CN106749563A - A kind of gene expression product BLSJ 3 of brucella diagnostic marker effect and preparation method thereof - Google Patents

Abstract

The invention relates to a gene expression product BLSJ-3 which functions as a diagnostic marker for Brucella and a preparation method thereof. The product is prokaryotically expressed by the gene number (GI) of Brucella S2 strain 490819668, and purified by glutathione S-transferase (GST) affinity chromatography column and glutathione gradient elution method. The product is used as a coating antigen, which can be used as a diagnostic antigen for distinguishing animal Brucella vaccine immunity and natural infection serum detection. Western blotting (Western-blot) was performed on the BLSJ-3 antigen, the vaccine immune antibody and the natural infection antibody respectively, and the difference was obvious. The BLSJ-3 antigen was used as an indirect enzyme-linked immunosorbent assay (ELISA) coating antigen to detect hundreds of brucellosis clinical serum samples, and the differential diagnosis effect was remarkable.

Description

A kind of gene expression product BLSJ-3 of Brucella diagnostic marker function and its preparation method

Technical field The present invention relates to a gene expression product BLSJ-3 with the function of diagnostic marker of Brucella and its preparation method, belonging to the field of veterinary microbiology diagnosis.

Background technique

Brucellosis (abbreviated as brucellosis) is a zoonotic infectious disease caused by Brucella. Human infection with brucellosis mainly comes from sick animals and their products. The source of infection related to human brucellosis in my country is mainly sick sheep and cattle, but Brucella melis is the most serious (Zhu Liangquan et al. Microbiology Bulletin, 2015(01): 171～177; Wu Qingmin. Veterinary Director Journal, 2011(09):46～47).

Serological methods are currently the main method for diagnosing animal brucellosis, and vaccination is an important means of preventing animal brucellosis. The existing serological diagnosis of brucellosis is mainly based on the detection of anti-brucella lipopolysaccharide antibodies. Since the existing animal brucellosis vaccine strains (S2, A19 and M5) and wild virus infection strains in China are smooth strains, they all induce the body to produce Anti-lipopolysaccharide antibodies, so it is impossible to distinguish vaccine immunity and natural infection by existing serological methods (Mao Kairong, et al. Diagnostic techniques for animal brucellosis. China Agricultural Press, 2014.).

However, there are significant differences in the serological antibody response after the infection of the host by virulent Brucella, which provides some background and clues for the serological screening of differential diagnostic antigens to distinguish natural infection and vaccine immunity. For example, existing studies have shown that after virulent infection with Brucella, antibodies can be detected 7 days later, the antibody level reaches the highest level on 15 days, and the agglutinated antibody titer reaches 1:3000, and then begins to decline, and the antibody level is lower than 60 days later. The antibody level at 7 days, and the antibody was negative after 240 days (Gao XL. et al Turkish Journal of Veterinary and Animal Sciences, 2015, 39: 271-278). After the sheep were immunized with Brucella vaccine S2, antibodies began to be detected 2 weeks later, and the antibody level reached the highest on the 30th day, with a titer of 1:120, and then began to decline, and the antibody was negative after 180 days. The difference information between immune and natural infection sera shown above opens up new ideas for the screening of differential diagnosis points.

According to reports, membrane proteins account for about 30% to 40% of the total protein of pathogenic microorganisms, and are not only the main target for the development of therapeutic drugs and vaccines, but also contain protective antigen components, thus becoming the diagnostic antigen and drug treatment target of Brucella Key objects of point research (Hu Jianfei, et al. Disease Surveillance, 2010(05): 380-389; Wu Shuo, et al. Journal of Immunology, 2008(04): 385-388). Since the immunoproteomics method can display the difference information of protein serological reactions, and use bioinformatics technology as a high-throughput screening technology platform to obtain a large number of possible differential diagnostic antigen information from membrane proteins, thereby deciphering the relationship between vaccine immunity and natural Infection problem.

Contents of the invention

The purpose of the present invention is to solve the difficult problem that existing serological methods cannot distinguish Brucella vaccine immune antibodies and natural infection antibodies, provide Brucella gene expression products with differential diagnosis value, and use immunological methods to realize vaccine immune antibodies and differential diagnosis of natural infection antibodies.

Technical scheme of the present invention

1. the expression product BLSJ-3 of a kind of brucella diagnostic mark action gene, it is characterized in that this product is the gene that is 490819668 by brucella S2 strain gene numbering (GI), and expressed protein is " hypothetical protein/31kDa Outer membrane immunogenic protein (hypothetical protein/31kDa outer-membrane immunogenic protein)"; the length of the gene: 723bp; the protein molecular weight of its expression product is about 25.3kDa; its gene sequence is gene sequence 1:

2. the preparation method of the expression product BLSJ-3 of a kind of brucella diagnostic marker action gene of the present invention is characterized in that the method may further comprise the steps:

(1) Gene screening: From the genome of Brucella S2 strain, through immunoproteomics method, screen and identify the gene of the above claim 1;

(2) Gene expression: through the designed primers (sequence 2 and sequence 3), through conventional PCR cloning and amplification, place it in the pGEX6p-1 vector to induce expression;

Upstream primer EcoRI: GAATTC: ttGAATTCat gaaatccgta attttggcg 29 (SEQ ID NO: 2)

Downstream primer XhoI: CTCGAG 5'-tttCTCGAGt tagaacttgt agttcagac-3'29 (SEQ ID NO: 3).

(3) Purification of the expression product: the expression product was purified by glutathione S-transferase (GST) affinity chromatography column and glutathione gradient elution method to obtain the desired antigen, which was named BLSJ-3.

3. the application of the expression product BLSJ-3 of a kind of Brucella diagnostic marker gene of the present invention, it is characterized in that this gene PCR clone is amplified, and its expression product BLSJ-3 is used as antigen. The antigen can be used as a detection antigen for distinguishing animal Brucella vaccine immunization and natural infection serum.

Detailed description of the invention

1. Screening of marker genes for differential diagnosis

(1) Using immunoproteomics technology, select the most widely used Brucella vaccine strain S2 in my country and the sheep serum seriously affected by brucellosis in my country as the research objects, and screen the differential diagnosis antigen gene information from the membrane protein of the S2 strain.

(2) 30 brucellosis-negative healthy sheep (Nm), 30 S2 immunized sheep (Sm), and 30 clinical brucellosis-infected sheep positive mixed sera (Zm) were respectively used on two-dimensional electrophoresis gels of membrane proteins of the S2 strain (2DE) performed Western-blot to search for differences in the reaction between S2 membrane protein and sheep serum in different states (infected/immune/negative), and a total of 113 differential protein points were found. Thirty protein spots with large differences in immune response were selected for matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry identification and bioinformatics analysis, and a total of 14 proteins were identified.

(3) Co-immunoprecipitate (IP) the three mixed sera (infected/immune/negative) with the S2 membrane protein, and perform high-throughput mass spectrometry identification (Q-Exactive mass spectrometer) and biological information on the IP conjugates Analyzed and obtained 182 candidate protein spots. Functional analysis was performed on all proteins identified by the two methods, and a total of 8 candidate targets for in vitro differential diagnosis were selected.

(4) Construct 8 prokaryotic expression plasmids of candidate target proteins, perform Western-blot and ELISA on the expressed proteins with 3 kinds of mixed sera, and screen out a gene with differential diagnosis effect ( Hypothetical protein/31kDa outer-membrane immunogenic protein, hypothetical protein/31kDa outer-membraneimmunogenic protein) differential diagnosis effect is obvious.

2. Expression of gene product BLSJ-3

(1) Cultivate and propagate Brucella S2 strain, and extract its genome.

(2) Using Sequence 2 and Sequence 3 as upstream and downstream primers respectively, the conventional PCR method is used for gene amplification,

Sequence 2: 5'-ttGAATTCat gaaatccgta attttggcg-3'29

Sequence 3: 5'-tttCTCGAGt tagaacttgt agttcagac-3'29.

(3) Gene amplified bands, which were detected in 1% agarose gel electrophoresis, were consistent with the expected size, and the obtained recombinant plasmids were sent to the company for sequencing and the genes published on the National Center for Biotechnology Information (NCBI) The sequences were aligned and the coincidence rate was 100%.

(4) After the screened positive recombinant plasmids were transformed into E.coLi BL21(DE3) competent cells, the expression was induced with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG), and then subjected to dodecyl Sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the recombinant protein is basically consistent with the theoretical size.

3. Purification of the expression product BLSJ-3

(1) The induced protein was identified as inclusion body by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

(2) The inclusion body was refolded by gradient dialysis with gradient urea (4.5M, 3.5M, 2.5M, 2M, 1.5M, 1M, 0.5M, 0M) dialysis buffer at 4°C according to conventional methods. sex.

(3) Use glutathione S-transferase (GST) fusion protein purification column to purify according to its instructions.

4. Application of the expression product BLSJ-3 as a diagnostic antigen

The target protein (hypothetical protein/31kDa outer membrane immunogenic protein, hypothetical protein/31kDaouter-membrane immunogenic protein) was purified by glutathione S-transferase (GST) affinity chromatography column and used as an enzyme-linked immunosorbent assay (ELISA) coated antigen, 30 infected sera and 656 immunized sheep farm samples were detected, and the detection rate of infected samples by the established enzyme-linked immunosorbent assay (ELISA) was 83%.

Description of drawings

Figure 1 Two-phase electrophoresis of S2 membrane protein and Western-blot results of sheep serum with different immune states Western-blot results of mixed serum from sheep (Nm), mixed serum from S2 immunized sheep (Sm), and mixed serum from naturally infected sheep (Zm). Figure E is the overlay analysis results of all points in Figures B-D.

Figure 2 S2 membrane protein co-immunoprecipitation results 1 is the IP mixture of negative serum (Nm); 2 is the IP mixture of S2 immune serum (Sm); 3 is the IP mixture of natural infection serum (Zm).

Fig. 3 PCR amplification result of antigen gene of Brucella suis S2 strain Note in the figure: Lane M: Marker. Lanes 1-8 are genes numbered 1-8.

Fig. 4 Induced expression map of 8 kinds of recombinant proteins: M, protein molecular weight marker; 1-8, 1-8# candidate protein; U, uninduced bacterial protein; W, induced whole bacterial protein; S, supernatant; P, pellet

Figure 5 Western-blot of 6 recombinant candidate proteins with three sera

Figure 6 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of 8# recombinant purified protein (BLSJ-3) hypothetical protein/31kDa outer-membrane immunogenic protein (hypothetical protein/31kDa outer-membrane immunogenic protein) In the result chart: M: protein molecular weight standard; 10, 8# candidate protein; P, precipitate; Pu, purified protein.

The present invention relates to biological material resource information

The microbial resources involved in the present invention are Brucella suis attenuated strain S2 strain (CVCC70502 strain), identified, kept and supplied by China Veterinary Medicine Supervision Institute (see China Veterinary Medicine Supervision Institute, China Veterinary Microbiological Strains Edited by the Preservation Management Center, Catalog of Chinese Veterinary Strains (Second Edition), China Agricultural Science and Technology Press, 2002, p145).

Positive effect of the present invention

The invention relates to a gene expression product BLSJ-3 which functions as a diagnostic marker for Brucella and a preparation method thereof. The product is prokaryotically expressed by the gene number (GI) of Brucella S2 strain 490819668, and purified by glutathione S-transferase (GST) affinity chromatography column and glutathione gradient elution method. The product is used as a coating antigen, which can be used as a diagnostic antigen for distinguishing animal Brucella vaccine immunity and natural infection serum detection. Western-blotting (Western-blot) was performed on the BLSJ-3 antigen, the vaccine immune antibody and the natural infection antibody respectively, and the difference was obvious. The BLSJ-3 antigen is used as an indirect enzyme-linked immunosorbent assay (ELISA) coating antigen to detect hundreds of brucellosis clinical serum samples, and the differential diagnosis effect is remarkable.

Example

The following examples illustrate the present invention better, but do not limit the present invention.

Example 1

——Screening research of differential diagnosis antigen protein spot

Differential diagnostic antigens were screened from membrane proteins of S2 strain by immunoproteomics method. Each of 30 brucellosis-negative healthy sheep, 30 S2 immunized sheep, and 30 clinical brucellosis-infected sheep-positive mixed sera were used for western-blotting (Western-blotting) with S2 strain membrane protein two-dimensional electrophoresis gel (2D), respectively. Looking for the difference between the S2 membrane protein and the serum reaction of sheep with different brucellosis status (infected/immune/negative), a total of 113 differential protein spots were found. A total of 30 protein spots with large differences in immune response were selected for matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry identification and bioinformatics analysis. A total of 14 proteins were identified. These proteins undertake 13 types of molecular functions and participate in the composition of 6 categories of cellular components and 13 categories of biological processes. Immunoprecipitation (IP) was performed on three mixed sera (infection/immunity/negative) and S2 membrane protein, and the combination was identified by high-throughput mass spectrometry (Q-Exactive), and 182 candidate protein spots were obtained. 129 types of molecular functions, participating in the composition of 91 types of cellular components and 137 types of biological processes. Functional analysis was performed on all the proteins identified by the two methods, and a total of 8 candidate targets for in vitro differential diagnosis were selected (numbering 1#-8#).

Eight candidate target protein prokaryotic expression plasmids were constructed, except for 1# (transporter) and 3# (cell division protein FtsH/cell division protein FtsH), 6 proteins were successfully expressed. The expressed protein was detected by Western-blot and ELISA with three kinds of mixed sera, and 8#BLSJ-3 (hypothetical protein/31kDa outer membrane immunogenic protein, hypothetical protein/ 31kDa outer-membraneimmunogenic protein) differential diagnosis effect is obvious.

1. Brucella S2 strain bacterial liquid preparation, membrane protein extraction and quantification

Brucella S2 strain seed liquid is prepared with reference to " the People's Republic of China Veterinary Biological Products Regulations " ((the Veterinary Biological Products Regulations Committee of the Ministry of Agriculture. The People's Republic of China Veterinary Biological Products Regulations (2000 edition). Beijing: Chemistry Industrial Publishing House, 2000, the present invention is hereinafter referred to as " regulation ") method is carried out, and membrane protein extraction is carried out with reference to the method recommended by Qu Qing etc. The membrane protein concentration was quantified according to the method of Coomassie brilliant blue (Bradford) protein quantification (Thermo Pierce product number: 23236 protein quantification kit).

2. Two-phase electrophoresis and western-blot results of membrane protein of S2 strain

The matching rate of gel protein spots between two different batches of experiments was over 80%. In the preliminary experiment, the immobilized pH 3-10 gradient gel strips were used to separate consistently, and it was found that the proteins were mainly concentrated at pH 4-7. Therefore, gel strips with an immobilized pH 4-7 gradient were selected for one-way separation, two-dimensional electrophoresis was performed with 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1A), and different sera were incubated Western-blot (Figure 1B-1D). After the electrophoresis images were scanned, the images of Western-blot exposures incubated with different sera were compared using PDquest 8.0 software to determine the differences in immunoreactive proteins. According to the results in Figure 1E, combined with the PDquest 8.0 software analysis results, a total of 262 protein differences can be identified. 30 protein spots with significant differences in gray value were selected and identified by in-gel digestion and mass spectrometry.

3. Two-phase electrophoresis (2DE) and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis and identification data list

The gray value list (Table 1) reflected in the 30 points is as follows. Nm is the gray value of the immune reaction between membrane protein and normal sheep serum, Sm is the gray value of membrane protein and immune sheep serum, and Zm is the gray value of membrane protein and naturally infected sheep serum. The ratio is the ratio of the gray value between immunity and natural infection.

Table 1 List of gray value differences between 30 protein spots and 3 different sera

4. Identification results by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry

The results showed that a total of 29 spots were identified, of which 9 spots (including 3 involved in transcriptional regulatory activity, 3 in DNA replication, 1 in the cytoplasmic large ribosomal subunit, and 2 inactive), due to low C.I. Unbelievable at 90. 20 points are in the credible zone, which can be divided into 3 categories according to the ontology of bioinformatics, 7 points of molecular function (Molecular Function), 3 points of cellular component (Cellular Component), 12 points of biological process (Biological Process) points. Among them, the function of oxidoreductase activity involved in two points can be classified as both molecular function and biological process classification.

5. Co-immunoprecipitation results

According to the characteristic that protein A/G magnetic beads (protein A/G beads) can carry out non-specific covalent binding with the F _C segment of IgG antibody, three different mixed serum antibodies were bound to protein A/G magnetic beads (protein A/G beads) respectively. G beads), and then combine the above mixture with S2 membrane protein. According to the principle of antigen-antibody reaction, three different sera are combined with membrane protein to form a mixture. The electrophoresis diagram is shown in Figure 2.

6. Immunoprecipitation to obtain mass spectrometry point information and possible diagnostic target information obtained by bioinformatics functional analysis

Through the immunological reaction of natural antibodies and non-denatured antigens, there are more points captured, a total of 284 points. The abundance of the identified protein spots in different serum immunoprecipitation (IP) mixtures was compared, and 54 protein spots had non-specific reactions with negative serum (Nm). Among the remaining 230 protein spots that had no non-specific reaction with negative serum, 50 protein spots only reacted with S2 immune serum (Sm); 63 protein spots only reacted with natural infection serum (Zm); There were 117 protein spots that reacted to the natural infection serum, among which there were 12 protein spots with the abundance ratio (Sm/Zm)>1.5 in the S2 serum and the natural infection serum IP mixture, and 12 protein spots with Sm/Zm<0.67 57. It can be seen that a total of 182 possible differential diagnosis antigen proteins were identified.

The 182 possible differential diagnostic antigen proteins identified by immunoprecipitation/high-throughput mass spectrometry (IP/QE) through the search of the Uniprot Gene Ontology (Gene Ontology) database can be divided into 3 categories. It can be seen that 182 proteins perform 129 different molecular functions (MolecularFunction), participate in the composition of 91 types of cellular components (Cellular Component), and participate in 137 different biological processes (Biological Process). The list of possible diagnostic targets obtained by analyzing the function and location information is shown in Table 2.

Table 2 Information list of possible diagnostic targets analyzed by immunoprecipitation/high-throughput mass spectrometry (IP/QE)

*, Obtained by sequence alignment with Brucella suis S2.

Eight proteins were screened out, and their functions may be related to the immunogenicity, virulence, interaction between Brucella and the host, and the transport of extracellular membrane proteins (see Table 2). Therefore, these 8 proteins were selected as possible differential diagnosis targets for further verification.

According to the possible differential diagnosis antigen proteins obtained by two-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry and immunoprecipitation/high-throughput mass spectrometry, 8 protein spots were initially identified as promising in vitro diagnostics Among them, 2-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry approach obtained 1 point, which was a transport protein; immunoprecipitation/high-throughput mass spectrometry approach obtained 8 points, respectively are transporters, membrane proteins (26 kDa periplasmic luminal immune protein), cell division protein FtsH, cell membrane biosynthesis protein OmpA, hypothetical proteins (lipoproteins), membrane proteins (peptidoglycan-associated lipoproteins), lectins, hypothetical proteins ( 31 kDa outer membrane immune protein). Among them, two spots obtained by immunoprecipitation/high-throughput mass spectrometry overlap with one spot obtained by two-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry, that is, the transporter.

Example 2

——Determination of gene information of differential diagnosis antigen protein spot

The candidate protein spots obtained by the two methods were compared with the expressed proteins in the S2 genome information published on the website of the National Center for Biotechnology Information (NCBI), and the corresponding gene information of each candidate protein spot was obtained in Table 3 (including gene name information, Gene number, gene length, protein molecular weight, etc.), then design primers respectively and use PCR to carry out gene cloning, obtain prokaryotic expression products (Table 3, Fig. 3, Fig. 4), respectively with Brucella immune serum and Brucella natural infection The differential diagnosis value of serum was verified by Western-blot and indirect enzyme-linked immunosorbent assay (ELISA) (Figure 5, Table 4).

1. Eight candidate differential diagnosis antigens (Table 3), numbered #1-#8 respectively, and protein numbers are used instead of detailed protein names in the following expressions.

Table 3 Detailed information and upstream and downstream primers of 8 candidate gene targets

2. PCR amplification and prokaryotic expression

Using S2 genomic DNA as a template, the amplified PCR products of 8 genes were obtained. The results of electrophoresis in Figure 3 show that all 8 genes have specific amplification bands, and the target fragments are consistent with the expected size. The obtained recombinant plasmids are sent to the company for sequencing and compared with the respective gene sequences provided by the National Center for Biotechnology Information (NCBI). Yes, the coincidence rate is 100%, and the direction of insertion is correct, which can be used for further induction of expression.

After the positive recombinant plasmids screened were transformed into E.coLi BL21(DE3) competent cells, the expression was induced with 1mM IPTG. After the collected bacteria were disrupted by ultrasonic, the supernatant and precipitate were taken for SDS-PAGE respectively. It was found that 1# and 3# candidate proteins were not expressed, and the other 6 recombinant proteins were all expressed in the form of inclusion bodies. The molecular weights of each recombinant protein were basically consistent with the theoretical size (Figure 4).

3. Western-blotting of recombinant protein and three sera

The 6 kinds of recombinant proteins except 1# and 3# were centrifuged and the pellet (P) containing 6 kinds of recombinant proteins was mixed with brucellosis negative mixed serum (N _m ), S2 immune mixed serum (S _m ) and The natural infection mixed serum (Z _m ) was subjected to Western-blot (Figure 5), and it was found that there was a strong difference in the reaction intensity between the 8# candidate protein and the immune serum and the natural infection serum.

4. Reaction of recombinant protein with enzyme-linked immunosorbent assay (ELISA) of three kinds of sera

In addition to using Western-blot to detect the immunoreactivity of the candidate protein with the three sera, an enzyme-linked immunosorbent assay (ELISA) method was also used for further verification.

Table 4 Antigen protein serum sample detection results of different coatings

As can be seen from Table 4, 6 kinds of proteins except 1# and 3#, that is, the centrifuged precipitate (P) containing 6 kinds of recombinant proteins were dissolved and suspended with 6M urea, and diluted with pH9.6 carbonate buffer To 1 μg/mL, the indirect enzyme-linked immunosorbent assay method was used to detect that the OD _450nm of 8# in S2 immunity was basically the same as that of brucellosis-negative serum, but it was significantly different from that of natural infection serum, indicating that 8# protein had differential diagnosis effect and was accepted Named BLSJ-3. Therefore, the identification information of genes that can distinguish immune and natural infection sera and play a role in differential diagnosis is shown in Table 5.

Table 5 Detailed information table of the screened differential diagnosis marker genes

Example 3

——8# (hypothetical protein/31kDa outer membrane immunogenic protein, hypothetical protein/31kDaouter-membrane immunogenic protein) differential diagnosis effect verification

1. Utilize the glutathione S-transferase (GST) affinity chromatography column and the glutathione gradient elution method to purify the recombinant protein 8#, and obtain the purified target protein with a purity of more than 90% (Fig. 6).

2. The results of the purified 8# recombinant protein brucellosis antibody enzyme-linked immunosorbent assay (ELISA)

Purified 8# recombinant protein under conventional conditions: optional 3 single parts of negative/immune/infected sera and 1 part of mixed negative/immune/infected sera, respectively purified recombinant protein with pH9.6 carbonate buffer The protein antigen was diluted to 100ng/mL, coated overnight at 4°C, blocked with 10% skimmed milk for 2 hours as a blocking solution, diluted 1:50 with PBS, and 1:50 for the enzyme-labeled secondary antibody with phosphate buffered saline (PBS). 5000 dilution, other steps with the routine procedure, and then OD _450nm detection. The test results are shown in Table 6

Table 6 ELISA results of 8# recombinant protein on different sera

It can be seen from Table 6 that the response of 8# recombinant protein between immunization and infection does have obvious differences. Moreover, the reaction value between negative and S2 immune sera was low, and the value of natural infection reaction was high.

Example 4

——Establishment of indirect enzyme-linked immunosorbent assay method and detection of clinical serum samples

1. Optimization of reaction conditions

(1) Optimization results of coating solution, coating antigen concentration and serum dilution

8# (BLSJ-3) recombinant purified protein was used as antigen, and its coating solution was set as neutral phosphate buffer (PBS), alkaline carbonate buffer, and acidic citrate buffer. The antigen concentration was set to 3 dilutions of 20ng/mL, 100ng/mL, and 500ng/mL, and the serum concentration was set to 1:50, 1:100 and 1:200. Screening is carried out through a checkerboard test, and the lowest background value and the largest infection/immunity ratio are determined as the optimum concentration. See Table 7 for the optimization results of the antigen coating solution, antigen coating concentration and serum dilution of the purified 8# recombinant protein antigen.

Table 7 Screening results of 8# antigen protein coating solution, antigen concentration and antibody dilution

It can be seen from Table 7 that the suitable coating solution for 8# protein is carbonate buffer; the optimum antigen coating concentration is 100ng/mL, and the serum dilution ratio is 1:50.

(2) Optimization results of blocking solution and enzyme-labeled secondary antibody dilution

According to the above results, the concentrations of the blocking solution and the enzyme-labeled secondary antibody were further optimized. Bovine serum albumin (BSA), skimmed milk and fish gelatin were respectively set as the blocking solution, and the enzyme-labeled secondary antibody was prepared at 1:10000 and 1:20000 respectively. Dilution, screening by checkerboard test, with the lowest background value and the largest infection/immunity ratio, determined as the optimal concentration. The specific results are shown in Table 8.

Table 8 Screening results of different blocking solutions and enzyme-labeled secondary antibody concentrations

It can be seen from Table 8 that the optimal blocking solution for 8# protein is 10% skimmed milk, and the optimal enzyme-labeled secondary antibody concentration is 1:10000 respectively.

(3) The optimization of reaction time and color development time are shown in Table 9 and Table 10 respectively.

Set the reaction time as 0.5h and 1h for comparison, and compare the color development for 15min, 20min and 30min, and choose the larger value of Zm/Sm as the most suitable parameter.

Table 9 reaction time optimization

Table 10 Color development time optimization

As can be seen from Table 9 and Table 10, 8# (BLSJ-3) recombinant protein enzyme-linked immunosorbent assay reaction 0.5h is basically consistent with the reaction 1h, and its Zm/Sm is slightly higher in the other 0.5h reaction; 8# recombinant protein develops color for 30min, The effect is better than 15min and 20min.

(4) The cut-off value (Cut-off) for the detection of immune serum by indirect enzyme-linked immunosorbent assay for differential diagnosis of antigens

30 samples of S2 immune sera were detected by the indirect enzyme-linked immunosorbent assay method established using 8# recombinant protein as the antigen, and the average value and variance were calculated.

Table 11 Indirect ELISA method established for 8# recombinant antigen

2. Application in clinical sheep farm serum

Thirty single samples of known clinical infections for immunoproteomics and 656 samples from 4 sheep farms from immunizations were tested using an established indirect ELISA method. The results are shown in Table 12.

Table 12 Serum samples for clinical testing

Note: The denominator is the number of tested samples; the numerator is the number of positive samples. If the purified antigen is judged positive for differential diagnosis, it is a clinically infected sample; if the indirect enzyme-linked immunosorbent assay method for anti-lipopolysaccharide is judged positive, it is immune or natural infection serum.

It can be seen from Table 12 that the detection rate of 8# recombinant protein BLSJ-3 in detecting 30 sera of known infection reached 83.3%.

sequence listing

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<223> Description of the artificial sequence: the downstream primer NotI for amplifying the 490822055 gene: GCGGCCGC

<400> 9

tttGCGGCCG Cttattgcgg ctgcggtttc 30 (sequence 9)

<210> 10

<211> 30

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: Primer amplifies the upstream primer of the 490824872 gene EcoRI: GAATTC

<400> 10

ggGAATTCat gctgaagaaa accggtattg 30 (sequence 10)

<210> 11

<211> 26

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: Amplify the downstream primer of the 490824872 gene downstream XhoI: CTCGAG

<400> 11

tttCTCGAGt caggctccgc gtagcg 26 (sequence 11)

<210> 12

<211> 28

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: EcoRI: GAATTC upstream primer for amplifying the 489053693 gene

<400> 12

gaaGAATTCa tgcggaaaat ggcgcttg 28 (sequence 12)

<210> 13

<211> 32

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: the downstream primer XhoI for amplifying the 489053693 gene: CTCGAG

<400> 13

tttCTCGAGc tatcggcggc agcgtgctcg ag 32 (sequence 13)

<210> 14

<211> 31

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: EcoRI: GAATTC upstream primer for amplifying the 489055761 gene

<400> 14

ccGAATTCat gaacagcttc aggaaaactt g 31 (sequence 14)

<210> 15

<211> 30

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: the downstream primer XhoI for amplifying the 489055761 gene: CTCGAG

<400> 15

ccgCTCGAGt ttaacgagaa taaggcgaac 30 (sequence 15)

<210> 16

<211> 30

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: the upstream primer EcoRI: GAATTC for amplifying the 489056820 gene

<400> 16

ttGAATTCat gcgccgtatc cagtcgattg 30 (sequence 16)

<210> 17

<211> 28

<212>DNA

<213> Artificial sequence

<223> Description of the artificial sequence: the downstream primer XhoI for amplifying the 489056820 gene: CTCGAG

<400>17

tttCTCGAGt taccgtccgg ccccgttg 28 (sequence 17)

1

Claims (3)

1. a kind of expression product BLSJ-3 of brucella diagnostic marker effect gene, it is characterised in that：The product is by cloth Shandong Salmonella S2 pnca genes numbering (GI) are 490819668 gene, are expressed as " imaginary albumen/31kDa adventitia immunogenic proteins (hypothetical protein/31kDa outer-membrane immunogenic protein)”；The mrna length： 723bp；The molecular weight of albumen of its expression product about 25.3kDa；Its gene order is sequence 1.

2. a kind of preparation side of the expression product BLSJ-3 of brucella diagnostic marker effect gene as claimed in claim 1 Method, it is characterised in that：(1) genescreen：From brucella S2 pnca gene groups, by immunoproteomics method, screening mirror Make the gene of aforesaid right 1；(2) gene expression：By the primer (sequence 2 and sequence 3) for designing, cloned by Standard PCR Amplification, is placed on induced expression in pGEX6p-1 carriers；(3) expression product purifying：By glutathione s-transferase (GST) Affinity column and glutathione linear gradient elution method purified expression product, antigen needed for obtaining are named as BLSJ-3.

3. a kind of application of the expression product BLSJ-3 of brucella diagnostic marker effect gene as claimed in claim 1, its It is characterised by the gene prokaryotic product as antigen.Its antigen can as distinguish animal brucella vaccine immunity and from So infect Virus monitory antigen.