CN106749563B - Gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof - Google Patents
Gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a gene expression product BLSJ-3 with brucella diagnosis and identification functions and a preparation method thereof. The product is obtained by prokaryotic expression of a Brucella S2 strain gene with the gene number (GI) of 490819668 and purification through a Glutathione S Transferase (GST) affinity chromatographic column and a glutathione gradient elution method. The product is used as a coating antigen and can be used as a diagnostic antigen for distinguishing animal brucella vaccine immunity and natural infection serum detection. The BLSJ-3 antigen is respectively subjected to immunoblotting (Western-blot) with vaccine immune antibodies and natural infection antibodies, and the difference is obvious. The BLSJ-3 antigen is used as an indirect enzyme-linked immunosorbent assay (ELISA) envelope antigen to detect hundreds of clinical serum samples with the disease, and the differential diagnosis effect is obvious.
Description
The invention relates to a gene expression product BLSJ-3 with brucella diagnosis and identification functions and a preparation method thereof, belonging to the field of veterinary microbiology diagnosis.
Background
Brucellosis (Brucellosis, simply referred to as Brucellosis) is a zoonosis caused by Brucella. Human infectious diseases mainly come from sick animals and products thereof. The infectious sources related to human brucellosis in China are mainly diseased sheep and cattle, but the harm of brucella melitensis of sheep is the most serious (Zhuliang et al. microbiological report 2015(01): 171-177; Wu Qingmin, veterinary guidance 2011(09): 46-47).
Serological methods are currently the main methods for diagnosing the disease distribution of animals, while vaccination is an important means for preventing the disease distribution of animals. The existing serological diagnosis of brucellosis is mainly based on detection of anti-brucella lipopolysaccharide antibodies, and because the existing animal brucellosis vaccine strains (S2, A19 and M5) and wild virus infected strains in China all belong to smooth strains and induce organisms to generate anti-lipopolysaccharide antibodies, vaccine immunity and natural infection cannot be distinguished by the existing serological method (Mao Kai, et al.
However, the serological antibody response after the Brucella with strong and weak toxicity infects the host shows obvious difference, and provides some background and clues for screening differential diagnosis antigen for distinguishing natural infection and vaccine immunity from serology. As already shown, antibodies were detectable 7 days after virulent Brucella infection, antibody levels reached a maximum 15 days, their agglutinative antibody titer reached 1:3000 and then began to decline, and antibody levels were already lower than those at 7 days after 60 days, while antibodies were negative after 240 days (Gao XL. et al Turkish Journal of Industrial and Animal Sciences 2015,39: 271-278). Whereas antibodies were detected after 2 weeks after immunization of sheep with brucella vaccine S2, antibody levels reached the highest at day 30 with a titer of 1:120 and then began to decline until 180 days after which the antibodies were negative. The difference information between the displayed immunity and the natural infection serum opens up a new idea for screening the differential diagnosis points.
The membrane protein accounts for about 30-40% of total protein of pathogenic microorganisms, is a main target point of development of therapeutic drugs and vaccines, and contains protective antigen components, so that the membrane protein becomes a key object of research of brucella diagnostic antigens and drug therapy target points (Hoffia, et al. disease monitoring 2010(05): 380-389; Wushu, et al. J. immunology 2008(04): 385-388). The immune proteomics method can display protein serological reaction difference information, and can use the bioinformatics technology as a technical platform of high-throughput screening to obtain a large amount of possible differential diagnosis antigen information from the membrane protein, so that the difficult problems of vaccine immunity and natural infection are solved.
Disclosure of Invention
The invention aims to solve the problem that the conventional serological method cannot distinguish the brucella vaccine immune antibody from the natural infection antibody, provide a brucella gene expression product with differential diagnosis value, and realize the differential diagnosis of the vaccine immune antibody and the natural infection antibody by applying an immunological method.
Technical scheme of the invention
1. An expression product BLSJ-3 of brucella diagnostic identification effect gene, which is characterized in that the product is a gene with the gene number (GI) of 490819668 from brucella S2 strain, and the expressed protein is 'hypothetical protein/31kDa outer membrane immunogenic protein'; the length of the gene is as follows: 723 bp; the protein molecular weight of the expression product is about 25.3 kDa; the gene sequence is gene sequence 1:
2. the invention relates to a preparation method of an expression product BLSJ-3 of a brucella diagnosis and identification effect gene, which is characterized by comprising the following steps:
(1) gene screening: screening and identifying the gene of the claim 1 from the genome of the Brucella S2 strain by an immunoproteomics method;
(2) gene expression: through designed primers (sequence 2 and sequence 3), the DNA fragment is cloned and amplified through conventional PCR and is placed in a pGEX6p-1 vector for induced expression;
the upstream primer EcoRI: GAATTC: ttGAATTCat gaaatccgta attttggcg 29 (sequence 2)
Downstream primer XhoI: CTCGAG 5'-tttCTCGAGt tagaacttgt agttcagac-3' 29 (SEQ ID NO: 3).
(3) And (3) purifying an expression product: the expression product was purified by Glutathione S Transferase (GST) affinity column and glutathione gradient elution to obtain the desired antigen, which was named BLSJ-3.
3. The application of the expression product BLSJ-3 of the brucella diagnostic identification function gene is characterized in that the gene is subjected to PCR cloning amplification, and the expression product BLSJ-3 is used as an antigen. The antigen can be used as a serum detection antigen for distinguishing animal brucella vaccine immunity and natural infection.
Detailed description of the invention
1. Screening of differential diagnosis marker genes
(1) By adopting an immunoproteomics technology, brucella vaccine strain S2 which is most widely applied in China and sheep serum which is seriously affected by the brucella in China are selected as research objects, and differential diagnosis antigen gene information is screened from S2 strain membrane protein.
(2) 30 parts of disease distribution negative healthy sheep (Nm), 30 parts of S2 immune sheep (Sm) and 30 parts of clinical disease distribution infected sheep positive mixed serum (Zm) are respectively used for immunoblotting (Western-blot) with S2 membrane protein two-dimensional electrophoresis gel (2DE), reaction difference points of S2 membrane protein and sheep serum in different states (infection/immunity/negative) are searched, and 113 difference protein points are searched in total. 30 protein points with larger immunoreaction difference are selected to carry out matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum identification and bioinformatics analysis, and 14 proteins are identified in total.
(3) 3 kinds of mixed serum (infection/immunization/negative) and S2 membrane protein are respectively subjected to co-Immunoprecipitation (IP), and the IP conjugate is subjected to high-throughput mass spectrometry (Q-active mass spectrometer) and biological information analysis, so that 182 candidate protein spots are obtained. And (3) performing functional analysis on all the proteins identified by the 2 methods, and selecting 8 candidate targets for in-vitro differential diagnosis.
(4) Constructing 8 candidate target protein prokaryotic expression plasmids, respectively carrying out immunoblotting (Western-blot) and enzyme-linked immunosorbent assay (ELISA) on the expression protein and 3 mixed serums, and screening a gene (hypothetical protein/31kDa outer membrane immunogenic protein/31kDa outer-membrane immunogenic protein) with an obvious differential diagnosis effect.
2. Expression of Gene product BLSJ-3
(1) The Brucella S2 strain is cultured and propagated, and the genome thereof is extracted.
(2) Taking the sequence 2 and the sequence 3 as upstream and downstream primers respectively, carrying out gene amplification by a conventional PCR method,
sequence 2: 5'-ttGAATTCat gaaatccgta attttggcg-3' 29
And (3) sequence: 5'-tttCTCGAGt tagaacttgt agttcagac-3' 29, respectively.
(3) The gene amplification band is detected in 1% agarose gel electrophoresis to find the size of the fragment consistent with the expected size, and the obtained recombinant plasmid is sent to a company for sequencing to be compared with a gene sequence published on the National Center for Biotechnology Information (NCBI), so that the coincidence rate is 100%.
(4) after the screened positive recombinant plasmid is transformed into E.coLi BL21(DE3) competent cells, 1mM isopropyl- β -D-thiogalactoside (IPTG) is used for induction expression, and then sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is carried out, wherein the molecular weight of the recombinant protein is basically consistent with the theoretical size.
3. Purification of expression product BLSJ-3
(1) The protein expressed by induction was identified as inclusion bodies by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
(2) The inclusion bodies were renatured by gradient dialysis against a renaturation buffer of gradient urea (4.5M, 3.5M, 2.5M, 2M, 1.5M, 1M, 0.5M, 0M) at 4 ℃ by a conventional method.
(3) Glutathione S Transferase (GST) fusion protein purification column was used for purification according to the method described in the specification.
4. Application of expression product BLSJ-3 as diagnostic antigen
After a target protein (hypothetical protein/31kDa outer membrane immunogenic protein) is purified by a Glutathione S Transferase (GST) affinity chromatography column, the target protein is used as an enzyme-linked immunosorbent assay (ELISA) coating antigen to detect 30 infected sera and 656 immune sheep field samples, and the detection rate of the established enzyme-linked immunosorbent assay (ELISA) on the infected samples is 83 percent.
Drawings
FIG. 1 is a graph showing the results of biphasic electrophoresis of membrane proteins of S2 and immunoblotting (Western-blot) with sheep serum in different immune states: the result of the diphase electrophoresis of the membrane protein of S2 is shown in the diagram A, and the result of the Western blot (Western-blot) of the membrane protein respectively mixed with the mixed serum (Nm) of a disease-negative healthy sheep, the mixed serum (Sm) of an S2 immune sheep and the mixed serum (Zm) of a naturally infected sheep is shown in the diagrams B and D. And the graph E is the superposition analysis result of all the points in the graphs B-D.
FIG. 2S 2 result of co-immunoprecipitation with membrane proteins 1 is an IP mixture of negative sera (Nm); 2 is IP mixture (Sm) of S2 immune serum; 3 is an IP mixture of naturally infected serum (Zm).
FIG. 3 shows PCR amplification results of Brucella suis S2 strain antigen gene: lane M: and (5) Marker. Lanes 1-8 show the genes numbered 1-8.
FIG. 48 shows the induced expression profiles of recombinant proteins: m, Protein molecular weight standards (Protein molecular weight marker); 1-8, which is a candidate protein of 1-8 #; u, uninduced mycoprotein; w, inducing whole mycoprotein; s, supernatant fluid; p, precipitation
FIG. 56 immunoblots of recombinant candidate proteins with three sera, respectively (Western-blot)
FIG. 68 is a photograph showing the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of hypothetical protein of recombinant purified protein # BLSJ-3/31 kDa outer membrane immunogenic protein: m: protein molecular weight standards; 10, 8# candidate protein; p, precipitation; pu, purified protein.
The invention relates to biomaterial resource information
The microbial resource related to the invention is swine brucella attenuated strain S2 strain (CVCC70502 strain), which is identified, stored and supplied by Chinese veterinary medicine inspection institute (see the Chinese veterinary medicine inspection institute, the Chinese veterinary microbial strain preservation management center, the Chinese veterinary bacterial catalogue (second edition), the Chinese agricultural science and technology publishing company, 2002, p 145).
Positive effects of the invention
The invention relates to a gene expression product BLSJ-3 with brucella diagnosis and identification functions and a preparation method thereof. The product is obtained by prokaryotic expression of a Brucella S2 strain gene with the gene number (GI) of 490819668 and purification through a Glutathione S Transferase (GST) affinity chromatographic column and a glutathione gradient elution method. The product is used as a coating antigen and can be used as a diagnostic antigen for distinguishing animal brucella vaccine immunity and natural infection serum detection. The BLSJ-3 antigen is respectively subjected to immunoblotting (Western-blot) with vaccine immune antibodies and natural infection antibodies, and the difference is obvious. The BLSJ-3 antigen is used as an indirect enzyme-linked immunosorbent assay (ELISA) envelope antigen to detect hundreds of clinical serum samples with the disease, and the differential diagnosis effect is obvious.
Examples
The following examples are intended to better illustrate the invention without limiting it.
Example 1
Screening studies for differential diagnosis of antigenic protein spots
Screening and identifying diagnostic antigens from the membrane protein of the strain S2 by adopting an immunoproteomics method. 30 parts of disease distribution negative healthy sheep, 30 parts of S2 immune sheep and 30 parts of clinical disease distribution infected sheep positive mixed serum are respectively used for immunoblotting (Western-blot) with S2 membrane protein two-dimensional electrophoresis gel (2D), reaction differences of S2 membrane protein and sheep serum in different disease distribution states (infection/immunity/negative) are searched, and 113 differential protein points are searched in total. 30 protein points with larger immunoreaction difference are selected to carry out matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum identification and bioinformatics analysis, and 14 proteins are identified in total, bear 13 types of molecular functions and participate in 6 types of cell components and 13 types of biological processes. 3 kinds of mixed serum (infection/immunity/negative) and S2 membrane protein are respectively subjected to Immunoprecipitation (IP), and the compound is identified by high-throughput mass spectrum (Q-active) to obtain 182 candidate protein spots, wherein the proteins have the function of 129 molecular and participate in 91 cell components and 137 biological processes. All proteins identified by the 2 methods are subjected to functional analysis, and 8 candidate targets (number 1# -8#) for in-vitro differential diagnosis are selected.
8 candidate target protein prokaryotic expression plasmids are constructed, and 6 proteins are successfully expressed except 1# (transport protein/transporter) and 3# (cell division protein FtsH/cell division protein FtsH). The expressed protein is respectively carried out immunoblotting (Western-blot) and enzyme linked immunosorbent assay (ELISA) detection with 3 kinds of mixed serum, and 8# BLSJ-3 (hypothetical protein/31kDa outer membrane immunogenic protein, and transdermal protein/31kDa outer-membrane immunogenic protein) is screened out to have obvious differential diagnosis effect.
1. Preparation of brucella S2 strain bacterial liquid, extraction and quantification of membrane protein
The preparation of the brucella S2 seed liquid is carried out according to the method of 'veterinary biological product regulation of the people' S republic of China '(Committee for veterinary biological product regulation of animal living being in Ministry of agriculture, veterinary biological product regulation of the people' S republic of China (good quality version of two) ', Beijing: chemical industry Press, 2000, hereinafter referred to as' regulation 'of the invention), membrane protein extraction according to the recommended methods of' Ching 'Qing and the like (Ching Qing, et al, Ching university Committee (medical edition), 2009(05): 805-811)' membrane protein concentration quantification and Coomassie brilliant blue (Bradford) protein quantification (Thermo Pierce Cat number: 23236 protein quantification kit).
Results of two-phase electrophoresis and immunoblotting (western-blot) of membrane protein of strain S2
The gel protein point matching rate among 2 different batches of experiments reaches more than 80 percent. In a preliminary experiment, firstly, an immobilized adhesive tape with a gradient pH of 3-10 is adopted for one-way separation, and the proteins are mainly concentrated at the pH of 4-7. Therefore, the gel strips with immobilized pH 4-7 gradient were selected for one-dimensional separation, subjected to two-dimensional electrophoresis (FIG. 1A) by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subjected to immunoblotting (Western-blot) by incubation with different sera (FIGS. 1B-1D). After scanning the electrophorograms into images, the images exposed by immunoblots (Western-blot) incubated with different sera were aligned by PDquest 8.0 software to determine the differences in immunoreactive proteins. From the results of FIG. 1E, a total of 262 protein differences were resolved by analyzing the results with PDquest 8.0 software. Selecting 30 protein points with obvious gray value difference, and carrying out in-gel enzyme digestion and mass spectrum identification.
3. Identification data list of two-phase electrophoresis (2DE) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry
The gray scale values reflected in the 30 points are tabulated below (table 1). Nm is the gray value of the membrane protein and the serum of the normal sheep in immunoreaction, Sm is the gray value of the membrane protein and the serum of the immune sheep in immunoreaction, and Zm is the gray value of the membrane protein and the serum of the naturally infected sheep in immunoreaction. The ratio is the ratio of the gray values between the immunity and the natural infection.
Table 130 protein points with 3 different serum response gray value difference information list
4. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry identification result
The results showed that 29 points were identified in total, 9 of which (3 involved in transcriptional regulatory activity, 3 in DNA replication, 1 in cytoplasmic ribosomal subunit, 2 without function) and were not reliable because the c.i. value was less than 90. The 20 points are in the confidence region and can be divided into 3 classes according to bioinformatics ontology, 7 Molecular functions (Molecular Function), 3 cell components (Cellular Component) and 12 Biological processes (Biological Process). The functions of the redox enzyme activities involved in 2 of these points can be classified as molecular functions and biological processes.
5. Co-immunoprecipitation results
According to the fact that protein A/G magnetic beads (protein A/G beads) can be combined with IgG antibody FCThe property of non-specific covalent binding is that 3 different mixed serum antibodies are respectively bound into protein A/G magnetic beads (protein A/G beads), then the mixture is bound with S2 membrane protein, and 3 different sera are respectively bound with the membrane protein to form a mixture according to the principle of antigen-antibody reaction, and the electrophoresis chart is shown in figure 2.
6. Immunoprecipitation to obtain mass spectrometric point information and possible diagnostic target point information obtained via bioinformatics functional analysis
The natural antibody immunologically reacts with the non-denatured antigen, and the number of captured spots is large, and is 284 spots in total. The abundances of the identified protein spots in different serum Immunoprecipitation (IP) mixtures were compared, with non-specific reactions between 54 protein spots and negative sera (Nm). Of the remaining 230 protein spots that did not react non-specifically with negative serum, 50 protein spots reacted only with the S2 immune serum (Sm); 63 protein spots reacted only with naturally infected serum (Zm); 117 protein spots reacted with both immune and naturally infected sera, 12 protein spots with an abundance ratio (Sm/Zm) >1.5 in the IP mixture of S2 serum and naturally infected serum, and 57 protein spots with Sm/Zm < 0.67. It can be seen that 182 possible differential diagnosis antigen proteins are identified in total.
182 possible differential diagnosis antigen proteins identified by immunoprecipitation/high-throughput mass spectrometry (IP/QE) by searching Uniprot Gene Ontology (Gene Ontology) database can be classified into 3 types according to the bioinformatics Gene Ontology (Gene Ontology). It can be seen that 182 proteins perform 129 different molecular functions (molecular function), participate in the formation of 91 cell components (Cellular Component), and participate in 137 different Biological processes (Biological Process). The list of possible diagnostic target information obtained from the analysis of the functional and positional information is shown in table 2.
TABLE 2 List of possible diagnostic target information analyzed by immunoprecipitation/high-throughput mass spectrometry (IP/QE)
Obtained by sequence alignment with brucella suis 2.
8 proteins are screened, and the functions of the proteins are probably related to the immunogenicity, the virulence, the interaction between the Brucella and the host and the transport of the outer membrane protein of the cell (see table 2). These 8 proteins were thus selected for further validation as possible differential diagnostic targets.
Preliminarily determining 8 protein points to be expected to become targets for in-vitro diagnosis according to possible differential diagnosis antigen proteins respectively obtained by two-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum and immunoprecipitation/high-throughput mass spectrum, wherein 1 point is obtained by a two-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum approach and is used as a transport protein; the immunoprecipitation/high-throughput mass spectrometry pathway was obtained at 8 points, which were transporter, membrane protein (26kDa periplasmic cavity immunity protein), cell division protein FtsH, cell membrane biosynthesis protein OmpA, hypothetical protein (lipoprotein), membrane protein (peptidoglycan-related lipoprotein), lectin, hypothetical protein (31kDa outer membrane immunity protein), respectively. Wherein two points obtained by immunoprecipitation/high-throughput mass spectrometry are repeated with one point obtained by two-dimensional electrophoresis/immunoblotting/matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, i.e., the transporter.
Example 2
Determination of Gene information for differential diagnosis of antigenic protein spots
Comparing the candidate protein spots obtained by the 2 methods with the expression protein in S2 genome information published by the National Center for Biotechnology Information (NCBI) website, obtaining the corresponding gene information of each candidate protein spot as shown in Table 3 (containing gene name information, gene number, gene length, protein molecular weight and the like), then respectively designing primers and carrying out gene cloning by using PCR (polymerase chain reaction), obtaining prokaryotic expression products (shown in Table 3, FIG. 3 and FIG. 4), and respectively verifying the differential diagnosis value with brucella immune serum and brucella natural infection serum by using immunoblotting (Western-blot) and indirect enzyme-linked immunosorbent assay (ELISA) (shown in FIG. 5 and Table 4).
1.8 candidate differential diagnostic antigens (Table 3), numbered #1- #8, and the detailed protein names are replaced by protein numbers in the following description.
Detailed information of Table 38 candidate Gene targets and upstream and downstream primers
PCR amplification and prokaryotic expression
PCR products of the amplified 8 genes were obtained using S2 genomic DNA as a template. FIG. 3 shows the electrophoresis results of 8 genes all have specific amplified bands, the target fragment is consistent with the expected size, the obtained recombinant plasmid is sent to the company for sequencing and then is compared with the respective gene sequences provided by the National Center for Biotechnology Information (NCBI), the coincidence rate is 100%, the insertion direction is correct, and the recombinant plasmid can be used for further induced expression.
After the screened positive recombinant plasmid is transformed into E.coLi BL21(DE3) competent cells, 1mM IPTG is used for induction expression, collected thalli are subjected to ultrasonication, supernatant and precipitate are respectively taken for SDS-PAGE, and the results show that 1# and 3# candidate proteins are not expressed, and the rest 6 recombinant proteins are all expressed in the form of inclusion bodies. The molecular weight of each recombinant protein was substantially identical to the theoretical size (FIG. 4).
3. Immunoblotting (Western-blot) of recombinant protein with three sera
6 kinds of recombinant proteins except 1# and 3# were added to the centrifuged precipitates (P) containing 6 kinds of recombinant proteins, respectively, to a cloth disease negative mixed serum (N)m) S2 immune mixed serum (S)m) And natural infection mixed serum (Z)m) Western-blot (FIG. 5) was performed, and strong differences were found between the reaction intensities of the 8# candidate protein and immune serum and naturally infected serum.
4. Enzyme-linked immunosorbent assay (ELISA) reaction of recombinant protein and three kinds of serum
Besides the detection of immunoreactivity of the candidate protein and three kinds of serum by adopting immunoblotting (Western-blot), the detection method also adopts an enzyme-linked immunosorbent assay (ELISA) method for further verification.
TABLE 4 detection results of different coated antigen protein serum samples
As shown in Table 4, 6 proteins other than 1# and 3# i.e., the pellet (P) containing 6 recombinant proteins after centrifugation was dissolved and suspended in 6M urea, diluted to 1. mu.g/mL with a carbonate buffer solution of pH9.6, and subjected to an indirect ELISA method to detect OD 8# in S2 which is a reaction between immune and disease-negative serum450nmBasically consistent with the natural infection serum, has obvious difference with the natural infection serum, shows that the 8# protein has differential diagnosis effect and is named as BLSJ-3. Therefore, the identification information of the genes for differential diagnosis by screening the serum with the function of distinguishing immunity from natural infection is shown in Table 5.
TABLE 5 detailed information Table of differential diagnosis marker genes selected
Example 3
-8# (hypothetical protein/31kDa outer membrane immunogenic protein, hypo-molecular immunogenic protein/31kDaouter-membrane immunogenic protein) differential diagnosis effect verification
1. The recombinant protein No. 8 is purified by using a Glutathione S Transferase (GST) affinity chromatographic column and a glutathione gradient elution method to obtain a purified target protein with the purity of more than 90 percent (figure 6).
2. Detection result of enzyme-linked immunosorbent assay (ELISA) of purified 8# recombinant protein disease antibody
The purified recombinant protein # 8 was purified under conventional conditions: optionally selecting 3 single negative/immune/infectious sera and 1 negative/immune/infectious serum mixed serum, respectively diluting purified recombinant protein antigen to 100ng/mL with carbonate buffer solution of pH9.6, coating overnight at 4 deg.C, blocking with 10% skimmed milk as blocking solution for 2h, diluting serum with PBS 1: 50, diluting enzyme-labeled secondary antibody with Phosphate Buffer Solution (PBS) 1: 5000, performing other steps as conventional procedures, and performing OD450nmAnd (6) detecting. The results are shown in Table 6
TABLE 68 ELISA results of recombinant protein on different sera
As seen in Table 6, there was indeed a clear difference in the response of recombinant protein # 8 between immunization and infection. Moreover, the reaction value between the negative and S2 immune sera was low, and the value of the natural infection reaction was high.
Example 4
Establishment of indirect enzyme-linked immunosorbent assay method and clinical serum sample detection
1. Optimization of reaction conditions
(1) Coating solution, coating antigen concentration and serum dilution optimization result
The 8# (BLSJ-3) recombinant purified protein is used as antigen, and the coating solution is set to be neutral Phosphate Buffer Solution (PBS), alkaline carbonate buffer solution and acidic citric acid buffer solution. Antigen concentrations were set at 3 dilutions of 20ng/mL, 100ng/mL, 500ng/mL, etc., and serum concentrations were set at 1: 50, 1: 100, and 1: 200. Screening was performed by a checkerboard test, and the optimal concentration was determined as the lowest background and the greatest infection/immunity ratio. The antigen coating solution, the antigen coating concentration and the serum dilution condition optimization results of the purified 8# recombinant protein antigen are shown in Table 7.
TABLE 78 # antigen protein coating solution, antigen concentration and antibody dilution screening results
As seen from Table 7, the 8# protein was suitably coated with carbonate buffer; the optimal antigen coating concentration is 100ng/mL, and the serum dilution is 1: 50.
(2) Dilution optimization results of blocking solution and enzyme-labeled secondary antibody
According to the results, the concentrations of the blocking solution and the enzyme-labeled secondary antibody are further optimized, Bovine Serum Albumin (BSA), skim milk and fish gelatin are respectively set as the blocking solution, the enzyme-labeled secondary antibody is respectively diluted by 1: 10000 and 1: 20000, and the screening is carried out through a chessboard test, so that the optimal concentration is determined with the lowest background value and the largest infection/immunity ratio. The specific results are shown in Table 8.
TABLE 8 screening results for different concentrations of blocking solution and enzyme-labeled secondary antibody
As shown in Table 8, the optimal blocking solution for the No. 8 protein is 10% skimmed milk, and the optimal enzyme-labeled secondary antibody concentrations are 1: 10000 respectively.
(3) The reaction time and the development time were optimized as shown in tables 9 and 10, respectively.
The reaction time is set to 0.5h and 1h for comparison, the color development is carried out for 15min, 20min and 30min for comparison, and the larger value of Zm/Sm is selected as the optimal parameter.
TABLE 9 reaction time optimization
TABLE 10 color development time optimization
As shown in tables 9 and 10, the enzyme-linked immunosorbent assay of 8# (BLSJ-3) recombinant protein was substantially the same as that of 1 hour in 0.5 hour, and slightly higher in Zm/Sm in the other 0.5 hour; the 8# recombinant protein develops color for 30min, and the effect is better than 15min and 20 min.
(4) Method for detecting critical value (Cut-off) of immune serum by differential diagnosis antigen indirect enzyme-linked immunosorbent assay method
An indirect enzyme-linked immunosorbent assay method established by using the 8# recombinant protein as an antigen is used for respectively detecting 30S 2 immune sera and calculating the average value and the variance of the immune sera.
Indirect enzyme-linked immunosorbent assay method established by recombinant antigen in Table 118 #
2. Application in clinical sheep farm serum
30 single samples for immunoproteomics known clinical infection and 656 samples from 4 sheep farms for immunization were tested by established indirect enzyme-linked immunosorbent assay. The results are shown in Table 12.
TABLE 12 serum samples for clinical testing
Note: the denominator is the number of the detection samples; the molecules are the number of positive samples. The antigen is judged to be positive for differential diagnosis, namely the clinical infection sample; the indirect enzyme-linked immunosorbent assay method for the anti-lipopolysaccharide is judged to be positive, namely immune or naturally infected serum.
As shown in Table 12, 30 sera with known infections were detected by the 8# recombinant protein BLSJ-3 at a detection rate of 83.3%.
Sequence listing
<110> China institute for veterinary drug inspection
<120> gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof
<130>
<160>17
<170>Patentin version 3.5
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<211>723
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: brucella S2 strain (GI) 490819668 gene
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atgaaatccg taattttggc gtccatcgcc gctatgttcg ccacgtccgc tatggctgcc 060
gacgtggttg tttctgaacc ttccgcccct actgctgctc ctgttgacac cttctcgtgg 120
accggcggct atatcggtat caacgccggt tacgcaggcg gcaagttcaa gcatccattt 180
tctagctttg acaaggaaga caacgaacag gtttccggtt cgctcgacgt aacagctggc 240
ggcttcgtcg gtggtgttca ggccggttac aactggcagc tcgacaacgg cgtcgtgctc 300
ggcgcggaaa ccgacttcca gggatcgagc gttacgggtt cgatttcagc cggtgccagc 360
ggtctcgaag gcaaagctga aaccaaggtc gagtggttcg gcacagttcg tgcccgtctt 420
ggctacacgg ctaccgaacg cctcatggtt tatggtaccg gcggtctggc ctatggtaag 480
gtcaagtctg cgttcaacct gggtgatgat gcaagtgccc tgcacacgtg gtccgacaag 540
acgaaagctg gctggaccct cggcgctggt gctgaatatg ccatcaacaa caactggacg 600
ctcaagtcgg aatacctcta caccgacctc ggcaagcgca acctcgtcga cgttgacaat 660
agcttccttg agagcaaggt caatttccac actgttcgcg tcggtctgaa ctacaagttc 720
taa 723
<210>2
<211>29
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for amplifying (GI) 490819668 gene
<400>2
ttGAATTCat gaaatccgta attttggcg 29 (sequence 2)
<210>3
<211>29
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI for amplifying (GI) 490819668 gene CTCGAG
<400>3
tttCTCGAGt tagaacttgt agttcagac 29 (sequence 3)
<210>4
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: amplifying upstream BamHI of 489056667 gene: GGATCC
<400>4
ggGGATCCat gaggtacacg gtgttcaaag 30 (sequence 4)
<210>5
<211>29
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI for amplifying 489056667 gene: CTCGAG
<400>5
aaaCTCGAGt cagcggccat caggcgtac 29 (sequence 5)
<210>6
<211>34
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for amplifying 489054409 gene
<400>6
aatGAATTCa tgaacactcg tgctagcaat tttc 34 (SEQ ID NO: 6)
<210>7
<211>34
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI CTCGAG for amplifying 489054409 gene
<400>7
cctCTCGAGt tacttgattt caaaaacgac attg 34 (SEQ ID NO: 7)
<210>8
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer SalI: GTCGAC for amplifying 490822055 gene
<400>8
gggGTCGACa tgaatccgaa ctatcgcaac 30 (SEQ ID NO: 8)
<210>9
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer NotI for amplification of 490822055 gene: GCGGCCGC
<400>9
tttGCGGCCG Cttattgcgg ctgcggtttc 30 (SEQ ID NO: 9)
<210>10
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for primer amplification of 490824872 gene
<400>10
ggGAATTCat gctgaagaaa accggtattg 30 (sequence 10)
<210>11
<211>26
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream XhoI of downstream primer for amplifying 490824872 gene CTCGAG
<400>11
tttCTCGAGt caggctccgc gtagcg 26 (sequence 11)
<210>12
<211>28
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for amplifying 489053693 gene
<400>12
gaaGAATTCa tgcggaaaat ggcgcttg 28 (SEQ ID NO: 12)
<210>13
<211>32
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI CTCGAG for amplifying 489053693 gene
<400>13
tttCTCGAGc tatcggcggc agcgtgctcg ag 32 (SEQ ID NO: 13)
<210>14
<211>31
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for amplifying 489055761 gene
<400>14
ccGAATTCat gaacagcttc aggaaaactt g 31 (SEQ ID NO: 14)
<210>15
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI CTCGAG for amplifying 489055761 gene
<400>15
ccgCTCGAGt ttaacgagaa taaggcgaac 30 (SEQ ID NO: 15)
<210>16
<211>30
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: upstream primer EcoRI: GAATTC for amplifying 489056820 gene
<400>16
ttGAATTCat gcgccgtatc cagtcgattg 30 (sequence 16)
<210>17
<211>28
<212>DNA
<213> Artificial sequence
<223> description of artificial sequences: downstream primer XhoI CTCGAG for amplifying 489056820 gene
<400>17
tttCTCGAGt taccgtccgg ccccgttg 28 (SEQ ID NO: 17)
1
Claims (2)
1. The application of BLSJ-3 as the expression product of brucella diagnosis and identification effect gene features that the expression product of the diagnosis and identification effect gene is prepared into the detection antigen for distinguishing brucella vaccine immunity from natural infection serum;
the expression product of the diagnosis and identification effect gene is a gene with the gene number of 490819668 of Brucella S2 strain and is expressed as a hypothetical protein/31kDa outer membrane immunogenic protein (hypo-synthetic protein/31kDa outer-membrane immunogenic protein); the length of the gene is as follows: 723 bp; the protein molecular weight of the expression product is 25.3 kDa; the gene sequence is sequence 1.
2. The use of the expression product BLSJ-3 of the Brucella diagnostic marker-acting gene according to claim 1, wherein the expression product BLSJ-3 is prepared by the following steps: (1) gene screening: screening and identifying the gene of the claim 1 from the genome of the Brucella S2 strain by an immunoproteomics method; (2) gene expression: primers of a sequence 2 and a sequence 3 are designed, and are subjected to conventional PCR cloning amplification, and are placed in a pGEX6p-1 vector for induced expression; (3) and (3) purifying an expression product: the expression product was purified by Glutathione S Transferase (GST) affinity column and glutathione gradient elution to obtain the desired antigen, which was named BLSJ-3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611078420.7A CN106749563B (en) | 2016-11-30 | 2016-11-30 | Gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611078420.7A CN106749563B (en) | 2016-11-30 | 2016-11-30 | Gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN106749563A CN106749563A (en) | 2017-05-31 |
| CN106749563B true CN106749563B (en) | 2020-05-19 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201611078420.7A Active CN106749563B (en) | 2016-11-30 | 2016-11-30 | Gene expression product BLSJ-3 with brucella diagnosis and identification functions and preparation method thereof |
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Families Citing this family (1)
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| CN107629951B (en) * | 2017-09-29 | 2021-03-02 | 深圳国际旅行卫生保健中心 | Micro-fluidic gene detection chip |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003101484A1 (en) * | 2002-05-31 | 2003-12-11 | Thomas Jefferson University | Compositions and methods for transepithelial molecular transport |
| CN101362800A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test strip for rapid detection of brucella |
| CN104166000A (en) * | 2014-07-03 | 2014-11-26 | 中国疾病预防控制中心传染病预防控制所 | A method of indentifying brucella natural infection or immunifaction for livestock |
-
2016
- 2016-11-30 CN CN201611078420.7A patent/CN106749563B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003101484A1 (en) * | 2002-05-31 | 2003-12-11 | Thomas Jefferson University | Compositions and methods for transepithelial molecular transport |
| CN101362800A (en) * | 2008-05-26 | 2009-02-11 | 北京庄笛浩禾生物医学科技有限公司 | Test strip for rapid detection of brucella |
| CN104166000A (en) * | 2014-07-03 | 2014-11-26 | 中国疾病预防控制中心传染病预防控制所 | A method of indentifying brucella natural infection or immunifaction for livestock |
Non-Patent Citations (2)
| Title |
|---|
| NONE.outer-membrane immunogenic protein [Brucella melitensis].《GenBank Database》.2013,ORIGIN部分. * |
| outer-membrane immunogenic protein [Brucella melitensis];NONE;《GenBank Database》;20130507;ORIGIN部分 * |
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| CN106749563A (en) | 2017-05-31 |
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