℃, the enzyme digestion products are connected, and the reaction system is as follows:

1 μ L of T4 ligase, 2 μ L of 10 XBuffer, 2 μ L of pZL1790 plasmid after digestion, and 15 μ L of green fluorescent gene after digestion. The reaction was placed in a 4 ℃ freezer overnight. The recombinant green fluorescent plasmid GFP-pZL1790 after connection is transformed into DH5 alpha competent cells by a heat shock transformation method for molecular cloning, screening is carried out by using a culture medium containing chloramphenicol, and sequencing is carried out to obtain a positive plasmid (figure 1).

(2) Three Brucella promoters and three common promoters

According to the Brucella RA343 complete geneGroup and gene function prediction analysis, three molecular chaperone promoters P1, P2 and P3 which can be expressed in brucella at sustainable high level are screened out, and the promoters P containing the molecular chaperone promoters are synthesized by Shanghai's pharmaceutical company after codon optimization₁，P₂，P₃And plasmids of the common promoter tac, trc and T7 sequences, the promoter P being amplified separately using the respective primers for the different promoters (Table 1)₁，P₂，P₃PCR products of tac, trc and T7 (addition of KpnI and EcoRI cleavage sites), the PCR reaction system and conditions were as follows:

PCR reaction (50. mu.L):

TABLE 1 promoter PCR amplification primer Table

(3) Promoter PCR product electrophoresis and purification

After fully mixing 100. mu.L of 6 XLoading Buffer with 20. mu.L of PCR amplification product, adding the mixture into a 0.8% agarose gel well, carrying out electrophoresis at 150V for about 25min, carrying out imaging analysis by using a gel imaging system, cutting an agarose gel block containing a target fragment, and carrying out recovery and purification of PCR products according to the instruction of a Takara gel recovery kit (Code No: 9762).

(4) Construction of Green fluorescent protein plasmid carrying promoter

After the PCR product is purified, the PCR product is cut by KpnI and EcoRI enzyme and then cloned to the plasmid GFP-pZL1790 cut by the same restriction enzyme respectively to construct 6 plasmids which can theoretically be cloned in BrucellaExpressing green fluorescent protein plasmid in the bacteria: p₁-GFP-pZL1790，P₂-GFP-pZL1790、P₃GFP-pZL1790, tac-GFP-pZL1790, trc-GFP-pZL1790 and T7-GFP-pZL 1790. After sequencing verification, the constructed plasmid is electrically transformed into Brucella RA343 strain.

(5) Electric conversion

Mu.g of YGT-pZL1790 (as a negative control), P₁-YGT-pZL1790、P₂-YGT-pBBR1mcs2、P₃The plasmids-YGT-pZL 1790, tac-YGT-pZL1790, trc-YGT-pZL1790 and T7-YGT-pBBR1mcs2 were added to 50. mu.L of RA343 competent bacteria, mixed well and transferred to a cuvette for electric shock. The shock voltage and time are respectively: 1.8kv, 3 ms. After the electric shock is finished, 1mL of TSB culture medium is immediately added, shaking culture is carried out for 4h at 37 ℃, then the bacterial liquid is coated into 2 TSA culture dishes containing 50 mu g/mL kanamycin, culture is carried out for more than 48h at 37 ℃, and the grown bacterial colony is the positive electrotransformation bacteria. The obtained recombinant gene engineering bacteria are named as RA343-YGT-pZL1790 (as negative control) and RA343-P₁-YGT-pZL1790、RA343-P₂-YGT-pZL1790、RA343-P₃-YGT-pZL1790, RA343-tac-YGT-pZL1790, RA343-trc-YGT-pZL1790 and RA343-T7-YGT-pZL 1790.

(6) Screening of strong promoters by brucella cell infection experiments

Mouse macrophage RAW264.7 is passed to six-hole plate, and respectively classified into RA343 group and RA343-P₁-YGT-pZL1790、RA343-P₂-YGT-pZL1790 and RA343-P₃Groups of-YGT-pZL 1790, RA343-tac-YGT-pZL1790, RA343-trc-YGT-pZL1790 and RA343-T7-YGT-pZL1790 were incubated until the bottom of the 6-well plate was confluent. Inoculating the corresponding strain into 20ml of TSB culture medium, and carrying out shaking culture at constant temperature of 180r/min and 37 ℃ until the logarithmic phase. And (5) collecting the bacteria, and repeatedly resuspending the bacteria by PBS. Respectively infecting with Brucella and RAW364.7 at infection ratios (MOI) of 50:1, 200:1, and 500:1, and placing in CO₂A 5% constant temperature incubator at 37 ℃. After the infection for 1h, gentamicin with the final concentration of 50 mug/ml is added into each hole of the six-hole plate, and the action is carried out for 45 min. Mouse macrophages after 24h, 48h, 72h infection were washed 3 times with PBS to stop the infection. Adding formaldehyde fixing liquid for fixing, and thenFluorescence detection is carried out by a fluorescence microscope under exciting light, and a strong promoter P is screened out according to the intensity of green fluorescence under the same exciting light intensity₃。

3. Codon optimization and traceless insertion of Chlamydia psittaci outer membrane protein MOMP gene

(1) Shuttle plasmid pUC/P₃-MOMP⁺Construction of

Promoter P artificially synthesized and codon-optimized by Shanghai bioengineering GmbH₃And a Chlamydia psittaci outer membrane protein MOMP gene sequence, using a cloned plasmid as a template, and using MOMP-F (sequence 13) and MOMP-R (sequence 14) as primers to amplify the MOMP gene; using Brucella RA343 genome DNA as a template, designing primers up-F (sequence 15), up-R (sequence 16), down-F (sequence 17) and down-R (sequence 18) to respectively amplify 941bp bases at the upstream of 814594 of No.1 chromosome as an upper homologous arm and 781bp bases at the downstream as a lower homologous arm (XbaI and BamHI restriction enzyme cutting sites are respectively added to the upper and lower homologous arms), and adding a promoter P into the upstream and downstream of the upper and lower homologous arms₃And the Chlamydia psittaci outer membrane protein MOMP gene is inserted into the brucella genome without trace, and the primers are shown in Table 2:

TABLE 2 upstream and downstream homology arm primers for wboA gene

The PCR reaction systems are as follows: in a 50. mu.L reaction system, 25. mu.L of 2 XBuffer, 2. mu.L of mixed primer stock solution, 1. mu.L of Taq enzyme, 1. mu.L of template DNA, and ddH₂O 21μL。

The PCR reaction procedures were as follows: 5min at 95 ℃; 31 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 60 s; 10min at 72 ℃.

Purifying and recycling PCR products of PCR amplified Brucella upstream and downstream homologous arms and MOMP gene segments of chlamydia psittaci outer membrane protein, and calculating the dosage of each template in fusion PCR according to a formula after determining the concentration:

and the above homology arm, MOMP gene and lower homology arm are fused together by fusion PCR.

And (3) PCR reaction system: in a 50. mu.L reaction system, 25. mu.L of 2 XBuffer, 2. mu.L of mixed primer stock solution, 1. mu.L of Taq enzyme, 2. mu.L of template DNA, and ddH₂O 20μL。

PCR reaction procedure: 5min at 95 ℃; 30s at 95 ℃, 30s at 56 ℃ and 3min at 72 ℃ for 31 cycles; 10min at 72 ℃.

Performing double digestion on PCR gel by XbaI and BamHI restriction enzymes to recover a product and a pUC19 plasmid, connecting the fusion fragment to a multiple cloning site of a shuttle plasmid pUC19 to obtain a recombinant shuttle plasmid named as pUC/MOMP⁺。

(2) Electrotransfer

The shuttle plasmid pUC/P prepared above₃-MOMP⁺Electrotransfer into the Brucella RA343 strain competent bacteria prepared in the step (1), wherein the specific method comprises the following steps:

1-10 ng of pUC/P₃-MOMP⁺The plasmid was added to 50. mu.L of the RA343 strain competent bacteria, and mixed well. After ice-cooling for 10min, the sample was pipetted into a 2nm cuvette for click. Setting parameters: 2.5kV, 5 ms. And after electric shock is finished, adding 1ml of TSB culture medium into the electric conversion cup, sucking the bacterial suspension, adding the bacterial suspension into a TSB culture medium triangular flask, placing the TSB culture medium triangular flask in the flask, and shaking and culturing for at least 4 hours at 37 ℃.

(3) Screening of positive recombinant strain RA343-MOMP strain

Screening ampicillin: the bacterial solution cultured with shaking at 37 ℃ for 4h is centrifuged at 6000r/min for 3min, part of the supernatant is gently aspirated, about 200. mu.L of the supernatant is reserved, the precipitated bacteria are gently blown to be uniform, and then all the colonies are spread on 1 TSA medium plate containing 50ug/ml ampicillin and cultured at 37 ℃ for at least 72 h. The recombinants containing ampicillin were screened.

Negative screening of sucrose: single colonies were picked on TSA plates containing ampicillin, inoculated into TSB liquid medium containing no ampicillin, cultured overnight with shaking at 37 ℃ for 24 hours, and then cultured with physiological saline at a ratio of 1: 10²、1:10³、1：10⁴Diluting bacterial suspension, and coating bacterial suspension 100 μ L/dilution with 5&Sucrose TSA plates were incubated at 37 ℃ for at least 72 h.

Randomly picking a single colony from a 5% sucrose plate, and carrying out colony PCR screening on positive recombinant bacteria by using the following pair of primers (sequences 19 and 20), wherein the size of the positive bacteria is 2148 bp;

the sequence 19F is that of ggagggacaa gcgtaaacca 20,

sequence 20R: ccagtattcg gaagcgtgag c 21

Simultaneous sequencing to verify promoter P₃And the MOMP gene is inserted into the genome of the Brucella without trace. The positive recombinant bacteria are recombinant Brucella ragutita (Brucella) RA343-MOMP strain (Brucella RA343-MOMP strain for short) capable of efficiently expressing chlamydia psittaci outer membrane protein (Cap MOMP), the strain is delivered to Beijing city of Beijing on No.1 institute of south china academy of sciences microorganism research institute China general microbiological culture Collection center, No. 3 of North Cheng West Lu province, on 03 and 14 days in 2019, and the preservation numbers are as follows: CGMCC No. 17330;

example 2 general biological Properties of recombinant Brucella RA343-MOMP Strain

1. Morphological and biochemical Properties

The colony edge of the recombinant bacterium is neat and round, the recombinant bacterium is in a drop shape, oblique light irradiation is carried out, and micro-strip blue opalescence is observed in a backlight mode. The staining morphology is coccobacillus, single scattered and does not form spores and capsules. The size is 0.3-0.6 μm. Gram staining was negative. The growth of the bacterium is independent of CO₂Capable of growing on medium containing thionine and basic rubine, H₂The S test is strong positive.

2. Thermal coagulation test

Inoculating the recombinant strain to a TSA culture medium, culturing at 37 ℃ for 46h, then hooking the culture into a test tube filled with about 100ml of normal saline, shaking uniformly to enable the culture to be turbidified to a bacterial suspension containing 10 hundred million/ml of live bacteria, dividing the bacterial suspension taken out into 2 tubes, wherein each tube is 4-5ml, heating the tubes in a 90 ℃ water bath for 1h, observing results at 30min and 60min, wherein the recombinant strain and the parent strain are both agglutination positive and rough, and the recombinant strain keeps the characteristic of RA343 rough.

3. Acridine yellow test

Preparing a 1:500 neutral acridine yellow solution by using distilled water, placing one drop of the acridine yellow solution on a glass slide, taking a little recombinant vaccine cultured for 48 hours by using an inoculating loop hook, uniformly mixing the recombinant vaccine with a parent strain, and immediately observing the result. Agglutination appeared within 3min, and the recombinant vaccine was a rough colony.

4. Colony crystal violet test

The recombinant strain is serially diluted by 10 times, inoculated on a TSA plate culture, cultured at 37 ℃ for 72-96 h, the crystal violet storage solution is diluted by 1:40 times by distilled water, a proper amount of diluted dye solution is absorbed to cover the TSA plate full of a single bacterial colony for dyeing for 15-20 s, the crystal violet dye solution is discarded in a disinfectant, and the coloration condition of the edges of the bacterial colony is observed by a magnifying glass or a low-power microscope. The recombinant colony has colored edges and unclear boundaries, and is rough.

5. Specificity of

(1) Serological specificity the culture is used to prepare antigen, and the antigen should not agglutinate with smooth type brucella positive serum and agglutinate with rough type serum.

(2) The specificity of the PCR specificity recombination brucella RA343-MOMP strain different from other bacteria is that a pair of specificity PCR primers (sequence 21, sequence 22) aiming at brucella genome and Chlamydia psittaci MOMP gene are adopted to amplify a specificity segment (sequence 23) with the size of 1529 bp; the amplified product was identified by electrophoresis on 1.5% agarose gel, and 1 specific PCR band (FIG. 1) was observed, which was 1529bp in size. This method is distinguished from other bacterial identification methods.

A pair of primers was synthesized and prepared as a primer mixture stock solution, each primer concentration being 25. mu.M.

(sequence 21) RA 343-MOMP-F: gagggatggc tttgcgtttc t 21

(sequence 22) RA 343-MOMP-R: tggctgatta gtattacttg cgtta 25

Template: RA343 genomic DNA extracted from colonies or kits cultured in RA 343.

And (3) PCR reaction system: mu.L of the reaction mixture contained 5. mu.L of 10 XBuffer, 8. mu.L of 2.5mM dNTPs, 2. mu.L of the mixed primer stock, 2U of Taq enzyme, and 1. mu.L of template DNA (or a small amount of picked colonies).

PCR reaction procedure: after 5min at 95 deg.C, 28 cycles at 94 deg.C for 1min, 60 deg.C for 1.5min, and 72 deg.C for 1.5min, and finally 72 deg.C for 10 min.

The amplified product was identified by electrophoresis on 1.5% agarose gel, and 1 specific PCR band of 1529bp appeared, while no band was generated using the genome of other strains (E.coli, Staphylococcus, Salmonella) as template (FIG. 2).

Sequence 23:

6. a culture cultured on a solid culture medium for 48-72 hours is washed down by using physiological saline for virulence, the culture is diluted into 10 billion suspension containing viable bacteria per milliliter, and 5 guinea pigs (Hartley strain) with the weight of 350-400 g are injected subcutaneously into the groin, wherein the injection amount is 1 ml/mouse. And (3) killing the cells after 14-15 days, taking spleens, mixing and weighing the spleens to prepare an emulsion, inoculating a TSA (TSA) plate, and calculating the bacteria content of the phagocytosed spleens according to the number of growing colonies, wherein the bacteria content of each 1g of the spleens is not more than 20 ten thousand.

7. Safety of mice

Dividing Balb/C mice with the weight of 20-20 g into 5 mice/group, and diluting the recombinant Brucella RA343-MOMP strain into 1 × 10¹²CFU/ml to 1X 10⁸CFU/ml bacterial suspension, inguinal injection of 0.1 ml/one, and the control group. The health of the mice was observed and recorded within 1 week after vaccination and the results showed 10 of the recombinant vaccine¹¹CFU/mice in the following immunization groups were all alive at 6 days. The results show that the recombinant vaccine has good safety when used for immunizing non-target animals at different doses.

Example 3 stability of recombinant bacterium RA343-MOMP Strain in target animals

The recombinant Brucella RA343-MOMP strain is used for culturing the Brucella in a culture medium of 1 multiplied by 10¹⁰CFU/first immunized adult sheep group, and 3d blood collection after immunization is inoculated in TSA culture mediumAnd (4) carrying out bacteria separation, carrying out PCR identification on the separated bacteria, and continuously passaging the non-immunized adult sheep for 5 generations according to the method. The phenotype of the recombinant vaccine strain after continuous passage is not changed, and the MOMP gene inserted into the chromosome 1 stably exists.

Example 4 expression verification of MOMP Gene in recombinant Brucella RA343-MOMP Strain

qPCR verification of MOMP Gene expression at transcriptional level

Absorbing a proper amount of recombinant brucella RA343-MOMP strain and RA343 parent strain liquid, killing the live bacteria liquid in a water bath at a constant temperature of 80 ℃ for more than 2h, extracting bacterial RNA by using a TAKARA bacterial genome extraction kit, carrying out reverse transcription, taking a 16s gene as an internal reference, and carrying out relative quantification on mRNA transcribed by MOMP gene through qPCR; the primers used are shown in table 3:

TABLE 3qPCR primer List

The reaction system is as follows: 2 × Premix Ex Taq 10 μ L, upstream and downstream primers 0.5 μ L (10 μmol/L), Rox 0.4 μ L, template 2 μ L, deionized water 6.6 μ L.

The reaction conditions were as follows: 30s at 95 ℃, 5s at 95 ℃ and 34s at 60 ℃ for 40 cycles.

The result shows that the relative expression quantity of mRNA of the MOMP gene of the recombinant strain RA343-MOMP strain is obviously higher than that of the parent strain RA343, and the MOMP gene has higher expression quantity at the transcription level (figure 3).

Western blot identification of expression of MOMP gene of recombinant Brucella RA343-MOMP strain

The Brucella recombinant RA343-MOMP strain and the parent strain RA343 are inoculated into a TSB culture medium for shaking culture for 24-48 h, and subjected to ultrasonic lysis, sample buffer solution is added, SDS-PAGE and Wsetron Blot analysis after boiling water bath for 10min, and the result shows that the recombinant RA343-MOMP has an obvious band at about 60kD relative to the parent strain RA343, and the MOMP protein expression is verified by a protein imprinting method (figure 4).

3. MOMP specific antibody of recombinant brucella RA343-MOMP strain stimulated mouse

The mice were randomly divided into 3 groups of 30 mice each. The first group of inguinal subcutaneous immune recombinant vaccines RA343-MOMP strain, 10⁹CFU/only; a second group of inguinal subcutaneous immune parent strain RA343 strain, 10⁹CFU/only; the third group was a blank, 1 ml/tube, immune saline.

Blood is collected 14 days after the immunization of the mice in the immunized group, serum is separated, and the MOMP antibody of Chlamydia psittaci is determined by an indirect ELISA method. The results show that MOMP antibody level OD of recombinant vaccine RA343-MOMP group Chlamydia psittaci₄₅₀Positive with a retention of 1.0 or more, parent strain RA343 group and blank group OD₄₅₀It remained below 0.5 and was negative (FIG. 5).

Example 5-screening and Effect verification of live vaccine of recombinant Brucella RA343-MOMP strain.

According to the characteristics and the eutectic performance of RA343-MOMP, 4 groups of heat-resistant protective agents (respectively marked as

formulas

1, 2, 3 and 4) are designed, freeze-drying is respectively carried out according to a conventional freeze-drying curve (figure 6), and through the aging-resistant test results (shown in table 4) of storage for 7 days at 37 ℃ before and after freeze-drying, the best effect of the freeze-drying RA343-MOMP live vaccine of the formula 1 of the heat-resistant protective agent is determined, the freeze-drying is not crystallized after freeze-drying, and the storage rate of freeze-drying bacteria reaches 90.4%; the product can be stored at 37 deg.C for 7 days without atrophy, and the viable bacteria reduction rate is 13.3%. Is obviously superior to the gelatin sucrose protectant control.

TABLE 4 Freeze drying results for different protectant formulations (viable count: 10)⁸CFU/ml)

The protectant formulations and methods of formulation are shown in table 5 below:

TABLE 5 Heat-resistant Freeze-drying protectant formulations

Note: preparation method

(1) Solution 1: adding proper amount of water for injection (100 ℃) into a clean beaker, sequentially weighing PVP, mannitol, trehalose and thiourea according to the formula, adding into the beaker while stirring by a glass rod to completely dissolve the PVP, the mannitol, the trehalose and the thiourea, adding the water for injection to a constant volume of 500ml, and sterilizing for 30min at 116 ℃.

(2) Solution 2: weighing BSA, 199 culture medium, ascorbic acid, vitamin C, potassium dihydrogen phosphate and dipotassium hydrogen phosphate in a beaker according to the formula in sequence, adding water for injection to fully dissolve the BSA, 199 culture medium, ascorbic acid, vitamin C, potassium dihydrogen phosphate and dipotassium hydrogen phosphate, metering the volume to 500ml, and filtering and sterilizing by a sterilizing filter with a 0.22 mu m filter membrane.

Example 6 determination of immunoprotective Effect of recombinant Brucella RA343-MOMP Strain live vaccine against Brucella and Chlamydia psittaci

1. Immune protection effect of recombinant vaccine on brucella

The 45 clean grade sheep were randomly divided into three groups of 15 sheep each. A group of RA343 vaccine strains for oral immunization of 50 hundred million CFU, a group of RA343-MOMP recombinant vaccine strains for oral immunization of 50 hundred million CFU, and a group of normal saline with the same dosage for oral immunization. Blood is collected at 0d, 7d, 14d, 28d, 42d and 60d after immunization, serum is separated, and brucella antibody IgG is detected by a microtubular agglutination method. The minimum infection dose at 8 weeks after immunization of 3 groups was 5X 10⁶CFU/single dose of Brucella melitensis (B.melitensis) BM28 strain was used to combat conjunctival infection. And observing and recording the ingestion condition, the weight, the mental state and the like. And (3) killing after 30d of challenge, taking spleen and inguinal lymph node, mashing and grinding, inoculating a TSA culture medium, culturing at 37 ℃ for at least 3d, separating bacteria and identifying. The results show that Brucella antibodies appear 7d after the sheep are immunized by RA343 and RA343-MOMP recombinant strains, the levels of the 7-28 d antibodies are continuously increased, the titer of the 28d antibodies reaches a peak after immunization, and then the titer starts to gradually decrease. The control group had no antibody production. The separation results of spleen and inguinal lymph node bacteria of sheep show that the RA343-MOMP strain recombinant vaccine and the parent strain RA343 vaccine can provide 87% of immune protection rate for sheep.

2. Immunoprotection effect of recombinant vaccine against chlamydia psittaci

40 clean-grade sheep are randomly divided into two groups, each group comprises 20 sheep, the experimental group is injected with recombinant vaccine subcutaneously according to the dose of 50 hundred million CFU bacteria to immunize adult sheep, and the control group is injected with normal saline with equal dose subcutaneously. After 30 days, Chlamydia psittaci was inoculated. After 14d, the infection rate of chlamydia psittaci of the two groups of sheep is observed, and the immune protection effect of the immune recombinant vaccine group is determined. Results the control group has 20 sheep with disease, and the infection rate of chlamydia psittaci is 100%; the experimental group had only 4 sheep with 80% chlamydia psittaci infection. The immune protection rate of the recombinant vaccine to the chlamydia psittaci is up to 80%, and the vaccine has ideal immune protection efficiency.

Sequence listing

<110> China institute for veterinary drug inspection

<120> Brucella ragrans of recombinant chlamydia psittaci outer membrane protein MOMP gene and vaccine production method thereof

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 31

<212> DNA

<213> Artificial Synthesis (P1-F)

<400> 1

cggggtaccc cgtgtcagcc cgccatccac a 31

<210> 2

<211> 29

<212> DNA

<213> Artificial Synthesis (P1-R)

<400> 2

ccggaattcg gtcacgccct cgaacttgc 29

<210> 3

<211> 33

<212> DNA

<213> Artificial Synthesis (P2-F)

<400> 3

cggggtaccc cgatgcggtg gtaagctcct tca 33

<210> 4

<211> 31

<212> DNA

<213> Artificial Synthesis (P2-R)

<400> 4

ccggaattcc ggcggcagcg cctcatacat t 31

<210> 5

<211> 32

<212> DNA

<213> Artificial Synthesis (P3-F)

<400> 5

cggggtaccc cgaaatgtgc cgctcttctc gc 32

<210> 6

<211> 31

<212> DNA

<213> Artificial Synthesis (P3-R)

<400> 6

ccggaattcc caggagttgg tcgttcccag a 31

<210> 7

<211> 28

<212> DNA

<213> Artificial Synthesis (tac-F)

<400> 7

cggggtgcct cgaatagctg acgtgcca 28

<210> 8

<211> 30

<212> DNA

<213> Artificial Synthesis (tac-R)

<400> 8

ccggaaaaag gacacacttt aacaataggc 30

<210> 9

<211> 25

<212> DNA

<213> Artificial Synthesis (trc-F)

<400> 9

cggggtttga caattaatca tccgg 25

<210> 10

<211> 27

<212> DNA

<213> Artificial Synthesis (trc-R)

<400> 10

ccggaacatt atacgagccg gatgatt 27

<210> 11

<211> 27

<212> DNA

<213> Artificial Synthesis (T7-F)

<400> 11

cggggttaat acgactcact ataggga 27

<210> 12

<211> 24

<212> DNA

<213> Artificial Synthesis (T7-R)

<400> 12

ccggaatctc cctatagtga gtcg 24

<210> 13

<211> 32

<212> DNA

<213> Artificial Synthesis (MOMP-F)

<400> 13

gatactaagc ataatcttta gaggtgagta tg 32

<210> 14

<211> 26

<212> DNA

<213> Artificial Synthesis (MOMP-R)

<400> 14

ctgatagcgg gacaaaaagt taggat 26

<210> 15

<211> 31

<212> DNA

<213> Artificial Synthesis (up-F)

<400> 15

ggctctagaa tacgcttatt gagaatgttc g 31

<210> 16

<211> 32

<212> DNA

<213> Artificial Synthesis (up-R)

<400> 16

agcggcacat tttcttaatg cgcgggaata gc 32

<210> 17

<211> 32

<212> DNA

<213> Artificial Synthesis (down-F)

<400> 17

tcaagcagtc gctgttcttg cttcagcttg tt 32

<210> 18

<211> 31

<212> DNA

<213> Artificial Synthesis (down-R)

<400> 18

ccggatccta agccgacgag caaatagaag g 31

<210> 19

<211> 20

<212> DNA

<213> Artificial Synthesis (F)

<400> 19

atcagttcta gagacaagga 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Synthesis (R)

<400> 20

attgttacac cgctaccctg g 21

<210> 21

<211> 21

<212> DNA

<213> Artificial Synthesis (RA343-MOMP-F)

<400> 21

gagggatggc tttgcgtttc t 21

<210> 22

<211> 25

<212> DNA

<213> Artificial Synthesis (RA343-MOMP-R)

<400> 22

tggctgatta gtattacttg cgtta 25

<210> 23

<211> 1529

<212> DNA

<213> Artificial Synthesis ()

<400> 23

gagggatggc tttgcgtttc tgcgctttga agatgttgaa attgggttag ggccgcaata 60

tggtggtgta agcctaccag catatgagtt tcgaaatttt gaggggttat ttcttcgccg 120

caccgaagcc actggattgg atggatatac agaccttgga tacgtcccag atgctgaaca 180

gcgggggttc atctttgcag acggagcagc cctccacatc aatagcctgt ttgcagataa 240

taacaagggt gatggcgtgt tttgccaaaa cgtccaatac gtagatggaa acgatctcaa 300

ttcatccatc gacggcggaa ctgggttcaa ttttatcaac gtagatcgca taaacatcaa 360

tacgatccgc agtggtggcc gccggaatat ggcaccagga aatcttaaca ctgtttccca 420

aggtatctct ttgaatgcaa attgtcagac tgtaattata ggcaacgcag ttacccacaa 480

ctggtgaagt cacggttttt atagccaagc tcaggacatt ttggttaatg gtctgatatc 540

acgtgataat ggcggaaggg ggtacgttgc agagggttca gcagggtcat ctctcctaaa 600

tggggccgtt ttcagagata atgtagcagg gaattatttt acaggaggga caagcgtaaa 660

ccatctcgcg aacctccaac ttcataactc tagcaccggg gggaaaactt ttgtggccaa 720

tgtcaccaca aatgggtctg cataacggtc cttgccattt taactataaa tgagctattc 780

ccgcgcatta agagtagaca cgggaaatca gtcgcggatc cgcgaaatgt gccgctcttc 840

tcgctggaaa attccagggc gaaccagaaa ttcatcgatg cgatttattc cgcgggcaga 900

agcggggtga gcgagactgt gtgacgcctg taaagagtgc ggggcaagga atctgttttt 960

gtctggctct tgtgacttgc agtgaaaaac tgcctttctt atatacgcct cgcacagggc 1020

tttgatgaca ggccggaaac cggtccgctt tctggaaccc aaccttgaag caggcattgt 1080

gaaaaccgat aggaactgcc catggaacgc tcggtcgagg tcttggcagt ttgctgaatg 1140

gagagaaata tggctaaggt tattggtatc gatcttggta cgaccaactc ctgggatact 1200

aagcataatc tttagaggtg agtatgaaaa aactcttgaa atcggcatta ttgtttgccg 1260

ctacgggttc cgctctctcc ttacaagcct tgcctgtagg gaacccagct gaaccaagtt 1320

tattaatcga tggcactatg tgggaaggtg cttcaggaga tccttgcgat ccttgcgcta 1380

cttggtgtga cgccattagc atccgcgcag gatactacgg agattatgtt ttcgatcgtg 1440

tattaaaagt tgatgtgaat aaaactttta gcggcatggc tgcaactcct acgcaggcta 1500

caggtaacgc aagtaatact aatcagcca 1529

<210> 24

<211> 17

<212> DNA

<213> Artificial Synthesis (16S-F)

<400> 24

cacaagcggt ggagcat 17

<210> 25

<211> 17

<212> DNA

<213> Artificial Synthesis (16S-R)

<400> 25

gcaactaagg gcgaggg 17

<210> 26

<211> 21

<212> DNA

<213> Artificial Synthesis (MOMP-F)

<400> 26

ctacatggaa cccaagcctt a 21

<210> 27

<211> 19

<212> DNA

<213> Artificial Synthesis (MOMP-R)

<400> 27

cgattaacgt tgcaccaac 19

<210> 28

<211> 30

<212> DNA

<213> Artificial Synthesis (GFP-F)

<400> 28

tgagtcgaca tggtctcgaa gggcgaagaa 30

<210> 29

<211> 25

<212> DNA

<213> Artificial Synthesis (GFP-R)

<400> 29

ataccgcggt tacttataca gttca 25

Claims

1. A recombinant live Brucella vaccine is characterized in that after the vaccine is used for immunizing animals, the vaccine can simultaneously realize double immune protection against Brucella disease and Chlamydia psittaci disease; the production strain is a recombinant brucella RA343-MOMP strain containing outer membrane protein capable of efficiently expressing chlamydia psittaci; the strain is prepared by adding P₃Carrying out codon optimization on the promoter and the MOMP sequence, artificially synthesizing a gene segment, and constructing a gene containing P₃-MOMP sequence and sucrose suicide plasmid vector pUC/P of upstream and downstream homology arms of Brucella₃-MOMP⁺Inserting part of an MOMP gene expression frame of the psittacosis chlamydia outer membrane protein into a brucella genome without trace, screening out positive clones by double screening of ampicillin and sucrose, carrying out subculture, identifying the stability of the clones by PCR and sequencing to obtain a recombinant brucella RA343-MOMP strain, and carrying out the cross-over in 2019, 03 and 14 days, wherein the strain is preserved in the general microorganism center of China Committee for culture Collection of microorganisms, China institute of microbiology, national institute of sciences, No. 3, North Cheng-Yang district, Beijing City, and the preservation numbers are as follows: CGMCC number 17330.

2. The recombinant brucella live vaccine according to claim 1, wherein the specificity of the recombinant brucella RA343-MOMP strain for production to other bacteria is that a pair of specific PCR primers respectively aiming at brucella genome and Chlamydia psittaci MOMP gene are adopted, as shown in sequence 21 and sequence 22, and a specific fragment with 1529bp in size is amplified as shown in sequence 23;

the expression verification of the MOMP gene is to verify the expression of MOMP gene transcription level in a real-time fluorescent quantitative PCR mode;

verifying the expression of the MOMP gene at the protein level by a western blotting method;

3. The live recombinant Brucella vaccine according to claim 1, which is produced by using tryptone soy broth as the culture medium of Brucella CGMCC No. 17330; after the culture medium is sterilized, brucella CGMCC No.17330 strain seed bacterial liquid is inoculated according to 1-2% of the amount of the culture medium, fermentation culture is carried out for 28-38 h at 37 ℃ according to a conventional method, a common freeze-drying protective agent for veterinary biological products is added, the mixture is fully and uniformly mixed, and the mixture is packaged in a vaccine bottle and then is frozen and dried in vacuum, so that the brucella RA343-MOMP strain live vaccine is obtained.

4. The live recombinant brucella vaccine according to claim 3, wherein the lyophilized protectant comprises the following formulation: 5-15 g of PVP, 5-15 g of BSA, 50-100 g of trehalose, 50g of mannitol, 10-15 g of thiourea, 4g of vitamin C, 2g of 199 culture medium, 1.25g of dipotassium hydrogen phosphate and 0.52g of potassium dihydrogen phosphate, and dissolving the mixture in 1000ml of water for injection.

5. The recombinant live Brucella vaccine according to claim 1, wherein the immune effect of the vaccine is evaluated by performing challenge protection test using Brucella and Chlamydia psittaci, respectively, after immunizing sheep with the vaccine.