CN110981947B - Preparation and application of treponema pallidum TP47 recombinant antigen - Google Patents
Preparation and application of treponema pallidum TP47 recombinant antigen Download PDFInfo
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Abstract
The invention provides a recombinant TP47 antigen for detecting Treponema Pallidum (TP) antibodies, a preparation method of the recombinant TP47 antigen and application of the recombinant TP47 antigen in preparation of a kit for detecting Treponema Pallidum (TP).
Description
Technical Field
The invention belongs to the field of biological medicine, in particular to the field of infectious disease diagnosis and detection.
Background
Syphilis is an infectious disease prevalent throughout the world, 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.
Syphilis is a chronic and systemic sexually transmitted disease caused by Treponema Pallidum (TP), 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. Latent syphilis accounts for the majority of reported syphilis, primary and secondary syphilis are 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.
Because the clinical symptoms and physical signs of a human body infected by syphilis have no obvious specificity, the diagnosis is mainly carried out clinically by depending on the result of a serological experiment. At present, the common immunological detection methods for treponema pallidum comprise non-treponema pallidum antigen serum tests, such as venereal disease research laboratory tests (VDRL), rapid plasma reactive protein circular card tests (RPR), toluidine red unheated serum reactive protein tests (TRUST) and the like, but the methods use normal bovine myocardial cardiolipin as an antigen, have low specificity, and both serum positive to anti-cardiolipin antibodies and hyperlipidemia can generate false positive reactions. 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 recent years, with the intensive research on TP functions and the disclosure of genome-wide sequences, a variety of functional proteins have been sequentially identified. In addition, researchers have used genetic engineering methods to recombinantly express membrane-bound polypeptide proteins such as TP15, TP17, and TP47, which are specific to TP strains and have strong antigen reactivity, to obtain antigenic TP Recombinant proteins, which have been used in diagnosis of Syphilis infection to achieve good results (ANNET GERBERR, SIEGFRIED KRELL, JOACHIM MORENZ; "Recombinant Treponema pallidum antibodies in Syphilis virology", immunology, 1996, 196 (5): 535 GREEN 549). At present, a relatively common method for applying TP specific protein to diagnosis and detection of syphilis infection is a chemiluminescence magnetic particle immunoassay, and the principle is that TP specific protein is used for coating magnetic beads, and a chemiluminescence instrument is matched to detect TP antibodies in serum, so that screening and judgment of syphilis infection are carried out. Therefore, a TP-specific protein having good stability and both specificity and sensitivity is the target of research by researchers, and is also the quality guarantee of TP antibody measurement kits.
TP47 is a TP integral membrane protein, has strong immunogenicity, is present in the whole course of syphilis patients, has important function in the immunity of treponema pallidum infection, and TP47 is an important specific protein in the detection of syphilis antibodies. Moreover, in AIDS virus and treponema pallidum infected patients, the content of anti-TP antibody is lower than that of general syphilis patients and can be detected by using TP 47. However, the wild type TP47 protein cannot be perfectly used in the detection of syphilis antibody, and the defect of poor stability exists (WO2013/107633A 1). In order to ensure the stability of the TP antibody determination kit, a recombinant TP47 antigen with excellent thermal stability is urgently needed, and the recombinant protein can maintain the specificity and the sensitivity and can be applied to the detection of the syphilis antibody.
Disclosure of Invention
The invention provides a recombinant TP47 antigen for detecting Treponema Pallidum (TP) antibody, wherein the recombinant TP47 antigen can be expressed in a soluble manner, has good thermal stability and higher activity; the invention also provides a preparation method of the recombinant TP47 antigen; the invention also provides application of the recombinant TP47 antigen in preparing a kit for detecting Treponema Pallidum (TP); and the application of the recombinant TP47 antigen in preparing a kit for diagnosing syphilis.
In a first aspect the present invention provides a recombinant TP47 antigen for the detection of Treponema Pallidum (TP), said recombinant TP47 antigen having an amino acid sequence selected from the group consisting of:
(1) SEQ ID NOs: 2-4, and the amino acid sequence shown in any one of the following items:
ETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLVVDTVGTVYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISCGINYGFDRFKGSGPGYYRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFT(SEQ ID NO:2)(DTP47，7-391aa)；
ETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLVVDTVGTVYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISSGINYGFDRFKGSGPGYYRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFT(SEQ ID NO:3)(C296S，7-391aa)；
ETHYGYATLSYADYWAGELGQSRDVLLAGNAEADRAGDLDAGMFDAVSRATHGHGAFRQQFQYAVEVLGEKVLSKQETEDSRGRKKWEYETDPSVTKMVRASASFQDLGEDGEIKFEAVEGAVALADRASSFMVDSEEYKITNVKVHGMKFVPVAVPHELKGIAKEKFHFVEDSRVTENTNGLKTMLTEDSFSARKVSSMESPHDLVVDTVGTVYHSRFGSDAEASVMLKRADGSELSHREFIDYVMNFNTVRYDYYGDDASYTNLMASYGTKHSADSWWKTGRVPRISAGINYGFDRFKGSGPGYYRLTLIANGYRDVVADVRFLPKYEGNIDIGLKGKVLTIGGADAETLMDAAVDVFADGQPKLVSDQAVSLGQNVLSADFT(SEQ ID NO:4)(C296A，7-391aa)；
(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).
In a second aspect, the present invention provides an isolated polynucleotide sequence encoding the recombinant TP47 antigen of the first aspect.
Preferably, the isolated polynucleotide has a nucleotide sequence selected from the group consisting of:
(1) SEQ ID NOs: 6-8, wherein:
GAGACGCACTATGGCTATGCGACGCTGAGCTATGCGGACTACTGGGCCGGGGAGTTGGGGCAGAGTCGCGACGTGCTTTTGGCGGGTAATGCCGAGGCGGACCGCGCGGGGGATCTCGACGCAGGCATGTTCGATGCAGTTTCTCGCGCAACCCACGGGCATGGCGCGTTCCGTCAGCAATTTCAGTACGCGGTTGAGGTATTGGGCGAAAAGGTTCTCTCGAAGCAGGAGACCGAAGACAGCCGCGGACGCAAAAAGTGGGAGTACGAGACTGACCCAAGCGTTACTAAGATGGTGCGTGCCTCTGCGTCATTTCAGGATTTGGGAGAGGACGGGGAGATTAAGTTTGAAGCAGTCGAGGGTGCAGTAGCGTTGGCGGATCGCGCGAGTTCCTTCATGGTTGACAGCGAGGAATACAAGATTACGAACGTAAAGGTTCACGGTATGAAGTTTGTCCCAGTTGCGGTTCCTCATGAATTAAAAGGGATTGCAAAGGAGAAGTTTCACTTCGTGGAAGACTCCCGCGTTACGGAGAATACCAACGGCCTTAAGACAATGCTCACTGAGGATAGTTTTTCTGCACGTAAGGTAAGCAGCATGGAGAGCCCGCACGACCTTGTGGTAGACACGGTGGGTACCGTCTACCACAGCCGTTTTGGTTCGGACGCAGAGGCTTCTGTGATGCTGAAAAGGGCTGATGGCTCTGAGCTGTCGCACCGTGAGTTCaTCGACTATGTGATGAACTTCAACACGGTCCGCTACGACTACTACGGTGATGACGCGAGCTACACCAATCTGATGGCGAGTTATGGCACCAAGCACTCTGCTGACTCCTGGTGGAAGACAGGAAGAGTGCCCCGCATTTCGTGTGGTATCAACTATGGGTTCGATCGGTTTAAAGGTTCAGGGCCGGGATACTACAGGCTGACTTTGATTGCGAACGGGTATAGGGACGTAGTTGCTGATGTGCGCTTCCTTCCCAAGTACGAGGGGAACATCGATATTGGGTTGAAGGGGAAGGTGCTGACCATAGGGGGCGCGGACGCGGAGACTCTGATGGATGCTGCAGTTGACGTGTTTGCCGATGGACAGCCTAAaCTTGTCAGCGATCAAGCGGTGAGCTTGGGGCAGAATGTCCTCTCTGCGGATTTCACT(SEQ ID NO：6)(DTP47)；
GAGACGCACTATGGCTATGCGACGCTGAGCTATGCGGACTACTGGGCCGGGGAGTTGGGGCAGAGTCGCGACGTGCTTTTGGCGGGTAATGCCGAGGCGGACCGCGCGGGGGATCTCGACGCAGGCATGTTCGATGCAGTTTCTCGCGCAACCCACGGGCATGGCGCGTTCCGTCAGCAATTTCAGTACGCGGTTGAGGTATTGGGCGAAAAGGTTCTCTCGAAGCAGGAGACCGAAGACAGCCGCGGACGCAAAAAGTGGGAGTACGAGACTGACCCAAGCGTTACTAAGATGGTGCGTGCCTCTGCGTCATTTCAGGATTTGGGAGAGGACGGGGAGATTAAGTTTGAAGCAGTCGAGGGTGCAGTAGCGTTGGCGGATCGCGCGAGTTCCTTCATGGTTGACAGCGAGGAATACAAGATTACGAACGTAAAGGTTCACGGTATGAAGTTTGTCCCAGTTGCGGTTCCTCATGAATTAAAAGGGATTGCAAAGGAGAAGTTTCACTTCGTGGAAGACTCCCGCGTTACGGAGAATACCAACGGCCTTAAGACAATGCTCACTGAGGATAGTTTTTCTGCACGTAAGGTAAGCAGCATGGAGAGCCCGCACGACCTTGTGGTAGACACGGTGGGTACCGTCTACCACAGCCGTTTTGGTTCGGACGCAGAGGCTTCTGTGATGCTGAAAAGGGCTGATGGCTCTGAGCTGTCGCACCGTGAGTTCaTCGACTATGTGATGAACTTCAACACGGTCCGCTACGACTACTACGGTGATGACGCGAGCTACACCAATCTGATGGCGAGTTATGGCACCAAGCACTCTGCTGACTCCTGGTGGAAGACAGGAAGAGTGCCCCGCATTTCGTCTGGTATCAACTATGGGTTCGATCGGTTTAAAGGTTCAGGGCCGGGATACTACAGGCTGACTTTGATTGCGAACGGGTATAGGGACGTAGTTGCTGATGTGCGCTTCCTTCCCAAGTACGAGGGGAACATCGATATTGGGTTGAAGGGGAAGGTGCTGACCATAGGGGGCGCGGACGCGGAGACTCTGATGGATGCTGCAGTTGACGTGTTTGCCGATGGACAGCCTAAaCTTGTCAGCGATCAAGCGGTGAGCTTGGGGCAGAATGTCCTCTCTGCGGATTTCACT(SEQ ID NO：7)(C296S)；
GAGACGCACTATGGCTATGCGACGCTGAGCTATGCGGACTACTGGGCCGGGGAGTTGGGGCAGAGTCGCGACGTGCTTTTGGCGGGTAATGCCGAGGCGGACCGCGCGGGGGATCTCGACGCAGGCATGTTCGATGCAGTTTCTCGCGCAACCCACGGGCATGGCGCGTTCCGTCAGCAATTTCAGTACGCGGTTGAGGTATTGGGCGAAAAGGTTCTCTCGAAGCAGGAGACCGAAGACAGCCGCGGACGCAAAAAGTGGGAGTACGAGACTGACCCAAGCGTTACTAAGATGGTGCGTGCCTCTGCGTCATTTCAGGATTTGGGAGAGGACGGGGAGATTAAGTTTGAAGCAGTCGAGGGTGCAGTAGCGTTGGCGGATCGCGCGAGTTCCTTCATGGTTGACAGCGAGGAATACAAGATTACGAACGTAAAGGTTCACGGTATGAAGTTTGTCCCAGTTGCGGTTCCTCATGAATTAAAAGGGATTGCAAAGGAGAAGTTTCACTTCGTGGAAGACTCCCGCGTTACGGAGAATACCAACGGCCTTAAGACAATGCTCACTGAGGATAGTTTTTCTGCACGTAAGGTAAGCAGCATGGAGAGCCCGCACGACCTTGTGGTAGACACGGTGGGTACCGTCTACCACAGCCGTTTTGGTTCGGACGCAGAGGCTTCTGTGATGCTGAAAAGGGCTGATGGCTCTGAGCTGTCGCACCGTGAGTTCaTCGACTATGTGATGAACTTCAACACGGTCCGCTACGACTACTACGGTGATGACGCGAGCTACACCAATCTGATGGCGAGTTATGGCACCAAGCACTCTGCTGACTCCTGGTGGAAGACAGGAAGAGTGCCCCGCATTTCGGCTGGTATCAACTATGGGTTCGATCGGTTTAAAGGTTCAGGGCCGGGATACTACAGGCTGACTTTGATTGCGAACGGGTATAGGGACGTAGTTGCTGATGTGCGCTTCCTTCCCAAGTACGAGGGGAACATCGATATTGGGTTGAAGGGGAAGGTGCTGACCATAGGGGGCGCGGACGCGGAGACTCTGATGGATGCTGCAGTTGACGTGTTTGCCGATGGACAGCCTAAaCTTGTCAGCGATCAAGCGGTGAGCTTGGGGCAGAATGTCCTCTCTGCGGATTTCACT (SEQ ID NO: 8) (C296A); or
(2) Hybridizes under stringent conditions to the nucleotide sequence defined in (1) and encodes a polynucleotide having the sequence shown in SEQ ID NOs: 2-4, or a pharmaceutically acceptable salt thereof.
In a third aspect, the present invention provides a polynucleotide construct comprising the polynucleotide sequence of the second aspect.
In a fourth aspect, the present invention provides a host cell transformed with or comprising a polynucleotide of the second aspect or a polynucleotide construct of the third aspect, which host cell is capable of producing an amino acid sequence of the first aspect.
In a fifth aspect, the present invention provides a method of making the recombinant TP47 antigen of the first aspect, the method comprising the steps of:
(1) culturing the host cell of the fourth aspect under conditions suitable for production of the recombinant TP47 antigen of the first aspect; and
(2) optionally isolating recombinant TP47 antigen from the culture obtained in step (1).
Preferably, the method of preparing the recombinant TP47 antigen of the first aspect comprises the steps of:
(1) transfecting the polynucleotide of the second aspect into a host cell of the fourth aspect, or a host cell of the fourth aspect with a polynucleotide construct of the third aspect;
(2) culturing the host cell of step (1) under conditions suitable for the production of the recombinant TP47 antigen of the first aspect; and
(3) optionally isolating recombinant TP47 antigen from the culture obtained in step (1).
Preferably, the method of preparing a truncated TP47 antigen (DTP47) comprises the steps of:
(1) the following primers were used to perform PCR amplification using the nucleotide sequence of wild type TP47 as a template to obtain SEQ ID NO: 6:
SEQ ID NO: 9, a forward primer TP 47-F; and
SEQ ID NO: 10, and a reverse primer TP 47-R;
(2) converting SEQ ID NO: 6 to escherichia coli competent cells for expression; and
(3) the truncated TP47 antigen (DTP47) was obtained from step (2).
Preferably, the method of preparing a truncated mutant TP47 antigen (C296S) comprises the steps of:
(1) the following primers were used to generate a primer comprising SEQ ID NO: 6 as a template to obtain the nucleotide sequence of SEQ ID NO: 7:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 12, reverse primer C296S-R; and
SEQ ID NO: 11, forward primer C296S-F, SEQ ID NO: 10, and a reverse primer TP 47-R;
(2) converting SEQ ID NO: 7 to Escherichia coli competent cells for expression; and
(3) obtaining a truncated mutant TP47 antigen (C296S) from step (2).
Preferably, the method of preparing a truncated mutant TP47 antigen (C296A) comprises the steps of:
(1) the following primers were used to generate a primer comprising SEQ ID NO: 6 as a template to obtain the nucleotide sequence of SEQ ID NO: 8, and the sequence of the polynucleotide shown in the specification:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 14, reverse primer C296A-R; and
a forward primer C296A-F shown by SEQ ID NO.13 and a reverse primer TP47-R shown by SEQ ID NO. 10;
(2) converting SEQ ID NO: 8, transforming the polynucleotide sequence shown in the specification into escherichia coli competent cells for expression; and
(3) obtaining a truncated mutant TP47 antigen (C296A) from step (2).
In a sixth aspect, the present invention provides a kit for detecting syphilis or a kit for detecting antibodies to syphilis, the kit comprising the recombinant TP47 antigen of the first aspect, or the recombinant TP47 antigen encoded by the polynucleotide sequence of the second aspect, or the recombinant TP47 antigen expressed by the host cell of the fourth aspect, or the recombinant TP47 antigen prepared by the method of the fifth aspect.
In a seventh aspect, the present invention provides the use of a recombinant TP47 antigen of the first aspect, or a recombinant TP47 antigen encoded by a polynucleotide sequence of the second aspect, or a recombinant TP47 antigen expressed by a host cell of the fourth aspect, or a recombinant TP47 antigen produced by a method of the fifth aspect, in the manufacture of a kit for the detection of syphilis or for the detection of syphilis antibodies.
In an eighth aspect, the present invention provides a method for amplifying SEQ ID NO: 6 of the nucleotide sequence of truncated DTP 47:
SEQ ID NO: 9, a forward primer TP 47-F; and
SEQ ID NO: 10, and reverse primer TP 47-R.
In a ninth aspect, the present invention provides a method for amplifying SEQ ID NO: 7, the primer set of the nucleotide sequence of truncated mutant C296S set forth in seq id no:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 12, reverse primer C296S-R; and
SEQ ID NO: 11, forward primer C296S-F, SEQ ID NO: 10, and reverse primer TP 47-R.
In a tenth aspect, the present invention provides a method for amplifying SEQ ID NO: primer set for the nucleotide sequence of truncated mutant C296A shown in fig. 8:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 14, reverse primer C296A-R; and
a forward primer C296A-F shown by SEQ ID NO.13 and a reverse primer TP47-R shown by SEQ ID NO. 10.
The invention aims to develop a recombinant TP47 antigen, which covers the dominant epitope region of TP47 antigen, has better solubility expression, better heat stability and immunoreactivity to a TP antibody from serum similar to the wild type TP47 antigen, sensitivity and specificity higher than the wild type TP47 antigen compared with the wild type TP47 antigen.
Furthermore, in the design of the recombinant TP47 antigen, through optimization of the dominant epitope region of the TP47 antigen, the full-length 26-410aa region of TP47 is selected, redundant amino acids at the N end and the C end are removed, and the solubility and the specificity of the antigen are improved without damaging the sensitivity.
Furthermore, in order to maximally improve the soluble expression and the thermal stability of the recombinant TP47 antigen, cysteine to serine mutation is carried out on the dominant epitope region of the TP47 antigen, so that the thermal stability of the antigen is improved without damaging the sensitivity of the antigen.
Meanwhile, the invention discloses the amino acid sequence and the nucleotide sequence of the screened recombinant TP47 antigen and a technical route for designing and constructing the whole antigen, and lays a foundation for the development of a high-quality treponema pallidum antibody determination kit (a chemiluminescence method).
Drawings
FIG. 1 shows the expression purification scheme of recombinant wild-type WTP47 antigen in example 1; from left to right, the following are in sequence: the inclusion body dissolves the supernatant and breaks the supernatant.
FIG. 2 shows the expression purification scheme of recombinant truncated DTP47 antigen in example 2; from left to right, the following are in sequence: supernatant of inclusion body lysate and supernatant of crushing liquid.
FIG. 3 shows a purification diagram for the expression of recombinant truncated, mutant C296S antigen in example 3; from left to right, the following are in sequence: supernatant of the disruption solution and supernatant of inclusion body lysate.
FIG. 4 shows a map of the expression purification of the recombinant truncated, mutant C296A antigen in example 4; from left to right, the following are in sequence: supernatant of the disruption solution and supernatant of inclusion body lysate.
FIG. 5 shows the results of activity assays for recombinant TP47 antigen in example 5.
FIG. 6 shows the results of the thermostability assay for recombinant TP47 antigen in example 6.
Description of sequence listing
SEQ ID NO: 1-the amino acid sequence of wild-type TP47 (i.e., the amino acid sequence of "WTP 47");
SEQ ID NO: 2-the amino acid sequence of recombinant truncated TP47 (i.e., the amino acid sequence of "DTP 47");
SEQ ID NO: 3-on the basis of the recombinant truncated TP47, the amino acid sequence of the S is mutated from the C at position 296 (namely the amino acid sequence of DTP47+ C296S);
SEQ ID NO: 4-on the basis of the recombinant truncated TP47, the amino acid sequence of A is mutated from C at position 296 (namely the amino acid sequence of DTP47+ C296A);
SEQ ID NO: 5-nucleotide sequence of wild type TP47 (i.e., "nucleotide sequence of WTP 47");
SEQ ID NO: 6-nucleotide sequence of recombinant truncated TP47 (i.e., "nucleotide sequence of DTP 47");
SEQ ID NO: 7-nucleotide sequence in which C at position 296 is mutated into S based on recombinant truncated TP47 (namely nucleotide sequence of "DTP 47+ C296S");
SEQ ID NO: 8-nucleotide sequence in which C at position 296 is mutated into A (namely nucleotide sequence of "DTP 47+ C296A") on the basis of recombinant truncated TP 47;
SEQ ID NO: 9-primer "TP 47-F" in the recombinant construction of truncated TP47 (i.e., "DTP 47");
SEQ ID NO: 10-primer "TP 47-R" in the recombinant construction of truncated TP47 (i.e., "DTP 47");
SEQ ID NO: 11-primer "C296 SF" for recombinant construction of "DTP 47+ C296S";
SEQ ID NO: 12-primer "C296 SR" for recombinant construction of "DTP 47+ C296S";
SEQ ID NO: 13-primer "C296 AF" for recombinant construction of "DTP 47+ C296A"; and
SEQ ID NO: 14-primer "C296 AR" for recombinant construction of "DTP 47+ C296A".
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that various changes and modifications based on the inventive concept herein will occur to those skilled in the art and are intended to be included within the scope of the present invention. The reagents used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1 construction and expression purification of recombinant wild-type TP47 antigen (WTP47)
The amino acids at 19 th to 434 th positions of TP47 are selected as an antigen region, and the nucleotide sequence WTP47(SEQ ID NO: 5) is obtained by a chemical synthesis method from the gene sequence after codon optimization. The WTP47 gene fragment was constructed into the pET28a vector according to the method described in the molecular cloning protocols (fourth edition) and the recombinant vector was named pET28a-WTP 47.
pET28a-WTP47 was transformed into E.coli BL21(Rosetta) competent cells and induced expression was performed as described in the molecular cloning protocols. Specifically, the cells were cultured at 37 ℃ at 200r/min until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 37 ℃ for 4 hours. The clone in which the desired protein was induced was named R-pET28a-WTP 47. The R-pET28a-WTP47 was subjected to mass fermentation under the induction conditions determined as described 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-WTP47 fermentation thalli by using a bacterium breaking buffer solution (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 20mM PBS containing 8M urea into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm for 20min at 4 ℃ after the completion of dissolving, and collecting the inclusion body dissolved substance supernatant. Taking the supernatant of the crushing liquid and the supernatant of the inclusion body dissolved matter respectively to carry out SDS-PAGE, and determining the expression form of the protein.
As shown in FIG. 1, pET28a-WTP47 was expressed as inclusion bodies. The inclusion body lysate supernatant was purified by NI column. The loading buffer solution is: 20mM PBS solution containing 8M urea, 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 a 4M urea-containing 10mM PBS solution and 1mM EDTApH7.4 at 2-8 ℃ while reducing the urea concentration to 0 in the absence of urea (10mM PBS, 1mM EDTA, pH7.4) twice. 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 protein was designated WTP 47.
Example 2 construction and expression purification of recombinant truncated DTP47 antigen
The nucleotide sequence of wild-type WTP47 in example 1, SEQ ID NO: 5 as a template, and a nucleotide sequence DTP47(SEQ ID NO: 6) is obtained by carrying out PCR amplification and gel recovery on a primer pair TP47-F (SEQ ID NO: 9) and TP47-R (SEQ ID NO: 10) according to the method in a molecular cloning experimental manual. The DTP47 gene fragment was constructed into pET28a vector, and the recombinant vector was named pET28a-DTP 47.
pET28a-DTP47 was transformed into E.coli BL21(Rosetta) competent cells and induced expression was performed as described in the molecular cloning protocols. Specifically, the cells were cultured at 37 ℃ at 200r/min until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 37 ℃ for 4 hours. The clone inducing the target protein was named R-pET28a-DTP 47. R-pET28a-DTP47 was subjected to bulk fermentation 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-DTP47 fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and carrying out ultrasonic crushing; after the disruption was completed, the mixture was centrifuged at 12000rpm at 4 ℃ for 20min, and the supernatant of the disruption solution was collected. Adding 20mM PBS solution of 8M urea into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm for 20min at 4 ℃ after the completion of dissolving, and collecting the inclusion body dissolved substance supernatant. Taking the supernatant of the crushing liquid and the supernatant of the inclusion body dissolved matter respectively to carry out SDS-PAGE, and determining the expression form of the protein.
As shown in FIG. 2, pET28a-DTP47 was expressed in both soluble and inclusion forms. 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; the impurity washing buffer solution is as follows: 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 at 2-8 deg.C with 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 protein was designated as DTP 47.
Example 3 construction and expression purification of recombinant truncated, mutant C296S antigen
The nucleotide sequence of DTP47 in example 2, SEQ ID NO: 6 as template, primer pair TP47-F (SEQ ID NO: 9), C296S-R (SEQ ID NO: 12); C296S-F (SEQ ID NO: 11), TP47-R (SEQ ID NO: 10) were subjected to over-lap PCR amplification and gel recovery according to the methods described in the molecular cloning protocols to obtain the nucleotide sequence C296S (SEQ ID NO: 7).
The C296S gene fragment was constructed into pET28a vector, and the recombinant vector was named pET28 a-C296S. pET28a-C296S was transformed into E.coli BL21(Rosetta) competent cells and induced expression was performed as described in the molecular cloning protocols. Specifically, the cells were cultured at 37 ℃ at 200r/min until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 37 ℃ for 4 hours. The clone inducing the target protein was named R-pET28a-DTP 47. R-pET28a-DTP47 was subjected to bulk fermentation 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-C296S fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and 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. The pellet was added with 8M urea 20mM PBS to fully dissolve the inclusion bodies, and after completion of the dissolution, the inclusion body lysate supernatant was collected by centrifugation at 12000rpm for 20min at 4 ℃. Taking the supernatant of the crushing liquid and the supernatant of the inclusion body dissolved matter respectively to carry out SDS-PAGE, and determining the expression form of the protein.
As shown in FIG. 3, pET28a-C296S is predominantly 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; the impurity washing buffer solution is as follows: 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 with 10mM PBS and 1mM EDTA ApH7.4 at 2-8 deg.C for two times. 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 protein was designated C296S.
Example 4 construction and expression purification of recombinant truncated and mutant C296A antigen
The nucleotide sequence of DTP47 in example 2, SEQ ID NO: 6 as template, primer pair TP47-F (SEQ ID NO: 9), C296A-R (SEQ ID NO: 14); C296A-F (SEQ ID NO: 13), TP47-R (SEQ ID NO: 10) were subjected to over-lap PCR amplification and gel recovery according to the methods described in the molecular cloning protocols to obtain the nucleotide sequence C296A (SEQ ID NO: 5).
The C296A gene fragment was constructed into the pET28a vector as described in the molecular cloning protocols, and the recombinant vector was named pET28 a-C296A.
pET28a-C296A was transformed into E.coli BL21(Rosetta) competent cells and induced expression was performed as described in the molecular cloning protocols. Specifically, the cells were cultured at 37 ℃ at 200r/min until OD600 became 0.8, IPTG was added to a final concentration of 1mM, and induction was carried out at 37 ℃ for 4 hours. The clone inducing the target protein was named R-pET28a-DTP 47. R-pET28a-DTP47 was subjected to bulk fermentation 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-C296A fermented thalli with a bacterium breaking buffer (20mM PBS pH7.4, 0.5M NaCl), mixing uniformly and 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 20mM PBS solution of 8M urea into the precipitate, fully dissolving the inclusion body, centrifuging at 12000rpm for 20min at 4 ℃ after the completion of dissolving, and collecting the inclusion body dissolved substance supernatant. Taking the supernatant of the crushing liquid and the supernatant of the inclusion body dissolved matter respectively to carry out SDS-PAGE, and determining the expression form of the protein.
As shown in FIG. 2, R-pET28a-C296A was expressed in both soluble form and inclusion body 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; the impurity washing buffer solution is as follows: 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 with 10mM PBS and 1mM EDTApH7.4 at 2-8 deg.C twice. 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 protein was designated C296A.
Example 5 Activity assay of recombinant TP47 antigen
Under the condition that other production processes are completely consistent, a double-antigen sandwich method is adopted, and the recombinant wild type TP47 antigen WTP47 prepared in the embodiment 1 of the invention is respectively used; recombinant truncated TP47 antigen DTP47 prepared in example 2; the recombinant truncated, mutant TP47 antigen C296S prepared in example 3; the recombinant truncated and mutant TP47 antigen C296A prepared in example 3 was coated with magnetic beads, and HRP enzyme was labeled with a TP15+17+47 chimeric antigen (Fei Peng Bio Inc., cat # CMIA-TP-Ag13), and after preparing a reagent, TP47 quality control was tested to evaluate the activity of the antigen prepared in the present invention. The results show that the truncated, mutant recombinant TP47 antigen C296S activity of the present invention is comparable to the recombinant wild-type TP47 antigen WTP47 (see fig. 5).
Example 6 thermostability assay of recombinant TP47 antigen
In the case of other production processes being completely identical, recombinant wild-type TP47 antigen WTP47 prepared in example 1 of the present invention was used; recombinant truncated TP47 antigen DTP47 prepared in example 2; the recombinant truncated, mutant TP47 antigen C296S prepared in example 3; the recombinant truncated and mutant TP47 antigen C296A prepared in example 3, was heat-accelerated at 37 ℃ for 3 days, 7 days, and 14 days, respectively; and (3) respectively coating the magnetic beads with the heat-accelerated antigens, applying the coated magnetic beads to a TP antibody determination kit (chemiluminescence method), and determining the quality control of TP 47. The results show that the signal values of the coated magnetic beads of the C296S of the invention have no obvious difference with the freshly prepared antigen after 3 days, 7 days and 14 days of thermal acceleration treatment at 37 ℃, while the signal values of the coated magnetic beads of the WTP47 are reduced by about 40% compared with those of the freshly prepared antigen after the thermal acceleration treatment at 37 ℃ (see figure 6).
Example 7 specificity, sensitivity detection of recombinant TP47 antigen
Under the condition that other production processes are completely consistent, a double-antigen sandwich method is adopted, and the recombinant wild type TP47 antigen WTP47 prepared in the embodiment 1 of the invention is respectively used; in example 3, freshly prepared recombinant truncated and mutant TP47 antigen C296S coated magnetic beads, TP15 antigen (Fenpeng biological corporation, cat # TP15-Ag2) coated magnetic beads, TP17 antigen (Fenpeng biological corporation, cat # TP17-Ag1) coated magnetic beads, and TP15+17+47 chimeric antigen (Fenpeng biological corporation, cat # CMIA-TP-Ag13) labeled HRP enzyme were prepared into a Treponema Pallidum (TP) antibody detection kit, and a national reference for detecting treponema pallidum antibody chemiluminescence reagents (China food and drug research institute, cat # 036-201370801) to evaluate the sensitivity of the antigen prepared by the present invention. The result shows that the C296S detection Treponema Pallidum (TP) antibody negative reference substance, positive reference substance and minimum detection limit reference substance all meet the requirements; when WTP47 detects national reference products of Treponema Pallidum (TP) antibody, the positive reference product and the minimum detection limit reference product meet the requirements, and the negative reference product has two false positive cases. Thus, the specificity of the truncated and mutant recombinant TP47 antigen C296S is improved, and the sensitivity is not influenced (see Table 3).
TABLE 3 sensitivity test results for recombinant TP47 antigen
Note: treponema pallidum antibody chemiluminescence reagent national reference substance, wherein N1-N20 are negative reference substances; 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 actual determination result recombines wild type TP47 antigen WTP47 to detect 2 cases of false positive, and the truncated and mutant type recombined TP47 antigen C296S is 20/20; the positive reference substance is 10/10, the actual detection result recombines wild type TP47 antigen WTP47, and the truncated and mutant recombined TP47 antigen C296S are 10/10; the minimum detection limit reference substance is required to be not less than 2/4(L4 needs to be negative), the actual detection result recombines the wild type TP47 antigen WTP47, the truncated and mutant recombinant TP47 antigen C296S is 3/4, and L4 is negative.
Example 8 clinical sample testing of recombinant TP47 antigen
Under the condition that other production processes are completely consistent, a double-antigen sandwich method is adopted, and the recombinant wild type TP47 antigen WTP47 prepared in the embodiment 1 of the invention is respectively used; the recombinant truncated and mutant TP47 antigen C296S coated magnetic beads prepared in example 3, the TP15 antigen (Fenpeng biological corporation, cat # TP15-Ag2) coated magnetic beads, the TP17 antigen (Fenpeng biological corporation, cat # TP17-Ag1) coated magnetic beads and the TP15+17+47 chimeric antigen (Fenpeng biological corporation, cat # CMIA-TP-Ag13) labeled HRP enzyme were used to prepare a Treponema Pallidum (TP) antibody detection kit, and 1153 clinical random samples were tested to evaluate the clinical sample compliance rate of the antigen prepared by the present invention. The result shows that in 1153 clinical random samples, 93 positive samples are detected by WTP47 and C296S antigens, but three false positives (determined as negative by the yapei kit) appear in wild type WTP47, and the other detection results are consistent, which indicates that the truncated and mutant recombinant TP47 antigen C296S has a positive influence on the clinical compliance rate of the recombinant TP47 antigen, and the clinical compliance rate of the truncated and mutant recombinant TP47 antigen C296S can meet the requirements of development of immunodiagnostic reagents (see Table 4).
TABLE 4 clinical sample testing of recombinant TP47 antigen
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
Preparation and application of <120> treponema pallidum TP47 recombinant antigen
<130> RYP1910505.3
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 416
<212> PRT
<213> Treponema pallidum
<400> 1
Gly Cys Gly Ser Ser His His Glu Thr His Tyr Gly Tyr Ala Thr Leu
1 5 10 15
Ser Tyr Ala Asp Tyr Trp Ala Gly Glu Leu Gly Gln Ser Arg Asp Val
20 25 30
Leu Leu Ala Gly Asn Ala Glu Ala Asp Arg Ala Gly Asp Leu Asp Ala
35 40 45
Gly Met Phe Asp Ala Val Ser Arg Ala Thr His Gly His Gly Ala Phe
50 55 60
Arg Gln Gln Phe Gln Tyr Ala Val Glu Val Leu Gly Glu Lys Val Leu
65 70 75 80
Ser Lys Gln Glu Thr Glu Asp Ser Arg Gly Arg Lys Lys Trp Glu Tyr
85 90 95
Glu Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala Ser Ala Ser Phe
100 105 110
Gln Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu Ala Val Glu Gly
115 120 125
Ala Val Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp Ser Glu
130 135 140
Glu Tyr Lys Ile Thr Asn Val Lys Val His Gly Met Lys Phe Val Pro
145 150 155 160
Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys Glu Lys Phe His
165 170 175
Phe Val Glu Asp Ser Arg Val Thr Glu Asn Thr Asn Gly Leu Lys Thr
180 185 190
Met Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser Met Glu
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Ser Pro His Asp Leu Val Val Asp Thr Val Gly Thr Val Tyr His Ser
210 215 220
Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met Leu Lys Arg Ala Asp
225 230 235 240
Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met Asn Phe
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Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr Thr Asn
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Leu Met Ala Ser Tyr Gly Thr Lys His Ser Ala Asp Ser Trp Trp Lys
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Thr Gly Arg Val Pro Arg Ile Ser Cys Gly Ile Asn Tyr Gly Phe Asp
290 295 300
Arg Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Arg Leu Thr Leu Ile Ala
305 310 315 320
Asn Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe Leu Pro Lys Tyr
325 330 335
Glu Gly Asn Ile Asp Ile Gly Leu Lys Gly Lys Val Leu Thr Ile Gly
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Gly Ala Asp Ala Glu Thr Leu Met Asp Ala Ala Val Asp Val Phe Ala
355 360 365
Asp Gly Gln Pro Lys Leu Val Ser Asp Gln Ala Val Ser Leu Gly Gln
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Asn Val Leu Ser Ala Asp Phe Thr Pro Gly Thr Glu Tyr Thr Val Glu
385 390 395 400
Val Arg Phe Lys Glu Phe Gly Ser Val Arg Ala Lys Val Val Ala Gln
405 410 415
<210> 2
<211> 385
<212> PRT
<213> Treponema pallidum
<400> 2
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
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Lys Met Val Arg Ala Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly
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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
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Thr Glu Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe
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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
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Ala Ser Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg
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Glu Phe Ile Asp Tyr Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr
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Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr
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Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile
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Ser Cys Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro
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Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val
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Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly
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Leu Lys Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu
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Met Asp Ala Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val
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Ser Asp Gln Ala Val Ser Leu Gly Gln Asn Val Leu Ser Ala Asp Phe
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Thr
385
<210> 3
<211> 385
<212> PRT
<213> Treponema pallidum
<400> 3
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
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Ala Asp Arg Ala Gly Asp Leu Asp Ala Gly Met Phe Asp Ala Val Ser
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Arg Ala Thr His Gly His Gly Ala Phe Arg Gln Gln Phe Gln Tyr Ala
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Val Glu Val Leu Gly Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp
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Ser Arg Gly Arg Lys Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr
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Lys Met Val Arg Ala Ser Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly
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Glu Ile Lys Phe Glu Ala Val Glu Gly Ala Val Ala Leu Ala Asp Arg
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Ala Ser Ser Phe Met Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val
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Lys Val His Gly Met Lys Phe Val Pro Val Ala Val Pro His Glu Leu
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Lys Gly Ile Ala Lys Glu Lys Phe His Phe Val Glu Asp Ser Arg Val
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Thr Glu Asn Thr Asn Gly Leu Lys Thr Met Leu Thr Glu Asp Ser Phe
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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
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Ala Ser Val Met Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg
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Glu Phe Ile Asp Tyr Val Met Asn Phe Asn Thr Val Arg Tyr Asp Tyr
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Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr
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Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile
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Ser Ser Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro
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Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val
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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 Ala Val Ser Leu Gly Gln Asn Val Leu Ser Ala Asp Phe
370 375 380
Thr
385
<210> 4
<211> 385
<212> PRT
<213> Treponema pallidum
<400> 4
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
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Ser Ala Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu Val Val
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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
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Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr Gly Thr
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Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile
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Ser Ala Gly Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro
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Gly Tyr Tyr Arg Leu Thr Leu Ile Ala Asn Gly Tyr Arg Asp Val Val
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Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp Ile Gly
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Leu Lys Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu
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Met Asp Ala Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val
355 360 365
Ser Asp Gln Ala Val Ser Leu Gly Gln Asn Val Leu Ser Ala Asp Phe
370 375 380
Thr
385
<210> 5
<211> 1251
<212> DNA
<213> Treponema pallidum
<400> 5
ggctgtggct cgtctcatca tgagacgcac tatggctatg cgacgctgag ctatgcggac 60
tactgggccg gggagttggg gcagagtcgc gacgtgcttt tggcgggtaa tgccgaggcg 120
gaccgcgcgg gggatctcga cgcaggcatg ttcgatgcag tttctcgcgc aacccacggg 180
catggcgcgt tccgtcagca atttcagtac gcggttgagg tattgggcga aaaggttctc 240
tcgaagcagg agaccgaaga cagccgcgga cgcaaaaagt gggagtacga gactgaccca 300
agcgttacta agatggtgcg tgcctctgcg tcatttcagg atttgggaga ggacggggag 360
attaagtttg aagcagtcga gggtgcagta gcgttggcgg atcgcgcgag ttccttcatg 420
gttgacagcg aggaatacaa gattacgaac gtaaaggttc acggtatgaa gtttgtccca 480
gttgcggttc ctcatgaatt aaaagggatt gcaaaggaga agtttcactt cgtggaagac 540
tcccgcgtta cggagaatac caacggcctt aagacaatgc tcactgagga tagtttttct 600
gcacgtaagg taagcagcat ggagagcccg cacgaccttg tggtagacac ggtgggtacc 660
gtctaccaca gccgttttgg ttcggacgca gaggcttctg tgatgctgaa aagggctgat 720
ggctctgagc tgtcgcaccg tgagttcatc gactatgtga tgaacttcaa cacggtccgc 780
tacgactact acggtgatga cgcgagctac accaatctga tggcgagtta tggcaccaag 840
cactctgctg actcctggtg gaagacagga agagtgcccc gcatttcgtg tggtatcaac 900
tatgggttcg atcggtttaa aggttcaggg ccgggatact acaggctgac tttgattgcg 960
aacgggtata gggacgtagt tgctgatgtg cgcttccttc ccaagtacga ggggaacatc 1020
gatattgggt tgaaggggaa ggtgctgacc atagggggcg cggacgcgga gactctgatg 1080
gatgctgcag ttgacgtgtt tgccgatgga cagcctaaac ttgtcagcga tcaagcggtg 1140
agcttggggc agaatgtcct ctctgcggat ttcactcccg gcactgagta cacggttgag 1200
gttaggttca aggaattcgg ttctgtgcgt gcgaaggtag tggcccagta g 1251
<210> 6
<211> 1155
<212> DNA
<213> Treponema pallidum
<400> 6
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 caagcggtga gcttggggca gaatgtcctc 1140
tctgcggatt tcact 1155
<210> 7
<211> 1155
<212> DNA
<213> Treponema pallidum
<400> 7
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 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 caagcggtga gcttggggca gaatgtcctc 1140
tctgcggatt tcact 1155
<210> 8
<211> 1155
<212> DNA
<213> Treponema pallidum
<400> 8
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 catttcggct 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 caagcggtga gcttggggca gaatgtcctc 1140
tctgcggatt tcact 1155
<210> 9
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 9
gagctcgaga cgcactatgg ctatgcgacg 30
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 10
aagcttctac tgggccacta ccttcgcac 29
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 11
ccgcatttcg tctggtatca actatg 26
<210> 12
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> CATAGTTGATACCagaCGAAATGCGG
<400> 12
catagttgat accagacgaa atgcgg 26
<210> 13
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 13
cccgcatttc ggctggtatc aactatgg 28
<210> 14
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 14
ccatagttga taccagccga aatgcggg 28
Claims (13)
1. Recombinant TP47 antigen for the detection of treponema pallidum TP, characterized in that the recombinant TP47 antigen is an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2-4.
2. An isolated polynucleotide sequence, characterized in that said polynucleotide sequence encodes the recombinant TP47 antigen of claim 1.
3. The polynucleotide sequence of claim 2, wherein the polynucleotide sequence has a polynucleotide sequence selected from the group consisting of:
(1) SEQ ID NOs: 6-8; or
(2) Hybridizing to the polynucleotide sequence defined in (1) and encoding a polypeptide having the sequence shown in SEQ ID NOs: 2-4, or a polynucleotide sequence of an amino acid sequence as set forth in any one of claims 2-4.
4. A polynucleotide construct comprising the polynucleotide sequence of claim 2 or 3.
5. Host cell, characterized in that it is transfected with or comprises a polynucleotide sequence according to claim 2 or 3 or with a polynucleotide construct according to claim 4, and in that it is capable of producing the recombinant TP47 antigen according to claim 1.
6. A method of preparing the recombinant TP47 antigen of claim 1, characterized in that the method comprises the steps of:
(1) culturing the host cell of claim 5 under conditions suitable for production of the recombinant TP47 antigen of claim 1; and
(2) optionally isolating recombinant TP47 antigen from the culture obtained in step (1).
7. Method according to claim 6, characterized in that it comprises the following steps:
(1) transfecting the polynucleotide sequence of claim 2 or 3 into the host cell of claim 5, or transfecting the polynucleotide construct of claim 4 into the host cell of claim 5;
(2) culturing the host cell of step (1) under conditions suitable for production of the recombinant TP47 antigen of claim 1; and
(3) optionally isolating recombinant TP47 antigen from the culture obtained in step (2).
8. Method according to claim 7, characterized in that it comprises the following steps:
(1) performing PCR amplification by using the nucleotide sequence of wild type TP47 as a template by using the following primers to obtain SEQ ID NO: 6:
SEQ ID NO: 9, a forward primer TP 47-F; and
SEQ ID NO: 10, and a reverse primer TP 47-R;
(2) mixing the amino acid sequence shown in SEQ ID NO: 6 to escherichia coli competent cells for expression; and
(3) the truncated TP47 antigen DTP47 was obtained from step (2).
9. Method according to claim 8, characterized in that it comprises the following steps:
(1) the following primers were used to generate a primer comprising SEQ ID NO: 6 as a template to obtain the nucleotide sequence of SEQ ID NO: 7:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 12, reverse primer C296S-R; and
SEQ ID NO: 11, forward primer C296S-F, SEQ ID NO: 10, and a reverse primer TP 47-R;
(2) converting SEQ ID NO: 7 to Escherichia coli competent cells for expression; and
(3) obtaining truncated mutant TP47 antigen C296S from step (2).
10. Method according to claim 8, characterized in that it comprises the following steps:
(1) the following primers were used to generate a primer comprising SEQ ID NO: 6 as a template to obtain the nucleotide sequence of SEQ ID NO: 8, and the sequence of the polynucleotide shown in the specification:
SEQ ID NO: 9, forward primer TP47-F, SEQ ID NO: 14, reverse primer C296A-R; and
SEQ ID NO: 13, forward primer C296A-F, seq id no: 10, and a reverse primer TP 47-R;
(2) converting SEQ ID NO: 8, transforming the polynucleotide sequence shown in the specification into escherichia coli competent cells for expression; and
(3) obtaining truncated mutant TP47 antigen C296A from step (2).
11. Kit for the detection of syphilis or for the detection of syphilis antibodies, characterized in that it comprises the recombinant TP47 antigen of claim 1, or the recombinant TP47 antigen encoded by the polynucleotide sequence of claim 2 or 3, or the recombinant TP47 antigen expressed by the host cell of claim 5, or the recombinant TP47 antigen prepared by the method of any one of claims 6 to 10.
12. Use of the recombinant TP47 antigen of claim 1, the recombinant TP47 antigen encoded by the polynucleotide sequence of claim 2 or 3, the recombinant TP47 antigen expressed by the host cell of claim 5, or the recombinant TP47 antigen prepared by the method of any one of claims 6 to 10 for the preparation of a kit for the detection of syphilis or for the detection of syphilis antibodies.
13. Primer set for amplifying a polynucleotide sequence according to claim 2 or 3, characterized in that said primer set is selected from the group consisting of:
(1) SEQ ID NO: 9, a forward primer TP 47-F; and
SEQ ID NO: 10, and a reverse primer TP 47-R;
(2) SEQ ID NO: 9, forward primer TP47-F, seq id NO: 12, reverse primer C296S-R; and
SEQ ID NO: 11, forward primer C296S-F shown in seq id NO: 10, and a reverse primer TP 47-R;
(3) SEQ ID NO: 9, forward primer TP47-F, seq id no: 14, reverse primer C296A-R; and
SEQ ID NO: 13, forward primer C296A-F, seq id no: 10, and reverse primer TP 47-R.
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