阅读说明：本技术 一种用于鉴别牛种布鲁氏菌与其它种布鲁氏菌的试剂盒 (Kit for identifying Brucella melitensis and Brucella melitensis of other species ) 是由 王文龙 王姝懿 陆静 毛晓伟 呼和巴特尔 刘春霞 于 2021-07-28 设计创作，主要内容包括：本发明提供一种用于鉴别牛种布鲁氏菌与其它种布鲁氏菌的试剂盒,该试剂盒包被有SEQ ID NO.2所示蛋白和布鲁氏菌BP26蛋白。本发明发现SEQ ID NO.2所述蛋白或其编码基因可用于区分牛种布鲁氏菌与其它种布鲁氏菌。本发明提供鉴别牛种布鲁氏菌与其它种布鲁氏菌感染血清的iELISA检测试剂,该试剂含有前述两种蛋白,或含有前述两种蛋白的N端或C端融合有His标签的重组蛋白。本发明提供的iELISA检测试剂能够快速鉴别家畜血清中牛种疫苗免疫或野毒与其它种(羊,猪,犬等)疫苗免疫或野毒的抗体,解决畜群中布鲁氏菌种型鉴定难的问题,为家畜布鲁氏菌病的病原鉴定、疫苗选择和畜群净化提供参考。(The invention provides a kit for identifying Brucella and other Brucella, which is coated with a protein shown in SEQ ID NO.2 and Brucella BP26 protein. The invention discovers that the protein described by SEQ ID NO.2 or the coding gene thereof can be used for distinguishing the Brucella melitensis from other Brucella melitensis. The invention provides an iELISA detection reagent for identifying bovine brucella and brucella infected serum of other species, which contains the two proteins, or recombinant protein containing His labels fused at the N end or the C end of the two proteins. The iELISA detection reagent provided by the invention can rapidly identify the antibodies of the bovine vaccine immunity or the wild virus and the vaccine immunity or the wild virus of other species (sheep, pigs, dogs and the like) in the livestock serum, solves the problem of difficult identification of the brucella species in the herd, and provides references for pathogen identification of brucella disease of livestock, vaccine selection and herd purification.)

1. A protein, comprising:

1) an amino acid sequence shown as SEQ ID NO. 2;

2) protein which is derived from the protein 1) and has the same activity and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

2. A gene encoding the protein of claim 1, having:

1) a nucleotide sequence shown as SEQ ID NO. 1; or

2) The nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides; or

3) Nucleotide sequences which hybridize under stringent conditions with the DNA sequences defined in 1).

3. A biological material comprising the gene of claim 2, wherein the biological material is a vector, a host cell or an expression cassette.

4. Use of the protein of claim 1 or the gene of claim 2 as a detection marker for identifying Brucella bovis from Brucella other species.

5. The use of the protein of claim 1 and brucella BP26 protein in the preparation of a kit or detection reagent for identifying brucella bovis and other brucella infections; the encoding gene of the Brucella BP26 protein is shown as SEQ ID NO. 3.

6. The application of a primer pair combination in identifying Brucella bovis and Brucella other species, wherein the primer pair combination comprises a primer pair for detecting the protein coding gene of claim 1 and a primer pair for detecting the Brucella BP26 protein coding gene.

7. The use of claim 6, wherein if the PCR result shows that both the antigen proteins have amplification bands of 879bp and 753bp, respectively, the sample to be tested contains Brucella non-bovine.

8. A kit, which comprises the protein of claim 1 and brucella BP26 protein, or comprises the protein of claim 1 and recombinant protein fused with His tag at the N-terminal or C-terminal of brucella BP26 protein.

9. A kit for identifying Brucella melitensis and other Brucella melitensis, which is an iELISA detection kit, wherein the coating concentration of the protein in claim 1 is 20 μ g/mL, the coating concentration of Brucella BP26 protein is 5.53 μ g/mL, the optimal dilution of the protein enzyme-labeled secondary antibody in claim 1 is 1: 5000, the optimal dilution of BP26 enzyme-labeled secondary antibody is 1: 7500;

the protein iELISA decision point of claim 1 was 0.390 and the BP26 protein iELISA decision point was 0.589.

10. The kit according to claim 8 or 9, wherein the serum to be tested is a serum of a non-bovine brucella-infected livestock, if both the antigenic proteins of the protein according to claim 1 and the BP26 protein are detected as positive by an elisa method; if the protein of claim 1 is detected as negative and the BP26 protein is detected as positive, the serum to be detected is the serum of domestic animals infected by Brucella. And if the two antigen proteins are negative, the serum to be detected is the livestock serum which is not infected with the Brucella.

Technical Field

The invention relates to the field of genetic engineering and immunology, in particular to a kit for identifying Brucella melitensis and other Brucella melitensis.

Background

Brucellosis (brucellosis) is a worldwide widespread zoonosis caused by brucella spp, and OIE is classified as a b-infectious disease. The brucella is divided into six classical species, namely, a cattle species (B.abortus), a sheep species (B.melitensis), a pig species (B.suis), a dog species (B.cains), a sheep species (B.ovis) and a forest rat species brucella (B.neotomae), and recently, the whale species brucella (B.ceti) and the fin species brucella (B.pinnipediis) are successively discovered, and the brucella is also found in animals such as human, rodent and bat, wherein the sheep species, the cattle species and the pig species are main epidemic pathogenic species of livestock and human. The brucellosis is infected by contacting with animal, non-sterilized meat product, and dairy product, or by invading into body via tissues and organs such as respiratory tract, digestive tract, and skin mucosa.

Once inside the host, brucella bacteria invade the blood and lymph vessels, multiply and are phagocytosed by macrophages. Common symptoms of livestock infected with brucellosis include female livestock abortion, testicular enlargement of male livestock, and liver and spleen enlargement of severe livestock. The livestock infected with the Brucella is mainly eliminated, so that the healthy development of the animal husbandry is seriously influenced, and the health of human beings is threatened. The pathogenicity of different types of brucella is different, the types of vaccines for immunization and prevention of the brucella are also different, and identification of the brucella in production practice has important significance for evaluating disease damage and selecting the vaccines; and the conventional common brucellosis diagnosis method cannot distinguish vaccine antibodies in immune animal serum and natural virulent strain infection antibodies, so that the development of the purification work of the livestock is seriously interfered, and the application of the serological differential diagnosis method is combined with the use of different types of brucella vaccines, so that the purification of the livestock is facilitated.

Disclosure of Invention

The invention aims to provide a kit for identifying Brucella melitensis and other Brucella melitensis.

Another objective of the invention is to provide an iELISA detection kit for identifying Brucella bovine infection serum and Brucella other infection serum.

It is still another object of the present invention to provide a protein characteristic for identifying Brucella species from other Brucella species.

In order to achieve the purpose of the invention, the invention aims at a brucella mainstream vaccine A19 vaccine commonly used in the market, and performs whole genome sequencing on the A19 vaccine, and performs comparative genomic analysis and bioinformatics analysis on the whole genomes of various brucella published on NCBI. Compared with a cattle/sheep/pig vaccine or a natural virulent strain of the Brucella, the deletion gene NC _017250 of the Brucella is found by taking A19 as a reference, and after the gene is recombined and expressed, the antigen protein with immunogenicity is screened out, and the iELISA identification method for infection of the Brucella and other Brucella is established.

The laboratory carries out whole genome sequencing on the A19 vaccine and submits the whole genome sequencing to an NCBI database for the first time, the whole genome sequence is compared and analyzed with the whole genome sequences of a plurality of Brucella vaccines and wild strains published on NCBI, the screened specific gene NC-017250 is compared with genome sequences of more than 240 Brucella of different types through blast comparison, the universal property is completely achieved, and PCR verification is carried out on the gene on a plurality of Brucella S2, A19, S19, rev.1 and M28 strains, and the specific deleted gene of the Brucella is determined to be a specific deleted gene of cattle.

The invention firstly provides a protein, namely an NC-017250 antigen protein deleted from Brucella, the amino acid sequence of which is shown as SEQ ID NO.2, or the amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence. The NC-017250 antigen protein is encoded by an S2 vaccine strain gene NC-017250 (SEQ ID NO. 1).

Since the NC _017250 gene is deleted from A19 (bovine species), the gene needs to be obtained by PCR amplification from other species of Brucella, S2 is Brucella in swine and contains the complete gene, and other Brucella in non-bovine species also have the complete gene, and the invention only serves as an exemplary selection of the S2 strain for relevant research.

In a first aspect, the present invention provides a protein encoded by NC _017250 gene, comprising:

1) an amino acid sequence shown as SEQ ID NO. 2;

2) protein or polypeptide which is derived from the protein or polypeptide in 1) and has equivalent activity and is obtained by substituting, deleting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

The NC _017250 gene provided by the present invention has:

1) a nucleotide sequence shown as SEQ ID NO. 1; or

2) The nucleotide sequence shown in SEQ ID NO.1 is substituted, deleted and/or added with one or more nucleotides; or

3) Nucleotide sequences which hybridize under stringent conditions with the DNA sequences defined in 1).

The NC-017250 gene or the protein coded by the gene can be used as an identification marker for distinguishing the Brucella bovis from other Brucella.

The biological material containing the NC-017250 gene belongs to the protection scope of the invention, and the biological material is a vector, a host cell or an expression cassette.

In a second aspect, the invention provides an application of the protein or NC _017250 gene as a detection marker in identifying Brucella bovis and Brucella other species.

The invention also provides application of the combination of the protein and the Brucella BP26 protein in preparing a kit or a detection reagent for identifying the infection of the Brucella of the cattle and the Brucella of other species; the encoding gene of the Brucella BP26 protein is shown as SEQ ID NO. 3.

In a third aspect, the invention provides application of a primer pair combination in identifying Brucella bovis and other Brucella, wherein the primer pair combination comprises a primer pair for detecting NC-017250 gene and a primer pair for detecting Brucella BP26 protein encoding gene.

Further, when the primers are used for PCR detection, if the PCR detection result shows that the coding genes of the two antigen proteins (NC _017250 gene coding protein and Brucella BP26 protein) have amplification bands, and the amplification bands are 879BP and 753BP respectively, the sample to be detected contains non-bovine Brucella.

In a fourth aspect, the invention provides a kit, which contains the protein and brucella BP26 protein, or contains recombinant protein in which the protein and brucella BP26 protein are fused with His tags at the N end or the C end.

The invention relates to an animal infection or vaccine immune antibody detection kit for identifying bovine brucella and other brucella, which is an iELISA detection kit, wherein the coating concentration of a protein shown by SEQ ID NO.2 is 20 mug/mL, the coating concentration of brucella BP26 protein is 5.53 mug/mL, and the optimal dilution of a protein enzyme-labeled secondary antibody is 1: 5000, and the optimal dilution of the BP26 enzyme-labeled secondary antibody is 1: 7500.

The protein iELISA decision point shown in SEQ ID NO.2 is 0.390, and the BP26 protein iELISA decision point is 0.589.

The iELISA detection kit disclosed by the invention is used for detecting the serum to be detected by using an iELISA method, and if the detection of two antigen proteins, namely the protein shown in SEQ ID NO.2 and the BP26 protein, is positive, the serum to be detected is the serum of the livestock infected by non-bovine brucella; if the protein shown in SEQ ID NO.2 is detected to be negative and the BP26 protein is detected to be positive, the serum to be detected is the serum of the cattle Brucella infected livestock. And if the two antigen proteins are negative, the serum to be detected is the livestock serum which is not infected with the Brucella.

Experiments show that the iELISA identification method established by the invention and the iELISA kit established based on the detection method have better repeatability and higher specificity and sensitivity.

The iELISA detection reagent provided by the invention can identify the Brucella melitensis and the cattle vaccine commonly used in the market, and can identify the wild virus of cattle or the cattle vaccine. Therefore, the antibody for infecting the bovine Brucella and other Brucella in the livestock serum can be identified in a short time.

Drawings

FIG. 1 shows the PCR detection result of the Brucella melitensis deletion gene NC-017250 in example 1 of the present invention; wherein M is DNA Marker DL 2000; 1 is negative control; 2 is a19 vaccine; 3 is S19 vaccine; 4 is S2 vaccine; 5 is M28 vaccine; and 6 is Rev.1 vaccine.

FIG. 2 is a PCR amplification electrophoretogram of NC-017250 and BP26 genes in example 1 of the present invention; wherein M is DNA Marker DL 2000; 1 is NC-017250 gene; 2 is BP26 gene; and 3 is blank control.

FIG. 3 shows the results of the double restriction enzyme identification of the recombinant cloning plasmid pMD-NC-017250 in example 1; wherein M is DNA Marker DL 2000; 1-3 are pMD-NC-017250 plasmids.

FIG. 4 shows the results of the double restriction enzyme identification of the recombinant clone plasmid pMD-BP26 in example 1 of the present invention; wherein M is DNA Marker DL 2000; 1-3 are pMD-BP26 plasmids.

FIG. 5 shows the results of the double restriction enzyme identification of the recombinant expression plasmid pETNC _017250 in example 1; wherein M1 is DNA Marker DL 2000; m2 is DNA Marker DL 10000; 1-3 are pETNC _017250 plasmids.

FIG. 6 shows the results of the double restriction enzyme identification of the recombinant expression plasmid pETBP26 in example 1; wherein M1 is DNA Marker DL 2000; m2 is DNA Marker DL 15000; 1-3 are pETBP26 plasmids.

FIG. 7 shows the result of SDS-PAGE electrophoresis of the expression induced by the recombinant expression bacterium BL21(pETNC _017250) in example 1 of the present invention; wherein M is protein marker; 1-3 are before BL21(pETNC _017250) induction; 4-5 BL21(pETNC _017250) induced expression.

FIG. 8 shows the result of SDS-PAGE electrophoresis of the expression form of the recombinant protein in example 1 of the present invention; wherein M is protein marker; 1 is BL21(pETNC _017250) whole bacterium; 2 is BL21(pETNC _017250) thallus lysate supernatant; BL21(pETNC _017250) cell lysate precipitate was named 3.

FIG. 9 shows SDS-PAGE results of the recombinant protein purified in example 1; wherein M is protein marker; 1 is rNC _017250 purified protein diluted 5 times; 2 is rNC _017250 purified protein diluted 10 times.

FIG. 10 shows the Western-blotting detection results of the recombinant protein His tag in example 1 of the present invention; wherein M is protein marker; 1 is rNC _ 017250.

FIG. 11 shows the results of the reaction of the purified recombinant protein rNC _017250 with bovine and non-bovine immune sera in example 1 of the present invention; wherein M is protein marker; 1 is rNC _017250 reacted with A19 serum; 2 rNC _017250 reacted with S2 serum.

FIG. 12 shows the results of the reaction between the purified protein rBP26 and S2 in the immune serum of example 1; wherein M is protein marker; 1 is BL 21; 2 is rBP26 natural infection serum reaction; rBP26 at 3 reacted with S2 immune sera.

FIG. 13 shows the sensitivity and specificity analysis of NC-017250 antigen iELISA in example 2 of the present invention.

FIG. 14 shows the sensitivity and specificity analysis of BP26 antigen iELISA in example 2 of the present invention.

FIG. 15 is a ROC graph of NC-017250 antigen iELISA test serum in example 2 of the present invention.

FIG. 16 is a ROC graph of BP26 antigen iELISA assay serum in example 2 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the molecular cloning protocol, or the conditions suggested by the manufacturer's instructions.

Example 1 cloning and prokaryotic expression of the deletion Gene NC-017250 of Brucella species and the BP26 Gene of Brucella

Through A19 vaccine strain whole genome sequencing, the predicted coding gene is compared and analyzed with published brucella genome data to find out the cattle deletion gene compared with other non-cattle brucella, the deletion gene NC-017250 (SEQ ID NO.1) is amplified from S2 genome by using PCR method, meanwhile, the brucella BP26 gene (SEQ ID NO.3) is used as a reference gene, after clone sequencing, the deletion gene is constructed into a prokaryotic expression vector pET30, and is transformed into E.coli BL21(DE3) for induced expression, and the immunogenicity of two recombinant proteins NC-017250 and BP26 is verified by using western-blotting method.

1. The experimental method comprises the following steps:

1.1 primer design

Primers were designed from NC-017250 and BP26 gene sequences using Oligo6.0, EcoRI and XhoI cleavage sites were inserted into the upstream and downstream primers, respectively, and the synthesis of the primers was performed by Huada Gene Co., Ltd, and the primer sequences are shown in Table 1.

TABLE 1 NC-017250 and BP26 Gene primer sequences

1.2 genomic DNA extraction

And extracting brucella genome DNA according to the steps of the Axygen nucleic acid extraction kit.

1.3 NC-017250 Gene PCR amplification

BLAST comparison analysis of the A19 vaccine strain NC-017250 gene with other Brucella species published by NCBI, PCR amplification and specificity verification. The PCR amplification reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5min, 35 cycles; further extension was carried out at 72 ℃ for 10 min. And after the PCR amplification is finished, carrying out agarose gel electrophoresis detection.

1.4 amplification of the Gene of interest

And carrying out PCR amplification on the target gene and carrying out gel agarose electrophoresis detection. Specific reaction conditions are described as 1.3.

1.5 PCR product recovery

The NC _017250 and BP26 gene PCR products were recovered following the Axygen PCR product recovery kit protocol procedures.

1.6 ligation of PCR-recovered product with pMD19-TSimple vector

Add 1. mu.L pMD19-T Simple Vector, 4. mu.L PCR recovery product and 5. mu.L Solution I into 200. mu.L centrifuge tube, centrifuge and mix well, connect overnight at 16 ℃.

1.7 transformation of recombinant plasmids

The ligation products were transformed into Trans1-T1 competent cells, plated on solid LB medium containing antibiotics (Amp), and cultured overnight at 37 ℃ according to the procedures described in the instructions for use in the Biotechnology Ltd, Trans1-T1, Beijing Omegal gold (TransGen Biotech).

1.8 PCR identification of recombinant clone bacteria liquid

And (4) selecting positive colonies, inoculating the positive colonies into a liquid LB culture medium containing antibiotics (Amp) to culture overnight, and performing PCR amplification identification on the culture.

1.9 double restriction enzyme identification of recombinant cloned plasmid

Recombinant plasmids are extracted according to the specification of the Axygen plasmid extraction kit, and the recombinant plasmids are subjected to double enzyme digestion electrophoresis detection by using restriction enzymes XhoI and EcoRI.

1.10 sequencing of recombinant clonal bacteria

The recombinant clone bacteria after PCR identification and plasmid double enzyme digestion identification are sent to Beijing Hua DageneCo company for bidirectional sequencing.

1.11 recovery of the Gene of interest and expression vector pET30a

The clone strain with correct sequencing and the bacterium containing pET30a plasmid are inoculated into LB culture medium and cultured overnight, the plasmid is extracted, and the target gene fragment and pET30a fragment are recovered.

1.12 ligation of the Gene of interest to expression vector pET30a

mu.L of the recovered target fragment, 2. mu.L of the pET30a expression vector fragment, 1. mu. L T4 of DNA ligase, and 1. mu.L buffer were added to a 200. mu.L centrifuge tube, and ligated at 16 ℃ overnight.

1.13 transformation of recombinant plasmids into competent cells

The ligation products were transformed into BL21(DE3) competent cells, plated on antibiotic (Kan) -containing solid LB medium at 37 ℃ for overnight culture according to the procedures described in the instructions for use in Beijing Whole gold (TransGen Biotech) Biotechnology, Inc. BL21(DE3) competent cells.

1.14 PCR identification of recombinant expression bacteria liquid

The specific steps are shown in 1.8.

1.15 double restriction enzyme identification of recombinant expression plasmid

The specific steps are shown in 1.9.

1.16 inducible expression of recombinant expression bacteria

1.16.1 conditions for inducible expression

The constructed BL21(pETNC _017250) recombinant expression bacteria are coated on an LB-Kan solid culture medium to be cultured for 12-16 h, a single colony is inoculated in 6mL of liquid LB-Kan culture medium, and the culture is carried out overnight at 37 ℃. Then inoculating into 20mL LB-Kan culture medium according to the ratio of 1: 100, and culturing with shaking (150r/min) to OD₆₀₀0.6 to 1.0. According to the optimization experiment of the induction expression condition performed in the earlier stage of the laboratory, the optimal induction time of selecting the recombinant expression bacterium BL21(pETNC _017250) is about 4h, when OD is less than OD₆₀₀When the final induction concentration of IPTG is 0.6-1.0, the optimum culture temperature is 37 ℃, SDS-PAGE electrophoresis detection is carried out, and OD is measured by 1mL₆₀₀。

1.16.2 detection of expression form of recombinant protein

According to the optimized optimal induction condition, the bacterial liquid and the culture medium are expanded and cultured according to the proportion of 1: 100, 300mL of recombinant bacteria are induced, 1,2000 Xg is centrifuged for 10min, PBS is used for washing and precipitating, after heavy suspension precipitation, lysozyme is added at room temperature for reaction for 1h, after repeated freeze thawing (-20 ℃) for several times, the bacterial liquid and the culture medium are placed in ultrasonic crushing treatment for 10min, 1,2000 Xg is centrifuged for 10min, precipitates and supernate are respectively collected, and the solubility of the recombinant protein is detected by 12% SDS-PAGE electrophoresis.

1.17 solubilization and purification of inclusion bodies of recombinant proteins

According to the optimized optimal conditions, 500mL of recombinant expression bacteria are induced and centrifuged at 1,2000 Xg for 10 min. Washing the precipitate with PBS, resuspending the thallus precipitate, adding lysozyme at room temperature for reaction for 1h, repeatedly freezing and thawing at (-20 ℃) for several times, performing ultrasonic disruption treatment for 10min, centrifuging for 10min at 1,2000 Xg, discarding the supernatant, dissolving the precipitate with 8mol/L urea, centrifuging for 10min at 1,2000 Xg, collecting the supernatant, filtering with a 0.45 mu m bacterial filter, and purifying the protein with a Ni + column (Shanghai Biotechnology).

1.18 recombinant protein western blotting assay

After SDS-PAGE electrophoresis of the recombinant protein, taking out the protein gel, placing the filter paper, the PVDF membrane, the protein gel and the filter paper on a clamp in sequence, and rotating the membrane at 100V for 60 min. After the membrane transfer is finished, the membrane is rinsed for 4min by TBST, repeated for 3 times, and sealed for 1h at room temperature. And repeatedly rinsing the PVDF membrane by using TBST for 3-4 times. S2 immune bovine serum (primary antibody) was diluted 1: 100, and the PVDF membrane was incubated overnight at 4 ℃ in the primary antibody dilution. And (3) rinsing the PVDF membrane for 3-4 times, diluting the rabbit anti-bovine IgG labeled by horseradish peroxidase according to a ratio of 1:7500, and incubating the PVDF membrane in a second antibody diluent for 1 h. PVDF membrane was rinsed 3 times with TBST, developed with ECL and analyzed imagewise.

2. Results of the experiment

2.1 NC-017250 Gene PCR amplification

The NC _017250 gene was BLAST aligned with the gene sequences in the NCBI database. The results showed that the NC _017250 gene was present in brucella melitensis of ovine, porcine, ovine, canine, whale and fin species, but not in brucella melitensis of bovine species (see table 2).

TABLE 2 NC-017250 alignment results

The NC-017250 gene was PCR amplified and detected by 1% agarose gel electrophoresis, and the results are shown in FIG. 1. The results show that about 879bp bands are amplified in the brucella melitensis nucleic acid of the sheep species and the brucella melitensis nucleic acid of the pig species, and bands with expected sizes are not amplified in the brucella melitensis nucleic acid of the cattle species, which indicates that the NC-017250 gene is a cattle species-specific deletion gene.

2.2 amplification of the Gene of interest

NC-017250 and BP26 were PCR amplified and detected by electrophoresis on a 1% agarose gel, the results are shown in FIG. 2. The results show that: bands of about 879BP and 753BP appeared in NC-017250 and BP26, respectively, and were consistent with the expected band sizes.

2.3 PCR identification of recombinant clonal bacteria

Connecting the target fragment with a pMD19-T carrier, transferring into a competent cell, culturing on a solid culture medium for 12-16 h, selecting 3 colonies, inoculating into an LB liquid culture medium, culturing for 12-16 h, performing PCR amplification by taking a bacterial liquid as a template, and performing 1% agarose gel electrophoresis detection. The NC-017250 and BP26 gene clone bacteria respectively amplify bands of about 879BP and 753BP, and target bands with expected sizes are obtained.

2.4 double restriction enzyme identification of recombinant cloned plasmids

The positive strain is identified by PCR of the bacterial liquid to extract plasmids, restriction enzymes (EcoRI and XhoI) are used for double enzyme digestion, and the electrophoresis detection of the enzyme digestion products is shown in figure 3 and figure 4. As a result, two bands, NC-017250 (0.8kb, 2.7kb) and BP26(0.7kb, 2.7kb) were observed, indicating that the two target gene sequences were successfully inserted into the pMD19-T vector sequence.

2.5 PCR identification of recombinant expression bacteria

And (3) inserting the target fragment into a pET30a vector, then transforming competent bacteria cells, coating the competent bacteria cells on a solid culture medium for culturing for 12-16 h, selecting 3 single colonies, shaking the bacteria for culturing for 12-16 h, carrying out PCR amplification by taking a bacterial solution as a template, and carrying out 1% agarose gel electrophoresis detection. NC-017250 and BP26 amplified bands of about 879BP and 753BP, respectively, which were consistent with the expected band sizes.

2.6 double restriction enzyme identification of recombinant expression plasmid

The plasmid extracted from the strain which is positive by PCR identification (according to the instructions of the Axygen plasmid extraction kit) is subjected to double enzyme digestion by restriction enzymes (EcoRI and XhoI), and the electrophoresis detection of the digestion product is shown in FIG. 5 and FIG. 6. As a result, two bands, NC-017250 (0.8kb, 5.4kb) and BP26(0.7kb, 5.4kb) were observed, and it was found that the two target fragments were successfully inserted into the expression vector pET30 a.

2.7 sequencing results and analysis

And (3) performing bidirectional sequencing on the positive clone bacteria and recombinant expression bacteria through PCR identification and double enzyme digestion identification, and selecting 3 independent positive strains for each gene to perform bidirectional sequencing in order to ensure the accuracy of a sequencing result. The aligned homology of the NC-017250 sequence obtained by bidirectional sequencing and the NC-017250 gene nucleotide sequence obtained by whole genome sequencing is 100%. The BP26 sequence obtained by bidirectional sequencing has 100% homology with the sequence published at NCBI. Thus, NC-017250 and BP26 are successfully cloned into a vector pMD19-T, and a recombinant expression vector is successfully constructed.

2.8 inducible expression of recombinant expression bacteria

2.8.1 Induction Condition optimization of recombinant expression bacteria

According to the experiment for optimizing the induced expression conditions in the earlier stage of the laboratory, the optimal induction time of selecting the recombinant expression bacterium BL21(pETNC _017250) is about 4h, the final induction concentration of IPTG is 0.5mmol/L when OD600 is 0.6-1.0, and the optimal culture temperature is 37 ℃. The results showed that the recombinant expression bacteria were expressed in large amounts under the above conditions, and were consistent with the expected product as detected by 15% SDS-PAGE (FIG. 7).

2.8.2 detection of recombinant protein expression Format under optimal Induction conditions

After the optimal expression condition of the recombinant expression bacteria is determined, inducing a large amount of recombinant bacteria according to the optimal condition, and collecting thalli at low temperature of 1,2000 Xg. Washing with PBS for several times, adding lysozyme, standing for 1h, repeatedly freezing and thawing at-20 deg.C, collecting the centrifugal precipitate and supernatant, and performing electrophoresis detection. The results show that the fusion protein is mainly present in the pellet, indicating that the recombinant protein is mainly expressed in the form of inclusion bodies, see FIG. 8.

2.9 recombinant protein inclusion body solubilization and protein purification

Culturing recombinant bacteria according to the optimal expression condition, centrifuging at high speed, discarding supernatant, collecting thallus, washing and suspending thallus precipitate, adding lysozyme for 1h at room temperature, repeatedly freezing and thawing for several times (-20 ℃), performing ultrasonic crushing treatment for 10min, centrifuging at 1,2000 Xg for 10min, discarding supernatant, dissolving precipitate with 8mmol/L urea, centrifuging at high speed and collecting supernatant. After filtration through a filter having a pore size of 0.45 μm, the recombinant protein was purified by a Ni + column (Shanghai Biotech) to obtain a purified recombinant protein (FIG. 9).

2.10 Western-blotting detection of recombinant protein

The recombinant protein rNC _017250 detected by WB reacted with bovine serum immunized with non-bovine S2 vaccine, and a specific band appeared at 40 kDa. The result shows that the recombinant protein can be specifically combined with the S2 antibody diluted by 1: 100 times, and the immunogenicity is better. Recombinant protein rNC _017250 did not react with A19 immune bovine serum diluted 1: 100 fold. The NC-017250 gene is not existed in the A19 vaccine strain, and the recombinant protein rNC-017250 has better immunogenicity. The BP26 recombinant protein can perform specific reaction with antibodies of bovine serum and non-bovine serum, and has better immunogenicity. The results of the deletion of NC-017250 gene in bovine species are shown in FIGS. 10, 11 and 12.

Example 2 establishment of a method for identifying Brucella and non-Brucella antibodies in cattle species and non-Brucella in livestock by iELISA

1. Experimental methods

1.1 determination of optimal coating concentration of antigen and optimal dilution of serum

According to the checkerboard titration method, two recombinant proteins purified in example 1 were diluted with the coating solution, and the initial antigen concentration of NC-017250 was 0.5. mu.g/. mu.L, and the dilution gradient was 1: 6.25, 1: 12.5, 1: 25. four gradients 1: 50; BP26 initial antigen concentration 0.553 μ g/μ L dilution gradient was 1: 25. 1: 50. 1: 100. and (3) four gradients of 1: 200, adding 100 mu L of the solution into each hole, coating the solution at 4 ℃ overnight, discarding the liquid in the holes the next day, washing the solution with the washing solution three times, standing the solution for 5min each time, and patting the liquid in the holes of the ELISA plate after the washing is finished. Adding 100 mu L of confining liquid into each hole, standing at 37 ℃ for 2h, washing with washing liquid for three times, standing for 5min each time, and patting off the liquid in the holes of the ELISA plate after washing. Known positive and negative sera were expressed as 1: 50. 1: 100. 1: 200. 1: diluting with four 400-degree gradients, adding 100 μ L diluted serum into each well, standing at 37 deg.C for 1h, washing with washing solution three times, standing for 5min each time, and patting off the liquid in the wells of the ELISA plate after washing. Each well of NC _017250 antigen plate was loaded with 1: 100 mul HRP rabbit anti-bovine IgG at 5000 dilution; BP26 antigen plate add 1 per well: 7000 dilution donkey anti-sheep IgG-HRP 100 u L. Standing at 37 deg.C for 30min, washing with washing solution for three times, standing for 5min each time, and drying the liquid in the wells of the ELISA plate after washing. 100 μ L of TMB developing solution was added to each well, and color development was performed at 37 ℃ for 10 min. After the color development is finished, 100 mu L of stop solution is added into each hole to stop the reaction, and OD is read on a microplate reader₄₅₀The value is obtained. According to OD₄₅₀Values were calculated for P/N for each group, OD positive serum/OD negative serum for the experimental/control group, and the antigen dilution and serum dilution corresponding to the highest P/N value were the optimal dilutions.

1.2 determination of optimal concentration of Secondary antibody

The optimal antigen coating concentration and serum dilution were selected according to 1.1. rNC — 017250 HRP-labeled rabbit anti-bovine IgG was added per well at a rate of 1: 2500. 1: 5000. 1: 7500. 1: 100004 gradients were diluted; rBP26 donkey anti-sheep IgG-HRP was added to each well at a ratio of 1: 5000. 1: 7500. 1: 10000. 1: 15000 four gradient dilutions. mu.L of each well was added and an iELISA assay was performed. According to OD₄₅₀And (4) calculating the P/N value of each secondary antibody dilution, wherein the corresponding secondary antibody dilution when the P/N value is maximum is the optimal secondary antibody dilution.

1.3 repeatability experiments

The elisa assay was performed on 2 positive sera and 2 sheep negative sera, each sample was repeated 3 times and the standard deviation was calculated for each sample.

1.4 determination of decision Point

22 parts of S2 immune serum and 20 parts of A19 immune serum are detected by using the recombinant protein rNC-017250 antigen, 20 parts of naturally infected serum with clear background and 30 parts of S2 immune serum are detected by using the recombinant BP26(rBP26) antigen, and two recombinant antigen iELISA decision points are confirmed by SPSS software ROC analysis.

1.5 sensitivity and specificity assays

rNC _017250 and rBP26 antigens 20 clear background sera from natural infections and 30S 2 immune sera were tested and analyzed by SPSS software to confirm the sensitivity and specificity of the two recombinant antigens iELISA.

1.6 field experiments

And (3) carrying out iELISA detection on the serum to be detected, and comparing the positive detection rates of the two antigens.

2. Results of the experiment

2.1 determination of optimal coating concentration and optimal serum dilution

The purified rNC-017250 and rBP26 antigens were diluted in four gradients as described in 1.1, and the known positive and negative sera were diluted in four gradients for the iELISA detection, and the color and OD after development by iELISA were determined₄₅₀The values of positive and negative serum color and OD₄₅₀The values are obviously different, and the detection results are shown in tables 3 and 4. When the coating concentration of the rNC _017250 antigen is 20 mug/mL, and the serum dilution is 1: when 100, the P/N value is maximum; the optimal coating concentration of PB26 antigen was 5.53. mu.g/mL and the optimal dilution of serum was 1: 100, as calculated from the P/N values, and the results are shown in tables 5 and 6.

TABLE 3 checkerboard titration rNC _017250 antigen OD₄₅₀Value of

TABLE 4 rBP26 checkerboard titration OD of antigens₄₅₀Value of

TABLE 5 rNC _017250 antigen detection P/N values

TABLE 6P/N values for 6 rBP26 antigens

2.2 determination of optimal dilution of enzyme-labeled Secondary antibody

According to the color of the iELISA color reaction and the calculation of the P/N value, determining that the optimal dilution of rNC-017250 enzyme-labeled secondary antibody is 1: the optimal dilution of 5000, rBP26 enzyme-labeled secondary antibody was 1:7500, and the results are shown in tables 7 and 8.

TABLE 7 rNC _017250 optimal secondary antibody dilutions

TABLE 8 determination of the optimum working concentration of the enzyme-labeled Secondary antibody 8 rBP26

2.3 repeat the experiment

2 positive and negative sera were selected, and two were usedRecombinant antigen elisa assays were repeated 3 times per sample. rNC-017250 results show that OD was obtained in 3 replicates₄₅₀The value difference changes insignificantly, and the standard deviation is close to 0, which indicates that the iELISA detection method established by the recombinant antigen has better repeatability, and the results are shown in Table 9. rBP26 the results show that OD was repeated 3 times₄₅₀The differences are not obvious, which indicates that the iELISA detection methods established by the recombinant antigens have better repeatability, and the results are shown in Table 10.

TABLE 9 rNC _017250 recombinant antigen repeat OD₄₅₀Value and SD value

TABLE 10 rBP26 recombinant antigen repeat experiment OD₄₅₀Value and SD value

2.4 determination of decision Point

rNC _017250 and rBP26 antigens serum, OD was detected by iELISA₄₅₀The results are shown in tables 11 and 12. According to OD using SPSS software₄₅₀The ROC analysis was performed to determine that the recombinant antigen rNC _017250 iiisa decision point was 0.390 and the recombinant antigen rBP26 iiisa decision point was 0.589, see fig. 13 and 14 for the decision point analysis. The area under the ROC analysis curve of the recombinant antigen rNC _017250 is 0.989, the area under the ROC analysis curve of the recombinant antigen rBP26 is 0.965, and the areas under the ROC analysis curves of the 2 recombinant antigens are all close to 1, which indicates that the reliability of the detection method of the 2 recombinant antigens iiiisa is high, and the results are shown in fig. 15 and fig. 16.

TABLE 11 rNC _017250 decision Point detection OD₄₅₀Value of

TABLE 12 rBP26 decision Point OD₄₅₀Value of

2.5 specific sensitivity assay

rNC-017250 recombinant antigen detection S2 immune serum 22 parts and A19 immune serum 20 parts, rBP26 antigen detection 20 parts of brucella natural infection serum with clear background and 30 parts of brucella S2 immune serum, SPSS software analysis shows that the sensitivity of the recombinant antigen rNC-017250 iELISA is 95.2%, the specificity is 90.5%, the sensitivity of the recombinant antigen rBP26 iELISA is 90%, and the specificity is 86.7%, and the specific results are shown in the figure 13 and the figure 14.

2.6 field experiments

The iELISA test was performed on 180 sera with positive SAT and RBPT test brucella infection, and rNC-017250 antigens were used to test 38 positives and rBP26 were used to test 176 positives, indicating that 38 of 180 sera were non-bovine brucella infection or antibodies to non-bovine vaccines. The rNC-017250 antigen iELISA test method detected 21.1% (38/180) positive rate, and the rBP26 antigen iELISA test method detected 97.8% (176/180) positive rate, indicating the presence of natural infection or bovine vaccine antibodies in cattle with positive SAT and RBPT test. rNC _017250 can be used to identify Brucella species from other Brucella species, and the results are shown in Table 13.

TABLE 13 results of field experiments

Antigens	Test of serum fraction	Fraction of positive	Negative fraction	Rate of positive detection
					rNC_017250	180	38	142	21.1％
rBP26	180	176	4	97.8％

Therefore, the iELISA identification and detection method established by the invention can identify the brucellosis infected by the bovine species and other species of brucella, and has better repeatability, higher specificity and sensitivity.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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<120> a kit for identifying Brucella melitensis and other Brucella melitensis

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Arg Leu Arg His Gly Ala Leu Leu Ala Ala Met Leu Phe Arg Phe His

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Gly Leu Ala Val Glu Lys Pro Ala Thr Gln Tyr Ile His Ile Glu Lys

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Ala Gln Ala Gly Pro Glu Arg Gln Ile Ile Gly Glu Arg Phe Gln Cys

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Ala Ala Pro Ala Leu Arg Phe Ala Gly Lys Ala Ala Pro Ile Asp Val

85 90 95

Ala Phe Gln Ala Leu Gly Gly Gln Val Cys Leu Asp Gly Gly Ile Thr

100 105 110

Val Val Leu Val Ile Pro Gly Gly Gln Pro Leu Val Gly Thr Val Met

115 120 125

Asp Glu Met Gly Leu Phe Ala Ala Arg Pro Thr Ala Cys Pro Arg Met

130 135 140

Asp Gly Ala Leu Glu Met His Thr Val Arg Pro His Glu Ala Gly Tyr

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Pro Gly Gly Ile Lys Ala Arg Met Ala Tyr Pro Ala Pro His Glu His

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Ile Leu Val Thr Arg Leu Glu Glu Asp Phe Arg Pro Val Gly Ile Gly

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Gly Tyr Arg Gln His Phe Ile Thr Gln Ile Arg Arg Gly Asp Phe Val

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Gly Ile Gly Cys Ile Asp Pro Gly Met Ser Glu Leu Asp Phe Gly Ser

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Ile His Ala Leu Ala Val Phe Gly Val Ile Gly Pro Leu Val Asp Ala

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Arg Ala Ser Arg Ser Gly Asn Leu Asp Arg Ala Val Gly Ala Glu Ile

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Gln His Asp Asp Ile Ile Ala Pro Val Glu Arg Ser Gln Thr Cys Arg

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Gln Val Gly Phe Leu Val Glu Arg His Asp Glu His Gly Asn Gly His

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Met Leu Val Gly Ala Phe Ser Leu Pro Ala Phe Ala Gln Glu Asn Gln

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Met Thr Thr Gln Pro Ala Arg Ile Ala Val Thr Gly Glu Gly Met Met

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Thr Ala Ser Pro Asp Met Ala Ile Leu Asn Leu Ser Val Leu Arg Gln

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Ala Lys Thr Ala Arg Glu Ala Met Thr Ala Asn Asn Glu Ala Met Thr

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Lys Val Leu Asp Ala Met Lys Lys Ala Gly Ile Glu Asp Arg Asp Leu

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Gln Thr Gly Gly Ile Asn Ile Gln Pro Ile Tyr Val Tyr Pro Asp Asp

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Lys Asn Asn Leu Lys Glu Pro Thr Ile Thr Gly Tyr Ser Val Ser Thr

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Ser Leu Thr Val Arg Val Arg Glu Leu Ala Asn Val Gly Lys Ile Leu

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Asp Glu Ser Val Thr Leu Gly Val Asn Gln Gly Gly Asp Leu Asn Leu

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Val Asn Asp Asn Pro Ser Ala Val Ile Asn Glu Ala Arg Lys Arg Ala

165 170 175

Val Ala Asn Ala Ile Ala Lys Ala Lys Thr Leu Ala Asp Ala Ala Gly

180 185 190

Val Gly Leu Gly Arg Val Val Glu Ile Ser Glu Leu Ser Arg Pro Pro

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Met Pro Met Pro Ile Ala Arg Gly Gln Phe Arg Thr Met Leu Ala Ala

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Ala Pro Asp Asn Ser Val Pro Ile Ala Ala Gly Glu Asn Ser Tyr Asn

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Val Ser Val Asn Val Val Phe Glu Ile Lys

245 250