Treponema pallidum recombinant antigen, nucleic acid molecule, recombinant expression vector, host cell and application

Abstract

The invention discloses a treponema pallidum recombinant antigen, a nucleic acid molecule, a recombinant expression vector, a host cell and application, and relates to the technical field of medical diagnosis, the treponema pallidum recombinant antigen comprises at least one TP47 antigen, and the amino acid of at least one of V50, F176, V196, L212, V222, F244, L291, M292, V345 and V404 in a corresponding TP47 amino acid sequence of the TP47 antigen is deleted or mutated into A, G, T, S, Y, N, Q, D, E, K, R, H or C. The treponema pallidum recombinant antigen provided by the invention is relatively good in thermal stability, and is relatively good in detection specificity and relatively accurate in detection result when being used for detecting an anti-treponema pallidum antibody.

Description

Technical Field

[0001] The present invention relates to the technical field of medical diagnosis, and particularly relates to a recombinant antigen of Treponema pallidum, a nucleic acid molecule, a recombinant expression vector, a host cell and applications thereof. Background Art

[0002] Treponema pallidum (TP), also known as Treponema pallidum, is the pathogen that causes syphilis, a sexually transmitted disease. The natural course of syphilis is divided into primary, secondary and latent stages. Primary syphilis usually presents as a painless ulcer (chancre), followed by systemic spread of Treponema pallidum, often manifested as extensive maculopapular rashes and non-specific systemic symptoms, which are most obvious in the secondary stage and can resolve spontaneously without treatment. The latent stage is divided into early (<2 years) and late (>2 years), and the disease shows no symptoms during this stage. When the disease reappears (tertiary stage), it can affect almost all organs, including the central nervous system (neurosyphilis), skin, bones (gummas) and cardiovascular system.

[0003] At present, there is still no specific vaccine against TP. Therefore, effective diagnosis, timely treatment and control of the source of infection are still important ways to control the spread of syphilis. Since Treponema pallidum is difficult to culture in vitro, the current effective strategy for diagnosing syphilis is based on immunological detection methods. The antigens used for immunological diagnosis mainly include TP15, TP17, and TP47. Among them, the TP47 antigen has high immunogenicity and can trigger an early humoral response three to six days after syphilis infection. Anti-TP47 antibodies can be detected in all stages of untreated syphilis.

[0004] However, the TP47 antigen has poor thermal stability and is extremely prone to forming aggregates, which affects its binding ability to antibodies and makes the test results inaccurate. The existing methods for improving the stability of the TP47 antigen mainly include two types: (1) fusion expression, that is, using the fusion expression scheme of chaperone proteins to increase the stability of the antigen, but the fusion expression method may cause potential epitope steric hindrance problems, leading to missed detection; (2) disulfide bond mutation, that is, mutating the disulfide bond may be able to improve the aggregation caused by intermolecular disulfide bonds, but after removing the disulfide bond, the hydrophobic instability state of the antigen itself cannot be completely improved. In addition, according to the results of the present invention, disulfide bonds do not play a dominant role in the instability of TP47. Therefore, how to ensure the thermal stability of the TP47 antigen remains a difficult point. Summary of the Invention

[0005] The main object of the present invention is to provide a recombinant antigen of Treponema pallidum, a nucleic acid molecule, a recombinant expression vector, a host cell and applications thereof, aiming to solve the problem of poor thermal stability of the TP antigen in the prior art.

[0006] To achieve the above object, the present invention provides a recombinant antigen of Treponema pallidum, comprising at least one TP47 antigen, wherein the TP47 antigen has an amino acid deletion at least at one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence, and the sequence of the TP47 antigen is shown in SEQ ID NO.1 to SEQ ID NO.3. In the technical solution of the present invention, the recombinant antigen of Treponema pallidum provided by the present invention has good thermal stability. When it is used to detect anti-Treponema pallidum antibodies, the detection specificity is good and the detection result is relatively accurate.

[0007] It should be noted that the recombinant antigen of Treponema pallidum in the present invention can be a TP47 antigen with an amino acid deletion at any one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence; it can also be a TP47 antigen with an amino acid deletion at any two or more of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence; it can also be two or more TP47 antigens, and the deletion sites of each TP antigen in the corresponding TP47 amino acid sequence can be the same or different, all within the protection scope of the present invention.

[0008] The TP47 recombinant antigen of the present invention can be obtained by designing the corresponding nucleotide sequence according to its amino acid sequence, ligating the nucleotide sequence to the corresponding expression vector, and stably expressing it in a host cell.

[0009] In some embodiments of the present invention, the TP47 antigen has an amino acid deletion at least at one of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence. That is, the TP47 antigen has an amino acid deletion at any one of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence, or it can be a TP47 antigen with an amino acid deletion at any two or more of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence. Compared with the TP47 antigens with deletions at other sites, the TP47 antigens with deletions at the above sites have better thermal stability and better detection accuracy.

[0010] The present invention provides a recombinant antigen of Treponema pallidum, comprising at least one TP47 antigen, wherein at least one amino acid of the TP47 antigen at V50, F176, V196, L212, V222, F244, L291, M292, V345, or V404 in the corresponding TP47 amino acid sequence is mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C, and the sequence of the TP47 antigen is as shown in SEQ ID NO.4 to SEQ ID NO.6.

[0011] In the technical solution of the present invention, the recombinant antigen of Treponema pallidum in the present invention may be a single TP47 antigen, in which any one of the amino acids at V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence is mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C; it may also be a single TP47 antigen, in which any two or more amino acids at V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence are mutated, and each site may be mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C; it may also be two or more TP47 antigens, and the mutation sites of each TP antigen in the corresponding TP47 amino acid sequence may be the same or different, all within the protection scope of the present invention. The recombinant antigen of Treponema pallidum provided by the present invention has good thermal stability. When it is used to detect antibodies against Treponema pallidum, the detection specificity is good and the detection result is relatively accurate.

[0012] In some embodiments of the present invention, the amino acid at at least one of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence of the TP47 antigen is mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C. That is, the amino acid at any one of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence of the TP47 antigen is mutated to A (alanine), G (glycine), T (threonine), S (serine), Y (tyrosine), N (asparagine), Q (glutamine), D (aspartic acid), E (glutamic acid), K (lysine), R (arginine), H (histidine), or C (cysteine). It can also be that the amino acids at any two or more of V50, V196, L212, V222, L291, V345, V404, L212, and L291 in the corresponding TP47 amino acid sequence of the TP47 antigen are mutated, and each site can be mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C. Compared with the TP47 antigens with mutations at other sites, the TP47 antigens with the above site mutations have better thermal stability and better detection accuracy.

[0013] The present invention provides a recombinant antigen of Treponema pallidum, comprising at least 1 TP47 antigen, wherein the amino acid at at least any one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence of the TP47 antigen is deleted; and, except for the deleted sites, the amino acid at at least one of the remaining amino acids among V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 is mutated to A, G, T, S, Y, N, Q, D, E, K, R, H, or C; the sequence of the TP47 antigen is as shown in SEQ ID NO.7 to SEQ ID NO.8.

[0014] In the technical solution of the present invention, the recombinant antigen of Treponema pallidum in the present invention may be a TP47 antigen, in which any one of the amino acids at positions V50, F176, V196, L212, V222, F244, L291, M292, V345 and V404 in the corresponding TP47 amino acid sequence is deleted, and except for the deletion site, any one of the remaining amino acids is mutated to A, G, T, S, Y, N, Q, D, E, K, R, H or C; it may also be a TP47 antigen, in which any one of the amino acids at positions V50, F176, V196, L212, V222, F244, L291, M292, V345 and V404 in the corresponding TP47 amino acid sequence is deleted, and except for the deletion site, any two or more of the remaining amino acids are mutated, and each mutation site can be mutated to A, G, T, S, Y, N, Q, D, E, K, R, H or C; it may also be a TP47 antigen, in which any two or more (less than ten) of the amino acids at positions V50, F176, V196, L212, V222, F244, L291, M292, V345 and V404 in the corresponding TP47 amino acid sequence are deleted, and except for the deletion site, any one or more of the remaining amino acids are mutated, and each mutation site can be mutated to A, G, T, S, Y, N, Q, D, E, K, R, H or C; it may also be two or more TP47 antigens, and the deletion and mutation sites of each TP antigen in the corresponding TP47 amino acid sequence may be the same or different, and all are within the protection scope of the present invention. The recombinant antigen of Treponema pallidum provided by the present invention has good thermal stability. When it is used to detect anti-Treponema pallidum antibodies, the detection specificity is good and the detection result is relatively accurate.

[0015] It should be noted that the recombinant antigen of Treponema pallidum provided by the present invention can also be used in combination with other recombinant antigens of Treponema pallidum such as TP15, TP17, and TP44.5, and the combination methods include but are not limited to covalent bond connection, chimeric expression or single fragment combination.

[0016] In some embodiments of the present invention, the TP47 antigen is mutated from a TP47 polypeptide comprising the TP47 amino acid sequence from position 1 to 434 or from position 26 to 434, and the sequence of the TP47 polypeptide is shown in SEQ ID NO.9 or SEQ ID NO.12.

[0017] The present invention also provides a recombinant expression vector, which comprises the aforementioned nucleic acid; preferably, the recombinant expression vector comprises an integrative pET-32a vector, pET-41a vector, pQE-30 vector, pBADHisA vector, PGS-21a vector or pSmartI vector. An integrative vector refers to a vector that can integrate an exogenous gene into the genome of a host cell. The above-mentioned recombinant expression vector can preferably integrate the nucleic acid sequence corresponding to the Treponema pallidum antigen into the genome of the host cell.

[0018] The present invention also provides a recombinant expression host cell, which comprises the aforementioned recombinant expression vector; preferably, the recombinant expression host cell comprises Escherichia coli, mammalian cells, insect cells or yeast. More preferably, the recombinant expression host cell is Escherichia coli. The above-mentioned recombinant expression host cell can stably express the Treponema pallidum antigen.

[0019] The present invention also provides a conjugate, which comprises the aforementioned recombinant Treponema pallidum antigen; preferably, the conjugate further comprises a solid support, a detectable label or a binding partner conjugated to the recombinant Treponema pallidum antigen; more preferably, the solid support comprises magnetic particles, microtiter plates or cellulose membranes; and / or, the detectable label comprises metal particles, fluorescent labels or chromophore labels; and / or, the binding partner comprises luciferase or horseradish peroxidase.

[0020] That is to say, the recombinant Treponema pallidum antigen in the detection reagent can be a single antigen or a conjugate formed by the antigen combined with other substances. The other substances can be solid supports such as magnetic particles, microtiter plates or cellulose membranes; detectable labels such as metal particles, fluorescent labels, chromophore labels; or binding partners such as luciferase or horseradish peroxidase. When the above-mentioned conjugate is used to detect anti-TP antibodies, it has good specificity and can reduce other types of non-specific binding.

[0021] The present invention also provides a detection reagent for anti-Treponema pallidum antibodies, which comprises the aforementioned recombinant Treponema pallidum antigen. The recombinant Treponema pallidum antigen includes the TP47 antigen with any amino acid site deletion or mutation among V50, F176, V196, L212, V222, F244, L291, M292, V345, V404, and also has a high immunogenicity of the TP47 antigen. Therefore, it can recognize anti-Treponema pallidum antibodies and is used to detect anti-Treponema pallidum antibodies in a sample. At the same time, the detection reagent of the present invention has all the beneficial effects of the above-mentioned recombinant Treponema pallidum antigen.

[0022] The present invention also provides the use of the aforementioned recombinant antigen, nucleic acid molecule, recombinant expression vector, recombinant expression host cell or conjugate of Treponema pallidum in the preparation of an in vitro diagnostic product for detecting anti-Treponema pallidum antibodies or antigens. The in vitro diagnostic products of the present invention include products for immunoassay, and are used in methods such as enzyme-linked immunosorbent assay (ELISA), fluorescence immunochromatography, colloidal gold immunochromatography, chemiluminescence assay, etc. Specific products include chromatographic test strips, magnetic beads, reagent kits, etc. The in vitro diagnostic products of the present invention have all the beneficial effects of the aforementioned recombinant antigen of Treponema pallidum. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0024] Figure 1 SDS-PAGE diagram of the recombinant antigen in Example 1 provided by the present invention;

[0025] Figure 2 SDS-PAGE diagram of the recombinant antigen in Example 358 provided by the present invention;

[0026] Figure 3 SDS-PAGE diagram of the wild-type TP47 antigen in Comparative Example 1 provided by the present invention.

[0027] The realization, functional features and advantages of the object of the present invention will be further described in conjunction with the embodiments and with reference to the drawings. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase. In addition, the meaning of "and / or" appearing throughout the text includes three parallel scenarios. Taking "A and / or B" as an example, it includes scenario A, or scenario B, or the scenario where both A and B are satisfied simultaneously. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions conflicts with each other or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] The following further details the technical solutions of the present invention in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0030] Experimental materials

[0031] LB medium: 1% tryptone, 0.5% yeast extract, 1% NaCl.

[0032] LB agar medium: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar.

[0033] LB medium containing 100 μg / mL kanamycin: After the above LB medium is sterilized and cooled to 45 - 50 °C, kanamycin with a final concentration of 100 μg / mL is added and mixed evenly.

[0034] LB resistance plate containing 100 μg / mL kanamycin (i.e., LB solid medium containing 100 μg / mL kanamycin): After the above LB agar medium is sterilized and cooled to 45 - 50 °C, kanamycin with a final concentration of 100 μg / mL is added, mixed evenly, and then poured into a petri dish to solidify naturally.

[0035] Example 1 Preparation of V50-deleted recombinant antigen

[0036] 1. Obtain the nucleic acid sequence corresponding to the TP47 antigen polypeptide

[0037] The synthetic sequence was constructed on the pET-28a vector based on the amino acid sequence of the natural Treponema pallidum TP47 antigen (derived from Uniprot Entry: P29723). The corresponding nucleotide sequence was constructed according to the above amino acid sequence, and its nucleic acid sequence is shown in SEQ ID NO.11 for PCR amplification.

[0038] 2. Design of deletion mutation primers

[0039] Based on the nucleotide sequence constructed in step 1, a pair of PCR mutation primers targeting the deletion of V50 were designed and named F1 and R1. The specific sequences are as follows:

[0040] F1: 5’-GATCTGCTGGCG-3’;

[0041] R1: 5’-CAGATCGCGGCTC-3’.

[0042] 3. PCR deletion mutation

[0043] PCR amplification mutation was carried out using the nucleotide sequence constructed in step 1 and the mutation primers in step 2. The amplification system was: 5 μL of 10×HIFI Buffer (high-fidelity buffer), 1 μL of template DNA (i.e., the nucleotide sequence constructed in step 1), 5 μL of dNTPs (2 mM each), 10 μM primer F1, 10 μM primer R1, 25 mM MgSO4, 1 U / μL HIFI, and the rest was ddH2O, with a total of 50 μL. The amplification conditions were: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C at 1 kb / min (the denaturation, annealing, and extension processes were cycled 15 times), extension at 72°C for 10 min, and holding at 16°C. HIFI was purchased from Beijing TransGen Biotech Co., Ltd., and the product number was AP131-01. After PCR amplification mutation, the mutant sequence was obtained.

[0044] 4. Construction of fusion tags and engineering bacteria

[0045] The plasmid with correct sequencing was transformed into the BL21(DE3) Escherichia coli host bacteria by the CaCl2-mediated method to obtain engineering bacteria.

[0046] 5. Fermentation of engineering bacteria

[0047] The engineering bacteria obtained in step 4 were spread on a kanamycin LB resistance plate with a concentration of 100 μg / mL and cultured overnight at 37°C. Subsequently, single colonies were selected from the plate and inoculated into an LB medium containing 100 μg / mL of kanamycin, and shaken at 220 rpm until the OD 600It is about 0.8. Isopropyl-β-D-thiogalactoside (IPTG) is added to a final concentration of 0.5 mM, and induction is carried out at 25 °C for 8 hours to obtain the induced engineered bacteria.

[0048] 6. Extract and purify the recombinant antigen

[0049] The induced engineered bacteria are centrifuged at 4 °C and 8000 rpm for 30 min to collect the bacterial cells. Each gram of the bacterial cells is resuspended in 10 mL of lysis buffer (1× phosphate buffer, 3 mM EDTA, pH = 7.4, 1 mM phenylmethylsulfonyl fluoride), and the cells are disrupted by high-pressure homogenization at 700 bar. Then, centrifugation is carried out at 4 °C and 18000 rpm for 30 minutes to collect the cell precipitate and supernatant. After verification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the target protein (recombinant antigen) is expressed in the cell supernatant. The obtained supernatant is filtered through a 0.45 μm microporous membrane to obtain the filtrate.

[0050] The specific steps for purifying the filtrate include: after balancing the Ni-IDA affinity column with the equilibration buffer (1× phosphate buffer, 3 mM EDTA, pH = 7.4), buffer I (1× phosphate buffer, 3 mM EDTA, 30 mM imidazole, pH 7.4) is added to remove the unbound miscellaneous proteins, and finally buffer II (1× phosphate buffer, 3 mM EDTA, 300 mM imidazole, pH 7.4) is used to elute the target protein to obtain the purified recombinant antigen.

[0051] Subsequently, the purified recombinant antigen is dialyzed into the storage buffer (1× phosphate buffer, 150 mM NaCl, 3 mM EDTA, pH 7.4) using a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da. After dialysis, the protein concentration is detected by the bicinchoninic acid (BCA) method. After verification by SDS-PAGE gel pattern, the purity of the dialyzed recombinant antigen can reach more than 90% (as Figure 1 shown).

[0052] Examples 2 - 357

[0053] Compared with Example 1, Examples 2 - 357 are different in that the deletion sites are different and the PCR primers are different (the primers are adjusted corresponding to the deletion sites).

[0054] The differences between the recombinant antigens obtained in Examples 1 - 357 and the original TP47 antigen (the sequence is shown in SEQ ID NO.11) are shown in Table 1. For multiple deletion sites, the method of multi-step PCR can be used.

[0055] Table 1 Differences between the recombinant antigens in Examples 1 - 357 and the original TP47 antigen

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Example 358 Preparation of F176A Mutant Recombinant Antigen

[0062] 1. Design of F176A Mutant

[0063] According to the amino acid sequence, the corresponding codon-optimized original nucleotide sequence such as SEQ ID NO.11 was constructed for PCR amplification.

[0064] 2. Design of Mutation Primers

[0065] According to the nucleotide sequence constructed in step 1, a pair of PCR mutation primers targeting F176A were designed and named F2 and R2. The specific sequences are as follows:

[0066] F2: 5’-GTGCCGCATGAACTGAAAGGCATTG-3’;

[0067] R2: 5’-ATGCGGCACCGCCAC-3’.

[0068] 3. PCR Mutation

[0069] Using the nucleotide sequence constructed in step 1 and the mutation primers in step 2 for PCR amplification and mutation, the amplification system was: 5μL 10×HIFI Buffer, 1μL Templet DNA, 5μL dNTPs (2mM each), 10μM primer F1, 10μM primer R1, 25mM MgSO4, 1U / μL HIFI, and the rest was ddH2O, with a total of 50μL. The amplification conditions were: pre-denaturation at 94℃ for 3min, denaturation at 94℃ for 30sec, annealing at 56℃ for 30sec, extension at 72℃ at 1kb / min (the denaturation, annealing, and extension processes were cycled 15 times), extension at 72℃ for 10min, and hold at 16℃. After PCR amplification and mutation, the mutant sequence was obtained.

[0070] 4. Construction of Fusion Tags and Engineering Bacteria

[0071] The correctly sequenced stable expression vector was transformed into the BL21(DE3) Escherichia coli host bacterium by the CaCl2-mediated method to obtain the engineering bacterium.

[0072] 5. Engineering bacteria fermentation

[0073] Spread the engineered bacteria obtained in step 4 on a kanamycin LB resistant plate with a concentration of 100 μg / mL and culture overnight at 37°C. Subsequently, pick single colonies from the plate and inoculate them into an LB medium containing 100 μg / mL of kanamycin, and culture with shaking at 220 rpm until the OD 600 is about 0.8. Then add IPTG to a final concentration of 0.5 mM and induce at 25°C for 8 hours to obtain the induced engineered bacteria.

[0074] 6. Extraction and purification of recombinant antigen

[0075] Centrifuge the induced engineered bacteria at 4°C and 8000 rpm for 30 minutes to collect the bacterial cells. Resuspend each gram of the bacterial cells with 10 mL of lysis buffer (1× phosphate buffer, 3 mM EDTA, pH = 7.4, 1 mM phenylmethylsulfonyl fluoride), and disrupt the cells by high-pressure homogenization at 700 bar. Then centrifuge at 4°C and 18000 rpm for 30 minutes to collect the cell pellet and the supernatant. After verification by SDS-PAGE, the target protein (recombinant antigen) is expressed in the cell supernatant. Filter the obtained supernatant through a 0.45 μm microporous membrane to obtain the filtrate.

[0076] The specific steps for purifying the filtrate include: after equilibrating the Ni-IDA affinity column with equilibration buffer (1× phosphate buffer, 3 mM EDTA, pH = 7.4), add buffer I (1× phosphate buffer, 3 mM EDTA, 30 mM imidazole, pH 7.4) to remove the unbound miscellaneous proteins, and finally elute the target protein with buffer II (1× phosphate buffer, 3 mM EDTA, 300 mM imidazole, pH 7.4) to obtain the purified recombinant antigen.

[0077] Subsequently, dialyze the purified recombinant antigen into the storage buffer (1× phosphate buffer, 150 mM NaCl, 3 mM EDTA, pH 7.4) using a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da. After dialysis, detect the protein concentration using the bicinchoninic acid (BCA) method. After verification by SDS-PAGE gel pattern, the purity of the dialyzed recombinant antigen can reach over 90% (as Figure 2 shown).

[0078] Example 359 - 901

[0079] Compared with Example 358, Example 359 - 901 is different in that the mutation sites are different and the mutation primers are different (the mutation primers are adjusted according to the corresponding mutation sites).

[0080] The differences between the recombinant antigens obtained in Examples 359 to 901 and the original TP47 antigen (the sequence is shown in SEQ ID NO.11) are shown in Table 2. For multiple mutation sites, the method of step-by-step mutation of mutants can be used.

[0081] Table 2 Differences between the recombinant antigens in Examples 359 to 901 and the original TP47 antigen

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 902 prepares a recombinant antigen with V50 deletion and F176 mutation

[0090] The preparation method is the same as the deletion and mutation methods of the recombinant antigens described in Example 1 and Example 358.

[0091] Examples 903 to 1040

[0092] Compared with Example 1, Examples 903 to 1041 are different in that: the deletion sites and mutation sites are different, and the corresponding primers for mutation are also changed accordingly. The differences between the recombinant antigens obtained in Examples 903 to 1041 and the original TP47 antigen (the sequence is shown in SEQ ID NO.11) are shown in Table 3.

[0093] Table 3 Differences between the recombinant antigens obtained in Examples 903 to 1041 and the original TP47 antigen

[0094]

[0095] Comparative Example 1

[0096] This comparative example provides a preparation method of wild-type TP47 antigen. The differences between this comparative example and Example 358 are as follows:

[0097] Steps 2 and 3 in Example 358 are not carried out, and "the mutant sequence obtained in step 3" in step 4 is replaced by "the nucleotide sequence constructed in step 1"; and,

[0098] In step 5, the final concentration of IPTG induction is 0.1 mM, the induction temperature is 16 °C, and the induction time is 16 h; and,

[0099] In step 6, purification is performed using lysis buffer (20 mM Tris 8.0, 500 mM NaCl, 3 mM EDTA, 1 mM phenylmethylsulfonyl fluoride), purification equilibrium buffer (20 mM Tris 8.0, 500 mM NaCl, 3 mM EDTA), buffer I (20 mM Tris 8.0, 500 mM NaCl, 3 mM EDTA, 30 mM imidazole), buffer II (20 mM Tris 8.0, 500 mM NaCl, 3 mM EDTA, 300 mM imidazole), and storage buffer (20 mM Tris 8.0, 500 mM NaCl, 3 mM EDTA);

[0100] Finally, dialysis yields a wild-type TP47 antigen (the SDS-PAGE results are as Figure 3 shown), and its sequence is as shown in SEQ ID NO.9.

[0101] Performance test

[0102] The thermal stability of the antigens prepared in some of the examples and Comparative Example 1 was tested. The test method was: directly measuring the Tm value of the antigen, and the detection instrument was an Uncle protein stability analyzer. The test results are shown in Table 4.

[0103] Table 4 Tm values of antigens in some of the examples and Comparative Example 1

[0104]

[0105]

[0106] As can be seen from Table 4, the Tm value of the recombinant antigen in the example was increased to varying degrees compared with the Tm value of the antigen in Comparative Example 1. This indicates that the mutagenesis of the TP47 antigen can improve the thermal stability of the antigen itself.

[0107] The antigen reactivity in the examples and Comparative Example 1 was compared. The test method was the indirect ELISA method: The antigens in Comparative Example 1 and the examples were respectively diluted to the same concentration of 1 μg / mL with carbonate buffer (pH 9.5) to obtain 10 groups of antigen solutions. The following operations were performed for each group of the above antigen solutions: Add 100 μL of the antigen solution to each well of a 96-well plate, coat overnight at 4°C, and then wash the plate three times with PBST (1× phosphate buffer, 0.01% Tween-20, pH 7.4) washing solution; then add the blocking solution (5% BSA, PBST) to each well and block at 37°C for 1 hour; then wash away the blocking solution, pat dry, add 3 positive controls and 3 negative controls of TP47, add 100 μL to each well, and incubate at 37°C for 1 hour; after incubation, wash the plate 5 times with PBST and then add the goat anti-human secondary antibody labeled with horseradish peroxidase (HRP), incubate at 37°C for half an hour; then wash the plate 5 times with PBST, add 50 μL of the chromogenic solution A (hydrogen peroxide was added to sodium phosphate buffer to a concentration of 0.74 mg / mL) and solution B (tetramethylbenzidine dissolved in dimethyl sulfoxide) as a 1:1 mixture, and develop color at 37°C in the dark for 5 minutes; then add 50 μL of 2M sulfuric acid termination solution to terminate the reaction; finally, use an enzyme-linked immunosorbent assay (ELISA) reader to read the OD value at a wavelength of 450 nm. The test results are shown in Table 5.

[0108] Table 5 ELISA detection of anti-TP antibodies by recombinant antigens in some examples and Comparative Example 1

[0109]

[0110] As can be seen from Table 5, the specificity of the mutant recombinant antigen did not change significantly compared with that of Comparative Example 1.

[0111] To evaluate the thermal accelerated stability, after antigen-coated magnetic beads, the coated magnetic beads were thermally accelerated at 37°C for 3 days, and then 3 positive controls and 3 negative controls of TP47 were evaluated by the indirect chemiluminescence method. The wild-type antigen of Comparative Example 1 without thermal acceleration was used as the control group. The test results are shown in Table 6.

[0112] Table 6 Detection results of antigens for quality control products in some examples and Comparative Example 1

[0113]

[0114]

[0115] As can be seen from Table 6, after thermal acceleration at 37°C for 3 days, the detection activity of the antigen in Comparative Example 1 decreased by nearly 77%. And the examples after mutation all had different degrees of improvement in the magnetic bead thermal acceleration.

[0116] The above are only the preferred embodiments of the present invention, and do not thus limit the patent scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the patent protection scope of the present invention.

Claims

1. A recombinant antigen of Treponema pallidum, characterized in that, Comprising at least one TP47 antigen, wherein the TP47 antigen has an amino acid deletion at at least one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence, and the sequence of the TP47 antigen is as shown in SEQ ID NO.1 to SEQ ID NO.

3.

2. A recombinant antigen of Treponema pallidum, characterized in that, Comprising at least one TP47 antigen, wherein the TP47 antigen has an amino acid mutation to A, G, T, S, Y, N, Q, D, E, K, R, H, or C at at least one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence, and the sequence of the TP47 antigen is as shown in SEQ ID NO.4 to SEQ ID NO.

6.

3. A recombinant antigen of Treponema pallidum, characterized in that, Comprising at least one TP47 antigen, wherein the TP47 antigen has an amino acid deletion at at least any one of V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 in the corresponding TP47 amino acid sequence; and Except for the deletion sites, at least one of the remaining amino acids among V50, F176, V196, L212, V222, F244, L291, M292, V345, and V404 has an amino acid mutation to A, G, T, S, Y, N, Q, D, E, K, R, H, or C; The sequence of the TP47 antigen is as shown in SEQ ID NO.7 to SEQ ID NO.

8.

4. The recombinant antigen of Treponema pallidum according to any one of claims 1 to 3, characterized in that, The TP47 antigen is mutated from a TP47 polypeptide comprising the TP47 amino acid sequence at positions 1-434 or 26-434, and the sequence of the TP47 polypeptide is as shown in SEQ ID NO.9 or SEQ ID NO.

12.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule is used to encode the Treponema pallidum recombinant antigen according to any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid according to claim 5; Preferably, the recombinant expression vector comprises an integrative pET-32a vector, pET-41a vector, pQE-30 vector, pBADHisA vector, PGS-21a vector, or pSmartI vector.

7. A recombinant expression host cell, characterized in that, The recombinant expression host cell comprises the recombinant expression vector according to claim 6; Preferably, the recombinant expression host cell comprises Escherichia coli, mammalian cells, insect cells, or yeast.

8. A conjugate, characterized in that, The conjugate comprises the Treponema pallidum recombinant antigen according to any one of claims 1 to 4; Preferably, the conjugate further comprises a solid support, a detectable label, or a binding partner conjugated to the Treponema pallidum recombinant antigen; More preferably, the solid support comprises magnetic particles, microtiter plates, or cellulose membranes; and / or, the detectable label comprises metal particles, fluorescent labels, or chromophore labels; and / or, the binding partner comprises luciferase or horseradish peroxidase.

9. A detection reagent for anti - Treponema pallidum antibody, characterized in that, The detection reagent for anti - Treponema pallidum antibody comprises the recombinant antigen of Treponema pallidum as described in any one of claims 1 to 4.

10. Use of a recombinant antigen of Treponema pallidum as described in any one of claims 1 to 4, or a nucleic acid molecule as described in claim 5, or a recombinant expression vector as described in claim 6, or a recombinant expression host cell as described in claim 7, or a conjugate as described in claim 8 in the preparation of an in vitro diagnostic product for detecting anti - Treponema pallidum antibody or antigen.

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