CN111575308A - Treponema pallidum recombinant chimeric antigen and preparation method and application thereof - Google Patents

Treponema pallidum recombinant chimeric antigen and preparation method and application thereof Download PDF

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CN111575308A
CN111575308A CN202010451493.6A CN202010451493A CN111575308A CN 111575308 A CN111575308 A CN 111575308A CN 202010451493 A CN202010451493 A CN 202010451493A CN 111575308 A CN111575308 A CN 111575308A
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干盈盈
韩新鹏
吕志强
罗琴
文丹华
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Sichuan Maccura Biological New Material Technology Co ltd
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Abstract

The invention relates to a recombinant chimeric antigen of treponema pallidum, which is formed by connecting an amino acid sequence shown as SEQ ID NO.12, a first flexible linker, an amino acid sequence shown as SEQ ID NO.9, a second flexible linker and an amino acid sequence shown as SEQ ID NO.13 in series. The recombinant chimeric antigen can be used for serological detection of treponema pallidum antibodies and has improved dissolving expression capacity, activity and thermal stability. The invention also relates to a method for preparing the recombinant chimeric antigen.

Description

Treponema pallidum recombinant chimeric antigen and preparation method and application thereof
Technical Field
The invention relates to the field of immunological detection, and in particular relates to a treponema pallidum recombinant chimeric antigen.
Background
Syphilis is a chronic and systemic sexually transmitted disease caused by Treponema pallidum (TP; also called Treponema pallidum), and is listed as a disease species for the prevention and management of b-type infectious diseases in the national common people's republic of china. The transmission route is mainly sexual transmission, and clinically syphilis includes primary syphilis, secondary syphilis, tertiary syphilis, latent syphilis, congenital syphilis (fetal syphilis) and the like.
Syphilis is an epidemic worldwide, estimated by the WHO, there are about 1200 million new cases worldwide each year, mainly concentrated in south asia, southeast asia and sub-saharan africa. Syphilis has grown rapidly in our country in recent years, and has become a venereal disease with the largest number of reported cases. Among the reported syphilis, latent syphilis is predominant, primary and secondary syphilis are also common, and the number of reported cases of congenital syphilis is also increasing. Syphilis is a unique disease for human beings, and both dominant and recessive syphilis patients are infectious agents.
Clinical symptoms and signs of a human body infected by treponema pallidum have no obvious specificity and are difficult to distinguish by the symptoms and the signs, so that the diagnosis is mainly carried out clinically by depending on the result of a serological experiment.
Currently, there are two main types of commonly used immunological detection methods for treponema pallidum. A non-treponema pallidum antigen serum test, such as venereal disease research laboratory test (VDRL), rapid plasma reactive hormone circular card test (RPR), toluidine red unheated serum reactive hormone test (TRUST), etc. Because the method adopts normal bovine myocardial cardiolipin as an antigen, the method has the problem of low specificity, and false positive reactions can be generated in serum positive to anticardiolipin antibodies and hyperlipidemia.
Another type of immunological detection method for treponema pallidum is treponema pallidum antigen serum test, such as treponema pallidum gelatin agglutination Test (TPPA), etc., which adopts Nichols strain treponema as antigen, but since treponema pallidum can not be artificially cultured in vitro so far, the antigen source difficulty is high, the test operation is complicated, and the method is not easy to popularize.
In order to provide a stable source of treponema pallidum antigens, several studies have been carried out to try to express antigenic proteins (such as TP15, TP17 and TP47) by fusion to provide a substitute for natural treponema pallidum. However, these recombinant antigens have some defects in the preparation process or in the process of immunoassay, which limits the application of the recombinant chimeric antigens in the detection of treponema pallidum.
Therefore, in the field of immunological detection of treponema pallidum, there is a strong demand for improvement of the recombinant chimeric antigen.
Disclosure of Invention
In view of the above, in a first aspect, the present invention provides a method for preparing a recombinant chimeric antigen of treponema pallidum, comprising:
1) introducing a polynucleotide encoding the recombinant chimeric antigen into a host cell, or transferring a polynucleotide construct comprising the polynucleotide into a host cell;
2) culturing the host cell of step 1) under conditions suitable for the production of a recombinant chimeric antigen; and
3) optionally isolating the recombinant chimeric antigen from the culture obtained in step 2),
wherein the recombinant chimeric antigen sequentially comprises an amino acid sequence of a TP17 fragment, a first flexible linker, a TP15 fragment, a second flexible linker and a TP47 fragment from an N end to a C end,
wherein the amino acid sequence of the TP17 fragment is shown as SEQ ID NO.12, the amino acid sequence of the TP15 fragment is shown as SEQ ID NO.9, and the amino acid sequence of the TP47 fragment is shown as SEQ ID NO. 13.
The TP17 fragment, the TP15 fragment and the TP47 fragment are firstly removed by signal peptide on the basis of wild-type antigen; subsequently, TP17 and TP15 were N-terminally truncated, and TP47 was N-terminally and C-terminally truncated; finally, for TP17, specific mutations were further introduced at positions 42 and 58, and for the TP47 fragment, specific mutations were further introduced at residue 315.
In a specific embodiment, the polynucleotide sequence comprises, in order from 5 'to 3', the nucleotide sequence shown in SEQ ID No.26, the nucleotide sequence encoding the first flexible linker, the nucleotide sequence shown in SEQ ID No.23, the nucleotide sequence encoding the second flexible linker, and the nucleotide sequence shown in SEQ ID No. 27.
In particular embodiments, the polynucleotide construct carries a SUMO tag thereon.
In a specific embodiment, the host cell carries a SUMO tag.
In some embodiments, the preparation method of the present invention additionally comprises a step of removing the SUMO tag. For example, the SUMO tag may be removed by SUMO protease.
In a second aspect, the invention provides a recombinant chimeric antigen of treponema pallidum, which sequentially comprises an amino acid sequence shown as SEQ ID No.12, a first flexible linker, an amino acid sequence shown as SEQ ID No.9, a second flexible linker and an amino acid sequence shown as SEQ ID No.13 from the N end to the C end.
In one variant of the recombinant chimeric antigen of the invention, the recombinant chimeric antigen additionally carries a SUMO tag.
In a third aspect, the present invention provides a polynucleotide which:
a) the code sequentially comprises an amino acid sequence shown as SEQ ID NO.12, a first flexible linker, an amino acid sequence shown as SEQ ID NO.9, a second flexible linker and an amino acid sequence shown as SEQ ID NO.13 from the N end to the C end; or
b) Consists of a nucleotide sequence shown as SEQ ID NO.26, a nucleotide sequence coding a first flexible linker, a nucleotide sequence shown as SEQ ID NO.23, a nucleotide sequence coding a second flexible linker and a nucleotide sequence shown as SEQ ID NO. 27; or
c) Complementary to a) or b).
In some embodiments, the polynucleotide of the invention encodes a recombinant chimeric antigen of the invention or a protein of the invention.
In a fourth aspect, the present invention provides a polynucleotide construct comprising a polynucleotide of the invention.
In particular embodiments, the polynucleotide construct carries a SUMO tag thereon.
In a fifth aspect, the invention provides a host cell comprising a polynucleotide construct of the invention or a polynucleotide of the invention.
In a sixth aspect, the present invention provides a composition for detecting anti-treponema pallidum antibodies, the composition comprising the recombinant chimeric antigen of the invention.
In a seventh aspect, there is provided the use of a recombinant chimeric antigen of the invention in the preparation of a kit for the detection of anti-treponema pallidum antibodies.
In an eighth aspect, the present invention provides a recombinant chimeric antigen produced by the method of producing a recombinant chimeric antigen of treponema pallidum of the present invention.
The invention successfully constructs an improved treponema pallidum recombinant chimeric antigen by removing, further truncating and introducing specific mutation of the signal peptide, and has the following beneficial effects:
on one hand, compared with the conventional triple recombinant antigen, the soluble expression degree during antigen production is obviously improved.
On the other hand, the recombinant chimeric antigen has higher stability and activity, thereby widening the applicability range of the recombinant chimeric antigen and prolonging the storage period.
On the other hand, through national reference substance detection and clinical sample detection, the recombinant chimeric antigen provided by the invention is fully proved to have higher activity and can meet the requirement of development of immunodiagnostic reagents.
Drawings
FIG. 1 shows the results of the expression purification of the recombinant chimeric antigen 32-WTP17+15+47 of Treponema pallidum in example 2; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 2 shows the results of the expression and purification of the recombinant chimeric antigen of Treponema pallidum CN-WTP17+15+47 in example 3; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 3 shows the results of the purification of the recombinant chimeric antigen 32-DTP17+15+47 of Treponema pallidum in example 5; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 4 shows the results of the purification of the recombinant chimeric antigen of Treponema pallidum CN-DTP17+15+47 in example 6; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 5 shows the results of the purification of the recombinant chimeric antigen 28-DSTP17+15+47 of Treponema pallidum in example 8; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 6 shows the results of the purification of the recombinant chimeric antigen 32-DSTP17+15+47 of Treponema pallidum in example 9; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 7 shows the results of the purification of the recombinant chimeric antigen of Treponema pallidum CN-DSTP17+15+47 in example 10; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 8 shows the cleavage of recombinant chimeric antigen 32-DSTP17+15+47 of Treponema pallidum in example 9; from left to right, the following are in sequence: before the enzyme digestion of 32-DSTP17+15+47, and after the enzyme digestion of 32-DSTP17+15+ 47.
FIG. 9 shows the cleavage of recombinant chimeric antigen CN-DSTP17+15+47 of Treponema pallidum in example 10; from left to right, the following are in sequence: before the enzyme digestion of CN-DSTP17+15+47, and after the enzyme digestion of CN-DSTP17+15+ 47.
FIG. 10 shows the results of activity assay of recombinant chimeric antigen of Treponema pallidum.
FIG. 11 shows the results of the thermostability assay for the recombinant chimeric antigen of Treponema pallidum.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
As previously mentioned, natural treponema pallidum is difficult to produce and to obtain, which limits the development of serological tests based on treponema pallidum antigens. Although some recombinant antigens are prepared at present, the antigens have certain defects in the production and detection links, so that the development of detection reagents for treponema pallidum antibodies is limited, and the improvement space exists. Based on this, the present invention provides a novel truncated and mutated recombinant TP17, TP15 and TP47 chimeric antigen.
The recombinant chimeric protein comprises a TP17 fragment, a TP15 fragment and a TP47 fragment.
For a TP17 fragment, taking wild type TP17(GenBank accession number: AAA27472.1) as an initial sequence, firstly removing a signal peptide sequence, and reserving 25-156aa to obtain an amino acid sequence shown as SEQ ID NO. 1; then further carrying out N-terminal truncation, and reserving 35-156aa to obtain an amino acid sequence shown as SEQ ID NO. 8; then, C42S and C58S mutations are further carried out, and finally the TP17 fragment shown as SEQ ID NO.12 is obtained.
For the TP15 fragment, taking wild type TP15(GenBank accession number: AAD00207.1) as an initial sequence, firstly removing a signal peptide sequence, and reserving 17-158aa to obtain an amino acid sequence shown as SEQ ID NO. 2; then, the N-terminal truncation is further carried out, and 36-158aa is reserved, so that a TP15 fragment shown as SEQ ID NO.9 is obtained.
For the TP47 fragment, taking wild type TP47(GenBank accession number: AAA75016.1) as an initial sequence, firstly removing a signal peptide sequence, and reserving 20-434aa to obtain an amino acid sequence shown as SEQ ID NO. 3; then further carrying out truncation at the N end and the C end, and reserving 26-396aa to obtain an amino acid sequence shown as SEQ ID NO. 10; then, C315S mutation is further carried out, and the TP47 fragment shown as SEQ ID NO.13 is finally obtained.
The recombinant chimeric protein of the invention sequentially comprises a TP17 fragment, a TP15 fragment and a TP47 fragment, and the TP antigen fragments are connected with each other through a linker.
The linker involved in the invention is a flexible linker which can ensure that the structure and activity of each TP antigen fragment are not affected while the TP antigen fragment is connected. The present invention is not particularly limited with respect to the kind of the flexible linker, and exemplary flexible linkers may be glycine-bearing flexible linkers, such as GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, ggggggs, SGG, and the like.
In a specific embodiment, the fragment TP17 is connected to the fragment TP15 by a first flexible linker, and the fragment TP15 is connected to the fragment TP47 by a second flexible linker. The first flexible connector and the second flexible connector may be the same or different.
Unless specifically stated otherwise, the terms "first", "second", and the like in the present application are used only to distinguish a plurality of elements, and are not intended to represent any difference in importance or the like between the elements.
In a specific embodiment, the amino acid sequence of the recombinant chimeric antigen of the invention is shown in SEQ ID No. 14.
In a specific embodiment, the coding sequence of the recombinant chimeric antigen of the invention is shown in SEQ ID NO. 28.
It will be appreciated by those skilled in the art that polynucleotide constructs comprising a nucleic acid molecule encoding the above-described fusion protein operably linked to expression control sequences, and additionally comprising genetic elements such as an origin of replication and/or a selectable marker gene for maintenance and propagation in the respective host cell, are useful for the production of recombinant chimeric antigens by recombinant expression.
The polynucleotide construct of the invention may be, for example, a phage, plasmid or cosmid vector expressed in a prokaryotic host cell such as a bacterium (e.g., E.coli, Bacillus subtilis or Listeria); vectors for expression in yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris); baculovirus vectors for expression in insect cell systems (e.g., Sf9 cells); viral and plasmid vectors for expression in plant cell systems (e.g., Ti plasmid, cauliflower mosaic virus CaMV; tobacco mosaic virus TMV); and viral and plasmid vectors for expression in higher eukaryotic cells or organisms. In general, such vectors are commercially available, or are available from depositories, or are available according to the teachings of publications describing the sequences, structures and methods of production of these vectors, thereby enabling their use by those skilled in the art.
Exemplary polynucleotide constructs may be pET series vectors, pColdTMI vector and the like.
In a preferred embodiment, the polynucleotide construct carries a SUMO tag.
In the case of using the polynucleotide construct with the tag of SUMO as demonstrated in the examples below, the recombinant chimeric antibody produced had better activity and thermostability, while the degree of soluble expression was better.
Depending on the polynucleotide construct used, the corresponding host cell can be determined by one skilled in the art. For example, when selecting pET series vectors or pColdTMIn the case of the I vector, the skilled worker knows that it is suitable for expression in an E.coli host.
The recombinant chimeric antigen can be used for detecting treponema pallidum antibodies.
For example, the recombinant chimeric antigens of the invention can be labeled with a detectable label, and any label and labeling method well known to those skilled in the art can be usedEnzymes, etc.), radioisotopes (e.g.32P or125I) Etc., biotin, digoxin, colloidal metals (e.g., colloidal gold, etc.), fluorescent dyes (e.g., fluorescein, rhodamine, texas red, etc.), chemiluminescent compounds, or bioluminescent compounds (e.g., dioxetane, luminol, acridinium, etc.). Any labeling step well known in the art may be used, such as covalent coupling of an enzyme or biotin group, iodination, phosphorylation, biotinylation, and the like.
As another example, the compounds of the present invention may be coated on a solid support. Wherein the solid support is a solid surface to which the recombinant chimeric antigen can be attached. The solid phase support used in the present invention is not particularly limited, and commercially available solid phase supports and any solid phase support that can be used for immunoassay can be used in the present invention. Exemplary solid supports can be magnetic strains, enzyme labeled plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates or microchips, and the like.
Example 1 sequence design of wild type Treponema pallidum recombinant chimeric antigen WTP17+15+47
Selecting a full-length sequence of a wild TP17 antigen after signal peptide removal, namely a 25-156aa region, wherein the amino acid sequence of the full-length sequence is shown as SEQ ID NO.1, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 15; selecting a full-length sequence of the wild TP15 antigen after signal peptide removal, namely a 17-158aa region, wherein the amino acid sequence of the full-length sequence is shown as SEQ ID NO.2, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 16; the full-length sequence of the wild TP47 antigen after signal peptide removal, namely the 20-434aa region, is selected, the amino acid sequence of the full-length sequence is shown as SEQ ID NO.3, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 17.
The three antigen epitopes are connected in series according to the sequence of TP17+ linker 1+ TP15+ linker 2+ TP47 through linker 1 (shown as amino acid sequence shown as SEQ ID NO.4 and nucleotide sequence shown as SEQ ID NO. 18) and linker 2 (shown as amino acid sequence shown as SEQ ID NO.5 and nucleotide sequence shown as SEQ ID NO. 19), the obtained amino acid sequence is named as WTP17+15+47 (shown as SEQ ID NO. 6), and the nucleotide sequence of the WTP17+15+47 after the corresponding codon is optimized is named as WTP17+15+47 (shown as SEQ ID NO. 20).
The wtp17+15+47 nucleotide sequence is obtained by a chemical synthesis method.
Example 2 construction of recombinant wild-type chimeric antigen 32-WTP17+15+47 and confirmation of expression form
Using wtp17+15+47 in example 1 as a template, TP17-F (SEQ ID NO.29) and TP47-R (SEQ ID NO.30) as a primer pair, PCR amplification and gel recovery were performed according to the method described in the molecular cloning protocol to obtain a wtp17+15+47 nucleotide sequence, and the wtp17+15+47 gene fragment was constructed into a pET32a vector, and the resulting recombinant vector was named pET32a-wtp17+15+ 47.
pET32a-wtp17+15+47 was transformed into E.coli BL21(Rossett) competent cells and inducible expression was performed as described in the molecular cloning protocol. Briefly stated, the method comprises the following steps: the cells were incubated at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 25 ℃ for 16 hours. The clone inducing the target protein is named as R pET32a-WTP17+15+ 47. A large number of fermentations were carried out with R pET32a-WTP17+15+47 under the induction conditions determined above. After the fermentation was completed, the cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.
Resuspending the R pET32a-TP17+15+47 fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and then carrying out ultrasonic disruption; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disruption solution and the supernatant of the inclusion body lysate were taken separately and subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 1.
The results showed that pET32a-WTP17+15+47 was expressed as inclusion bodies.
Example 3 construction of recombinant wild-type chimeric antigen CN-WTP17+15+47 and confirmation of expression form
The nucleotide sequence of the SUMO fusion tag (SEQ ID NO.21, wherein, the amino group of the SUMO fusion tag is obtained by a chemical synthesis methodThe sequence is shown as SEQ ID NO. 7), taking the sequence as a template, taking SUMO-F (SEQ ID NO.31) and SUMO-R (SEQ ID NO.32) as a primer pair, and carrying out PCR amplification and gel recovery according to the method of the molecular cloning experimental guidance to obtain the nucleotide sequence of the SUMO fusion tag. PCR amplification and gel recovery were carried out using wtp17+15+47 in example 1 as a template and SUMO-TP17-F (SEQ ID NO.33) and TP47-R (SEQ ID NO.30) as primer pairs according to the protocols described in the molecular cloning protocols to obtain the wtp17+15+47 nucleotide sequence. Then, with the nucleotide sequence of the wtp17+15+47 and SUMO fusion tag as a template and SUMO-F (SEQ ID NO.31) and TP47-R (SEQ ID NO.30) as primer pairs, carrying out PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance to obtain a method for constructing the SUMO + wtp17+15+47 gene segment into pColdTMAmong the vectors I, the resulting recombinant vector was named CN-wtp17+15+ 47.
CN-wtp17+15+47 was transformed into e.coli BL21(Rossett) competent cells and induced to express as described in the molecular cloning protocol. Briefly stated, the method comprises the following steps: the cells were cultured at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 16 ℃ for 24 hours. The clone in which the target protein was induced was named CN-WTP17+15+ 47. A large number of fermentations were carried out with CN-WTP17+15+47 under the induction conditions determined above. After the fermentation was completed, the cells were collected and washed twice with a buffer solution of 20mM PBS pH 7.4. The cells were frozen at-20 ℃.
Resuspending CN-WTP17+15+47 fermentation thalli with bacteria breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and carrying out ultrasonic crushing; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disruption solution and the supernatant of the inclusion body lysate were taken separately and subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 2.
The results indicated that CN-WTP17+15+47 was expressed as inclusion bodies.
Example 4 sequence design of the truncated Treponema pallidum recombinant chimeric antigen DTP17+15+47
The selected TP17 epitope is 35-156aa region, the amino acid sequence is shown as SEQ ID NO.8, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 22; the selected TP15 epitope is 36-158aa region, the amino acid sequence is shown as SEQ ID NO.9, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 23; the selected TP47 epitope is 26-396aa region, the amino acid sequence is shown as SEQ ID NO.10, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 24.
The three antigen epitopes are connected in series according to the sequence of TP17+ linker 1+ TP15+ linker 2+ TP47 by linker 1 (shown as SEQ ID NO. 4) and linker 2 (shown as SEQ ID NO. 5), the obtained amino acid sequence is named as DTP17+15+47 (shown as SEQ ID NO. 11), and the nucleotide sequence of DTP17+15+47 after the codon corresponding to DTP17+15+47 is optimized is DTP17+15+47 (shown as SEQ ID NO. 25).
The dtp17+15+47 was obtained by chemical synthesis.
Example 5 construction of the truncated Treponema pallidum recombinant chimeric antigen 32-DTP17+15+47 and confirmation of expression form
Using dtp17+15+47 in example 4 as a template, TP17-F2(SEQ ID NO.34) and TP47-R2(SEQ ID NO.35) as a primer pair, PCR amplification and gel recovery were performed according to the method described in the molecular cloning protocol to obtain a dtp17+15+47 nucleotide sequence, and the dtp17+15+47 gene fragment was constructed into a pET32a vector, and the resulting recombinant vector was named pET32a-dtp17+15+ 47.
pET32a-dtp17+15+47 was transformed into E.coli BL21(Rossett) competent cells and inducible expression was performed as described in the molecular cloning protocol. Briefly stated, the method comprises the following steps: the cells were incubated at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 25 ℃ for 16 hours. The clone inducing the target protein is named RpET32a-DTP17+15+ 47. The large scale fermentation was carried out on R pET32a-DTP17+15+47 under the induction conditions determined above. After the fermentation was completed, the cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.
Resuspending the R pET32a-DTP17+15+47 fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and then carrying out ultrasonic disruption; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disruption solution and the supernatant of the inclusion body lysate were taken separately and subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 3.
The results showed that pET32a-DTP17+15+47 was expressed as inclusion bodies.
Example 6 construction of the truncated Treponema pallidum recombinant chimeric antigen CN-DTP17+15+47 and confirmation of expression form
The nucleotide sequence of the SUMO fusion tag (SEQ ID NO.21) is obtained by adopting a chemical synthesis method, and is used as a template, SUMO-F (SEQ ID NO.31) and SUMO-R (SEQ ID NO.32) are used as a primer pair, and the nucleotide sequence of the SUMO fusion tag is obtained by carrying out PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance. Using dtp17+15+47 in example 4 as a template, SUMO-TP17-F2(SEQ ID NO.36) and TP47-R2(SEQ ID NO.35) as a primer pair, PCR amplification and gel recovery were carried out according to the methods described in the molecular cloning protocols to obtain the dtp17+15+47 nucleotide sequence. Then, with the nucleotide sequence of the dtp17+15+47 and SUMO fusion tag as a template, SUMO-F (SEQ ID NO.31) and TP47-R2(SEQ ID NO.35) as a primer pair, carrying out PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance to obtain a gene fragment of SUMO + dtp17+15+47 constructed to pColdTMThe resulting recombinant vector was named CN-dtp17+15+47 among the vectors I.
CN-dtp17+15+47 was transformed into E.coli BL21(Rossett) competent cells and induced expression was performed as described in the molecular cloning protocols. Briefly stated, the method comprises the following steps: the cells were cultured at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 16 ℃ for 24 hours. The clone in which the desired protein was induced was named CN-DTP17+15+ 47. CN-DTP17+15+47 was subjected to bulk fermentation under the induction conditions identified above. After the fermentation was completed, the cells were collected and washed twice with a buffer solution of 20mM PBS pH 7.4. The cells were frozen at-20 ℃.
Resuspending CN-DTP17+15+47 fermentation thalli with bacteria breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing well and performing ultrasonic breaking; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disrupted solution and the supernatant of the solubilized inclusion body were separately subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 4.
The results show that CN-DTP17+15+47 is expressed in the form of partial supernatant and partial inclusion body.
Example 7 sequence design of the mutated Treponema truncatum recombinant chimeric antigen DSTP17+15+47
The selected antigenic epitope of TP17 is 35-156aa region, and 42 th and 58 th cysteine are mutated into serine, the amino acid sequence is shown as SEQ ID NO.12, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 26; the selected TP15 epitope is 36-158aa region, the amino acid sequence is shown as SEQ ID NO.9, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 23; the selected TP47 epitope is 26-396aa region, the 315 th cysteine is mutated into serine, the amino acid sequence is shown as SEQ ID NO.13, and the corresponding codon optimized nucleotide sequence is shown as SEQ ID NO. 27.
The three antigen epitopes are connected in series according to the sequence of TP17+ linker 1+ TP15+ linker 2+ TP47 by linker 1 (shown as SEQ ID NO. 4) and linker 2 (shown as SEQ ID NO. 5), the obtained amino acid sequence is named as DSTP17+15+47 (shown as SEQ ID NO. 14), and the nucleotide sequence of the TP17+15+47 after the codon optimization is DSTP17+15+47 (shown as SEQ ID NO. 28).
The above-mentioned dstp17+15+47 was obtained by chemical synthesis.
Example 8 construction and expression purification of the mutated Treponema truncatum recombinant chimeric antigen 28-DSTP17+15+47
The method comprises the steps of carrying out PCR amplification and gel recovery by using the dstp17+15+47 in the example 7 as a template and TP17-F2(SEQ ID NO.34) and TP47-R2(SEQ ID NO.35) as primer pairs according to the method of a molecular cloning experimental manual to obtain a dstp17+15+47 nucleotide sequence, constructing a dstp17+15+47 gene fragment into a pET28a vector, and naming the obtained recombinant vector as pET28a-dstp17+15+ 47.
pET28a-dstp17+15+47 was transformed into E.coli BL21(Rossett) competent cells and inducible expression was performed as described in the molecular cloning protocols. Briefly stated, the method comprises the following steps: the cells were incubated at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 25 ℃ for 16 hours. The clone inducing the target protein is named as R pET28a-DSTP17+15+ 47. The large scale fermentation was carried out on R pET28a-DSTP17+15+47 under the induction conditions determined above. After the fermentation was completed, the cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.
Resuspending the R pET28a-DSTP17+15+47 fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and then carrying out ultrasonic disruption; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disruption solution and the supernatant of the inclusion body lysate were subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 5.
The results showed that pET28a-DSTP17+15+47 was expressed as inclusion bodies.
The inclusion body was taken to dissolve the supernatant and purified by NI column. The loading buffer solution is: 8M Urea 20mM PBS, 0.5M NaCl, pH 7.4; the equilibrium buffer was: 8M urea, 20mM PBS, 0.5M NaCl, pH 7.4; washing with a miscellaneous buffer solution: 8M urea, 20mM PBS, 0.5M NaCl, 30mM imidazole, pH 7.4; the elution buffer was: 8M Urea 20mM PBS, 300mM imidazole, pH 7.4.
The eluate was collected, placed in a dialysis bag with molecular weight of 3000-3500 Dalton, and dialyzed against 4M urea 10mM PBS, 1mM EDTA pH7.4 at 2-8 ℃ while reducing the urea concentration to 0 by changing the solution, and dialyzed twice in the absence of urea (10mM PBS, 1mM EDTA, pH 7.4). The dialyzed target protein was centrifuged at 12000rpm at 2-8 ℃ for 20min, and the supernatant was collected in a 50mL centrifuge tube and the antigen protein concentration was measured. The recombinant chimeric antigen was named 28-DSTP17+15+ 47.
Example 9 construction and expression purification of the mutated Treponema truncatum recombinant chimeric antigen 32-DSTP17+15+47
The method comprises the steps of carrying out PCR amplification and gel recovery by using the dstp17+15+47 in the example 7 as a template and TP17-F2(SEQ ID NO.34) and TP47-R2(SEQ ID NO.35) as primer pairs according to the method of a molecular cloning experimental manual to obtain a dstp17+15+47 nucleotide sequence, constructing a dstp17+15+47 gene fragment into a pET32a vector, and naming the obtained recombinant vector as pET32a-dstp17+15+ 47.
pET32a-dstp17+15+47 was transformed into E.coli BL21(Rossett) competent cells and inducible expression was performed as described in the molecular cloning protocols. Briefly stated, the method comprises the following steps: the cells were incubated at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 25 ℃ for 16 hours. The clone inducing the target protein is named as R pET32a-DSTP17+15+ 47. The large scale fermentation was carried out on R pET32a-DSTP17+15+47 under the induction conditions determined above. After the fermentation was completed, the cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.
Resuspending the R pET32a-DSTP17+15+47 fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and then carrying out ultrasonic disruption; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disrupted solution and the supernatant of the solubilized inclusion body were separately subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 6.
The results show that R pET32a-DSTP17+15+47 is expressed in a partially soluble, partially inclusion form.
And (4) taking the supernatant of the crushing liquid to perform NI column purification. The loading buffer solution is: 20mM PBS, 0.5M NaCl, pH 7.4; the equilibrium buffer was: 20mM PBS, 0.5M NaCl, pH 7.4; washing with a miscellaneous buffer solution: 20mM PBS, 0.5M NaCl, 30mM imidazole, pH 7.4; the elution buffer was: 20mM PBS, 300mM imidazole, pH 7.4.
Collecting eluate, loading into 3000-3500 Dalton dialysis bag, and dialyzing the target protein eluate twice with 10mM PBS and 1mM EDTA pH7.4 at 2-8 deg.C. The dialyzed target protein was centrifuged at 12000rpm at 2-8 ℃ for 20min, and the supernatant was collected in a 50mL centrifuge tube and the antigen protein concentration was measured. The recombinant chimeric antigen was named 32-DSTP17+15+ 47.
Adding thrombin at a ratio of 1U/mg, performing enzyme digestion at 25 deg.C, sampling, and performing SDS-PAGE to determine the effect of the enzyme digestion, wherein the result is shown in FIG. 8, wherein the protein is non-specifically cleaved and the target protein is fragmented.
Example 10 construction, expression and purification of recombinant chimeric antigen CN-DSTP17+15+47 of Treponema pallidum
The nucleotide sequence of the SUMO fusion tag (SEQ ID NO.21) is obtained by adopting a chemical synthesis method, and is used as a template, SUMO-F (SEQ ID NO.31) and SUMO-R (SEQ ID NO.32) are used as a primer pair, and the nucleotide sequence of the SUMO fusion tag is obtained by carrying out PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance. The nucleotide sequence of dstp17+15+47 is obtained by PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance by using the dstp17+15+47 in example 7 as a template and SUMO-TP17-F2(SEQ ID NO.36) and TP47-R2(SEQ ID NO.35) as a primer pair. Subsequently, with the nucleotide sequence of the dstp17+15+47 and SUMO fusion tag as a template, SUMO-F (SEQ ID NO.31) and TP47-R2(SEQ ID NO.35) as a primer pair, carrying out PCR amplification and gel recovery according to the method described in the molecular cloning experimental guidance to obtain a gene fragment for constructing SUMO + dstp17+15+47 into pColdTMOf the vectors I, the resulting recombinant vector was named CN-dstp17+15+ 47.
CN-dstp17+15+47 was transformed into e.coli BL21(Rossett) competent cells and induced expression was performed as described in the molecular cloning protocols. Briefly stated, the method comprises the following steps: the cells were cultured at 37 ℃ and 200rpm until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 16 ℃ for 24 hours. The clone in which the desired protein was induced was named CN-DSTP17+15+ 47. The bulk fermentation was carried out under the induction conditions defined above for CN-DSTP17+15+ 47. After the fermentation was completed, the cells were collected and washed twice with 20mM PBS pH7.4 buffer. The cells were frozen at-20 ℃.
Resuspending CN-DSTP17+15+47 fermentation thalli with bacteria breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and carrying out ultrasonic crushing; after the completion of disruption, centrifugation was carried out at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 8M urea 20mM PBS into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm and 4 ℃ for 20min after the completion of dissolving, and collecting the inclusion body dissolving supernatant. The supernatant of the disruption solution and the supernatant of the inclusion body lysate were subjected to SDS-PAGE to determine the expression pattern of the protein, and the results are shown in FIG. 7.
The results show that CN-DSTP17+15+47 is mainly expressed in soluble form.
And (4) taking the supernatant of the crushing liquid to perform NI column purification. The loading buffer solution is: 20mM PBS, 0.5M NaCl, pH 7.4; the equilibrium buffer was: 20mM PBS, 0.5M NaCl, pH 7.4; washing with a miscellaneous buffer solution: 20mM PBS, 0.5M NaCl, 30mM imidazole, pH 7.4; the elution buffer was: 20mM PBS, 300mM imidazole, pH 7.4.
Collecting eluate, loading into 3000-3500 Dalton dialysis bag, and dialyzing the target protein eluate twice with 10mM PBS and 1mM EDTA pH7.4 at 2-8 deg.C. The dialyzed target protein was centrifuged at 12000rpm at 2-8 ℃ for 20min, and the supernatant was collected in a 50mL centrifuge tube and the antigen protein concentration was measured. The recombinant chimeric antigen was named CN-DSTP17+15+ 47.
Adding SUMO protease according to the proportion of 1U/mg, carrying out enzyme digestion at the temperature of 2-8 ℃, after the enzyme digestion is finished, sampling and carrying out SDS-PAGE to determine the enzyme digestion effect of the protein, wherein the enzyme digestion efficiency is high, and the target protein is complete.
Taking the enzyme digestion solution for NI column purification. The loading buffer solution is: 20mM PBS, pH 7.4; the equilibrium buffer was: 20mM PBS, 0.5M NaCl, pH 7.4; washing with a miscellaneous buffer solution: 20mM PBS, 0.5M NaCl, 30mM imidazole, pH 7.4; the elution buffer was: 20mM PBS, 300mM imidazole, pH 7.4. Collecting the permeate, filling into a dialysis bag with molecular weight of 3000-3500 Dalton, and dialyzing the target protein eluate twice with 10mM PBS and 1mM EDTA pH7.4 at 2-8 deg.C. The dialyzed target protein was centrifuged at 12000rpm at 2-8 ℃ for 20min, and the supernatant was collected in a 50mL centrifuge tube and the antigen protein concentration was measured. The recombinant chimeric antigen is named CN-DSTP17+15+47 enzyme cutting.
Example 11 Activity assay of recombinant chimeric antigens of Treponema pallidum
Under the condition that other production processes are completely consistent, the enzyme digestion and HRP labeling are respectively carried out by using the freshly prepared 28-DSTP17+15+47 in example 8, the freshly prepared 32-DSTP17+15+47 in example 9, the freshly prepared CN-DSTP17+15+47 and the freshly prepared CN-DSTP17+15+47 in example 10, and the chemiluminescence immunoassay is carried out by adopting the principle of a double antigen sandwich method.
Specifically, the magnetic particles coated with the TP15 antigen, the TP17 antigen and the TP47 antigen are mixed with the HRP-labeled treponema pallidum recombinant chimeric antigen and a TP quality control product (michael bio-ltd), and a "coated antigen-antibody-HRP-labeled antigen" complex is formed because the TP quality control product contains an anti-TP antibody; and then adding a chemiluminescence substrate solution, performing chemiluminescence immunoassay on an IS 1200 full-automatic chemiluminescence determinator of Mike organisms, and determining chemiluminescence intensity (RLU), wherein the higher the RLU value IS, the more complexes of the coating antigen-antibody-HRP labeled antigen are, namely the stronger the binding capacity of the HRP-labeled treponema pallidum recombinant chimeric antigen and the TP antibody IS, and the higher the activity of the reaction chimeric antigen IS. The results are shown in FIG. 10.
The results show that the enzyme digestion activity of CN-DSTP17+15+47 is the highest.
Example 12 detection of thermostability of recombinant chimeric antigen of Treponema pallidum
In the case of complete agreement of other production processes, the four recombinant antigens were digested with the freshly prepared 28-DSTP17+15+47 in example 8, the freshly prepared 32-DSTP17+15+47 in example 9, the freshly prepared CN-DSTP17+15+47 in example 10, and the freshly prepared CN-DSTP17+15+47 in example 10, and subjected to 37-degree thermal acceleration treatment for 3 days, 7 days, and 14 days, respectively. Subsequently, the antigen subjected to the thermal acceleration treatment was labeled with HPR, and a TP quality control product (a mixed quality control product containing TP15 antibody, TP17 antibody, and TP47 antibody) was subjected to chemiluminescence immunoassay using a double-antigen sandwich principle with a magnetic bead coated with TP15, TP17, and TP47, and protein activity was evaluated based on chemiluminescence intensity (RLU) in an IS 1200 full-automatic chemiluminescence analyzer from michael organisms, and the results are shown in fig. 11.
The result shows that the signal value of the marked HRP of the recombinant antigen cut by CN-DSTP17+15+47 enzyme after being subjected to 37-degree thermal acceleration treatment for 3 days, 7 days and 14 days is not obviously different from that of the freshly prepared antigen, and the signal values of other antigens are reduced in different degrees, which indicates that the thermal stability of the recombinant antigen cut by CN-DSTP17+15+47 enzyme is relatively better.
Example 13 evaluation of specificity and sensitivity of recombinant chimeric antigen of Treponema pallidum
In the embodiment 10 of the present invention, CN-DSTP17+15+47 IS used for enzyme digestion of recombinant antigen labeled HRP, and together with magnetic beads coated with TP15, TP17 and TP47, a double-antigen sandwich principle IS adopted to perform chemiluminescence immunodetection on a national reference product (China food and drug testing institute, [ batch No. ] 370036-201801) of a TP treponema pallidum antibody chemiluminescence reagent on an IS 1200 full-automatic chemiluminescence determinator of Mike biology, and sensitivity evaluation IS performed according to chemiluminescence intensity (RLU) and a calculated S/CO value, and the results are shown in the following table 1.
TABLE 1 results of sensitivity detection
Figure BDA0002507739790000141
Figure BDA0002507739790000151
Note: national reference products of Treponema Pallidum (TP) antibody diagnostic reagents, wherein N1-N20 are negative reference products; P1-P10 are positive reference substances, and L1-L4 are minimum detection limit reference substances. The negative reference substance requires that the sample with positive determination result is less than or equal to 3 cases; the requirement of a positive reference substance is 10/10, and the requirement of a minimum detection limit reference substance is more than or equal to 2/4(L4 needs to be negative). And (4) judging the standard: when S/CO <1, the product is negative (-), and when S/CO ≧ 1, the product is positive (+).
The result shows that CN-DSTP17+15+47 enzyme digestion recombinant antigen detection Treponema Pallidum (TP) antibody negative national reference substance, positive national reference substance and sensitivity national reference substance all meet the requirements.
Example 14 clinical sample testing for recombinant chimeric antigens of Treponema pallidum
HRP IS enzyme-labeled by recombinant chimeric antigen CN-DSTP17+15+47 in the embodiment 10 of the invention, and 1200 clinical samples (200 confirmed syphilis positive samples and 1000 syphilis negative samples) are subjected to chemiluminescence immunoassay on an IS 1200 full-automatic chemiluminescence determinator of Mike biology by using a double-antigen sandwich principle together with magnetic beads coated with TP15, TP17 and TP47, and the results are shown in the following table 2.
TABLE 2 detection results of clinical sample compliance rates
Figure BDA0002507739790000152
The detection result is completely consistent with the diagnosis result of a clinical sample, which shows that the clinical coincidence rate of the CN-DSTP17+15+47 enzyme digestion recombinant antigen can meet the requirement of the development of an immunodiagnostic reagent.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Sichuan Mike BioNew Material technology Co., Ltd
<120> treponema pallidum recombinant chimeric antigen and preparation method and application thereof
<130>2020
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<170>PatentIn version 3.5
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Val Leu Gly Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp Ser Arg
355 360 365
Gly Arg Lys Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr Lys Met
370 375 380
Val Arg Ala Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly Glu Ile
385 390 395 400
Lys Phe Glu Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg Ala Ser
405 410 415
Ser Phe Met Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val Lys Val
420 425 430
His Gly Met Lys Phe Val Pro Val Ala Val Pro His Glu Leu Lys Gly
435 440 445
Ile Ala Lys Glu Lys Phe His Phe Val Glu Asp Ser Arg Val Thr Glu
450 455 460
Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe Ser Ala
465 470 475 480
Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu Val Val Asp Thr
485 490495
Val Gly Thr Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser
500 505 510
Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe
515 520 525
Ile Asp Tyr Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr Tyr Gly
530 535 540
Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr Lys His
545 550 555 560
Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile Ser Cys
565 570 575
Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro Gly Tyr
580 585 590
Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val Ala Asp
595 600 605
Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly Leu Lys
610 615 620
Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu Met Asp
625 630 635 640
Ala Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val Ser Asp
645 650655
Gln Ala Val Ser Leu Gly Gln Asn Val Leu Ser Ala Asp Phe Thr Pro
660 665 670
Gly Thr Glu Tyr Thr Val Glu Val Arg Phe Lys Glu Phe Gly Ser Val
675 680 685
Arg Ala Lys Val Val Ala Gln
690 695
<210>7
<211>100
<212>PRT
<213> Artificial Synthesis
<220>
<223>SUMO
<400>7
Gly Ser Leu Gln Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val
1 5 10 15
Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp
20 25 30
Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg
35 40 45
Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser
50 55 60
Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro
65 70 75 80
Glu Asp Leu Asp Met Glu AspAsn Asp Ile Ile Glu Ala His Arg Glu
85 90 95
Gln Ile Gly Gly
100
<210>8
<211>122
<212>PRT
<213> treponema pallidum
<400>8
Ala Lys Ala Glu Lys Val Glu Cys Ala Leu Lys Gly Gly Ile Phe Arg
1 5 10 15
Gly Thr Leu Pro Ala Ala Asp Cys Pro Gly Ile Asp Thr Thr Val Thr
20 25 30
Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu Lys
35 40 45
Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg
50 55 60
Glu Asp Gly Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys
65 70 75 80
Ala Pro His Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn Ser Val
85 90 95
Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu Met Ala
100 105 110
Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys
115 120
<210>9
<211>123
<212>PRT
<213> treponema pallidum
<400>9
Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp
1 5 10 15
Phe Asp Glu Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly
20 25 30
Gly Lys Met Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly Arg
35 40 45
Phe Lys Ser Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser Ser
50 55 60
Gly Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu
65 70 75 80
Glu Lys Gly Asn Pro Glu Met Val Asp Val Val Thr Gly Ala Thr Val
85 90 95
Ser Ser Gln Ser Phe Arg Arg Leu Gly Ala Ala Leu Leu Gln Ser Ala
100 105 110
Arg Arg Gly Glu Lys Glu Ala Ile Ile Ser Arg
115 120
<210>10
<211>371
<212>PRT
<213> treponema pallidum
<400>10
Glu Thr His Tyr Gly Tyr Ala Thr Leu Ser Tyr Ala Asp Tyr Trp Ala
1 5 10 15
Gly Glu Leu Gly Gln Ser Arg Asp Val Leu Leu Ala Gly Asn Ala Glu
20 25 30
Ala Asp Arg Ala Gly Asp Leu Asp Ala Gly Met Phe Asp Ala Val Ser
35 40 45
Arg Ala Thr His Gly His Gly Ala Phe Arg Gln Gln Phe Gln Tyr Ala
50 55 60
Val Glu Val Leu Gly Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp
65 70 75 80
Ser Arg Gly Arg Lys Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr
85 90 95
Lys Met Val Arg Ala Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly
100 105 110
Glu Ile Lys Phe Glu Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg
115 120 125
Ala Ser Ser Phe Met Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val
130 135 140
Lys Val His Gly Met Lys Phe Val Pro Val Ala Val Pro His Glu Leu
145 150 155 160
Lys Gly Ile Ala Lys Glu Lys Phe His Phe Val Glu Asp Ser Arg Val
165 170 175
Thr Glu Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe
180 185 190
Ser Ala Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu Val Val
195 200 205
Asp Thr Val Gly Thr Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu
210 215 220
Ala Ser Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg
225 230 235 240
Glu Phe Ile Asp Tyr Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr
245 250 255
Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr
260 265 270
Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile
275 280 285
Ser Cys Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro
290 295 300
Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val
305 310 315 320
Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly
325 330 335
Leu Lys Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu
340 345 350
Met Asp Ala Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val
355 360 365
Ser Asp Gln
370
<210>11
<211>622
<212>PRT
<213> treponema pallidum
<400>11
Ala Lys Ala Glu Lys Val Glu Cys Ala Leu Lys Gly Gly Ile Phe Arg
1 5 10 15
Gly Thr Leu Pro Ala Ala Asp Cys Pro Gly Ile Asp Thr Thr Val Thr
20 25 30
Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu Lys
35 40 45
Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg
50 55 60
Glu Asp Gly Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys
65 70 75 80
Ala Pro His Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn Ser Val
85 90 95
Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu Met Ala
100 105 110
Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys Gly Ser Gly Ser Phe Ser
115 120 125
Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp Phe Asp Glu
130 135 140
Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly Gly Lys Met
145 150 155 160
Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly Arg Phe Lys Ser
165 170 175
Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser Ser Gly Ile Gly
180 185 190
Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly
195 200 205
Asn Pro Glu Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser Gln
210 215 220
Ser Phe Arg Arg Leu Gly Ala Ala Leu Leu Gln Ser Ala Arg Arg Gly
225 230 235 240
Glu Lys Glu Ala Ile Ile Ser Arg Ser Gly Gly Glu Thr His Tyr Gly
245 250 255
Tyr Ala Thr Leu Ser Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln
260 265 270
Ser Arg Asp Val Leu Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly
275 280 285
Asp Leu Asp Ala Gly Met Phe Asp Ala Val Ser Arg Ala Thr His Gly
290 295 300
His Gly Ala Phe Arg Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly
305 310 315 320
Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys
325 330 335
Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala
340 345 350
Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu
355 360 365
Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met
370 375 380
Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val Lys Val His Gly Met
385 390 395 400
Lys Phe Val Pro Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys
405 410 415
Glu Lys Phe His Phe Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn
420 425 430
Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val
435 440 445
Ser Ser Met Glu Ser Pro His Asp Leu Val Val Asp Thr Val Gly Thr
450 455 460
Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu
465 470 475 480
Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr
485 490 495
Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala
500 505 510
Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp
515 520 525
Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile Ser Cys Gly Ile Asn
530 535 540
Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu
545 550 555 560
Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe
565 570 575
Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val
580 585 590
Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val
595 600 605
Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val Ser Asp Gln
610 615 620
<210>12
<211>122
<212>PRT
<213> treponema pallidum
<400>12
Ala Lys Ala Glu Lys Val Glu Ser Ala Leu Lys Gly Gly Ile Phe Arg
1 5 10 15
Gly Thr Leu Pro Ala Ala Asp Ser Pro Gly Ile Asp Thr Thr Val Thr
20 25 30
Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu Lys
35 40 45
Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg
50 5560
Glu Asp Gly Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys
65 70 75 80
Ala Pro His Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn Ser Val
85 90 95
Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu Met Ala
100 105 110
Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys
115 120
<210>13
<211>371
<212>PRT
<213> treponema pallidum
<400>13
Glu Thr His Tyr Gly Tyr Ala Thr Leu Ser Tyr Ala Asp Tyr Trp Ala
1 5 10 15
Gly Glu Leu Gly Gln Ser Arg Asp Val Leu Leu Ala Gly Asn Ala Glu
20 25 30
Ala Asp Arg Ala Gly Asp Leu Asp Ala Gly Met Phe Asp Ala Val Ser
35 40 45
Arg Ala Thr His Gly His Gly Ala Phe Arg Gln Gln Phe Gln Tyr Ala
50 55 60
Val Glu Val Leu Gly Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp
6570 75 80
Ser Arg Gly Arg Lys Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr
85 90 95
Lys Met Val Arg Ala Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly
100 105 110
Glu Ile Lys Phe Glu Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg
115 120 125
Ala Ser Ser Phe Met Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val
130 135 140
Lys Val His Gly Met Lys Phe Val Pro Val Ala Val Pro His Glu Leu
145 150 155 160
Lys Gly Ile Ala Lys Glu Lys Phe His Phe Val Glu Asp Ser Arg Val
165 170 175
Thr Glu Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe
180 185 190
Ser Ala Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu Val Val
195 200 205
Asp Thr Val Gly Thr Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu
210 215 220
Ala Ser Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg
225230 235 240
Glu Phe Ile Asp Tyr Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr
245 250 255
Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr
260 265 270
Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile
275 280 285
Ser Ser Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro
290 295 300
Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val
305 310 315 320
Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly
325 330 335
Leu Lys Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu
340 345 350
Met Asp Ala Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val
355 360 365
Ser Asp Gln
370
<210>14
<211>622
<212>PRT
<213> treponema pallidum
<400>14
Ala Lys Ala Glu Lys Val Glu Ser Ala Leu Lys Gly Gly Ile Phe Arg
1 5 10 15
Gly Thr Leu Pro Ala Ala Asp Ser Pro Gly Ile Asp Thr Thr Val Thr
20 25 30
Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu Lys
35 40 45
Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg
50 55 60
Glu Asp Gly Ile Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys
65 70 75 80
Ala Pro His Glu Lys Glu Leu Tyr Glu Leu Ile Asp Ser Asn Ser Val
85 90 95
Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu Met Ala
100 105 110
Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys Gly Ser Gly Ser Phe Ser
115 120 125
Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp Phe Asp Glu
130 135 140
Asn Gly Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly Gly Lys Met
145 150155 160
Val Gln Val Val Tyr Asp Tyr Gln His Lys Glu Gly Arg Phe Lys Ser
165 170 175
Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser Ser Gly Ile Gly
180 185 190
Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly
195 200 205
Asn Pro Glu Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser Gln
210 215 220
Ser Phe Arg Arg Leu Gly Ala Ala Leu Leu Gln Ser Ala Arg Arg Gly
225 230 235 240
Glu Lys Glu Ala Ile Ile Ser Arg Ser Gly Gly Glu Thr His Tyr Gly
245 250 255
Tyr Ala Thr Leu Ser Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln
260 265 270
Ser Arg Asp Val Leu Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly
275 280 285
Asp Leu Asp Ala Gly Met Phe Asp Ala Val Ser Arg Ala Thr His Gly
290 295 300
His Gly Ala Phe Arg Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly
305 310 315320
Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys
325 330 335
Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala
340 345 350
Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu
355 360 365
Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met
370 375 380
Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val Lys Val His Gly Met
385 390 395 400
Lys Phe Val Pro Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys
405 410 415
Glu Lys Phe His Phe Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn
420 425 430
Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val
435 440 445
Ser Ser Met Glu Ser Pro His Asp Leu Val Val Asp Thr Val Gly Thr
450 455 460
Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu
465 470 475480
Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr
485 490 495
Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala
500 505 510
Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp
515 520 525
Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile Ser Ser Gly Ile Asn
530 535 540
Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu
545 550 555 560
Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe
565 570 575
Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val
580 585 590
Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val
595 600 605
Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val Ser Asp Gln
610 615 620
<210>15
<211>396
<212>DNA
<213> treponema pallidum
<400>15
tgcacaaccg tgtgtccgca cgccgggaag gccaaagcgg aaaaggtaga gtgcgcgttg 60
aagggaggta tctttcgggg tacgctgcct gcggccgatt gcccgggaat cgatacgact 120
gtgacgttca acgcggatgg cactgcgcaa aaggtagagc ttgcccttga gaagaagtcg 180
gcaccttctc ctcttaccta tcgcggtacg tggatggtac gtgaagacgg aattgtcgaa 240
ctctcgcttg tgtcctcgga gcaatcgaag gcaccgcacg agaaagagct gtacgagctg 300
attgacagta actccgttcg ctacatgggc gctccgggcg caggaaagcc ttcaaaggag 360
atggcgccgt tttacgtgct caaaaaaaca aagaaa 396
<210>16
<211>429
<212>DNA
<213> treponema pallidum
<400>16
atggtgaaaa gaggtggcgc gttcgcgctg tgtcttgcgg tgttgcttgg ggcgtgttca 60
tttagttcta tcccgaatgg cacgtaccgg gcgacgtatc aggattttga tgagaatggt 120
tggaaggact ttctcgaggt tacttttgat ggtggcaaga tggtgcaggt ggtttacgat 180
tatcagcata aagaagggcg gtttaagtcc caggacgctg actaccatcg ggtcatgtat 240
gcatcctcgg gcattggtcc tgaaaaggcc ttccgtgagc tcgccgatgc tttgcttgaa 300
aagggtaatc cggagatggt ggatgtggtc accggtgcaa ctgtttcttc ccagagtttc 360
cgtcgtttgg gtgcggcgct tctgcagagt gcgcggcgcg gcgagaagga agccattatt 420
agccgataa 429
<210>17
<211>1248
<212>DNA
<213> treponema pallidum
<400>17
tgtggctcgt ctcatcatga gacgcactat ggctatgcga cgctgagcta tgcggactac 60
tgggccgggg agttggggca gagtcgcgac gtgcttttgg cgggtaatgc cgaggcggac 120
cgcgcggggg atctcgacgc aggcatgttc gatgcagttt ctcgcgcaac ccacgggcat 180
ggcgcgttcc gtcagcaatt tcagtacgcg gttgaggtat tgggcgaaaa ggttctctcg 240
aagcaggaga ccgaagacag ccgcggacgc aaaaagtggg agtacgagac tgacccaagc 300
gttactaaga tggtgcgtgc ctctgcgtca tttcaggatt tgggagagga cggggagatt 360
aagtttgaag cagtcgaggg tgcagtagcg ttggcggatc gcgcgagttc cttcatggtt 420
gacagcgagg aatacaagat tacgaacgta aaggttcacg gtatgaagtt tgtcccagtt 480
gcggttcctc atgaattaaa agggattgca aaggagaagt ttcacttcgt ggaagactcc 540
cgcgttacgg agaataccaa cggccttaag acaatgctca ctgaggatag tttttctgca 600
cgtaaggtaa gcagcatgga gagcccgcac gaccttgtgg tagacacggt gggtaccgtc 660
taccacagcc gttttggttc ggacgcagag gcttctgtga tgctgaaaag ggctgatggc 720
tctgagctgt cgcaccgtga gttcatcgac tatgtgatga acttcaacac ggtccgctac 780
gactactacg gtgatgacgc gagctacacc aatctgatgg cgagttatgg caccaagcac 840
tctgctgact cctggtggaa gacaggaaga gtgccccgca tttcgtgtgg tatcaactat 900
gggttcgatc ggtttaaagg ttcagggccg ggatactaca ggctgacttt gattgcgaac 960
gggtataggg acgtagttgc tgatgtgcgc ttccttccca agtacgaggg gaacatcgat 1020
attgggttga aggggaaggt gctgaccata gggggcgcgg acgcggagac tctgatggat 1080
gctgcagttg acgtgtttgc cgatggacag cctaaacttg tcagcgatca agcggtgagc 1140
ttggggcaga atgtcctctc tgcggatttc actcccggca ctgagtacac ggttgaggtt 1200
aggttcaagg aattcggttc tgtgcgtgcg aaggtagtgg cccagtag 1248
<210>18
<211>9
<212>DNA
<213> Artificial Synthesis
<220>
<223> linker
<400>18
ggtagtggt 9
<210>19
<211>9
<212>DNA
<213> Artificial Synthesis
<220>
<223> linker
<400>19
tccggcggc 9
<210>20
<211>2088
<212>DNA
<213> treponema pallidum
<400>20
tgcacaaccg tgtgtccgca cgccgggaag gccaaagcgg aaaaggtaga gtgcgcgttg 60
aagggaggta tctttcgggg tacgctgcct gcggccgatt gcccgggaat cgatacgact 120
gtgacgttca acgcggatgg cactgcgcaa aaggtagagc ttgcccttga gaagaagtcg 180
gcaccttctc ctcttaccta tcgcggtacg tggatggtac gtgaagacgg aattgtcgaa 240
ctctcgcttg tgtcctcgga gcaatcgaag gcaccgcacg agaaagagct gtacgagctg 300
attgacagta actccgttcg ctacatgggc gctccgggcg caggaaagcc ttcaaaggag 360
atggcgccgt tttacgtgct caaaaaaaca aagaaaggta gtggtatggt gaaaagaggt 420
ggcgcgttcg cgctgtgtct tgcggtgttg cttggggcgt gttcatttag ttctatcccg 480
aatggcacgt accgggcgac gtatcaggat tttgatgaga atggttggaa ggactttctc 540
gaggttactt ttgatggtgg caagatggtg caggtggttt acgattatca gcataaagaa 600
gggcggttta agtcccagga cgctgactac catcgggtca tgtatgcatc ctcgggcatt 660
ggtcctgaaa aggccttccg tgagctcgcc gatgctttgc ttgaaaaggg taatccggag 720
atggtggatg tggtcaccgg tgcaactgtt tcttcccaga gtttccgtcg tttgggtgcg 780
gcgcttctgc agagtgcgcg gcgcggcgag aaggaagcca ttattagccg atccggcggc 840
tgtggctcgt ctcatcatga gacgcactat ggctatgcga cgctgagcta tgcggactac 900
tgggccgggg agttggggca gagtcgcgac gtgcttttgg cgggtaatgc cgaggcggac 960
cgcgcggggg atctcgacgc aggcatgttc gatgcagttt ctcgcgcaac ccacgggcat 1020
ggcgcgttcc gtcagcaatt tcagtacgcg gttgaggtat tgggcgaaaa ggttctctcg 1080
aagcaggaga ccgaagacag ccgcggacgc aaaaagtggg agtacgagac tgacccaagc 1140
gttactaaga tggtgcgtgc ctctgcgtca tttcaggatt tgggagagga cggggagatt 1200
aagtttgaag cagtcgaggg tgcagtagcg ttggcggatc gcgcgagttc cttcatggtt 1260
gacagcgagg aatacaagat tacgaacgta aaggttcacg gtatgaagtt tgtcccagtt 1320
gcggttcctc atgaattaaa agggattgca aaggagaagt ttcacttcgt ggaagactcc 1380
cgcgttacgg agaataccaa cggccttaag acaatgctca ctgaggatag tttttctgca 1440
cgtaaggtaa gcagcatgga gagcccgcac gaccttgtgg tagacacggt gggtaccgtc 1500
taccacagcc gttttggttc ggacgcagag gcttctgtga tgctgaaaag ggctgatggc 1560
tctgagctgt cgcaccgtga gttcatcgac tatgtgatga acttcaacac ggtccgctac 1620
gactactacg gtgatgacgc gagctacacc aatctgatgg cgagttatgg caccaagcac 1680
tctgctgact cctggtggaa gacaggaaga gtgccccgca tttcgtgtgg tatcaactat 1740
gggttcgatc ggtttaaagg ttcagggccg ggatactaca ggctgacttt gattgcgaac 1800
gggtataggg acgtagttgc tgatgtgcgc ttccttccca agtacgaggg gaacatcgat 1860
attgggttga aggggaaggt gctgaccata gggggcgcgg acgcggagac tctgatggat 1920
gctgcagttg acgtgtttgc cgatggacag cctaaacttg tcagcgatca agcggtgagc 1980
ttggggcaga atgtcctctc tgcggatttc actcccggca ctgagtacac ggttgaggtt 2040
aggttcaagg aattcggttc tgtgcgtgcg aaggtagtgg cccagtag 2088
<210>21
<211>300
<212>DNA
<213> Artificial Synthesis
<220>
<223>SUMO
<400>21
gggtccctgc aggactcaga agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc 60
aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagagat cttcttcaag 120
atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag acagggtaag 180
gaaatggact ccttaagatt cttgtacgac ggtattagga ttcaagctga tcagacccct 240
gaagatttgg acatggagga taacgatatt attgaggctc accgcgaaca gattggaggt 300
<210>22
<211>366
<212>DNA
<213> treponema pallidum
<400>22
gccaaagcgg aaaaggtaga gtgcgcgttg aagggaggta tctttcgggg tacgctgcct 60
gcggccgatt gcccgggaat cgatacgact gtgacgttca acgcggatgg cactgcgcaa 120
aaggtagagc ttgcccttga gaagaagtcg gcaccttctc ctcttaccta tcgcggtacg 180
tggatggtac gtgaagacgg aattgtcgaa ctctcgcttg tgtcctcgga gcaatcgaag 240
gcaccgcacg agaaagagct gtacgagctg attgacagta actccgttcg ctacatgggc 300
gctccgggcg caggaaagcc ttcaaaggag atggcgccgt tttacgtgct caaaaaaaca 360
aagaaa 366
<210>23
<211>369
<212>DNA
<213> treponema pallidum
<400>23
tcatttagtt ctatcccgaa tggcacgtac cgggcgacgt atcaggattt tgatgagaat 60
ggttggaagg actttctcga ggttactttt gatggtggca agatggtgca ggtggtttac 120
gattatcagc ataaagaagg gcggtttaag tcccaggacg ctgactacca tcgggtcatg 180
tatgcatcct cgggcattgg tcctgaaaag gccttccgtg agctcgccga tgctttgctt 240
gaaaagggta atccggagat ggtggatgtg gtcaccggtg caactgtttc ttcccagagt 300
ttccgtcgtt tgggtgcggc gcttctgcag agtgcgcggc gcggcgagaa ggaagccatt 360
attagccga 369
<210>24
<211>1113
<212>DNA
<213> treponema pallidum
<400>24
gagacgcact atggctatgc gacgctgagc tatgcggact actgggccgg ggagttgggg 60
cagagtcgcg acgtgctttt ggcgggtaat gccgaggcgg accgcgcggg ggatctcgac 120
gcaggcatgt tcgatgcagt ttctcgcgca acccacgggc atggcgcgtt ccgtcagcaa 180
tttcagtacg cggttgaggt attgggcgaa aaggttctct cgaagcagga gaccgaagac 240
agccgcggac gcaaaaagtg ggagtacgag actgacccaa gcgttactaa gatggtgcgt 300
gcctctgcgt catttcagga tttgggagag gacggggaga ttaagtttga agcagtcgag 360
ggtgcagtag cgttggcgga tcgcgcgagt tccttcatgg ttgacagcga ggaatacaag 420
attacgaacg taaaggttca cggtatgaag tttgtcccag ttgcggttcc tcatgaatta 480
aaagggattg caaaggagaa gtttcacttc gtggaagact cccgcgttac ggagaatacc 540
aacggcctta agacaatgct cactgaggat agtttttctg cacgtaaggt aagcagcatg 600
gagagcccgc acgaccttgt ggtagacacg gtgggtaccg tctaccacag ccgttttggt 660
tcggacgcag aggcttctgt gatgctgaaa agggctgatg gctctgagct gtcgcaccgt 720
gagttcatcg actatgtgat gaacttcaac acggtccgct acgactacta cggtgatgac 780
gcgagctaca ccaatctgat ggcgagttat ggcaccaagc actctgctga ctcctggtgg 840
aagacaggaa gagtgccccg catttcgtgt ggtatcaact atgggttcga tcggtttaaa 900
ggttcagggc cgggatacta caggctgact ttgattgcga acgggtatag ggacgtagtt 960
gctgatgtgc gcttccttcc caagtacgag gggaacatcg atattgggtt gaaggggaag 1020
gtgctgacca tagggggcgc ggacgcggag actctgatgg atgctgcagt tgacgtgttt 1080
gccgatggac agcctaaact tgtcagcgat caa 1113
<210>25
<211>1866
<212>DNA
<213> treponema pallidum
<400>25
gccaaagcgg aaaaggtaga gtgcgcgttg aagggaggta tctttcgggg tacgctgcct 60
gcggccgatt gcccgggaat cgatacgact gtgacgttca acgcggatgg cactgcgcaa 120
aaggtagagc ttgcccttga gaagaagtcg gcaccttctc ctcttaccta tcgcggtacg 180
tggatggtac gtgaagacgg aattgtcgaa ctctcgcttg tgtcctcgga gcaatcgaag 240
gcaccgcacg agaaagagct gtacgagctg attgacagta actccgttcg ctacatgggc 300
gctccgggcg caggaaagcc ttcaaaggag atggcgccgt tttacgtgct caaaaaaaca 360
aagaaaggta gtggttcatt tagttctatc ccgaatggca cgtaccgggc gacgtatcag 420
gattttgatg agaatggttg gaaggacttt ctcgaggtta cttttgatgg tggcaagatg 480
gtgcaggtgg tttacgatta tcagcataaa gaagggcggt ttaagtccca ggacgctgac 540
taccatcggg tcatgtatgc atcctcgggc attggtcctg aaaaggcctt ccgtgagctc 600
gccgatgctt tgcttgaaaa gggtaatccg gagatggtgg atgtggtcac cggtgcaact 660
gtttcttccc agagtttccg tcgtttgggt gcggcgcttc tgcagagtgc gcggcgcggc 720
gagaaggaag ccattattag ccgatccggc ggcgagacgc actatggcta tgcgacgctg 780
agctatgcgg actactgggc cggggagttg gggcagagtc gcgacgtgct tttggcgggt 840
aatgccgagg cggaccgcgc gggggatctc gacgcaggca tgttcgatgc agtttctcgc 900
gcaacccacg ggcatggcgc gttccgtcag caatttcagt acgcggttga ggtattgggc 960
gaaaaggttc tctcgaagca ggagaccgaa gacagccgcg gacgcaaaaa gtgggagtac 1020
gagactgacc caagcgttac taagatggtg cgtgcctctg cgtcatttca ggatttggga 1080
gaggacgggg agattaagtt tgaagcagtc gagggtgcag tagcgttggc ggatcgcgcg 1140
agttccttca tggttgacag cgaggaatac aagattacga acgtaaaggt tcacggtatg 1200
aagtttgtcc cagttgcggt tcctcatgaa ttaaaaggga ttgcaaagga gaagtttcac 1260
ttcgtggaag actcccgcgt tacggagaat accaacggcc ttaagacaat gctcactgag 1320
gatagttttt ctgcacgtaa ggtaagcagc atggagagcc cgcacgacct tgtggtagac 1380
acggtgggta ccgtctacca cagccgtttt ggttcggacg cagaggcttc tgtgatgctg 1440
aaaagggctg atggctctga gctgtcgcac cgtgagttca tcgactatgt gatgaacttc 1500
aacacggtcc gctacgacta ctacggtgat gacgcgagct acaccaatct gatggcgagt 1560
tatggcacca agcactctgc tgactcctgg tggaagacag gaagagtgcc ccgcatttcg 1620
tgtggtatca actatgggtt cgatcggttt aaaggttcag ggccgggata ctacaggctg 1680
actttgattg cgaacgggta tagggacgta gttgctgatg tgcgcttcct tcccaagtac 1740
gaggggaaca tcgatattgg gttgaagggg aaggtgctga ccataggggg cgcggacgcg 1800
gagactctga tggatgctgc agttgacgtg tttgccgatg gacagcctaa acttgtcagc 1860
gatcaa 1866
<210>26
<211>366
<212>DNA
<213> treponema pallidum
<400>26
gccaaagcgg aaaaggtaga gagcgcgttg aagggaggta tctttcgggg tacgctgcct 60
gcggccgata gcccgggaat cgatacgact gtgacgttca acgcggatgg cactgcgcaa 120
aaggtagagc ttgcccttga gaagaagtcg gcaccttctc ctcttaccta tcgcggtacg 180
tggatggtac gtgaagacgg aattgtcgaa ctctcgcttg tgtcctcgga gcaatcgaag 240
gcaccgcacg agaaagagct gtacgagctg attgacagta actccgttcg ctacatgggc 300
gctccgggcg caggaaagcc ttcaaaggag atggcgccgt tttacgtgct caaaaaaaca 360
aagaaa 366
<210>27
<211>1113
<212>DNA
<213> treponema pallidum
<400>27
gagacgcact atggctatgc gacgctgagc tatgcggact actgggccgg ggagttgggg60
cagagtcgcg acgtgctttt ggcgggtaat gccgaggcgg accgcgcggg ggatctcgac 120
gcaggcatgt tcgatgcagt ttctcgcgca acccacgggc atggcgcgtt ccgtcagcaa 180
tttcagtacg cggttgaggt attgggcgaa aaggttctct cgaagcagga gaccgaagac 240
agccgcggac gcaaaaagtg ggagtacgag actgacccaa gcgttactaa gatggtgcgt 300
gcctctgcgt catttcagga tttgggagag gacggggaga ttaagtttga agcagtcgag 360
ggtgcagtag cgttggcgga tcgcgcgagt tccttcatgg ttgacagcga ggaatacaag 420
attacgaacg taaaggttca cggtatgaag tttgtcccag ttgcggttcc tcatgaatta 480
aaagggattg caaaggagaa gtttcacttc gtggaagact cccgcgttac ggagaatacc 540
aacggcctta agacaatgct cactgaggat agtttttctg cacgtaaggt aagcagcatg 600
gagagcccgc acgaccttgt ggtagacacg gtgggtaccg tctaccacag ccgttttggt 660
tcggacgcag aggcttctgt gatgctgaaa agggctgatg gctctgagct gtcgcaccgt 720
gagttcatcg actatgtgat gaacttcaac acggtccgct acgactacta cggtgatgac 780
gcgagctaca ccaatctgat ggcgagttat ggcaccaagc actctgctga ctcctggtgg 840
aagacaggaa gagtgccccg catttcgtct ggtatcaact atgggttcga tcggtttaaa 900
ggttcagggc cgggatacta caggctgact ttgattgcga acgggtatag ggacgtagtt 960
gctgatgtgc gcttccttcc caagtacgag gggaacatcg atattgggtt gaaggggaag 1020
gtgctgacca tagggggcgc ggacgcggag actctgatgg atgctgcagt tgacgtgttt 1080
gccgatggac agcctaaact tgtcagcgat caa 1113
<210>28
<211>1866
<212>DNA
<213> treponema pallidum
<400>28
gccaaagcgg aaaaggtaga gagcgcgttg aagggaggta tctttcgggg tacgctgcct 60
gcggccgata gcccgggaat cgatacgact gtgacgttca acgcggatgg cactgcgcaa 120
aaggtagagc ttgcccttga gaagaagtcg gcaccttctc ctcttaccta tcgcggtacg 180
tggatggtac gtgaagacgg aattgtcgaa ctctcgcttg tgtcctcgga gcaatcgaag 240
gcaccgcacg agaaagagct gtacgagctg attgacagta actccgttcg ctacatgggc 300
gctccgggcg caggaaagcc ttcaaaggag atggcgccgt tttacgtgct caaaaaaaca 360
aagaaaggta gtggttcatt tagttctatc ccgaatggca cgtaccgggc gacgtatcag 420
gattttgatg agaatggttg gaaggacttt ctcgaggtta cttttgatgg tggcaagatg 480
gtgcaggtgg tttacgatta tcagcataaa gaagggcggt ttaagtccca ggacgctgac 540
taccatcggg tcatgtatgc atcctcgggc attggtcctg aaaaggcctt ccgtgagctc 600
gccgatgctt tgcttgaaaa gggtaatccg gagatggtgg atgtggtcac cggtgcaact 660
gtttcttccc agagtttccg tcgtttgggt gcggcgcttc tgcagagtgc gcggcgcggc 720
gagaaggaag ccattattag ccgatccggc ggcgagacgc actatggcta tgcgacgctg 780
agctatgcgg actactgggc cggggagttg gggcagagtc gcgacgtgct tttggcgggt 840
aatgccgagg cggaccgcgc gggggatctc gacgcaggca tgttcgatgc agtttctcgc 900
gcaacccacg ggcatggcgc gttccgtcag caatttcagt acgcggttga ggtattgggc 960
gaaaaggttc tctcgaagca ggagaccgaa gacagccgcg gacgcaaaaa gtgggagtac 1020
gagactgacc caagcgttac taagatggtg cgtgcctctg cgtcatttca ggatttggga 1080
gaggacgggg agattaagtt tgaagcagtc gagggtgcag tagcgttggc ggatcgcgcg 1140
agttccttca tggttgacag cgaggaatac aagattacga acgtaaaggt tcacggtatg 1200
aagtttgtcc cagttgcggt tcctcatgaa ttaaaaggga ttgcaaagga gaagtttcac 1260
ttcgtggaag actcccgcgt tacggagaat accaacggcc ttaagacaat gctcactgag 1320
gatagttttt ctgcacgtaa ggtaagcagc atggagagcc cgcacgacct tgtggtagac 1380
acggtgggta ccgtctacca cagccgtttt ggttcggacg cagaggcttc tgtgatgctg 1440
aaaagggctg atggctctga gctgtcgcac cgtgagttca tcgactatgt gatgaacttc 1500
aacacggtcc gctacgacta ctacggtgat gacgcgagct acaccaatct gatggcgagt 1560
tatggcacca agcactctgc tgactcctgg tggaagacag gaagagtgcc ccgcatttcg 1620
tctggtatca actatgggtt cgatcggttt aaaggttcag ggccgggata ctacaggctg 1680
actttgattg cgaacgggta tagggacgta gttgctgatg tgcgcttcct tcccaagtac 1740
gaggggaaca tcgatattgg gttgaagggg aaggtgctga ccataggggg cgcggacgcg 1800
gagactctga tggatgctgc agttgacgtg tttgccgatg gacagcctaa acttgtcagc 1860
gatcaa 1866
<210>29
<211>27
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer TP17-F
<400>29
gagctctgca caaccgtgtg tccgcac 27
<210>30
<211>33
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer TP47-R
<400>30
aagcttttac tactgggcca ctaccttcgc acg 33
<210>31
<211>28
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer SUMO-F
<400>31
gagctcgggt ccctgcagga ctcagaag 28
<210>32
<211>26
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer SUMO-R
<400>32
acctccaatc tgttcgcggt gagcct 26
<210>33
<211>42
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer SUMO-TP17-F
<400>33
gggtccctgc aggactcaga atgcacaacc gtgtgtccgc ac 42
<210>34
<211>23
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer TP17-F2
<400>34
gagctcgcca aagcggaaaa ggt 23
<210>35
<211>37
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer TP47-R2
<400>35
aagcttttat tgatcgctga caagtttagg ctgtcca 37
<210>36
<211>41
<212>DNA
<213> Artificial Synthesis
<220>
<223> primer SUMO-TP17-F2
<400>36
gctcaccgcg aacagattgg aggtgccaaa gcggaaaagg t 41

Claims (10)

1. A method of preparing a recombinant chimeric antigen of treponema pallidum comprising:
1) culturing the host cell under conditions suitable for production of the recombinant chimeric antigen; and
2) optionally isolating the recombinant chimeric antigen from the culture obtained in step 1),
wherein the host cell has introduced therein or transferred thereto a polynucleotide construct comprising the polynucleotide, the polynucleotide sequence encoding an amino acid sequence consisting of the sequence of TP17 fragment, first flexible linker, TP15 fragment, second flexible linker and TP47 fragment,
wherein the amino acid sequence of the TP17 fragment is shown as SEQ ID NO.12, the amino acid sequence of the TP15 fragment is shown as SEQ ID NO.9, and the amino acid sequence of the TP47 fragment is shown as SEQ ID NO. 13.
2. The method of claim 1, wherein the polynucleotide sequence consists of the nucleotide sequence set forth in SEQ ID No.26, the nucleotide sequence encoding the first flexible linker, the nucleotide sequence set forth in SEQ ID No.23, the nucleotide sequence encoding the second flexible linker, and the nucleotide sequence set forth in SEQ ID No. 27.
3. The method of claim 1 or 2, wherein the host cell carries a SUMO tag or the polynucleotide construct carries a SUMO tag.
4. The method of claim 3, which, after expression, comprises the additional step of removing the SUMO tag; for example, the SUMO tag is removed by enzymatic cleavage.
5. A protein having an amino acid sequence selected from the group consisting of:
(1) is composed of an amino acid sequence shown as SEQ ID NO.12, a first flexible linker, an amino acid sequence shown as SEQ ID NO.9, a second flexible linker and an amino acid sequence shown as SEQ ID NO.13 which are connected in series;
(2) and (2) a derivative amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence in (1), wherein the derivative amino acid sequence has the activity of the amino acid sequence shown in (1).
6. A polynucleotide, said polynucleotide:
a) encoding a protein consisting of an amino acid sequence shown as SEQ ID NO.12, a first flexible linker, an amino acid sequence shown as SEQ ID NO.9, a second flexible linker and an amino acid sequence shown as SEQ ID NO. 13;
b) consists of a nucleotide sequence shown as SEQ ID NO.26, a nucleotide sequence coding a first flexible linker, a nucleotide sequence shown as SEQ ID NO.23, a nucleotide sequence coding a second flexible linker and a nucleotide sequence shown as SEQ ID NO. 27; or
c) Complementary to a) or b).
7. A polynucleotide construct comprising the polynucleotide of claim 6.
8. A host cell comprising the polynucleotide construct of claim 7 or the polynucleotide of claim 6.
9. A composition for detecting antibodies to treponema pallidum, which contains the protein according to claim 5; preferably, the protein is additionally labeled with a detectable label, or the protein is coated on a solid support.
10. Use of a recombinant chimeric antigen in the preparation of a kit for detecting an anti-treponema pallidum antibody, wherein the recombinant chimeric antigen consists of an amino acid sequence shown as SEQ ID No.12, a first flexible linker, an amino acid sequence shown as SEQ ID No.9, a second flexible linker and an amino acid sequence shown as SEQ ID No. 13.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113004380A (en) * 2021-02-18 2021-06-22 青岛硕景生物科技有限公司 Treponema pallidum recombinant antigen, preparation and application
CN113024642A (en) * 2021-03-08 2021-06-25 珠海丽禾医疗诊断产品有限公司 Protein based on mycoplasma pneumoniae and preparation method and application thereof
CN114163539A (en) * 2021-12-14 2022-03-11 青岛硕景生物科技有限公司 TP recombinant antigen and preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103298941A (en) * 2011-01-13 2013-09-11 奥索临床诊断有限公司 Treponema pallidum triplet antigen
CN105542014A (en) * 2015-12-24 2016-05-04 菲鹏生物股份有限公司 TP recombinant antigen and preparing method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103298941A (en) * 2011-01-13 2013-09-11 奥索临床诊断有限公司 Treponema pallidum triplet antigen
CN105542014A (en) * 2015-12-24 2016-05-04 菲鹏生物股份有限公司 TP recombinant antigen and preparing method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113004380A (en) * 2021-02-18 2021-06-22 青岛硕景生物科技有限公司 Treponema pallidum recombinant antigen, preparation and application
CN113024642A (en) * 2021-03-08 2021-06-25 珠海丽禾医疗诊断产品有限公司 Protein based on mycoplasma pneumoniae and preparation method and application thereof
CN114163539A (en) * 2021-12-14 2022-03-11 青岛硕景生物科技有限公司 TP recombinant antigen and preparation method and application thereof

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