CN113004380B

CN113004380B - Treponema pallidum recombinant antigen, preparation and application

Info

Publication number: CN113004380B
Application number: CN202110187951.4A
Authority: CN
Inventors: 刘万建; 杨帆; 李林; 王婷; 杜金芳; 李文
Original assignee: Qingdao Shuojing Biotechnology Co ltd
Current assignee: Qingdao Shuojing Biotechnology Co ltd
Priority date: 2021-02-18
Filing date: 2021-02-18
Publication date: 2022-06-28
Anticipated expiration: 2041-02-18
Also published as: CN113004380A

Abstract

The invention belongs to the technical field of treponema pallidum immunodetection. In the detection of treponema pallidum, the gold-labeled chromatography detection method has the problems of low sensitivity and poor specificity, and the existing solution has the problems of missed detection and interference of detection results, so the invention provides the treponema pallidum recombinant antigen TP34S, the preparation method and the application thereof, and the invention takes TP17 outer membrane protein as the basis, carries out site-specific amino acid mutation on the protein, connects double gene sequences to a carrier, and carries out prokaryotic expression, so that the TP17 mutant sequence completes repeated expression, the epitope can be fully and effectively exposed, the missed detection is prevented, and the specificity of the detection and the stability of the product are obviously improved.

Description

Treponema pallidum recombinant antigen, preparation and application

Technical Field

The invention belongs to the technical field of treponema pallidum immunodetection, and particularly relates to a treponema pallidum recombinant antigen, and preparation and application thereof.

Background

Treponema Pallidum (TP) is also known as treponemiallidium pallidum and is the causative agent of syphilis, a venereal disease, and serological methods are commonly used clinically to detect the infection of treponema pallidum. Conventionally, detection of anti-cardiolipin antibodies using non-specific lipid antigens (cardiolipin) has been used as a marker for treponema pallidum infection, such as the Venereal Disease Research Laboratory (VDRL) method, the unheated serum response test (USR), and the rapid plasma response cycle test (RPR). These methods are sensitive but not specific and have false positive reactions, so that they require further confirmation tests.

Specific detection methods using treponema pallidum as antigen include treponema pallidum fluorescent antibody adsorption test (FTA-ABS) and treponema pallidum hemagglutination Test (TPHA), and the specificity and the sensitivity are high. However, because of the difficulty in culturing treponema pallidum, the cost of the reagent is high, and because the treponema pallidum antigen is complex, the treponema pallidum antigen can still have a certain non-specific reaction with other pathogens, especially similar spirochete diseases (such as yasi disease).

In view of the above, scientists have begun to use recombinant proteins as specific detection methods for antigens. With the development of scientific technology, the whole genome sequence of syphilis has been determined, and with the development of gene cloning technology, immunoassay methods using syphilis recombinant proteins as antigens have been applied successively, such as immunocapture eia (ce) assay, double antigen sandwich assay, and the like.

At present, immunological test methods for treponema pallidum mainly utilize enzyme-linked immunosorbent assay, chemiluminescence assay, gold-labeled chromatography and the like based on the principle of a double-antigen sandwich method.

The principle of the TP double antigen sandwich method is as follows: coating a first antigen (namely a coating antigen) on a solid phase support, adding a sample to be detected, introducing a second antigen (namely a labeled antigen) labeled with a special label in a proper mode, and if TP antibodies exist in the sample, forming a sandwich structure of coating antigen-TP antibody-labeled antigen by the TP antibodies and the two antigens, and then amplifying a signal by the label on the labeled antigen to obtain a final judgment result.

The enzyme linked immunosorbent assay and the chemiluminescence assay have the advantages of high sensitivity and high accuracy, but are not suitable for point of care (POCT) because of long detection time, complex operation, multiple instruments and high quality requirement of detected samples.

Therefore, the gold-labeled chromatography detection method which is simple, rapid, specific and high in sensitivity is developed and applied in the detection of the treponema pallidum.

However, the gold-labeled chromatography method is high in sensitivity and good in specificity, and the key is the quality of the coating antigen, and the performance of the coating antigen has a decisive influence on the accuracy of the immunoassay. Therefore, in the case of the solid phase carrier and the coating method, the quality of the prepared coated antigen is determined by the characteristics of the antigen, and the high quality antigen needs to have the following conditions:

the protein is adsorbed on a solid phase carrier in the immunochromatography detection, and as a capture reagent of a sample to be detected, the detection result greatly depends on the good adsorption effect of the capture reagent on the solid phase carrier, so that the uniform and good adsorption of the protein on the solid phase carrier is very important for the detection result. The different structures and charges of the antigens have great influence on the adsorption effect.

And (II) the specificity is good, the antigen specificity greatly depends on the selected antigen and epitope, whether the antigen is correctly folded in the expression process and the antigen purity, and once the epitope is improperly selected or the protein purity cannot reach the standard, the detection result can be missed or false positive.

(III) the stability is good, and the antigen with poor stability is usually reduced in activity due to aggregation or degradation; the antigen with good stability is not easy to reduce the activity no matter stored in a buffer system or coated on a solid phase carrier under certain extreme conditions.

The problems that the detection result is not ideal, the detection line dispersion phenomenon caused by poor adsorption effect exists, the sensitivity is low, the specificity is poor, and the stability experiment is unqualified are generally solved at present.

The above problems are usually solved by selecting different antigen dominant epitopes for chimeric expression or by adding various stabilizers or protective agents to the buffer system. However, both methods have disadvantages, and various epitopes are chimeric and expressed, so that inclusion bodies are easily formed in the expression process, which causes wrong protein folding, and the reduction of activity causes missed detection of results or complete loss of activity; when a plurality of stabilizing agents or protective agents are added to coat the solid phase carrier, the membrane surface of the solid phase carrier is not clean in the climbing process, or the problems of false positive and the like are caused, so that the detection result is interfered.

Disclosure of Invention

In the detection of treponema pallidum, the gold-labeled chromatography detection method has the problems of low sensitivity and poor specificity, the existing solution scheme is generally to select different antigen dominant epitopes for chimeric expression, or to add various stabilizers or protective agents in a buffer system, the two methods have the problems of missed detection and interfered detection results, therefore, the invention provides the treponema pallidum recombinant antigen, the preparation method and the application thereof, the application of the treponema pallidum recombinant antigen to the detection of TP antibody by a gold-labeled chromatography platform obviously improves the specificity of detection and the stability of products.

In a first aspect, the invention provides a treponema pallidum recombinant antigen TP34S, which has an amino acid sequence shown in SEQ ID No.3, and specifically comprises the following steps:

VSCTTVCPHAGKAKAEKVESALKGGIFRGTLPAADSPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKKEFVSCTTVCPHAGKAKAEKVESALKGGIFRGTLPAADSPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKKHHHHHH

the second aspect of the present invention provides a method for preparing the treponema pallidum recombinant antigen TP34S, comprising the following steps:

(1) referring to an amino acid sequence of a GenBank database WP _010881883.1, removing signal peptide 1-22aa, and obtaining treponema pallidum antigen TP17 through codon optimization, wherein the corresponding nucleotide sequence is shown in SEQ ID NO. 7;

(2) amino acid mutations were performed on the basis of TP 17: the 20 th cysteine and the 36 th cysteine of the N end are mutated into serine, namely: C20S, C36S; the target gene product is TP17S, and the corresponding nucleotide sequence is shown in SEQ ID NO. 5;

(3) Respectively designing and amplifying two sections of upstream and downstream primers of TP17S by taking TP17S as a template, adding an enzyme cutting site and a protective base in front of the primers, introducing a histidine tag into the downstream primer of the amplified second section of TP17S, wherein the nucleotide sequences of the primers P1, P2, P6 and P7 respectively correspond to SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.13 and SEQ ID NO. 14;

using TP17S as a template, P1 as an upstream primer and P6 as a downstream primer to amplify a first section of a target gene sequence TP17S of TP34S, using P7 as an upstream primer and P2 as a downstream primer to amplify a second section of a target gene sequence TP17S, recovering amplification products, respectively carrying out double enzyme digestion, connecting after recovering enzyme digestion products, connecting the connected product TP34S to a plasmid vector pGEX-4T-1 after enzyme digestion of BamH I and Xho I to form a recombinant plasmid;

(4) transferring the recombinant plasmid in the step (3) into a host cell to form a transformant cell;

(5) culturing the transformant cell of step (4) under suitable conditions to express the recombinant antigen;

(6) separating and purifying to obtain the recombinant antigen.

In a third aspect, the invention provides a gold-labeled chromatography test strip for detecting syphilis, and the coating antigen of the test strip is treponema pallidum recombinant antigen TP 34S.

The invention is based on TP17 outer membrane protein with strong immunogenicity, carries out site-specific amino acid mutation, connects twice gene sequences to a carrier, carries out prokaryotic expression, ensures that the TP17 mutation sequence finishes repeated expression, ensures that the epitope can be fully and effectively exposed, prevents omission, and simultaneously avoids the aggregation caused by disulfide bonds due to the mutation of cysteine, thereby solving the false positive problem caused by protein aggregation, having obvious advantages in the aspect of antigen specificity and simultaneously bringing more than expected effects in the aspect of stability when being coated on a solid phase carrier. The protein is used as a coating antigen to be applied to a gold-labeled chromatography platform to detect TP antibodies, and the specificity of detection and the stability of products are obviously improved.

Drawings

FIG. 1 is a flow chart of construction of a recombinant plasmid in example 1;

FIG. 2 is a flow chart of point mutation PCR of example 1;

FIG. 3 is the gene tandem recombinant plasmid construction procedure of example 1;

FIG. 4 shows the SDS-PAGE and SEC-HPLC detection results of the recombinant antigen TP17 in example 2;

FIG. 5 shows the SDS-PAGE and SEC-HPLC detection results of the recombinant antigen TP17 in example 2;

FIG. 6 shows the SDS-PAGE and SEC-HPLC detection results of the recombinant antigen TP17 in example 2;

FIG. 7 is a diagram of the structure of a gold-labeled test strip and a result description thereof in example 3;

FIG. 8 example 4 shows the result of color development of positive quality control material.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings.

Example 1: construction of recombinant plasmids

(1) Construction of TP17 recombinant plasmid

The nucleotide sequence of TP17(23-156aa) (synthesized by Suzhou Honghen Biotech, Ltd.) of SEQ ID NO.7 was synthesized by codon optimization with reference to the amino acid sequence of GenBank database WP _010881883.1, with the signal peptides 1-22aa removed. By using primer5 primer design software, according to the primer design and the enzyme cutting site design principle, combining with a pGEX-4T-1 vector enzyme cutting site map, respectively adding an enzyme cutting site and a protective base before an upstream primer and a downstream primer, and simultaneously introducing a histidine tag into the downstream primer, the design primer is as follows:

P1：5’-CGGGATCCGTTTCTTGCACCACCGTTT-3’(BamH I)；

P2：5’-CCCTCGAGGTGGTGGTGGTGGTGGTGTTTTTTGGTTTTTTTCAGAACGTA-3’(Xho I)

the primer P1 is shown as a sequence table SEQ ID NO.8, the primer P2 is shown as a sequence table SEQ ID NO.9, and the amplification sequence is shown as a sequence table SEQ ID NO. 4.

The reaction system was added as in Table 1 and amplified by the amplification procedure of Table 2

TABLE 1 PCR reaction System

TABLE 2 amplification procedure

After the PCR amplification product is recovered, the product is subjected to double enzyme digestion by BamH I and XhoI (various enzymes for molecular biology adopted by the invention are purchased from Boehringer Biotechnology, Inc.), then the enzyme digestion product is recovered, the product is connected to a vector pGEX-4T-1 after the enzyme digestion of the BamH I and the XhoI, a positive transformant is obtained by transforming DH5 alpha and identifying the recombinant plasmid by PCR, and sequencing is carried out, and the sequencing result is compared with an amino acid sequence SEQ ID NO.1 of a GenBank database WP _010881883.1, and the coincidence rate is 100%. Extracting recombinant plasmids, transforming an escherichia coli expression strain Rosetta, and obtaining a TP17 recombinant antigen expression strain: R/pGEX-4T-1-TP 17. The specific process is shown in figure 1.

(2) Construction of TP17S recombinant plasmid

TP17S was amino acid mutated based on TP 17: mutating the N-terminal 20 th cysteine and 36 th cysteine to serine, namely: C20S, C36S. Mutant primers P3, P4 and P5 were designed using primer5 primer design software. The 5' end of the P3 primer contains a BamH I cleavage site and a Ser mutation site at position 20. P4 and P5 are complementary primers, including a Ser mutation site at position 36. Primers were designed as follows:

P3:5’-CGGGATCCGTTTCTTGCACCACCGTTTGCCCGCACGCTGGTAAAGCTAAAGCTGAAA-3’(BamH I)

P4：5’-TGCTGACTctCCGGGTATCGACACCA-3’

P5：5’-TGGTGTCGATACCCGGagAGTCAGCA-3’

the primer sequences correspond to SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively. The primers P2, P3, P4 and P5 are used for carrying out point mutation PCR by taking TP17 as a template, the obtained target gene product is TP17S, and the corresponding nucleic acid sequence is SEQ ID NO. 5. The specific process is shown in figure 2.

After the PCR amplification product is recovered, carrying out double enzyme digestion through BamH I and XhoI, then recovering the enzyme digestion product, connecting the enzyme digestion product to a vector pGEX-4T-1 after the BamH I and XhoI are subjected to enzyme digestion, carrying out conversion on DH5 alpha, carrying out PCR identification on the recombinant plasmid to obtain a positive transformant, carrying out sequencing, comparing the sequencing result with an amino acid sequence of a GenBank database WP _010881883.1, wherein the coincidence rate is 98%, and Cys at the 20 th position and the 36 th position is successfully mutated into Ser. Extracting recombinant plasmids, transforming an escherichia coli expression strain Rosetta, and obtaining a TP17S recombinant antigen expression strain: R/pGEX-4T-1-TP 17S.

(3) Construction of TP34S recombinant plasmid

Taking TP17S as a template, utilizing primer5 primer design software, combining pGEX-4T-1 vector enzyme cutting site maps according to primer design and enzyme cutting site design principles, respectively designing and amplifying upstream and downstream primers of two sections of TP17S, adding enzyme cutting sites and protective bases in front of the primers, and simultaneously introducing histidine tags into downstream primers for amplifying a second section of TP17S, wherein the design primers are as follows:

P1：5’-CGGGATCCGTTTCTTGCACCACCGTTT-3’(BamH I)；

P2:5’-CCCTCGAGGTGGTGGTGGTGGTGGTGTTTTTTGGTTTTTTTCAGAACGTA-3’(Xho I)

P6：5’-CGGAATTCTTTTTTGGTTTTTTTCAGAACGTA-3’(EcoR I)

P7：5’-CGGAATTCGTTTCTTGCACCACCGTTTGCCCGCA-3’(EcoR I)

p6 and P7 correspond to SEQ ID NO.13 and SEQ ID NO.14, respectively.

TP17S is taken as a template, P1 is taken as an upstream primer, P6 is taken as a downstream primer to amplify a first section of target gene sequence TP17S (BamH I-EcoR I) of TP34S, P7 is taken as an upstream primer, and P2 is taken as a downstream primer to amplify a second section of target gene sequence TP17S (EcoR I-Xho I). And after the amplification products are recovered, carrying out double enzyme digestion respectively, connecting the enzyme digestion products after recovery, connecting the connected product TP34S (BamH I-TP17S-EcoR I-TP17S-Xho I) to a vector pGEX-4T-1 after BamH I and Xho I enzyme digestion, converting DH5 alpha, identifying recombinant plasmids by PCR to obtain positive transformants, sequencing, comparing the sequencing result with an amino acid sequence SEQ ID NO.2 of TP17S, and ensuring that the coincidence rate is 100%. And the TP17S sequence was repeated for two segments. Extracting recombinant plasmids, transforming an escherichia coli expression strain Rosetta, and obtaining a TP17S recombinant antigen expression strain: R/pGEX-4T-1-TP 34S. The specific process is shown in figure 3.

Example 2: recombinant antigen expression purification

Inducing expression:

mu.l of the recombinant plasmid-expressed strain was inoculated into 5ml of LB medium containing 200ug/ml of ampicillin sodium (AMP) (purchased from Beijing Solebao technologies Co., Ltd.), cultured overnight (about 16 hours) at 37 ℃ under shaking at 200rpm, 200. mu.l of the resulting bacterial solution was added to 60ml of LB medium containing the same concentration of AMP, cultured overnight at 37 ℃ under shaking at 200rpm, and all the bacterial solutions were inoculated to the same mediumDetermining OD after 2-2.5h in 1LLB medium with AMP concentration at 28 deg.C and 150rpm with shaking₆₀₀Adding inducer IPTG (purchased from AMRESCO) with final concentration of 0.5mM into the mixture of 0.5-0.8, continuously performing shaking culture at 28 deg.C and 150rpm for 4.5h, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the pellet was resuspended in 50ml of 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet collected.

Ultrasonic crushing: the pellet was resuspended in 30ml of 20mM PBS (pH 7.4), and subjected to cryosonication, sonication settings: the total time is 14min and exceeds 2s, the time is stopped for 4s, and the force is 8 percent. After the completion of sonication, the disrupted product was centrifuged at 7500rpm at low temperature for 30min, and the supernatant was collected.

Protein purification: carrying out affinity chromatography on GST-Bestarose-4FF chromatographic column (purchased from Bogelong (Shanghai) Biotechnology Co., Ltd.), and balancing the chromatographic column with ultrapure water of 10 times of column volume; then, the chromatographic column is equilibrated with 10 times of column volume of equilibration buffer (20mM PBS pH7.4), and then a protein sample is added; after the loading, unbound protein was washed off with 10 column volumes of equilibration buffer (20mM PBS pH7.4); finally, the target protein was eluted with an elution buffer (20mM Tris-HCl,50mM GSH pH 8.0).

The collected protein samples were placed in dialysis bags and placed in a beaker containing dialysate (20mM PB, pH8.0) and dialyzed overnight at 4 ℃.

Purifying the dialyzed crude protein product by using a Q-Bestarose-HP anion protein chromatographic column (purchased from Booglong (Shanghai) Biotechnology Co., Ltd.), balancing the anion chromatographic column by using 10 times of column volume of ultrapure water, balancing by using 10 times of column volume of balancing buffer solution (20mM PB, pH8.0), adding the crude protein product after balancing, and washing out unbound protein by using 10 times of column volume of balancing buffer solution (20mM PB, pH 8.0); finally, the target protein was eluted with an elution buffer (20mM PB, 200mM NaCl pH 7.4).

And (4) carrying out SDS-PAGE and SEC-HPLC detection on the purified protein respectively. The TP17 results are shown in FIG. 4, the TP17S results are shown in FIG. 5, and the TP34S results are shown in FIG. 6. As can be seen from the figure, the purity of all three recombinant proteins is above 95%, but HPLC results show that TP17 exists in aggregates, and TP17S and TP34S are both monomers.

Example 3: preparation of gold-labeled test strip for detecting treponema pallidum antibody by double-antigen sandwich method

The recombinant antigen prepared in the present invention was diluted to a working concentration with a coating solution (20mM PBS 5% sucrose pH7.4) as a detection line (T line) coating material, and a quality control line (C line) coating material (goat anti-mouse IgG, available from Shandong Shuojing Biotech Co., Ltd.) was streaked on an NC membrane using a streaking apparatus, followed by baking at 37 ℃ for 30mi to complete the immobilization of the material.

The method comprises the steps of assembling the solid-phase NC film with a sample pad, a gold label pad, a PVC base plate, absorbent paper and a Mark paste (the sample pad, the gold label pad, the NC film, the PVC base plate, the absorbent paper and the Mark paste related by the invention are purchased from Shandong Kanghua biomedical science and technology Co., Ltd.), and cutting into test strips with the thickness of 3-4 mm by a slitter for later use.

The test strip for comparison is a treponema pallidum antibody detection test strip purchased from Shandong Kanghua biology, registration card number: national mechanical Standard 20153401404.

Before use, the test strip and the sample are returned to room temperature, and the test strip should be used within 1 hour of being placed at room temperature.

The test strip is flatly placed on a table, 70ul of serum sample to be tested is added, and the experimental result is observed in about 20 min. Only the T line color development detection result is negative, the T line and the C line color development show that the result is positive, and no color development indicates that the test strip is invalid and the detection result is invalid. The structure and the result of the test strip are illustrated in figure 7.

Example 4: recombinant antigen TP34S as coating antigen for detecting TP antibody by gold-labeled chromatography platform

(1) Sensitivity of the probe

The optimal coating concentration is optimized by the same method, quality control serum of strong positive, medium positive and weak positive is detected, the color development of the quality control serum reaches the level of a contrast strip of a treponema pallidum antibody detection test strip, the working concentration of the three recombinant antigen coatings is shown in a table 3, the result is shown in a figure 8, from the viewpoint of the working concentration of the coatings, TP34S can reach the level equivalent to the color development of the contrast test strip only by coating 0.5mg/ml, and the sensitivity of TP34S is superior to that of TP17 and TP 17S.

TABLE 3 recombinant antigen coating concentration

(2) Specificity of

The recombinant antigens TP17, TP17S and TP34S are used as coating antigens and matched with a gold-labeled pad to prepare a finished gold-labeled chromatographic test strip by the same method respectively, and 2000 clinical negative serums and 100 positive serums are detected by comparing the finished gold-labeled chromatographic test strip with the Kanghua biological treponema pallidum antibody test strip. From the following table, it can be seen that the false positive rate of TP17 is 0.5%, the false positive rate of TP17S is 0.45%, the false positive rate of TP34S is 0.15%, the detection rate of positive sample TP17 is 98%, the detection rate of TP17S is 98%, and the detection rate of TP34S is 100%. Specific sorting: TP34S is more than or equal to that of the contrast bar, and more than or equal to TP17S is more than or equal to that of TP 17. The pGEX-4T-1 vector has a GST tag, the GST tag can cover a part of antigen epitope, and the TP34S adds a TP17S sequence on the basis of TP17S, so that the covered antigen epitope can be supplemented, the omission can be reduced, the positive detection rate can be improved, and meanwhile, the cysteine forming a disulfide bond is mutated through self design, and the false positive problem caused by protein aggregation is reduced. TP34S specificity was significantly improved over the other recombinant antigens and was superior to the coating antigen used in the control strips.

TABLE 4 results of specific detection

Name of antigen	Number of negatives	Number of positive	Number of false positive	Rate of false positive	Rate of positive detection
						TP17	1990(2000)	98(100)	10	0.5％	98％
TP17S	1991(2000)	98(100)	9	0.45％	98％
						TP34S	1997(2000)	100(100)	3	0.15％	100％
Contrast bar	1994(2000)	100(100)	6	0.3％	100％

(3) Stability of

The recombinant antigen is diluted to working concentration, examined at 37 ℃ for 7 days respectively, taken out and coated on an NC membrane, and prepared into a colloidal gold test strip together with an outsourcing gold label pad, and then the colloidal gold test strip and the recombinant antigen stored at 4 ℃ simultaneously detect the same negative and positive quality control serum under the same condition, and the result is shown in table 5. After the recombinant antigen is coated on an NC membrane, a destructive experiment is carried out for 7 days at 55 ℃, test paper strips are prepared and then placed at room temperature for comparison and detection of the same negative and positive quality control serum, and the results are shown in Table 6. Experiments show that the recombinant antigen has good liquid stability at 37 ℃ and no obvious difference, and the disruption experiment of immobilizing the recombinant antigen on an NC membrane at 55 ℃ shows that the recombinant antigen TP34S is superior to TP17 and TP 17S.

TABLE 5 detection results of 37 ℃ stability of recombinant antigens

TABLE 6 results of 55 ℃ disruption of recombinant antigen coated onto NC membrane

Note: in tables 5 and 6, the signal intensity + -represents very weak positive, + represents medium positive, + represents strong positive, -represents negative.

The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Sequence listing

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aaatctgctc cgtctccgct gacctaccgt ggtacctgga tggttcgtga agacggtatc 240

gttgaactgt ctctggtttc ttctgaacag tctaaagctc cgcacgaaaa agaactgtac 300

gaactgatcg actctaactc tgttcgttac atgggtgctc cgggtgctgg taaaccgtct 360

aaagaaatgg ctccgttcta cgttctgaaa aaaaccaaaa aa 402

<210> 6

<211> 831

<212> DNA

<213> Artificial Sequence

<400> 6

gtttcttgca ccaccgtttg cccgcacgct ggtaaagcta aagctgaaaa agttgaatct 60

gctctgaaag gtggtatctt ccgtggtacc ctgccggctg ctgactctcc gggtatcgac 120

accaccgtta ccttcaacgc tgacggtacc gctcagaaag ttgaactggc tctggaaaaa 180

aaatctgctc cgtctccgct gacctaccgt ggtacctgga tggttcgtga agacggtatc 240

gttgaactgt ctctggtttc ttctgaacag tctaaagctc cgcacgaaaa agaactgtac 300

gaactgatcg actctaactc tgttcgttac atgggtgctc cgggtgctgg taaaccgtct 360

aaagaaatgg ctccgttcta cgttctgaaa aaaaccaaaa aagaattcgt ttcttgcacc 420

accgtttgcc cgcacgctgg taaagctaaa gctgaaaaag ttgaatctgc tctgaaaggt 480

ggtatcttcc gtggtaccct gccggctgct gactctccgg gtatcgacac caccgttacc 540

ttcaacgctg acggtaccgc tcagaaagtt gaactggctc tggaaaaaaa atctgctccg 600

tctccgctga cctaccgtgg tacctggatg gttcgtgaag acggtatcgt tgaactgtct 660

ctggtttctt ctgaacagtc taaagctccg cacgaaaaag aactgtacga actgatcgac 720

tctaactctg ttcgttacat gggtgctccg ggtgctggta aaccgtctaa agaaatggct 780

ccgttctacg ttctgaaaaa aaccaaaaaa caccaccacc accaccacta a 831

<210> 7

<211> 402

<212> DNA

<213> Artificial Sequence

<400> 7

gtttcttgca ccaccgtttg cccgcacgct ggtaaagcta aagctgaaaa agttgaatgc 60

gctctgaaag gtggtatctt ccgtggtacc ctgccggctg ctgactgccc gggtatcgac 120

accaccgtta ccttcaacgc tgacggtacc gctcagaaag ttgaactggc tctggaaaaa 180

aaatctgctc cgtctccgct gacctaccgt ggtacctgga tggttcgtga agacggtatc 240

gttgaactgt ctctggtttc ttctgaacag tctaaagctc cgcacgaaaa agaactgtac 300

gaactgatcg actctaactc tgttcgttac atgggtgctc cgggtgctgg taaaccgtct 360

aaagaaatgg ctccgttcta cgttctgaaa aaaaccaaaa aa 402

<210> 8

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 8

cgggatccgt ttcttgcacc accgttt 27

<210> 9

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 9

ccctcgaggt ggtggtggtg gtggtgtttt ttggtttttt tcagaacgta 50

<210> 10

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 10

cgggatccgt ttcttgcacc accgtttgcc cgcacgctgg taaagctaaa gctgaaa 57

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 11

tgctgactct ccgggtatcg acacca 26

<210> 12

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 12

tggtgtcgat acccggagag tcagca 26

<210> 13

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 13

cggaattctt ttttggtttt tttcagaacg ta 32

<210> 14

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 14

cggaattcgt ttcttgcacc accgtttgcc cgca 34

Claims

1. A treponema pallidum recombinant antigen is characterized in that the amino acid sequence of the treponema pallidum recombinant antigen is shown as SEQ ID NO. 3.

2. The method for preparing a recombinant antigen of treponema pallidum according to claim 1, comprising the steps of:

(2) amino acid mutations were performed on the basis of TP 17: mutating the N-terminal 20 th cysteine and 36 th cysteine to serine, namely: C20S, C36S; the target gene product is TP17S, and the corresponding nucleotide sequence is shown in SEQ ID NO. 5;

(6) separating and purifying to obtain the recombinant antigen.

3. A gold-labeled chromatography test strip for detecting syphilis, which is characterized in that the coating antigen of the test strip is the treponema pallidum recombinant antigen of claim 1.