Treponema pallidum recombinant antigen and method for preparing the same

Abstract

The application discloses a Treponema pallidum recombinant antigen and a preparation method thereof, and belongs to the technical field of Treponema pallidum antibody detection. In order to solve the technical problems of low sensitivity and low specificity in syphilis antibody detection, the application provides a Treponema pallidum recombinant antigen, the amino acid sequence of the recombinant antigen is shown as SEQ ID NO. 1, and a Treponema pallidum antibody detection kit, the kit comprising the Treponema pallidum recombinant antigen. The Treponema pallidum recombinant protein antigen of the application can specifically recognize the antibody of the serum species of Treponema pallidum, and due to the four different protein fragments, the sensitivity and specificity are greatly improved, and the Treponema pallidum recombinant protein antigen is the best candidate antigen of the Treponema pallidum antibody.

Description

Technical Field

[0001] This invention belongs to the field of antibody detection technology for Treponema pallidum, specifically relating to a recombinant Treponema pallidum antigen and its preparation method. Background Technology

[0002] Treponema pallidum is the pathogen of syphilis. Because it is transparent and difficult to stain, it is also known as the pale spirochete. Syphilis is a widespread sexually transmitted disease, and its incidence has rebounded in China. Treponema pallidum can only infect humans and is classified as acquired syphilis or congenital syphilis. Acquired syphilis is mainly transmitted through sexual contact; congenital syphilis is transmitted from the spirochete to the fetus through the placenta and umbilical cord blood circulation, causing systemic infection in the fetus. The spirochete multiplies extensively in the fetal internal organs and tissues, which can cause fetal death or miscarriage.

[0003] Syphilis has a long incubation period, slow disease progression, and a low positive rate of Treponema pallidum isolation from lesions. Therefore, serological tests for Treponema pallidum-specific antibodies have become a common method for the clinical laboratory diagnosis of syphilis. Currently, there are numerous research reports on serological antibody detection methods using single recombinant expressed antigens of Treponema pallidum, which have the most promising clinical application prospects. However, the sensitivity and specificity of single antigens are relatively low. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems of low sensitivity and low specificity in syphilis antibody detection.

[0005] This invention provides a recombinant antigen of Treponema pallidum, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The present invention provides a gene encoding the above-mentioned Treponema pallidum recombinant antigen.

[0007] The present invention provides an antibody detection kit for Treponema pallidum, the kit comprising the above-mentioned recombinant antigen of Treponema pallidum or the above-mentioned encoding gene.

[0008] Further specifying, the kit also includes coating solutions: pre-coated plate, enzyme-labeled antibody, standards, chromogenic agent, stop solution, washing solution, and sample diluent.

[0009] Further specified: Coating solution: pH 9.6 carbonate buffer, 50 ml; Pre-coated plate: 96-well polystyrene plate; Enzyme-labeled antibody: HRP-labeled mouse anti-IgG antibody, 1 mg; Standard: Polyclonal antibody corresponding to Treponema pallidum recombinant antigen, 1 mg; ELISA buffer: 2% BSA (bovine serum albumin), 0.1% Tween 20, PBS solution, 200 ml; Chromogenic agent: TMB substrate, 1.5 ml; Stop solution: 1 mol sulfuric acid, 1.5 ml; Washing buffer: 2% BSA (bovine serum albumin), 0.1% Tween 20, 0.1X PBS solution, 300 ml; Sample dilution buffer: 0.1X PBS solution, 100 ml.

[0010] This invention provides a method for preparing the above-mentioned Treponema pallidum recombinant antigen. The specific steps of the preparation method are as follows: the gene sequence shown in SEQ ID NO.2 is ligated with the linearized pET-15b plasmid to obtain a recombinant vector; the recombinant vector is transformed into BL21 competent cells to obtain bacterial culture; and the expressed protein is obtained by induction with an inducer.

[0011] Further specifying, the primer sequence for amplifying the gene sequence shown in SEQ ID NO.2 is F5'TACAAATGGTGCGCGCGA3', and the reverse R5'CGGATCTCAGTGGTGGTGGTGGT3'.

[0012] To further specify, the restriction enzyme sites for the gene sequence shown in SEQ ID NO.2 and the linearized pET-15b plasmid are EcoRI and HindIII.

[0013] To further specify, the inducer is IPTG.

[0014] This invention provides the application of the above-mentioned recombinant antigen, the above-mentioned gene, or the above-mentioned kit in the preparation of antibody detection kits after syphilis vaccine immunization, wildlife syphilis epidemic monitoring kits, drug screening kits, and vaccine research and evaluation kits.

[0015] Beneficial effects: The recombinant Treponema pallidum protein of this invention differs from other single protein sequences by incorporating fragments of four basic proteins from Treponema pallidum, linked together in series. This recombinant antigen can specifically recognize antibodies against Treponema pallidum serum. Furthermore, due to the presence of four different protein fragments, its sensitivity and specificity are greatly enhanced, making it an optimal candidate antigen for Treponema pallidum antibodies. Attached Figure Description

[0016] Figure 1 This is a graph showing the results of SDS-PAGE detection of the target protein.

[0017] Figure 2 Image of pET-15b plasmid vector; Detailed Implementation

[0018] Example 1. Preparation method of recombinant Treponema pallidum antigen

[0019] This invention uses gene cloning technology to transfer the required gene fragment (synthesized by Shanghai Langjing Biotechnology Co., Ltd.) into the pET-15b plasmid vector and then into the BL21(D3) strain. Using IPTG as an inducer, the bacteria are induced to express a large amount of the target protein, which is then purified by nickel chelate affinity chromatography.

[0020] TpNl7 protein 120-156: ELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK (SEQ ID NO.5);

[0021] TpN47 protein 123-185:

[0022] MVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPV AVPHE(SEQID NO.6);

[0023] TpN47 protein 332-368: YRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLT (SEQ IDNO.7);

[0024] FLaA protein 190-221: FVGFRIRTSPSERVDDFYVFFDQFKALANMHI (SEQ ID NO. 8).

[0025] I. Methods for constructing the carrier:

[0026] The amino acid sequence described in SEQ ID NO.1 was synthesized, and the gene sequence is SEQ ID NO.2. The gene template was synthesized by a professional gene synthesis company. Then, using primers F 5'TACAAATGGTGCGCGCGA3' (SEQ ID NO.9) in the forward direction and R 5'CGGATCTCAGTGGTGGTGGTGGT3' (SEQ ID NO.10) in the reverse direction, PCR amplification was performed at 95℃ for 5 min, followed by 35 cycles of (98℃, 30 s, 55℃, 30 s, 72℃, 30 s), and then at 72℃ for 5 min, to obtain a sufficient amount for plasmid ligation.

[0027] The gene sequence (amplified fragment) described in SEQ ID NO.2 was combined with the linearized pET-15b plasmid (sequence shown in SEQ ID NO.3, vector diagram shown in...). Figure 2 To ligate the pET-15b plasmid and the amplified gene sequence fragment described in SEQ ID NO.1, the pET-15b plasmid and the gene sequence fragment described in SEQ ID NO.1 were digested with EcoRI and HindIII at 37°C for 2 hours. After digestion, the linearized pET-15b plasmid and the gene sequence fragment described in SEQ ID NO.1 were recovered and ligated. The ligation conditions were the addition of ligase and incubation at 16°C for half an hour to obtain the recombinant vector (sequence shown in SEQ ID NO.4).

[0028] The results of the identification: Sequencing of the recombinant vector showed that the entire vector sequence matched the modified sequence.

[0029] II. Obtaining Recombinant Proteins

[0030] 1. The specific method for transforming the recombinant vector obtained in step one into the BL21(D3) strain to obtain recombinant Escherichia coli and inducing the acquisition of recombinant proteins is as follows:

[0031] 2. Equilibrium solution: 20mM PB, 50mM Tris-HCl, 500mM NaCl, 5% imidazole, pH 7.4;

[0032] Washing solution: 20mM PB, 50mM Tris-HCl, 500mM NaCl, 10% imidazole, pH 7.4;

[0033] Eluent: 20 mM PB, 50 mM Tris-HCl, 500 mM NaCl, 25% imidazole, pH 7.4;

[0034] Recombinant Escherichia coli was cultured, inoculated with 1:100 volume of LB medium, and cultured overnight at 37°C with shaking (200 pm). Amplification was continued with 1:10 volume of LB medium, cultured at 37°C with shaking (200 pm) for 1 h, and then induced with 1 mM IPTG for 2 h. The bacteria were recovered by centrifugation at 30°C and 3000 g. Equilibration buffer was added to the recovered bacteria and lysed. The supernatant was collected and equilibrated with a HIS-tagged affinity resin column using equilibration buffer, rinsing twice. The sample-containing buffer was added to the equilibrated resin column. After loading, the column was rinsed to baseline using equilibration buffer. The target protein (Treponema pallidum-specific multisite recombinant protein) adsorbed by the resin column was washed with 10 times the amount of washing buffer, repeated three times, to remove impurity proteins. Then, elution was performed with 3 times the amount of elution buffer to elute the target protein (Treponema pallidum-specific multisite recombinant protein) adsorbed by the resin column, which was then recovered.

[0035] The target protein (molecular weight: 20 kDa) was detected by SDS-PAGE gel chromatography. The results are as follows: Figure 1 As shown: The results indicate that the target protein band quality meets the requirement of (20 kDa), the band is single, there are basically no other impurities, and the purity of the obtained protein can reach (99%).

[0036] Example 2. Application of recombinant Treponema pallidum antigen

[0037] The Treponema pallidum-specific multisite recombinant protein contains three important antigenic determinant fragments of Treponema pallidum. This series of fragments effectively activates the body and releases corresponding antibodies. Therefore, it can be used as a raw material for the targeted detection of Treponema pallidum antibodies formed in the blood. It can be used as an antigen for detection in ELISA, colloidal gold, immunofluorescence, etc., specifically for the detection of Treponema pallidum antibodies. It can also be used as a raw material for the preparation of related antibodies (mouse, rabbit, etc.).

[0038] Example 3. Reagent kit for detecting Treponema pallidum antibodies

[0039] I. Preparation of the reagent kit:

[0040] Coating solution (bottle): pH 9.6 carbonate buffer, 50 ml;

[0041] Pre-coated substrate (tube): Treponema pallidum recombinant antigen (lyophilized powder), filtered and purified protein, 1 mg;

[0042] Pre-coated board: 96-hole polystyrene board;

[0043] Enzyme-labeled antibody (tube): HRP-labeled mouse anti-IgG antibody, 1 mg;

[0044] Standard (tube): Recombinant polyclonal antibody corresponding to Treponema pallidum antigen, 1 mg;

[0045] ELISA buffer (bottle): 2% BSA (bovine serum albumin), 0.1% Tween 20, PBS solution, 200 ml;

[0046] Colorimetric reagent (tube): TMB substrate, 1.5 ml;

[0047] Stop solution (tube): 1 mol sulfuric acid, 1.5 ml;

[0048] Washing buffer (bottle): 2% BSA (bovine serum albumin), 0.1% Tween 20, 0.1X PBS solution, 300ml. Sample dilution buffer (bottle): 0.1X PBS solution, 100ml.

[0049] II. Detection Operation Steps (1) Bring all components of the kit to room temperature.

[0050] (2) Add sample diluent, 50 μL / well; add positive and negative control, 2 wells each, 50 μL / well; add serum to be tested, 50 μL / well; record the order of sample addition; place in a sealed box or sealed bag; incubate at room temperature for 2 hours.

[0051] (3) Wash 3 times with washing solution, 300 μL / well, and leave the washing solution in the well for 2 minutes each time before patting it dry.

[0052] (4) Add enzyme-labeled reagent, 100 μL / well; incubate at room temperature for 30 minutes;

[0053] (5) Wash 3 times with washing solution, 300 μL / well, and leave the washing solution in the well for 2 minutes each time before patting it dry.

[0054] (6) Add colorimetric solution to develop color, 100 μL / well, and develop color at room temperature in the dark for 10 min;

[0055] (7) Add stop solution, 100 μL / well; measure OD450 value within 10 min;

[0056] (8) Result judgment: The judgment result is greater than the highest negative value.

[0057] The test results are shown in Table 1: OD450 values ​​are calculated. Negative results are determined by testing, and each test is different, generally between 0 and 0.3. A positive result is defined as a value greater than the highest negative value. This is usually determined visually first; positive results are generally bluish, while negative results are colorless and transparent.

[0058] Table 1

[0059] Color development results Qualitative testing Positive sample 1 2.61 Positive Positive sample 2 1.59 Positive Positive sample 3 1.82 Positive Negative sample 1 0.20 Negative Negative sample 2 0.15 Negative negative sample 3 0.14 Negative PBS buffer 0.20 Negative PBS buffer 0.19 Negative PBS buffer 0.19 Negative

[0060] The results showed that the Treponema pallidum-specific multi-site recombinant protein can be used as a substrate for detecting Treponema pallidum-specific antibodies. The efficacy data demonstrates high sensitivity and specificity: Currently, the sensitivity of commercially available ELISA-related tests is around 93%, and the specificity is around 95%. Because their antigens are not screened and contain a large number of non-antigenic epitope protein sequences, a large number of non-specific antibody linkages occur, leading to a certain amount of error. This product uses a specific gene fragment, which is also a protein sequence with a specific antigenic epitope, and only screens for specific antibodies produced by the syphilis virus. By removing non-specific fragments, the possibility of non-specific antibody linkage is eliminated. The antigen only binds to specific antibodies that recognize the antigenic epitope of this sequence, thereby improving detection sensitivity and specificity, achieving a 100% detection rate and 98% specificity, improving accuracy and reducing false positive rates.

[0061] Protein sequence (SEQ ID NO.1)

[0062] MVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAV

[0063] PHEGGGGGSSSSGELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKKGGGGGSSSSG

[0064] YRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTGGGGGSSSSGFVGFRIRTSPSERVDDFYVFFDQFKALANMHI;

[0065] (SEQ ID NO.2):

[0066] ATGGTGCGCGCGAGCGCGAGCTTTCAGGATCTGGGCGAAGATGGCGAAATTAAATTT

[0067] GAAGCGGTGGAAGGCGCGGTGGCGCTGGCGGATCGCGCGAGCAGCTTTATGGTGGAT

[0068] AGCGAAGAATATAAAATTACCAACGTGAAAGTGCATGGCATGAAATTTGTGCCGGTG

[0069] GCGGTGCCGCATGAAGGCGGCGGCGGCGGCAGCAGCAGCAGCGGCGAACTGTATGA

[0070] ACTGATTGATAGCAACAGCGTGCGCTATATGGGCGCGCCGGGCGCGGGCAAACCGAG

[0071] CAAAGAAATGGCGCCGTTTTATGTGCTGAAAAAAACCAAAAAAGGCGGCGGCGGCGG

[0072] CAGCAGCAGCAGCGGCTATCGCCTGACCCTGATTGCGAACGGCTATCGCGATGTGGTG

[0073] GCGGATGTGCGCTTTCTGCCGAAATATGAAGGCAACATTGATATTGGCCTGAAAGGCA

[0074] AAGTGCTGACCGGCGGCGGCGGCGGCAGCAGCAGCAGCGGCTTTGTGGGCTTTCGCA

[0075] TTCGCACCAGCCCGAGCGAACGCGTGGATGATTTTTATGTGTTTTTTGATCAGTTTAAAGCGCTGGCGAACATGCATATT;

[0076] pET-21(a) plasmid vector sequence (SEQ ID NO.3):

[0077] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTAC

[0078] GCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC

[0079] CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT

[0080] TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTG

[0081] ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGA

[0082] GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT

[0083] CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT

[0084] GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT

[0085] CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATA

[0086] CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT

[0087] GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG

[0088] GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG

[0089] AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA

[0090] TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG

[0091] CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCA

[0092] TACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC

[0093] GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC

[0094] TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG

[0095] CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA

[0096] GCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTG

[0097] CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT

[0098] GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

[0099] GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

[0100] CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG

[0101] CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC

[0102] ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT

[0103] TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC

[0104] TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT

[0105] TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT

[0106] ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

[0107] GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCC

[0108] ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA

[0109] GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT

[0110] TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT

[0111] TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG

[0112] CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

[0113] CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG

[0114] TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCG

[0115] GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

[0116] CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC

[0117] GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA

[0118] GTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG

[0119] GTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGT

[0120] TAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACAC

[0121] CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA

[0122] GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

[0123] GAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACA

[0124] GATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCT

[0125] GGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCC

[0126] TCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGG

[0127] ATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGG

[0128] GTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAAT

[0129] GCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGC

[0130] GATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTAC

[0131] GAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGC

[0132] AGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCA

[0133] ACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGG

[0134] GGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA

[0135] GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCG

[0136] ATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCC

[0137] GGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA

[0138] GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATC

[0139] GGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCAC

[0140] TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACG

[0141] CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

[0142] GACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCG

[0143] GTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGG

[0144] ATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAA

[0145] CGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGC

[0146] AACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0147] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAG

[0148] TGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGC

[0149] CCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCG

[0150] CGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCA

[0151] AGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCA

[0152] TCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCG

[0153] CCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACC

[0154] CAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGC

[0155] CAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC

[0156] ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTT

[0157] CGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACC

[0158] GGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGA

[0159] CTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTC

[0160] CGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAG

[0161] GTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCC

[0162] CAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCAT

[0163] GAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCC

[0164] AGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGAT

[0165] CGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGAT

[0166] AACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGGC

[0167] TAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCGAGCTCCGTCGACA

[0168] AGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACA

[0169] AAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAAC

[0170] CCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT;

[0171] Sequence of the recombinant vector (SEQ ID NO.4):

[0172] TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTAC

[0173] GCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC

[0174] CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT

[0175] TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTG

[0176] ATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGA

[0177] GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT

[0178] CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT

[0179] GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT

[0180] CAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATA

[0181] CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT

[0182] GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCG

[0183] GCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG

[0184] AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA

[0185] TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG

[0186] CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCA

[0187] TACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC

[0188] GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC

[0189] TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG

[0190] CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA

[0191] GCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTG

[0192] CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACT

[0193] GGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG

[0194] GTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA

[0195] CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGG

[0196] CAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGC

[0197] ATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT

[0198] TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC

[0199] TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT

[0200] TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCT

[0201] ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACT

[0202] GGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCC

[0203] ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA

[0204] GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT

[0205] TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT

[0206] TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG

[0207] CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA

[0208] CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG

[0209] TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCG

[0210] GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

[0211] CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC

[0212] GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCA

[0213] GTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG

[0214] GTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGT

[0215] TAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACAC

[0216] CCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA

[0217] GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

[0218] GAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACA

[0219] GATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCT

[0220] GGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCC

[0221] TCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGG

[0222] ATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGG

[0223] GTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAAT

[0224] GCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGC

[0225] GATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTAC

[0226] GAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGC

[0227] AGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCA

[0228] ACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGG

[0229] GGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCA

[0230] GTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCG

[0231] ATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCC

[0232] GGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATA

[0233] GTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATC

[0234] GGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCAC

[0235] TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACG

[0236] CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGA

[0237] GACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCG

[0238] GTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGG

[0239] ATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAA

[0240] CGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGC

[0241] AACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA

[0242] CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAG

[0243] TGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGC

[0244] CCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCG

[0245] CGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCA

[0246] AGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCA

[0247] TCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCG

[0248] CCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACC

[0249] CAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGC

[0250] CAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC

[0251] ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTT

[0252] CGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACC

[0253] GGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGA

[0254] CTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTC

[0255] CGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAG

[0256] GTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCC

[0257] CAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCAT

[0258] GAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCC

[0259] AGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGAT

[0260] CGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGAT

[0261] AACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAT ACCTA

[0262] CAAATGGTGCGCGCGAGCGCGAGCTTTCAGGATCTGGGCGAAGATGGCGAAATTAAA

[0263] TTTGAAGCGGTGGAAGGCGCGGTGGCGCTGGCGGATCGCGCGAGCAGCTTTATGGTG

[0264] GATAGCGAAGAATATAAAATTACCAACGTGAAAGTGCATGGCATGAAATTTGTGCCG

[0265] GTGGCGGTGCCGCATGAAGGCGGCGGCGGCGGCAGCAGCAGCAGCGGCGAACTGTAT

[0266] GAACTGATTGATAGCAACAGCGTGCGCTATATGGGCGCGCCGGGCCGGGCAAACCG

[0267] AGCAAAGAAATGGCGCCGTTTTATGTGCTGAAAAAAACCAAAAAAGGCGGCGGCGGC

[0268] GGCAGCAGCAGCAGCGGCTATCGCCTGACCCTGATTGCGAACGGGCTATCGCGATGTG

[0269] GTGGCGGATGTGCGCTTTCTGCCGAAATATGAAGGCAACATTGATATTGGCCTGAAAG

[0270] GCAAAGTGCTGACCGGCGGCGGCGGCGGCAGCAGCAGCAGCAGCGGCTTTGTGGGCTTTC

[0271] GCATTCGCACCAGCCCGAGCGAACGCGTGGATGATTTTTATGTGTTTTTTGATCAGTTT

[0272] AAAGCGCTGGCGAACATGCATATT CACCACCACCACCACCACTGAGATCCGGCTGCTA

[0273] ACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCAT

[0274] AACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT。

Claims

1. A recombinant antigen of Treponema pallidum, characterized in that, The amino acid sequence of the recombinant antigen is shown in SEQ ID NO.

1.

2. The gene encoding the Treponema pallidum recombinant antigen as described in claim 1.

3. An antibody detection kit for Treponema pallidum, characterized in that, The kit includes the recombinant Treponema pallidum antigen of claim 1 or the encoding gene of claim 2.

4. The antibody detection kit according to claim 3, characterized in that, The kit also includes coating solution, pre-coated plate, enzyme-labeled antibody, standards, chromogenic agent, stop solution, washing solution and sample diluent.

5. The antibody detection kit according to claim 4, characterized in that, Coating buffer: pH 9.6 carbonate buffer; Pre-coated plate: 96-well polystyrene plate; Enzyme-labeled antibody: HRP-labeled mouse anti-IgG antibody; Standard: polyclonal antibody corresponding to Treponema pallidum recombinant antigen; ELISA buffer: 2% BSA, 0.1% Tween 20, PBS solution; Colorimetric reagent: TMB substrate; Stop solution: 1 mol sulfuric acid, 1.5 ml; Washing solution: 2% BSA, 0.1% Tween 20, 0.1X PBS solution; Sample diluent: 0.1×PBS solution.

6. A method for preparing the recombinant Treponema pallidum antigen according to claim 1, characterized in that, The specific steps of the preparation method are as follows: the gene sequence shown in SEQ ID NO.2 is ligated with the linearized pET-15b plasmid to obtain a recombinant vector, the recombinant vector is transformed into BL21 competent cells to obtain bacterial culture, and the expressed protein is obtained by induction with an inducer.

7. The preparation method according to claim 6, characterized in that, The primer sequences for amplifying the gene sequence shown in SEQ ID NO.2 are TACAAATGGTGCGCGCGA and CGGATCTCAGTGGTGGTGGTGGT.

8. The preparation method according to claim 6, characterized in that, The restriction enzyme sites for the gene sequence shown in SEQ ID NO.2, which are linked to the linearized pET-15b plasmid, are EcoRI and HindIII.

9. The preparation method according to claim 6, characterized in that, The inducer was IPTG.

10. The application of the recombinant antigen of claim 1, the gene of claim 2, or the kit of claim 3 or 4 in the preparation of antibody detection kits after syphilis vaccine immunization, wildlife syphilis epidemic monitoring kits, drug screening kits, and vaccine research and evaluation kits.

Images