Recombinant adenovirus based on 33 protective genes of Brucella

Abstract

The invention discloses a recombinant adenovirus based on 33 protective genes of Brucella, 33 most important protective antigen genes are screened from 89 whole genes of Brucella through a bioinformatics technology, and are recombined into a human type 5 replication-deficient adenovirus vector through a genetic engineering technology. Therefore, the recombinant adenovirus vaccine based on 33 protective genes of Brucella is obtained. The invention effectively overcomes the defects of virus dispersion, virulence recombination, virulence reversion, virulence residue and the like of attenuated vaccine strains such as conventional brucella vaccine strains, gene deletion vaccine strains and the like, is a safe and effective vaccine, has the effect reaching the brucella vaccine evaluation standard formulated by the world animal hygiene organization OIE, and can be applied to prevention of brucellosis.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and genetic engineering vaccines, and in particular to a recombinant adenovirus based on 33 protective genes of Brucella. Background Art

[0002] Brucellosis is a zoonotic infectious disease caused by bacteria of the genus Brucella. Among them, Brucella melitensis, Brucella abortus and Brucella suis are considered to be the most pathogenic Brucella, causing most human and livestock brucellosis, causing serious impacts on animal husbandry and public health. Animal infection with Brucella can cause miscarriage in female animals and infertility in male animals. Human infection with Brucella can cause joint pain, affecting labor and fertility. In recent years, brucellosis has shown a rebound trend worldwide. Due to the increase in the number of pigs, cattle and sheep raised in my country, disorderly transportation between regions, outdated inspection and quarantine technology and other reasons, the brucellosis epidemic in my country is particularly serious. At present, the prevention and control of brucellosis mainly involves the following two types of vaccinations.

[0003] Category 1: Traditional attenuated live vaccines. Traditional attenuated live vaccines are the main vaccines currently used to prevent and control brucellosis, including the traditional pig S2 strain, cattle A19 strain and sheep M5 (M5-90) strain, as well as the new Brucella gene-deleted marker live vaccine. The pig S2 strain was first obtained by the China Veterinary Drug Inspection Institute in 1952 by isolating pig aborted embryos; the cattle A19 strain was obtained by my country through the Soviet Union in the 1950s; the sheep M5 (M5-90) strain was obtained by using the streptomycin-resistant culture of the sheep Brucella 6056 strain; the Brucella gene-deleted marker live vaccine is a vaccine developed with the sheep M5 (M5-90) strain as the parent and the bp26 gene deleted. Both traditional attenuated live vaccines and new Brucella gene-deleted vaccines are attenuated live vaccines, and these vaccines are obtained through in vitro culture.

[0004] Although the existing attenuated live vaccines can play an effective role in the prevention and control of brucellosis, their use also brings the following risks. (1) There is an inevitable risk of spreading the virus: Although the attenuated vaccine strains are weak in virulence and can produce protection after being inoculated into the animal body, the attenuated vaccine strains are still live viruses. During inoculation and in the inoculated animals, they will inevitably be scattered into the environment, causing environmental pollution. In addition, the attenuated strains of Brucella are bacteria and can reproduce themselves under suitable conditions, increasing the risk of disease spread. (2) The risk of virulence recombination increases the difficulty of prevention and control: Whether the attenuated vaccine is scattered in the environment or inoculated into animals, it can replicate itself. If it comes into contact with wild Brucella in nature, gene exchange may occur and recombination may occur, resulting in mutated Brucella, which increases the difficulty of brucellosis prevention and control. (3) The risk of virulence reversion increases the difficulty of prevention and control: The attenuated strains of Brucella can gradually restore or even become more virulent through genetic recombination and repeated passage of susceptible animals, further increasing the difficulty of prevention and control. (4) They are pathogenic to humans and animals: Both attenuated strains and gene-deficient strains are attenuated live vaccines. Although they have low virulence, they still have some residual virulence, causing miscarriage, orchitis, and infertility in humans and animals. In addition, it is difficult to distinguish infected animals from vaccinated animals through serological testing.

[0005] Category 2: New vaccines

[0006] Since traditional attenuated vaccines have defects that they cannot overcome, scientific researchers are exploring new vaccines to solve these problems, mainly DNA vaccines, subunit vaccines and recombinant live vector vaccines.

[0007] (1) DNA vaccine: The pcDNA3 plasmid driven by the cytomegalovirus promoter was inserted into the Brucella abortus lumazine synthase (BLS) gene to construct a pcDNA-BLS plasmid carrying the BLS gene. After immunization of mice, although the bacterial load was reduced, it did not meet the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE) (Infect Immun. 2002 May; 70(5): 2507-11. doi: 10.1128 / IAI.70.5.2507-2).

[0008] (2) Subunit vaccines: Although the Brucella protein P39 provided some protection after inoculation into mice, it was far from the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE). (Infect Immun. 2001Aug; 69(8): 4816-22.doi: 10.1128 / IAI.69.8.4816-4822.2001.) Single subunit vaccines of four proteins aspC, dps, yaeC and inpB can improve their protective effect, but they also did not meet the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE). (Vaccine. 2018May 17; 36(21): 3027-3033.doi: 10.1016 / j.vaccine.2018.04.019.Epub 2018Apr17.)

[0009] (3) Recombinant influenza virus vector vaccine: Although the use of influenza virus vectors to express Brucella outer membrane proteins (Omp) 16 and 19, ribosomal L7 / L12, and Cu-Zn superoxide dismutase (SOD) protein can protect mice from Brucella virulent attacks in the range of 1.6 to 2.97 Log, it does not meet the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE). (Biomed Res Int. 2020Nov 19:2020:1438928.doi:10.1155 / 2020 / 1438928.eCollection 2020.)

[0010] (4) Recombinant adenovirus vector vaccine

[0011] Since the discovery of adenovirus, especially human type 5 replication-deficient adenovirus (Ad5), it has become an important carrier for vaccine development due to its many advantages: first, the modified adenovirus genome is very stable and easy to operate in vitro; second, adenovirus vectors can accommodate large fragments of foreign genes and can be used to construct vaccines with multiple fragments of antigenic epitopes; third, replication-deficient adenovirus has good safety. Replication-deficient adenovirus can only proliferate in human embryonic kidney cells and can only reproduce transiently in other types of cells. The reproduced virus is defective and cannot reproduce again, so there is no defect of conventional attenuated vaccine strains such as virus shedding, virulence recombination and virulence reversion; fourth, adenovirus vectors can produce strong and lasting humoral and cellular immune responses to surface antigens, and the adenovirus's own hexon protein is an effective adjuvant for activating innate immunity, so adenovirus vectors are highly immunogenic; fifth, adenovirus is easy to prepare and store. Adenovirus can be cultured using serum-free suspension cells, with rapid production and high yield, which can meet the surging market demand, and its immunogenicity is maintained for at least 1 year using conventional freeze-drying methods. These advantages of adenovirus vectors make up for the defects of attenuated vaccines.

[0012] All along, Brucella antigens have been screened based on experience, such as Omp16, Omp19, Omp25, Omp31, SurA, Dnak, trigger factor (TF), ribosomal protein L7 / L12, bacterial ferritin (BFR) P39 and lumazine synthase BLS, such as the patent with publication number CN117717611A "A Brucella Bp26 gene recombinant adenovirus vector vaccine and its preparation and application", and the patent with publication number CN102703505A "Co-expression Brucella recombinant adenovirus and its preparation method and use", expressing Brucella L7 / L12 and BCSP31 genes, and the journal recombinant adenovirus expressing Brucella P39 and lumazine genes, although they can reduce the number of Brucella in mouse models, they have not reached the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE). (https: / / pubmed.ncbi.nlm.nih.gov / 29492808 / )

[0013] In fact: because the Brucella genome is large and complex, the vaccines prepared by existing technologies using one or two Brucella antigen genes cannot fully neutralize the Brucella virulence genes, and therefore cannot prepare effective vaccines; secondly, the Brucella protective genes selected based on experience cannot reflect the essence of the Brucella protective genes, and their effectiveness in epidemic prevention needs to be further improved and verified. Summary of the invention

[0014] The purpose of the present invention is to provide a recombinant adenovirus based on 33 protective genes of Brucella, so as to simultaneously improve the safety and effectiveness of preventing brucellosis and meet the Brucella vaccine evaluation standards established by the World Organization for Animal Health (OIE).

[0015] To achieve the above purpose, the present invention successfully constructed a human type 5 replication-deficient adenovirus vector through genome biological function analysis, design, screening and cloning of 33 kinds of Brucella key antigen genes, and expressed the corresponding antigen protein; the main antigenic epitopes of Brucella are analyzed and identified by computer for Brucella whole genome data using a reverse vaccine development strategy, and potential antigen scores are performed. After different optimization combinations, 33 high-potential antigenic epitopes are finally obtained, and expressed in 4 adenovirus vectors in series. Animal experiments have verified that its protective effect is equivalent to that of conventional attenuated vaccine A19 strain. The present invention can not only stimulate the body to produce an effective immune response, but also overcome the problems of attenuated vaccine shedding, toxicity recombination, toxicity re-strengthening and toxicity residual. The specific technical scheme is as follows.

[0016] The recombinant adenovirus pADV-mCMV-ABCD based on 33 protective genes of Brucella in the present invention was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 27, 2024, with the deposit number CGMCCNO.46170, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0017] The recombinant adenovirus comprises 33 target genes of Brucella that have been screened and optimized through the whole genome and a linearized adenovirus expression vector; the construction process of the recombinant adenovirus is as follows:

[0018] Process 1: Screening of protective genes: 33 genes with the most potential antigens were screened from 89 whole genomes of 54 Brucella melitensis, 17 Brucella abortus and 18 Brucella suis; most of the antigens are related to bacterial adhesion, invasion, escape and adaptation to the internal environment of macrophages;

[0019] Process 2, recombinant adenovirus construction, specifically includes the following steps:

[0020] Step 2.1, genes, expression system and cells: The 33 Brucella main protective genes screened were divided into four groups of target genes, A sequence, B sequence, C sequence and D sequence, after expression verification, and then cloned into the pMd18-T vector after serial connection and adding corresponding restriction sites; the human type 5 replication-deficient adenovirus expression system and human embryonic kidney HEK 293T cells were optimized and preserved;

[0021] The nucleotide sequence of the A sequence is shown in SEQ ID NO.1, the nucleotide sequence of the B sequence is shown in SEQ ID NO.2, the nucleotide sequence of the C sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the D sequence is shown in SEQ ID NO.4;

[0022] Step 2.2, target gene digestion: extract the pMd18-T vector containing the target gene, and digest the plasmid containing the target gene with a restriction endonuclease; perform agarose gel electrophoresis on the digestion product to detect the digestion effect, and cut the target gene band from the gel after agarose gel electrophoresis, perform gel recovery, and obtain the target gene fragment;

[0023] Step 2.3, linearization of adenovirus expression vector: digest the human type 5 replication-deficient adenovirus expression vector with restriction endonucleases, perform agarose gel electrophoresis on the digestion products to detect the digestion effect, and cut the target vector band from the gel after agarose gel electrophoresis, perform gel recovery, and obtain the linearized adenovirus expression vector pADV-mCMV;

[0024] Step 2.4, constructing the target gene into a linearized adenovirus expression vector: adding the target gene fragment and the linearized adenovirus at a molar ratio of 2:1 into a centrifuge tube for a recombination ligation reaction to obtain a recombinant ligation product;

[0025] Step 2.5, transformation and identification: The recombinant ligation products were transformed into E. coli DH5α competent cells, PCR was used to identify positive transformants, and sequencing was used to verify that the transformants were correct, thereby constructing recombinant adenovirus expression vectors pADV-mCMV-Asequence, pADV-mCMV-Bsequence, pADV-mCMV-C sequence, and pADV-mCMV-Dsequence;

[0026] Step 2.6, recombinant adenovirus packaging and protein expression detection: the recombinant adenovirus expression vector was cut with Pme I to obtain a linearized recombinant adenovirus expression vector, and the linearized recombinant adenovirus expression vector was transfected into HEK 293T cells to package recombinant adenovirus pADV-mCMV-A, recombinant adenovirus pADV-mCMV-B, recombinant adenovirus pADV-mCMV-C and recombinant adenovirus pADV-mCMV-D.

[0027] Process 3: Immune effect verification

[0028] Step 3.1, animal grouping: 150 Balb / C mice were divided into 5 groups, namely, commercial vaccine group, adenovirus vaccine group, empty adenovirus group, DMEM control group and blank control group, with 30 mice in each group;

[0029] Step 3.2, Immunization: The commercial vaccine group was vaccinated for 10 days according to the instructions. 5 CFU / dose, 0.1ml; adenovirus vaccine group and empty adenovirus inoculation group, 10 6 PFU / mouse, 0.1ml; DMEM control was inoculated with 0.1ml DMEM; immunization was repeated once 21 days after the first immunization, i.e. the second immunization;

[0030] Step 3.3, virulent challenge: 28 days after the second immunization, mice were inoculated with standard Brucella M28 virulent virus at a dose of 1×10 4 CFU / piece;

[0031] Step 3.4, sample collection and evaluation: observe the clinical manifestations of mice every day after vaccination with strong virus, and detect the vaccine residues on the 7th, 15th, 30th and 45th days; kill the mice on the 7th, 14th and 21st days after the virus attack, separate and weigh the spleen aseptically, count the colonies, and calculate the protection units; the results showed that the Brucella recombinant adenovirus vaccine had no residues in the animals, and the protection effect met the protection standards of the OIE Brucella vaccine, and passed the immune effect verification.

[0032] The present invention has beneficial effects. The present invention is based on the analysis of a large number of Brucella whole genomes, screens out all the antigenic genes of Brucella, and identifies 89 important protective potential antigens in the whole genome of Brucella by combining reverse vaccinology with pan-genome analysis. After objective evaluation, scoring and verification one by one, the permutation and combination are re-optimized, and finally 33 key protective genes are screened out, and expressed in human type 5 replication-deficient adenovirus through 4 combinations of A, B, C, and D, thereby obtaining the corresponding recombinant adenovirus vector vaccine. The vaccine of the present invention is verified to be effective according to the Brucella vaccine evaluation standards of the Ministry of Agriculture and Rural Affairs, and reaches the Brucella vaccine evaluation standards formulated by the World Organization for Animal Health OIE; according to the evaluation method of the national standard, the commercialized Brucella A19 vaccine is used as the standard to evaluate the Brucella recombinant adenovirus vaccine of the present invention, and the results confirm that the vaccine of the present invention is not only safe but also effective.

[0033] The present invention uses human type 5 replication-deficient adenovirus as a vector to express the 33 most important protective genes of Brucella to prepare a vaccine: avoids using traditional strong strains to weaken or gene deletion methods to prepare vaccines, so that the present invention overcomes the defects of attenuated vaccine strains such as traditional Brucella vaccine strains and gene deletion vaccine strains, such as toxin shedding, virulence recombination, virulence reversion, and virulence residue; at the same time, it overcomes the problem that vaccines prepared using one or two antigen genes of Brucella in the prior art cannot fully neutralize Brucella virulence genes, and that Brucella protective genes selected based on experience cannot reflect the essence of Brucella protective genes. The Brucella vaccine successfully developed by the present invention is not only safe but also effective, and provides a new way for the safe and effective control of Brucella, and provides a strong guarantee for the public health security of my country and even the world.

[0034] The deposit information of the microorganisms involved in the present invention is as follows:

[0035] Depository: China General Microbiology Culture Collection Center (CGMCC);

[0036] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0037] Classification and nomenclature: Brucella recombinant adenovirus pADV-mCMV-ABCD;

[0038] Deposit number: CGMCC NO.46170;

[0039] Date of preservation: August 27, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1-A This is the map of the recombinant adenovirus A backbone vector;

[0041] Figure 1-B This is the map of the recombinant adenovirus B backbone vector;

[0042] Figure 1-C This is the map of the recombinant adenovirus C backbone vector;

[0043] Figure 1-D This is the map of the recombinant adenovirus D backbone vector;

[0044] Figure 1-E is the map of the empty adenoviral backbone vector;

[0045] Figure 2-A for protein A expression;

[0046] Figure 2-B for protein B expression;

[0047] Figure 2-C for protein C expression;

[0048] Figure 2-D Protein D expression. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0050] A recombinant adenovirus based on 33 protective genes of Brucella, including protective gene screening, recombinant adenovirus construction, and immune effect verification.

[0051] Example 1. Screening of protective genes

[0052] Reverse vaccinology was combined with pan-genomic analysis to identify cross-protective potential antigens of pathogenic Brucella. A reverse vaccinology strategy was used to analyze the core proteome from 89 whole genomes of 54 Brucella melitensis, 17 Brucella abortus, and 18 Brucella suis, using a subcellular localization prediction pipeline to identify surface-associated proteins. The analysis yielded 1939 core proteins, 177 surface-associated proteins, and 87 proteins with protective antigenic potential. Based on the cumulative scores obtained from protein antigenicity, MHC-I and MHC-II epitope density, MHC allele coverage, and B cell epitope density scores, 33 genes with the most potential antigens were finally screened out. Most of these antigens are related to bacterial adhesion, invasion, escape, and adaptation to the internal environment of macrophages. The specific steps are as follows.

[0053] (1) Data retrieval (NCBI): 89 whole genome complete protein sequences in FASTA format were downloaded from the National Center for Biotechnology Information Microbial Genome Resource - NCBI (https: / / www.ncbi.nlm.nih.gov / genome) of 54 Brucella melitensis, 17 Brucella abortus and 18 Brucella suis. The strain names of the 89 Brucella genomes are shown in Table 1.

[0054] Table 1. Strain names of 89 Brucella genomes

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] (2) Pan-genome analysis (BPGA): The whole-genome proteome of 89 Brucella species was analyzed using the Bacterial Pan-genome Analysis (BPGA) tool to identify core proteins. The analysis tool parameters were the default values, and the cut-off value was 50%. The number of core proteins in 17 Brucella abortus strains was 2840, the number of core proteins in 54 Brucella melitensis strains was 2578, and the number of core proteins in 18 Brucella suis strains was 2484. The number of common core proteins was 1939.

[0064] (3) Subcellular localization (SCL) analysis: It was divided into three stages. In the first stage, PSORTb v3.0.2, CELLOv.2.5 and SOSUI-GramN software were used to analyze and screen out outer membrane (OM) and / or extracellular membrane (P) proteins. A total of 151 proteins were screened, and 1639 inner membrane (IM), cytoplasm (CYT) and extracellular (EX) proteins were excluded. 149 proteins were marked as uncertain and entered the second stage of analysis. In the second stage, ClubSub-P and ngLoc tools were used to analyze subcellular localization, further excluding inner membrane (IM), cytoplasm (CYT) and extracellular (EX) proteins, 16 proteins were screened, 104 proteins were excluded, and the remaining 29 uncertain proteins entered the third stage of analysis. In the third stage, the meta-prediction tool MetaLoc was used to divide proteins into two categories: proteins included in the OM and P sites and proteins excluded in other sites. 10 of the 29 proteins were screened and 19 were excluded. A total of 177 proteins were screened in three stages.

[0065] (4) Adhesion rate analysis: The proteins predicted by SCL were further analyzed by the Vaxign tool to obtain the adhesion probability of surface-related proteins. Proteins with an adhesion probability greater than 0.5 were selected for further analysis. Among the 177 surface-related proteins, there were 38 high-potential proteins with adhesion scores ≥ 0.5, 49 proteins with adhesion scores between 0.4 and 0.5, and the remaining adhesion scores were all less than 0.4. Among these 38 proteins, one protein was excluded because it was homologous to the host, three proteins were excluded because their molecular weight was less than 10 kDa and their immunogenicity was poor, and one protein was excluded because it could not be effectively expressed during the verification process. Finally, 33 high-potential antigenic proteins were obtained.

[0066] (5) Protein optimization combination: AntigenPro and VaxiJen were used to analyze protein antigenicity, ProPred1 and ProPred were used to analyze T cell epitopes, and BCPred and AAPred were used to analyze B cell epitopes. Then, based on the principle of controlling the adenovirus packaging capacity at 8 kb, the 33 protein gene groups were further divided into 4 combinations according to priority, namely A, B, C, and D. The 33 protein gene groups are shown in Table 2.

[0067] Table 2. Grouping of 33 protein genes

[0068]

[0069]

[0070] Example 2. Construction of recombinant adenovirus

[0071] 2.1 Genes, expression systems and cells

[0072] (1) Genes: The 33 main protective genes of Brucella that were screened were divided into four groups, A, B, C, and D, after expression verification. The target genes were concatenated in the order of Appendix 2 and the corresponding restriction sites were added. They were synthesized, sequenced, and the corresponding restriction sites were added by Shanghai Shenggong Biotechnology Co., Ltd. and cloned into the pMd18-T vector. The target A sequence, B sequence, C sequence, and D sequence genes containing restriction sites were obtained.

[0073] The nucleotide sequence of A sequence is shown in SEQ ID NO.1,

[0074] The nucleotide sequence of B sequence is shown in SEQ ID NO.2.

[0075] The nucleotide sequence of C sequence is shown in SEQ ID NO.3,

[0076] The nucleotide sequence of D sequence is shown in SEQ ID NO.4.

[0077] (2) Expression system and cells: The human adenovirus type 5 replication-deficient expression system and human embryonic kidney HEK 293T cells were optimized and maintained by our laboratory.

[0078] 2.2 Target gene digestion

[0079] Extract the pMd18-T vector containing the target gene, and digest the plasmid containing the target gene with restriction endonucleases. The digestion reaction system is: 2μg of plasmid, 5μl of 10× reaction buffer, 1μl of each restriction endonuclease, and make up to 50μl with water. Incubate in a 37℃ water bath for more than 2h. The digestion product is subjected to agarose gel electrophoresis to detect the digestion effect, and the target gene band is cut out from the gel after agarose gel electrophoresis, and the gel is recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction KitVer.3.0.

[0080] 2.3 Linearized expression vector

[0081] The expression vector was digested with restriction endonucleases. The digestion reaction system was: 2 μg of plasmid, 5 μl of 10× reaction buffer, 1 μl of each restriction endonuclease, and 50 μl of water was added. The mixture was incubated in a 37°C water bath for more than 2 hours. The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the target vector band was cut out from the gel after agarose gel electrophoresis and recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0.

[0082] 2.4 Construction of target gene into adenoviral expression vector

[0083] The target gene fragment and the linearized adenovirus expression vector were added to a centrifuge tube at a molar ratio of 2:1 for recombination reaction, as shown in Table 3.

[0084] Table 3. Recombination ligation reaction system

[0085]

[0086] Optimal amount of insert fragment used = [0.04 x number of bases in the insert fragment] ng (0.03 pmol)

[0087] The optimal amount of linearized vector used = [0.02 x the number of linearized vector bases] ng (0.03 pmol)

[0088] After mixing, incubate at 37℃ for 30min, then transfer to ice for 5min. Transform directly or store at -20℃ and thaw when needed.

[0089] 2.5 Transformation and identification

[0090] The ligation product was transformed into E. coli DH5α competent cells, and the positive transformants were identified by PCR. The cells were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing verification. The adenovirus expression vector and the recombinant adenovirus expression vectors pADV-mCMV-A sequence, pADV-mCMV-B sequence, pADV-mCMV-C sequence and pADV-mCMV-D sequence were successfully constructed. The recombinant adenovirus backbone vector maps are shown in the following table. Figure 1-A , 1-B , 1-C, 1-C; the empty adenovirus backbone vector pADV-mCMV is shown in Figure 1-E shown.

[0091] The sequencing result of A sequence is the same as the nucleotide sequence shown in SEQ ID NO. 1. The sequencing result of B sequence is the same as the nucleotide sequence shown in SEQ ID NO. 2. The sequencing result of C sequence is the same as the nucleotide sequence shown in SEQ ID NO. 3. The sequencing result of D sequence is the same as the nucleotide sequence shown in SEQ ID NO. 4.

[0092] 2.6 Recombinant adenovirus packaging and protein expression detection

[0093] 2.6.1 Recombinant adenovirus packaging

[0094] According to Invitrogen's Lipo According to the instructions for use of 2000 transfection reagent, the linearized expression vector was transfected into HEK 293T cells to package recombinant adenovirus pADV-mCMV-A, adenovirus pADV-mCMV-B, adenovirus pADV-mCMV-C and adenovirus pADV-mCMV-D.

[0095] 2.6.2 Total protein extraction

[0096] A. Take out Western, cell lysate and protease inhibitor PMSF from -20℃, add 10μl PMSF to every 990μl cell lysate and place on ice;

[0097] B. Take out the sterilized EP tube, mark it, and place it on ice at 4℃;

[0098] C. Remove cells from the incubator, discard the cell culture medium, and wash with PBS 1-2 times;

[0099] D. Discard PBS, add appropriate amount of pre-cooled Western and cell lysis buffer (including PMSF), lyse cells on ice for 30 min, scrape off cells, and transfer samples into EP tubes;

[0100] E. Transfer the centrifuge tube containing the lysed cells to a pre-cooled high-speed low-temperature centrifuge and centrifuge at 4°C, 12,000 rpm for 5-10 minutes.

[0101] 2.6.3 Sample preparation

[0102] Add loading buffer according to the actual amount of sample mother solution at a ratio of 1:4 (loading buffer: cell solution), mix, boil at 95°C for 5 min, place on a dry ice box, and then centrifuge at 4°C, 12,000 g for 5 min.

[0103] 2.6.4 SDS-PAGE gel preparation

[0104] A. Align two clean glass plates, put them into the clamp, clamp them, and fix them on the glue filling bracket;

[0105] B. Prepare the separation gel liquid and stir gently to mix; see Table 4 for the preparation system.

[0106] C. Immediately add the separation gel liquid to the glass plate sandwich until the gel is about 5 cm high;

[0107] D. Slowly add deionized water to the top of the liquid surface of the interlayer. Allow the gel to polymerize at room temperature for 30 minutes. After polymerization, a clear refractive line can be seen between the interface of the top deionized water and the gel;

[0108] E. Pour off the top layer of deionized water and try to dry it with absorbent paper;

[0109] F. Prepare 5% concentrated glue liquid and add the liquid to the glass plate interlayer until it reaches the top of the interlayer; the preparation system is detailed in Table 5.

[0110] G. Insert a 1.5 mm thick comb into the concentrated glue liquid in the sandwich. If necessary, add more concentrated glue liquid to fill the remaining space. Allow the concentrated glue to polymerize at room temperature for 30 minutes.

[0111] Table 4. SDS-PAGE separation gel formula

[0112]

[0113] Table 5. SDS-PAGE stacking gel (5% Acrylamide) recipe

[0114]

[0115] 2.6.5 SDS-PAGE gel electrophoresis

[0116] A. Fix the gel plate to the inner tank of the electrophoresis device, put the fixed gel plate into the outer tank, add an appropriate amount of 1×SDS electrophoresis buffer to the outer tank, and add electrophoresis buffer to the inner tank until it just covers the gel loading hole;

[0117] B. Carefully pull out the comb to avoid tearing the polyacrylamide gel loading wells. After pulling out the comb, rinse and fill the loading wells with 1× SDS electrophoresis buffer;

[0118] C. Load the sample and perform SDS-PAGE electrophoresis. Connect the power supply and run the electrophoresis at 80V for 30min until the bromophenol blue dye enters the separation gel from the stacking gel, then adjust the voltage to 120V and continue the electrophoresis for 70min until the bromophenol blue reaches the bottom of the gel;

[0119] D. Turn off the power supply and remove the connected wires, discard the electrophoresis buffer, and remove the gel sandwich.

[0120] 2.6.6 Transfer

[0121] A. Before the end of electrophoresis, prepare a PVDF membrane and 6 filter papers of the same size as the gel. Activate the PVDF membrane with methanol for 5 minutes, then equilibrate in the buffer for 15 minutes; equilibrate the filter paper in the transfer buffer for 15 minutes;

[0122] B. Place the transfer clips, two sponge pads, filter paper and the soaked membrane in the plate with transfer solution;

[0123] C. Open the clamp to keep the black side horizontal. Place a sponge pad on top. Place three layers of filter paper on the pad to remove air bubbles.

[0124] D. Remove the glass plate and gently scrape off the concentrated gel. 2 Rinse once in 1× 2% O, and then rinse once in 1× transfer buffer. Carefully cover the separation gel on the filter paper and adjust it by hand to align it with the filter paper. Cover the membrane on the gel, covering the entire gel and removing bubbles. Cover the membrane with 3 pieces of filter paper and remove bubbles. Finally, cover it with another sponge pad and close the clamp;

[0125] E. Place the clip into the transfer tank, with the black side of the clip facing the black side of the tank, and the white side of the clip facing the red side of the tank. Electric transfer will generate heat, so place a piece of ice on one side of the tank to cool it down. Generally, transfer with 300mA for 2h.

[0126] 2.6.7 Immune response (see Table 3 for antibody information)

[0127] A. Move the membrane to a TBST incubation box containing 5% skim milk powder and block it on a decolorizing shaker at room temperature for 1 hour;

[0128] B. Dilute the primary antibody to an appropriate concentration with TBST containing 5% skim milk powder; remove the membrane from the blocking solution, place the membrane with the protein side facing up on the antibody liquid surface, and shake the four corners of the membrane to drive out the remaining bubbles; shake and incubate on a decolorizing shaker at 4℃ overnight;

[0129] C. Wash three times with TBST on a decolorizing shaker at room temperature, 10 min each time;

[0130] D. Dilute the secondary antibody as above and contact it with the membrane. After incubation at room temperature for 1-2 hours, wash it three times with TBST on a decolorizing shaker at room temperature for 10 minutes each time. Place the membrane on a white plate with the protein side facing up, add the luminescent liquid droplets on the membrane to fully cover the membrane surface, and then put it into the imager to take pictures. Antibody information is shown in Table 6.

[0131] Table 6. Antibody information

[0132]

[0133]

[0134] 2.6.8 Protein detection results

[0135] The A sequence protein was predicted to be approximately 147 KDa. The experimental results showed that protein bands were detected between Marker 55-180 KDa, proving that the A sequence protein was expressed. Figure 2-A As shown.

[0136] The B sequence protein was predicted to be approximately 147 KDa. The experimental results showed that a protein band was detected near the marker 180 KDa, proving that the B sequence protein was expressed. Figure 2-B shown.

[0137] The B sequence protein was predicted to be approximately 147 KDa. The experimental results showed that protein bands were detected near the marker 70-180 KDa, proving that the C sequence protein was expressed. Figure 2-C As shown.

[0138] The B sequence protein was predicted to be approximately 255 KDa. The experimental results showed that a protein band was detected near Marker>180 KDa, proving that the D sequence protein was expressed. Figure 2-D shown.

[0139] Example 3. Immune effect verification

[0140] 3.1 Evaluation of immune protection in mice

[0141] 3.1.1 Vaccines, virulent viruses, animals, culture media, and equipment.

[0142] Brucella vaccine A19 was purchased from Xinjiang Tiankang Animal Husbandry Biotechnology Co., Ltd.; Malta Brucella virulent strain M28 was prepared and preserved by Harbin Veterinary Research Institute; 6- to 8-week-old female SPF Balb / C mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in P3 laboratory IVC. Tryptic soy agar (TSA) medium was purchased from BD. Brucella selective antibiotics (OXOID) were purchased from Thermo; Retsch MM400 tissue grinder; 37°C constant temperature incubator.

[0143] 3.1.2 Animal Experiment Design

[0144] According to the experimental design, 150 6-8 week old female Balb / C mice were divided into 5 groups, 30 mice in each group, including commercial Brucella A19 vaccine positive control group, adenovirus vaccine group, empty adenovirus group, DMEM control group and blank control group. The specific groups are shown in Table 7.

[0145] Table 7 Mouse grouping, immunization and challenge

[0146]

[0147] 3.1.3 Observation of mice after inoculation with virulent virus and detection of in vivo residues

[0148] The mental state, coat, exercise, diet and drinking water of all mice were observed daily. The results showed that there were no abnormalities in the mental state, coat, exercise, diet and drinking water of the mice in the five groups.

[0149] On the 7th, 15th, 30th and 45th days after immunization, three mice were taken from each group, and the cervical vertebrae were broken and killed. Tissues, organs and lymph nodes were taken for in vivo residual determination. The A19 vaccine group was tested for Brucella residues. The samples were bacteriologically separated. As long as one Brucella was isolated from each tissue organ, it was judged as positive. The adenovirus vaccine group and the empty adenovirus group were tested for adenovirus active substance residues. As long as the adenovirus gene was detected in any tissue organ, it was judged as positive.

[0150] The results showed that the residual rate of Brucella A19 vaccine in animals was 100% within one month after vaccination, and it did not decrease until the 45th day. This also proved that the attenuated vaccine strain had the defect of residual virulence, and also increased the risk of virulence reversion and virulence recombination. In the adenovirus group, no adenovirus bioactive substances were detected in the four tests, whether it was the vaccine group or the empty vector virus group, and there was no safety problem at all. See Table 8 for specific data.

[0151] Table 8 Residue determination in mice after immunization

[0152]

[0153] 3.1.4 Preparation of Brucella virulent challenge strain M28

[0154] After the Brucella M28 stored at -80℃ is thawed in an ice water bath, it is streaked onto a TSA plate and cultured in a 37℃ incubator for 96h. A single colony is picked and placed in 100μL DMEM. After rinsing three times, 100μL of bacterial suspension is spread on a TSA plate and cultured for 96h. The cultured bacterial moss on the plate is washed with 2ml DMEM, collected into a 15mL centrifuge tube, mixed by pipetting, and then dispensed into 1.5ml centrifuge tubes. Centrifuge at 8000g for 5min, wash three times with DMEM, and add TSB containing 10% glycerol. The bacterial solution is diluted with DMEM in a 1:10 ratio to 1:10. -8 , absorb 10 -7 and 10 -8 The diluted solution was applied to Brucella TSA plates and cultured in an incubator. The colonies were counted after 96 hours and the concentration of 1×10 5 CFU / ml of M28 strong strain bacterial solution.

[0155] 3.1.5 Attack

[0156] Take 0.1 ml of the prepared highly virulent M28 bacterial solution and perform intraperitoneal injection to challenge the virus 28 days after immunization.

[0157] 3.1.6 Isolation and counting of M28 challenge bacteria

[0158] Mice were killed by cervical vertebrae dissection on days 7, 14, and 21 after challenge, and spleens were aseptically isolated, weighed, added with 0.9 ml PBS, and ground (Retsch MM400 tissue grinder, 25 Hz, 10 min). The suspension was appropriately diluted and plated, and colonies were counted after incubation at 37°C for 72-96 h.

[0159] 3.1.7 Calculation

[0160] Spleen weight: It is an important indicator reflecting the inflammatory response of mice. The net weight of the spleen was aseptically weighed after the mice were killed, and the weights of the spleens were compared among the groups.

[0161] Spleen bacterial count: It is the most important indicator for measuring vaccine protection. After grinding the spleen of each mouse, it is appropriately diluted. The colonies grown after 72-96 hours multiplied by the dilution factor is the spleen bacterial count of the mouse. The log of the bacterial count of each mouse is taken and the average value is calculated.

[0162] Protection unit: average value of Log(bacterial count in spleen of DMEM group) - average value of Log(bacterial count in spleen of immune group). According to OIE standard: average value of Log(bacterial count in DMEM control group) should be above 4.5, and protection unit of immune group should be above 2.5 to be qualified.

[0163] 3.1.5 Protection results of mice after challenge

[0164] The results of bacterial separation on day 7 showed that the mean values ​​of the A19 vaccine group and the adenovirus vaccine group were 3.59 and 3.6 respectively, and the mean values ​​of the empty adenovirus and DMEM were 5.57 and 6.12. The protection index of the A19 vaccine group and the adenovirus vaccine group were 2.53 and 2.52 respectively. Detailed spleen weight and bacterial separation results are shown in Table 9.

[0165] Table 9 Bacteria and spleen weight results 7 days after challenge

[0166]

[0167] Notes A: average spleen weight of each group of mice; B: average Log (spleen mycobacterial count) of each group of mice; C: protection unit = average Log (spleen mycobacterial count of DMEM group) - average Log (spleen mycobacterial count of immunized group).

[0168] Bacterial results on day 14: The mean values ​​of the A19 vaccine group and the adenovirus vaccine group after taking the log were 2.95 and 2.97, respectively, and the mean values ​​of the empty adenovirus and DMEM were 5.15 and 5.59. The protection index of the A19 vaccine group and the adenovirus vaccine group were 2.64 and 2.62, respectively. Detailed spleen weight and bacterial results are shown in Table 10.

[0169] Table 10 Bacteria and spleen weight results 14 days after challenge

[0170]

[0171]

[0172] Notes A: Average spleen weight of each group of mice B: Average Log (spleen mycobacterial count) of each group of mice; C: Protection unit = Average Log (spleen mycobacterial count of DMEM group) - Average Log (spleen mycobacterial count of immune group)

[0173] Bacterial results on day 21: The mean values ​​of the A19 vaccine group and the adenovirus vaccine group were 3.18 and 3.16 after taking the log, and the mean values ​​of the empty adenovirus and DMEM were 5.61 and 5.88. The protection index of the A19 vaccine group and the adenovirus vaccine group were 2.71 and 2.72, respectively. Detailed spleen weight and bacterial results are shown in Table 11.

[0174] Table 11 Bacteria and spleen weight results 21 days after challenge

[0175]

[0176] Notes A: Average spleen weight of each group of mice; B: Average Log (spleen mycobacterial count) of each group of mice; C: Protection unit = Average Log (spleen mycobacterial count of DMEM group) - Average Log (spleen mycobacterial count of immune group)

[0177] The results showed that there was no difference in the protective efficacy between the Brucella recombinant adenovirus vaccine and the commercialized A19 vaccine, and the protection units of the immunization groups were both greater than 2.5, which met the protection standards of the OIE Brucella vaccine.

Claims

1. A recombinant adenovirus based on 33 protective genes of Brucella, pADV-mCMV-ABCD, Features: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on August 27, 2024, with the deposit number CGMCCNO.46170.

2. A recombinant adenovirus based on 33 protective genes of Brucella according to claim 1, Features: The recombinant adenovirus comprises 33 target genes of Brucella that have been screened and optimized through the whole genome and a linearized adenovirus expression vector; the construction process of the recombinant adenovirus is as follows: A. Screening of protective genes: 33 genes with the most potential antigens were screened from 89 whole genomes of 54 Brucella melitensis, 17 Brucella abortus and 18 Brucella suis; most of the antigens are related to bacterial adhesion, invasion, escape and adaptation to the internal environment of macrophages; B. Construction of recombinant adenovirus a. Genes, expression systems and cells: The 33 main protective genes of Brucella that were screened were divided into four groups of target genes, A sequence, B sequence, C sequence and D sequence, after expression verification. They were serially connected and cloned into the pMd18-T vector after adding corresponding restriction sites. The human type 5 replication-deficient adenovirus expression system and human embryonic kidney HEK 293T cells were optimized and preserved; b. Target gene digestion: Extract the pMd18-T vector containing the target gene, and digest the plasmid containing the target gene with restriction endonucleases; Perform agarose gel electrophoresis on the digested products to detect the digestion effect, and cut the target gene band from the gel after agarose gel electrophoresis, perform gel recovery, and obtain the target gene fragment; c. Linearized adenovirus expression vector: Use restriction endonucleases to digest the human type 5 replication-deficient adenovirus expression vector, perform agarose gel electrophoresis on the digestion product to detect the digestion effect, and cut the target vector band from the gel after agarose gel electrophoresis, perform gel recovery, and obtain the linearized adenovirus expression vector pADV-mCMV; d. Constructing the target gene into a linearized adenovirus expression vector: Add the target gene fragment and the linearized adenovirus at a molar ratio of 2:1 into a centrifuge tube for a recombination ligation reaction to obtain a recombinant ligation product; e. Transformation and identification: The recombinant ligation products were transformed into E. coli DH5α competent cells, PCR was used to identify positive transformants, and sequencing was used to verify that they were correct, thereby constructing the recombinant adenovirus expression vectors pADV-mCMV-A sequence, pADV-mCMV-B sequence, pADV-mCMV-C sequence, and pADV-mCMV-D sequence; f. Recombinant adenovirus packaging and protein expression detection: The recombinant adenovirus expression vector was cut with Pme I to obtain a linearized recombinant adenovirus expression vector, which was transfected into HEK293T cells to package recombinant adenovirus pADV-mCMV-A, recombinant adenovirus pADV-mCMV-B, recombinant adenovirus pADV-mCMV-C and recombinant adenovirus pADV-mCMV-D.

3. A recombinant adenovirus based on 33 protective genes of Brucella according to claim 2, Features: The nucleotide sequence of the Asequence is shown in SEQ ID NO.1, the nucleotide sequence of the B sequence is shown in SEQ ID NO.2, the nucleotide sequence of the C sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the D sequence is shown in SEQ ID NO.

4.

4. A recombinant adenovirus based on 33 protective genes of Brucella according to any one of claims 1 to 3, Features: The immune effect verification process of the recombinant adenovirus is as follows: A. Animal grouping: 150 Balb / C mice were divided into 5 groups, namely commercial vaccine group, adenovirus vaccine group, empty adenovirus group, DMEM control group and blank control group, with 30 mice in each group; B. Immunization: The commercial vaccine group was vaccinated for 10 days according to the instructions. 5 CFU / dose, 0.1ml; adenovirus vaccine group and empty adenovirus inoculation group, 10 6 PFU / mouse, 0.1ml; DMEM control was inoculated with 0.1ml DMEM; immunization was repeated once 21 days after the first immunization, i.e. the second immunization; C. Virulent challenge: 28 days after the second immunization, mice were inoculated with standard Brucella M28 virulent virus at a dose of 1×10 4 CFU / piece; D. Sample collection and evaluation: The clinical manifestations of mice were observed every day after vaccination with the strong virus, and the vaccine residues were detected on the 7th, 15th, 30th and 45th days respectively. The mice were killed on the 7th, 14th and 21st days after the virus attack, the spleens were aseptically separated and weighed, and the colony counts were performed to calculate the protection units. The results showed that the Brucella recombinant adenovirus vaccine had no residues in the animals, and the protection effect met the protection standards of the OIE Brucella vaccine, and passed the immune effect verification.

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