CN1156491C - Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit - Google Patents

Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit Download PDF

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CN1156491C
CN1156491C CNB998154245A CN99815424A CN1156491C CN 1156491 C CN1156491 C CN 1156491C CN B998154245 A CNB998154245 A CN B998154245A CN 99815424 A CN99815424 A CN 99815424A CN 1156491 C CN1156491 C CN 1156491C
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antigen
protein
antibody
enzyme
treponema pallidum
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CN1332750A (en
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建 汪
汪建
牟峰
李志杰
高存记
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Beijing Gbi Biotechnology Co ltd
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Abstract

The present invention discloses a protein having a general formula (Xaa [n] Tpp (Xaa)[m]), wherein Tpp is selected from syphilis spiral extrasomatic membrane proteins Tpp15, Tpp17 andTpp47 or segments or derivates with immunoreactivity for combining human immunoglobulin; Xaa represents natural amino acid residues, wherein at least one Xaa and at least one end Xaa of the general formula protein proximal to each side of Tpp are selected from the amino acid residues of Lys, Arg and Trp; n and m respectively present integral numbers from 0 to 10, and the condition is that n plus m is not smaller than 2. The present invention also discloses a method for detecting an antibody of human treponema pallidum by using a general formula protein immunization method, particularly a method for detecting an antibody of human treponema pallidum by using a dual-antigen sandwich method. The present invention also discloses a detection kit for detecting the antibody of human treponema pallidum by using the methods.

Description

Modified treponema pallidum outer membrane protein, its test kit is used and contained to its immunodetection
Invention field
The present invention relates to some modified treponema pallidum outer membrane proteins, relate to immunological method that utilizes these protein detection syphilis helicoid antibodies and the test kit that contains them.
Background of invention
Syphilis is a kind of disease that is caused by treponema pallidum (Treponema pallidum is hereinafter to be referred as Tp).Along with the development of purified technology of protein, DNA recombinant technology and monoclonal antibody technique, for exploitation high specific, highly sensitive treponema pallidum detection technique provide favourable condition.US4514498,4740467 has prepared and has carried out the SD ELISA test kit of syphilis, but its sensitivity and specificity still can not be satisfactory.Immunogenicity treponema pallidum outer membrane protein Tpp15, Tpp17, Tpp47 are known.B.K.Purcell etc., molecular microbiology (Molecular Microbiology) (1990) 4 (8), 1371-1379; R A.Darrin etc., infect and immunity (Infection and Immunity) 1993 (4), 1202-1210 and L.M.Weigel etc. infect and immunity 1992 (4), 1568-1576 has illustrated structure and the composition of treponema pallidum outer membrane protein Tpp15, Tpp17, Tpp47 basically and has provided their aminoacid sequence and their nucleotide sequence of encoding.Japanese Patent Application Publication Hei-2-500403 described a kind of 47kDa of preparation treponemal antigen method and utilize this antigen immune to detect the method for anti-syphilis helicoid antibody.US5578456 has prepared according to the DNA recombinant technology on the basis of fusion rotein of treponema pallidum outer membrane protein Tpp15, Tpp17, Tpp47 and GST S transferring enzyme, propose a kind of utilize with treponema pallidum outer membrane protein Tpp15-and or Tpp17-GST S transferring enzyme fusion rotein as the method for Detection of antigen people syphilis helicoid antibody, it is said and utilize the antigenic method of 47kDa to compare to have improved the SD sensitivity of syphilis.But the part in the above-mentioned fusion rotein beyond the treponemal antigen accounts for major part, and this certainly will increase antigenic non-specific immunity in conjunction with activity, reduces the detection specificity to syphilis helicoid antibody.Thereby still need a kind of immunological method with higher sensitivity and specific detection syphilis helicoid antibody.
The present invention is based on following surprising discovery: treponema pallidum outer membrane protein antigen Tpp15, Tpp17, Tpp47 are modified, significantly improved the sensitivity of immunodetection people's syphilis helicoid antibody under the situation that does not influence its immune binding specificity substantially; In addition, and can keep the binding specificity of natural antigen, use dual-antigen sandwich method can obtain higher detection specificity.
Summary of the invention
One aspect of the present invention relates to a kind of protein with general formula (Xaa) nTpp (Xaa) m, and wherein Tpp is selected from treponema pallidum outer membrane protein Tpp15, Tpp17, Tpp47 or its and has immunocompetent fragment or derivative in conjunction with human normal immunoglobulin; Xaa represents the natural amino acid residue, is the amino-acid residue that is selected from Lys, Arg and Trp with immediate at least one Xaa of the every side of Tpp and the proteic terminal Xaa of at least one general formula wherein; N and m are respectively the integer of 0--10, and condition is n+m 〉=2.
Another aspect of the present invention relates to the method for utilizing this general formula protein immunodetection people syphilis helicoid antibody, particularly utilizes the method for dual-antigen sandwich method detection by quantitative people syphilis helicoid antibody.The present invention also comprise utilize this general formula protein as antigen with conventional ELISA method detection by quantitative people syphilis helicoid antibody.
Another aspect of the present invention relates to the test kit that is used for detecting with above-mentioned immunization method syphilis helicoid antibody.
Description of drawings
Aminoacid sequence and the nucleotide sequence of the treponema pallidum outer membrane protein KTpp15 that Fig. 1 is modified.
Aminoacid sequence and the nucleotide sequence of the treponema pallidum outer membrane protein KTpp17 that Fig. 2 is modified.
Aminoacid sequence and the nucleotide sequence of the treponema pallidum outer membrane protein KTpp47 that Fig. 3 is modified.
Fig. 4 contains the construction of recombinant plasmid synoptic diagram of the nucleotide sequence of the modified treponema pallidum outer membrane protein KTpp15 of coding.
Fig. 5 contains the construction of recombinant plasmid synoptic diagram of the nucleotide sequence of the modified treponema pallidum outer membrane protein KTpp17 of coding.
Fig. 6 contains the construction of recombinant plasmid synoptic diagram of the nucleotide sequence of the modified treponema pallidum outer membrane protein KTpp47 of coding.
The SDS-PAGE result of modified treponema pallidum outer membrane protein KTpp behind Fig. 7 purifying, coomassie brilliant blue staining.Wherein: swimming lane 1 is the low molecular protein standard; Swimming lane 2 is for containing the cellular lysate thing of KTpp15 before the purifying; Swimming lane 3 is the KTpp15 behind the purifying; Swimming lane 5 is for containing the cellular lysate thing of KTpp17 before the purifying; Swimming lane 6 is the KTpp17 behind the purifying; Swimming lane 8 is for containing the cellular lysate thing of KTpp47 before the purifying; Swimming lane 9 is the KTpp47 behind the purifying.
Fig. 8 utilizes the Western Western blotting to detect modified treponema pallidum outer membrane protein KTpp of the present invention and syphilis helicoid antibody bonded activity behind the purifying.Wherein: the right side is the painted molecular weight standard of amino black; 1 is the KTpp15 that reacts with people's treponema pallidum positive serum; 2 is the KTpp17 that reacts with people's treponema pallidum positive serum; 3 is the KTpp47 that reacts with people's treponema pallidum positive serum.
Fig. 9 HRP mark is modified the comparison of recombinant syphilis spirochete antigen and unmodified treponemal antigen labeling effciency.The Tpp17 that wherein last figure is the HRP mark passes through the spectrogram behind the Superdex75 gel chromatography column, and figure below is the spectrogram after the KTpp17 of HRP mark passes through the Superdex75 gel chromatography column.
Figure 10 utilizes dual-antigen sandwich method, recombinant syphilis spirochete outer membrane protein KTpp and the unmodified recombinant syphilis spirochete outer membrane protein Tpp that modifies with the present invention is envelope antigen respectively, and relatively the recombinant protein HR-Tpp of enzyme mark modification recombinant syphilis spirochete outer membrane protein HRP-KTpp and enzyme mark unmodified detects the result of people's syphilis helicoid antibody as enzyme-labelled antigen.Go up the comparison of marking the recombinant protein HRP-Tpp15 of unmodified for the recombinant syphilis spirochete outer membrane protein HRP-KTpp15 and the enzyme of the modification of enzyme mark; In the comparison of marking the recombinant protein HRP-Tpp17 of unmodified of the recombinant syphilis spirochete outer membrane protein HRP-KTpp17 that modifies for enzyme mark and enzyme; The comparison of marking the recombinant protein HRP-Tpp47 of unmodified down for the recombinant syphilis spirochete outer membrane protein HRP-KTpp47 and the enzyme of the modification of enzyme mark;
Detailed Description Of The Invention
A kind of have a general formula (Xaa)nTpp(Xaa) mProtein, wherein Tpp is selected from microspironema pallidum EMP Tpp15, Tpp17, Tpp47 or its and has immunocompetent fragment or derivative in conjunction with human immunoglobulin(HIg); Xaa represents the natural amino acid residue, and wherein the terminal Xaa with immediate at least one Xaa of the every side of Tpp and at least one general formula albumen is the amino acid residue that is selected from Lys, Arg and Trp; N and m are respectively the integer of 0--10, and condition is n+m 〉=2. Modified recombinant syphilis spirochete EMP of the present invention, below be sometimes referred to as KTpp, refer to the protein N end and the C end of amino acid sequence modified in conjunction with the active fragment basis in known microspironema pallidum EMP Tpp15, Tpp17, Tpp47 or its immunity, thus improved described protein or its fragment to the immunity of people's syphilis helicoid antibody in conjunction with activity. Microspironema pallidum EMP Tpp15 of the present invention, Tpp17, Tpp47 comprise the whole protein that contains respectively separately self secreting signal peptide, also comprise the protein that does not contain secretion signal peptide sequence mature form. In addition, microspironema pallidum EMP Tpp15 of the present invention, Tpp17, Tpp47 yet can respectively carry the allos signal peptide that is used for the protein secreting, expressing that the those skilled in the art from multiple other source commonly use.
In the preferred embodiment of the present invention, KTpp is MetLysLys-Tpp-LysLysLeuGluLys, and Tpp is that microspironema pallidum EMP Tpp15 or its have immunocompetent fragment or the derivative in conjunction with human immunoglobulin(HIg).
In preferred another embodiment of the present invention, KTpp is MetLysLys-Tpp-LysLysLeuGluLys, and Tpp is that microspironema pallidum EMP Tpp17 or its have immunocompetent fragment or the derivative in conjunction with human immunoglobulin(HIg).
In preferred another embodiment of the present invention, KTpp is MetLysLys-TPP-LysLysLeuGluLys, and Tpp is that treponema pallidum outer membrane protein Tpp47 or its have immunocompetent fragment or the derivative in conjunction with human normal immunoglobulin.
Term used herein " immunity in conjunction with active " is meant antigen protein identification syphilis helicoid antibody and specificity bonded ability with it.
In order to obtain modified recombinant syphilis spirochete outer membrane protein KTpp, can pass through known pcr amplification technology and the recombinant DNA technology of those skilled in the art.Design corresponding PCR primer, in used PCR primer, add the base of coding respective number amino-acid residue correspondence simultaneously, respectively the above-mentioned three kinds of protein DNA fragments of amplification from clinical treponema pallidum type strain (Nichols).Gained PCR product is imported suitable expression vector by the known method of those skilled in the art, make up the recombinant DNA construction body.Transform proper host cell with the gained DNA construct, under the condition that is suitable for the target protein expression, cultivate transformed host cells like this, and reclaim and purification of target protein by the known technology in this area.
These expression vectors comprise chromosomal, achromosomal and the synthetic dna sequence dna, for example, the derivative of SV40, bacterial plasmid, phage DNA, yeast plasmid, be derived from plasmid and phage DNA bonded carrier, viral DNA, baculovirus, vaccinia virus, adenovirus, fowlpox virus and Pseudorabies virus.But, as long as can duplicate in the host and survive, other carrier also can utilize.
Suitable DNA row can be inserted in the carrier by many methods.Usually, dna sequence dna is inserted on the suitable restriction endonuclease sites with method as known in the art.These methods are believed in the ken that is in those skilled in the art.
DNA in the expression vector is listed as and is connected together with a suitable expression control sequenc (promotor) effectively, thereby instructs synthesizing of mRNA.Cited below is other promotor of genetic expression in the PL promotor of the representative of this promotor: LTR or SV40 promotor, colibacillary Lac or trp promotor, phage and known control protokaryon or eukaryotic cell or its virus.Expression vector also comprises needed ribosome bind site of translation initiation and transcription terminator.Carrier can also have the proper sequence that strengthens expression.
In addition, expression vector preferably comprises one or more selectable marker genes that phenotypic characteristic can be provided, so that the screening of transformed host cell, available Tetrahydrofolate dehydrogenase of for example eukaryotic cultivation or neomycin resistance, then available tsiklomitsin of intestinal bacteria or amicillin resistance.
Comprise above-mentioned suitable DNA row, suitable promotor or the carrier of control sequence and can be used to transform appropriate host, allow this albumen of host expresses.
What enumerate below is the representative of suitable host: bacterial cell, such as intestinal bacteria, streptomycete, Salmonella typhimurium; The fungal cell is as yeast; Insect cell is as fruit bat S2 and fall army worm Sf9; Zooblast is as CHO, COS (monkey kidney fibroblast) or Bowes melanoma; Adenovirus; Vegetable cell or the like.From these instruction, the selection of suitable host should be in those skilled in the art's ken.
The method that construct is imported host cell has: transfection, electroporation or the particle gun method of the DNA conversion of PEG-mediation, calcium phosphate transfection, the mediation of DEAE-dextran.
Usually, recombinant expression vector comprises replication orgin and is used for the selective marker of transformed host cell, for example colibacillary ampicillin resistance gene and cereuisiae fermentum TRP1 gene also comprise the promotor of coming from efficiently expressing gene, thereby mediate transcribing of downstream configurations sequence.These promotors can get from the operon of coding glycolytic ferment, such as glycerol 3-phosphate acid kinase (PGK), alpha factor, acid phosphatase or heat shock protein(HSP).Allogenic structure sequence is assembled together with suitable orientation and translation initiation and terminator sequence and other preferred sequence.Selectable situation is, the dna sequence dna fusion rotein of can encoding, and this albumen contains a N-terminal confirms peptide, shows the feature of expection, the stabilization of for example expressed recombinant products and purify and simplify.
The effective expression carrier that bacterium is suitable for makes up like this: the structural DNA sequence of the target protein of will encoding is inserted in the steerable reading frame that has function in company with suitable translation initiation and termination signal.Carrier should comprise one or more Phenotypic Selection marks and replication orgin, and replication orgin has guaranteed that carrier can remain in the host, and better is to make carrier obtain amplification.The prokaryotic hosts that is suitable for transforming has the various bacteriums of intestinal bacteria, subtilis, Salmonella typhimurium and Pseudomonas, streptomyces and Staphylococcus, and other host also can be selected.
Transforming after appropriate host bacterial strain and host strain grow into appropriate cell density, induce selected promotor with appropriate means (for example temperature transition or pharmaceutical chemicals are induced), and cell is cultivated for some time again.
Usually use the centrifugation method harvested cell, the method smudge cells with physics or chemistry keeps crude extract to be further purified.The microorganism cells that is used for marking protein can comprise freeze-thaw cycle, ultrasonic wave, Mechanical Crushing with the fragmentation of any method easily, perhaps uses cell lytic agent, and these methods all are well-known to those having ordinary skill in the art.
The culture systems of various mammalian cells also can be used for express recombinant protein matter.The example of mammals expression system have Gluzman (Cell 23:175,1981) the fibroblastic COS-7 clone of the monkey kidney of describing, and other can express the clone of compatible carrier, for example C127,3T3, CHO, HeLa and bhk cell are.The mammals expression vector comprises replication orgin, suitable promotor, enhanser and any essential ribosome bind site, polyadenous glycosidation site, splicing donor and acceptor site, transcription termination sequence and 5 ' flank non-transcribed sequence.The DNA row and the polyadenous glycosidation site that are derived from SV40 splicing sequence can be used for providing needed non-transcribed genetic elements.
Expressed can reclaim from the reconstitution cell culture and purifying comes out reclaimed and the method for purifying has ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and phytohemagglutinin chromatography.High performance liquid chromatography (HPLC) is applied to last purification step.
In the embodiment preferred of the present invention, utilized expression vector GB-sy1 plasmid construction recombinant DNA construction body,, utilized IPTG to induce target protein to express its transformed into escherichia coli ER2566.
In a preferred embodiment of the invention, the escherichia coli host by the conversion after the broken cultivation of supersound process utilizes metallic nickel-Sepharose affinity chromatography column separating purification target protein.
The present invention also comprises having protein or the polypeptide that immunocompetent treponema pallidum outer membrane protein fragment or derivative in conjunction with human normal immunoglobulin obtain after modification of the present invention.
Modified recombinant syphilis spirochete outer membrane protein KTpp of the present invention obtains after can also being modified Tpp15, Tpp17, Tpp47 or its fragment of treponema pallidum outer membrane protein by chemical method.
Find that now modifying protein of the present invention compares with the albumen of unmodified, not only have higher mark, and have higher immunodetection activity surprisingly.
" height mark " is meant that the Chemical Crosslinking Methods that adopts identical routine is with using the protein of not modifying to significantly improve the productive rate of labeled complex through the protein ratio of modifying.This point can be confirmed in embodiment 3.
" high immunodetection activity " is meant and utilizes sensitivity and/or the specificity that has significantly improved the syphilis helicoid antibody immunodetection through the protein ratio of modifying with the protein of not modifying.This point can be confirmed in the dual-antigen sandwich method of the indirect elisa method of embodiment 10 and embodiment 11.
Therefore, the present invention also relates to a kind of method of utilizing modified recombinant syphilis spirochete outer membrane protein KTpp immunodetection people syphilis helicoid antibody of the present invention.
The antigenic determinant of known treponema pallidum mainly is positioned on outer membrane protein Tpp47, Tpp17 and the Tpp15.Utilize at least a modified recombinant syphilis spirochete outer membrane protein KTpp of the present invention as antigen, form immunity in conjunction with mixture by antigen-antibody immunity association reaction, thereby detect people's syphilis helicoid antibody in the idiobiology sample.
In one embodiment of the invention, this method may further comprise the steps:
1) utilize the modifying protein bag at least a of the present invention of purifying by a kind of solid support;
2) biological sample to be detected is added on the solid support of bag quilt, be fit to hatch for some time under the condition of formation antigen-antibody immunity in conjunction with mixture;
3) appropriateness is cleaned and is removed any unconjugated syphilis helicoid antibody;
4) adding is hatched for some time through the second antibody at people's antibody of mark under the condition that is fit to formation antigen-antibody immunocomplex;
5) appropriateness is cleaned and is removed any unconjugated second antibody;
6) in the detection by quantitative mixtures incubated antigen-antibody immunity in conjunction with the existence of mixture.
In the such scheme, can be (being RIA) with labelled with radioisotope at the second antibody of people's antibody, also can be (being ELISA) of carrying out mark with horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase.
Described second antibody can be anti-people-IgG or/and-IgM antibody.These second antibody can be buied from the market, as sheep anti people-IgG or/and-IgM, the anti-people-IgG of goat or/and-IgM, mouse anti human-IgG or/and-IgM, rat anti people-IgG or/and-IgM, the anti-people-IgG of rabbit or/and-IgM etc.
Utilize different second antibody can detect antibody specific in the sample and whether exist, and the relative amount of different antibodies.Since different in the intravital relative quantity of people at the different times IgG of syphilis with IgM, utilize different second antibody to carry out the course of disease that repeated detection helps to judge the patient.
Biological sample described in this specification sheets is meant from experimenter's blood of (comprising patient and negative control), serum, blood plasma, urine sample, body fluid, saliva and other secretory product or movement and tissue or cell extract.
In the embodiment preferred of the present invention, adopt so-called dual-antigen sandwich method immunodetection (DAGS EIA) to detect people's syphilis helicoid antibody.Typically, this method may further comprise the steps:
1) the modified recombinant syphilis spirochete outer membrane protein KTpp with purifying wraps by a kind of solid support;
2) in advance mark and bag by the corresponding modified recombinant syphilis spirochete outer membrane protein KTpp of the used antigen KTpp of solid support kind;
3) with the biological sample of individuality to be detected and by step 2) protein of mark adds on the solid support of bag quilt, hatches for some time being fit to form under the condition of antigen-antibody immunity in conjunction with mixture;
4) appropriateness is cleaned and is removed any unconjugated syphilis helicoid antibody;
5) existence of antigen-antibody immunocomplex in the detection by quantitative mixtures incubated.
Described " purifying antigen " is meant that the purity as antigenic modified treponema pallidum outer membrane protein is higher than 95%, preferredly is higher than 98%.
Described " solid support " comprises the organic and inorganic polymer that this area is known, such as including, but not limited to: dextran, natural or modified Mierocrystalline cellulose, polyethylene, polystyrene, polyacrylamide, agarose, latex, container inner wall such as test tube, titer plate, glass cylinder etc.
Can use the known several different methods bag in this area by solid support.For example: utilize the bifunctional reagent activation, referring to United States Patent (USP) 5399501.
In the embodiment that adopts dual-antigen sandwich method, bag is preferably included not only a kind of modified recombination outer membrane protein matter by the modified treponema pallidum outer membrane protein of solid support especially.That is, use and to be selected from two or three of KTpp15, KTpp17, KTpp47 and to carry out solid-phase coating.
Accordingly, by the kind of the modified treponema pallidum outer membrane protein of solid support, utilize the modified treponema pallidum outer membrane protein of the corresponding kind of this area common technology mark according to the step 1) bag.Preferred utilization can be carried out mark by the enzyme of colorimetry, spectrophotometry or fluorescence spectrometry, including, but not limited to redox enzymes, as katalaze enzyme, peroxidase, horseradish peroxidase, glucose oxidase, beta-galactosidase enzymes; Alkaline phosphatase; The enzyme of acetylcholinesterase etc. carries out mark.Embodiment preferred of the present invention is the antigen with the corresponding kind of horseradish peroxidase-labeled.Can use labelled with radioisotope as antigenic modified treponema pallidum outer membrane protein in addition.
The method of above-mentioned enzyme of mark and modified treponema pallidum outer membrane protein of the present invention is that this area is known, for example utilizes the method for known peptide chemistry to carry out mark by forming amido linkage.The known method in also available this area utilizes radio isotope to carry out mark.
Described " being fit to form the condition of antigen-antibody immunocomplex " is that this area is known, be meant the mixture of under suitable temperature and time condition, hatching antigen and containing the sample of antibody, for example at about 0-50 degree centigrade, under preferred about 4-30 degree centigrade, mark about 30 minutes to about 48 hours, preferred mark about 30 minutes to about 4 hours.
After hatching formation antigen-antibody immunocomplex, with this area suitable washing soln washing check-out console several commonly used, to remove unconjugated antigen through mark.Used scavenging solution is generally the about 5-8 of pH, is preferably the phosphate buffer soln of pH about 7.4.
Can adopt the formation of the known method detection by quantitative immune complex in multiple this area.The treponema pallidum outer membrane protein employed method modified according to mark selected suitable detection by quantitative scheme.In embodiment preferred of the present invention, use horseradish peroxidase during labelled antigen, be A liquid and the diphenyl amine material substrate and the antigen-antibody immunocomplex effect that has the horseradish peroxidase that contains mark as B liquid so use with peroxide solutions, the colour developing back is that 450nm (reference wavelength 630nm) microplate reader reads light absorption value at wavelength.The reading of gained reading and negative control is compared, judged institute's test sample result originally.
Utilize modified recombinant syphilis spirochete outer membrane protein KTpp of the present invention as antigen, the method that detects people's syphilis helicoid antibody in the idiobiology sample by immunization comprises that also the conventional specific antigen that utilizes that uses detects other method of antibody in this area.
The present invention also relates to be used for the detection kit that these methods detect people's syphilis helicoid antibody.The detection kit that detects people's syphilis helicoid antibody according to the present invention comprises: with the modified recombinant syphilis spirochete outer membrane protein KTpp of the present invention of purifying as antigen coated solid support; With the explanation of using this test kit.
In one embodiment, test kit of the present invention also comprises the second antibody of mark as indicated above.This second antibody can be anti-IgG antibody and/or anti-IgM antibody.
When being used for dual-antigen sandwich method, this test kit also comprise with step 1) in bag by the protein KTpp kind of solid support accordingly through the recombinant syphilis spirochete outer membrane protein KTpp of mark.
In above-mentioned detection kit, through the treponema pallidum outer membrane protein of mark can liquid packaged in proper container, also can be so that the form of the lyophilized products of reconstruct places suitable packing.In the later case, also preferably contain in the detection kit and be useful on the suitable damping fluid of reconstruct through the treponema pallidum outer membrane protein of mark.
In above-mentioned detection kit, can also contain the corresponding scope of judging detected result that provides.
It is example explanation the present invention that the following example is represented the protein of SEQ ID No.1, SEQ ID No.2 and SEQ IDNo.3 with KTpp, but this and do not mean that and limit the scope of the invention.
Embodiment
Embodiment 1 utilizes PCR method amplification coding treponema pallidum to rise the gene of membrane protein KTpp
Experiment material and method
Treponema pallidum Nichols strain, intestinal bacteria ER2566 strain is Microbe Inst., Chinese Academy of Sciences provides.The GB-Sy1 plasmid is so kind as to give by Chinese Academy of Sciences's heredity, and this plasmid contains NdeI, XhoI single endonuclease digestion site, is connected with the distinctive histidine-tagged of reorganization and is used to cut histidine-tagged aspartic acid sequence label at XhoI restriction enzyme site one end.Restriction enzyme NdeI, XhoI enzyme and T4DNA ligase enzyme are provided by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, the TaqDNA polysaccharase, and the digestible protein enzyme, enteropeptidase provides by heredity institute of Chinese Academy of Sciences molecular biosciences laboratory.IPTG is a PROMEGA company product.
The preparation of treponema pallidum DNA and purifying
The cultivation of treponema pallidum Nichols strain and the preparation of chromosomal DNA, purifying infect and immunity 58 with reference to Zhampionce etc., 1697-1704, the method for mentioning in 1990.
The preparation of plasmid DNA and purifying
The preparation purifying of e. coli plasmid dna with reference to Sambrook " molecular cloning, laboratory manual " the 2nd edition, Cold Spring Harbour Laboratory Press, the method that provides in 1989.
The design of pcr amplification primer
Coding Tpp15, Tpp17 and the proteinic nucleotide sequence design of Tpp47 PCR primer (B.K.Purcell etc., molecular microbiology (1990) 4 (8), 1371-1379 according to the known references report; R.A.Darrin etc. infect and immunity 1993 (4), and 1202-1210 and L.M.Weigel etc. infect and immunity 1992 (4), and 1568-1576), wherein interpolation is used to introduce the restriction enzyme site NdeI of expression vector and the codon of XhoI and coding lysine residue.
The primer sequence that is used for pcr amplification is as follows:
KTpp15:SEQ ID NO:4 and SEQ ID NO:5
Upstream primer: 5 ' GGG TCGT CATATG AAG AAGATG GTG AAA AGA GGT 3 '
NdeI Lys Lys
Downstream primer: 5 ' TTCG CTCGAG TTT TTTCCT GCT AAT AAT GGC 3 '
XhoI Lys Lys
Tpp15:SEQ ID NO:6 and SEQ ID NO:7
Upstream primer: 5 ' GGG TCGT CATATGATG GTG AAA AGA GGT 3 '
NdeI
Downstream primer: 5 ' TTCG CTCGAGCCT GCT AAT AAT GGC 3 '
XhoI
KTpp17:SEQ ID NO:8 and SEQ ID NO:9
Upstream primer: 5 ' GATTGAG CATATG AAG AAGATG AAA GGA TCT GTC3 '
NdeI Lys Lys
Downstream primer: 5 ' GTCAG CTCGAG TTT TTTTTT CTT TGT TTT TTT3 '
XhoI Lys Lys
Tpp17:SEQ ID NO:10 and SEQ ID NO:11
Upstream primer: 5 ' GATTGAG CATATGATG AAA GGA TCT GTC3 '
NdeI
Downstream primer: 5 ' GTCAG CTCGAGTTT CTT TGT TTT TTT3 '
XhoI
KTpp47:SEQ ID NO:12 and SEQ ID NO:13
Upstream primer: 5 ' GTACTAG CATATG AAG AAGATG TTC GAT GCA GTT 3 '
NdeI Lys Lys
Downstream primer: 5 ' CTGT CTCGAG TTT TTTCTG GGC GAC TAC CTT3 '
XhoI Lys Lys
Tpp47:SEQ ID NO:14 and SEQ ID NO:15
Upstream primer: 5 ' GTACTAG CATATGATG TTC GAT GCA GTT 3 '
NdeI
Downstream primer: 5 ' CTGT CTCGAG_ CTG GGC GAC TAC CTT3 '
XhoI
The pcr amplification of the gene of coding for antigens protein KTpp
With the total DNA of treponema pallidum Nichels strain as above-mentioned acquisition is template, adds the 50pmmol primer, and 2.5mmol/L dNTP and 1U Taq archaeal dna polymerase add water to reaction cumulative volume 100 μ l, the surface coverage paraffin oil.In the PCR-90AD type PCR instrument that Chinese Academy of Sciences's heredity is produced, increase according to following condition:
95 ℃ of sex change 60 seconds
60 seconds (KTpp15) of 55 ℃ of annealing, 60 seconds (KTpp17) of 58 ℃ of annealing, 60 seconds (KTpp47) of 60 ℃ of annealing
72 ℃ were extended totally 30 circulations 90 seconds.
After finishing, last circulation extended 10 minutes in 72 ℃ again.4 ℃ of depot reaction mixed solutions are stand-by.With equal-volume phenol/chloroform extractive reaction mixed solution, remove paraffin oil.Get reaction solution that 5 μ l contain the PCR product with 1% agarose gel electrophoresis inspection amplification situation, add the 3M liquor kalii acetici of 0.1 times of volume in all the other products, dehydrated alcohol is deposited in-20 ℃, centrifugal collection amplified production.
The pcr amplification product that obtains know through 1% agarose gel electrophoresis inspection: corresponding to the reaction product of the nucleotide sequence of encoded K Tpp, K15Tpp17 and the KTpp47 DNA band of corresponding have an appointment 0.4Kb, about 0.5Kb and about 1.1Kb respectively, show that design of primers is correct, obtained the corresponding target gene fragment.
Embodiment 2 construction of recombinant plasmid and evaluation
The clone of pcr amplification product
The GB-Sy1 plasmid DNA of 1 μ g under 37 ℃ through NdeI and XhoI enzyme complete degestion, phenol/chloroform extracting ,-20 ℃ of following dehydrated alcohols precipitations are spent the night.Centrifuged deposit is dissolved in 13 μ l H 2O.Be connected with the plasmid GB-Sy1 that above-mentioned pre-enzyme is cut with the PCR product that the abundant enzyme of XhoI enzyme is cut through NdeI, be converted into intestinal bacteria ER2566, the LB that coating contains 200 μ g/ml penbritins selects dull and stereotyped.37 ℃ of incubated overnight.Building process is referring to Fig. 4, Fig. 5 and Fig. 6.The method of described connection and conversion and condition very wait " molecular cloning experiment guide " (the Molecular Cloning:A LaboratoryManual) P672-849 (Science Press, 1998) of work referring to, J. Sa nurse mine-laying
Recombinant plasmid screening and evaluation
From transforming the dull and stereotyped picking resistance bacterium colony of going up, extract plasmid and electrophoretic examinations, the screening recon.By restriction enzyme NdeI and XhoI restriction analysis, screening contains the recon of the goal gene of correct insertion.Gained is contained the segmental recombinant plasmid order-checking of insertion, and conclusive evidence target gene fragment in the PCR process does not morph or goes wrong.
The expression of target protein and purifying
In the LB substratum that contains 200 μ g/ml penbritins, place the constant temperature shaking table to cultivate selected recon inoculation, 37 ℃ are cultured to about 0.6 back of bacterium liquid OD value and add 1mMIPTG and induce, and 30 ℃ are continued to cultivate 2 hours.Then with the centrifugal collection bacterial sediment of fermenting culture.With ultrasonic wave with gained bacterium bacterial cell disruption after recentrifuge, collect supernatant liquor.Utilize the affinity column of metallic nickel-Sepharose (Pharmacia company) to carry out proteic separation and purification (elution requirement 0.02M pH7.4 phosphate buffer soln (PBS), elution speed 1ml/ minute).Remove the protein of non-specific binding through thorough washing after, separation and collection and metallic nickel-Sepharose bonded expressing protein carries out SDS-PAGE electrophoresis and purity and identifies and the ELISA serologic test.Purification process reference literature Arnild, FH (1991) biotechnology (Bio/Technology) 9,151-156 and Dekker, N. (1992) nature (Nature) 362,852-855.
Collection contains histidine-tagged target protein, adds the 1U enteropeptidase Histidine is cut.Target protein passes through DEAE-Sepharose post (Pharmacia company) and Superdex 75 posts (Pharmacia company) again and is further purified and obtains.Specific operation process is as follows: collect 6 the histidine-tagged target protein solution that are connected with under cutting on the Ni-Sepharose post, the ratio that adds the 1U enteropeptidase in IL adds nickase, 4 ℃ are spent the night, protein soln after will cutting then is splined on uses pH7.2,0.01M the DEAE-Sepharose post of Tris-HCl damping fluid pre-equilibration, collect each component.Method by ELISA is identified the target protein immunocompetence: each component that will collect is diluted to bag by working fluid with 0.05M pH9.6 carbonate buffer solution (CB) 1:10, and 4 ℃ of bags of 96 hole enzyme plates are spent the night.The 0.02MpH7.4 phosphate buffered saline buffer (PBS) that adds 1% bovine serum albumin (BSA) and 1% skim-milk then, 4 ℃ of following sealings discarded after 2 hours.Add treponema pallidum positive patients serum 10 microlitres/hole again, the solution that contains the PBS of 1%BSA with 100 microlitres is sample diluting liquid, 37 ℃, after 30 minutes, discards.Wash twice with the 0.05M PBS washings (PBS-T) that contains 0.5%TW-20, add with enzyme labelled antibody diluent (20% lowlenthal serum (the sharp blood products in Zhejiang three company produces), 1% casein (Sigma), 0.02MPBS pH7.4)) goat anti-human igg's (production of DAKO company) 100 microlitres/hole that dilute with 1:5000,37 ℃, 30 minutes, wash twice with the 0.02MpH7.4 PBS washings (PBS-T) that contains 0.5% polysorbas20 (TW-20) again, add zymolyte A liquid that 50 μ l are made up of the acetate buffer of 0.06% urea peroxide (Sigma) 0.01M pH4.5 and 50 μ l in last every hole by 0.06%3,3, `5,5`-tetramethyl benzidine (TMB) (Sigma), the zymolyte B that the citrate buffer of 0.01MpH4.5 is formed, 37 ℃, developed the color 10 minutes, and added 1M H at last 2SO 4Stop, read light absorption value (reference wavelength 630nm) at the 450nm place, if the light absorption value height illustrates in the fraction of collecting and contains target recombinant syphilis spirochete albumen.After these high light absorption value fraction cryoconcentration, pass through Superdex75 post (adopting 0.02M pH7.4 PBS balance cylinder) purifying again, collect one-component, further adopt the ELISA method to carry out the immunocompetence evaluation, method is the same.
The evaluation of recombinant antigen
The gene of the treponema pallidum outer membrane protein KTpp that the code book invention is modified is respectively behind escherichia coli expression, behind affinity chromatography separation and purifying, identify through the SDS-PAGE electrophoresis, modified treponema pallidum outer membrane protein KTpp15, KTpp17 and the molecular weight of KTpp47 are about 15KD, 17KD and 47KD respectively, (referring to Fig. 7); By the Western Western blotting, confirm that the protein of gained has and people's syphilis helicoid antibody bonded activity (referring to Fig. 8).
The enzyme process mark of embodiment 3 recombinant antigen protein matter KTpp
It is good to confirm that through the known indirect elisa method in this area institute obtains the protein immunocompetence by the KTpp protein of aforesaid method purifying preparation.The horseradish peroxidase (HRP) that is used for enzyme labelling, NaIO 4, NaBH 3CN, thanomin, methyl-α-D-mannoside is all available from Sigma company.Enzyme process mark recombinant antigen protein matter KTpp adopts the periodates method of improvement, and with reference to Tijssien (Analytical Biochem 136,451-457,1984), concrete steps are as follows:
1) activates HRP: with 100 μ l 0.1M NaIO 4The HRP of adding 1ml 5mg/ml, abundant mixing under the room temperature places fully reaction on the oscillator, and 150 rev/mins of lucifuge concussions 20 minutes, were fully dialysed 24 hours for 4 ℃ to the acetate buffer of 1mM pH4.4;
2) crosslinked with treponemal antigen: the 0.2M pH9.6 carbonate buffer solution (CB) that adds 1/4 volume among the HRP after the above-mentioned activation of 1ml is regulated pH value to 9.6, to treponemal antigen (KTpp15, KTpp17 or KTpp47 3mg/ml) the 0.2M pH9.6CB that adds 1/4 volume among the 1ml regulates pH value to 9.6, then with above-mentioned two kinds of abundant mixings of liquid, place on the oscillator 200 rev/mins, lucifuge was fully reacted 2 hours under the room temperature;
3) reduction of two key Shiff alkali: will add the NaBH of 25 μ l 5M in the above-mentioned product after crosslinked 3CN, fully 4 ℃ of lucifuges left standstill 2 hours behind the mixing;
4) sealing unreacted aldehyde radical: add the sealing of 125 μ l 1M thanomins not with the crosslinked HRP of treponemal antigen on unnecessary aldehyde radical, fully 4 ℃ of lucifuges left standstill 1 hour behind the mixing;
5) small-molecule substance in the removal cross-linking products: the PBS to 0.02M pH7.4 fully dialysed 24 hours with above-mentioned cross-linking products;
6) remove uncrosslinked resolvase: take out liquid from dialysis tubing and add isopyknic 50% saturated (NH4) 2SO 4, fully behind the mixing 4 ℃ left standstill 24 hours, centrifugal 2000 rev/mins 20 minutes, collecting precipitation with the PBS dilution precipitation of 0.02M pH7.4, was fully dialysed 24 hours to 0.02M pH7.4 PBS;
7) remove uncrosslinked free antigen: the ConA-Sepharose purification column (production of Pharmaca company) of preparing a 2mi, adopt the PBS balance cylinder of 0.02M pH7.4, add the enzyme-labelled antigen after dialysing, use the abundant wash-out of PBS of 0.02M pH7.4 earlier, PBS with the 0.02M pH7.4 that contains methyl-α-D-mannoside is an elutriant again, collects the enzyme-labelled antigen elution peak that contains glycosylated protein;
8) purifying enzyme is marked antigenic preservation: the enzyme-labelled antigen of collecting is added equal-volume glycerine, and-20 ℃ of preservations promptly make enzyme mark treponemal antigen stoste.
Adopt the labeling effciency of aforesaid method mark for more modified recombinant syphilis spirochete antigen and not modified treponemal antigen, KTpp17 and Tpp17 antigen (3mg/ml) with same concentrations are example, fully according to the amount that adopts in the above-mentioned marking method, carry out parallel laboratory test, the product (before not adding glycerine) of purified mark is adopted AKTO purifier protein purification analyser (production of Armersham pharmaca company) analysis via Superdex75 gel column (applied sample amount 100 μ tl) (production of Pharmaca company), detect the variation of antigen protein amount in the binding substances with the UV-detector of 280nm, detect variation (reference Tijssien (the Analytical Biochem136 of horseradish peroxidase amount in the binding substances with the 403nm UV-detector, 451-457,1984), detecting the recombinant syphilis spirochete antigen molecule with the ratio with main peak peak area 280nm 403nm can be by the degree of horseradish peroxidase-labeled, and product goes out the peak result referring to accompanying drawing 9.Now integral result is listed as follows:
Table 1 HRP mark is modified recombinant syphilis spirochete antigen KTpp17 and unmodified treponema pallidum
The comparison of antigen Tpp17
Project Tpp17-HRP KTpp17-HRP
280nm 403nm 280nm 403nm
Climax post bed (ml) 8.10 8.11 8.05 8.06
Main peak halfwidth (ml) 0.64 0.57 0.54 0.55
Main peak peak height (mAU) 275.357 45.673 279.252 108.728
The main peak area (mAU * ml) 142.2093 27.1652 206.1923 63.3682
Purity (main peak area/total peak area 100%) 55.57％ 97.62％ 98.96％ 99.68％
Crosslinked mole ratio (Tpp17mg/HRPmg) 3∶5 3∶5
Joint efficiency (main peak 403nm area/280nm area 100%) 19.10％ 30.73％
By experimental result, the labeling effciency of the treponemal antigen KTpp17 of modification is apparently higher than the treponemal antigen Tpp17 of unmodified.
Embodiment 4 definite bags are antigenic by the antigen of solid support and process enzyme process mark
The preferred compatibility of optimum concn and used antigen type
Bag is by the optimized choice of the concentration of the antigen of solid support and enzyme-labelled antigen
96 orifice plate bars select for use the good polystyrene board of adsorptivity (production of Shenzhen Li Ji company) (between the hole, between plate and difference between batch CV＜I0%), selecting 0.05M pH9.6 carbonate buffer solution (CB) for use is coating buffer, the adding of every hole prepares antigenic coating buffer 100 microlitres according to experimental design concentration, and the room temperature bag is spent the night.Discard antigen liquid then, bag is washed twice by plate with the 0.02MPBS washings (PBS-T) that contains 0.5%TW-20, pats dry, and 0.02M pH7.4 phosphate buffered saline buffer (PBS) room temperature that adds 1% bovine serum albumin (BSA) and 1% skim-milk was sealed 2 hours.Discard confining liquid, air-dry bag is by plate, the vacuum-packed 4 ℃ of preservations of aluminium foil bag.By selecting concentration for use is that the antigen coated concentration of KTpp15 of 5,12.5,25,50,75,100 μ g/ml is wrapped quilt, and selecting concentration for use is that the enzyme-labelled antigen HRP-KTpp15 of 0,10,20,30,40,60,80,100 μ g/ml carries out the dual-antigen sandwich method experiment according to the working method of describing in detail among the embodiment 7.The result shows, the KTpp15 bag is 50 μ g/ml by concentration, positive matter was commented serum (this positive matter is commented serum to be Tpp15 through Western Blot conclusive evidence when enzyme-labelled antigen was 40 μ g/ml, the syphilis positive serum of Tpp17 and Tpp47 band) reach maximum OD value, negative matter comments the reaction average of serum less than 0.05.In view of the above we select for use 50 μ g/ml be KTpp15 bag by concentration, 40 μ g/ml are enzyme-labelled antigen concentration.Adopt identical method to determine that the bag of KTpp17 is 20 μ g/ml by concentration, enzyme-labelled antigen concentration is 40 μ g/ml; The bag of determining KTpp47 is 40 μ g/ml by concentration, and enzyme-labelled antigen concentration is 40 μ g/ml.
Determine the selection of used antigen type compatibility
Ratio referring to envelope antigen as shown in following table and enzyme-labelled antigen is passed through suitable compatibility concentration between each antigen of square formation test and Selection.
Table 2. double antigens sandwich result (S/CO)
Figure C9981542400221
By above data, we select antigenic compatibility concentration as follows: the bag of KTpp15, KTpp17, KTpp47 is respectively 50,20,40 μ g/ml by concentration; The concentration of enzyme-labelled antigen is respectively 20,40,20 μ g/ml.This compatibility selects to have given prominence to the effect of KTpp17, has also taken into account the possible complementary action of KTpp15, KTpp47.
Envelope antigen composition after the optimization is called syphilis S-3 series antigen, and the enzyme-labelled antigen after the optimization is referred to as enzyme mark S-3 antigen
Determining of antigen antibody reaction condition that embodiment 5 is suitable and time
Under envelope antigen and enzyme-labelled antigen concentration that the foregoing description is determined, adopt enzyme plate and reaction buffer as the aforementioned, research is suitable for antigen antibody reaction and forms the condition of immunity in conjunction with mixture.Select for use weak, in, strong 3 positive serums and negative control, carry out the dual-antigen sandwich method experiment according to the working method of describing in detail among the embodiment 7.By under 37 ℃ of conditions, reacting respectively 20,40,60,90,120 minutes, developed the color 15 minutes, the OD value of being surveyed reached maximum in the time of 60 minutes.So the selective reaction time is 60 minutes.
Determining of the positive threshold value of embodiment 6 dual-antigen sandwich method immunodetection samples
According to the method for immunodetection as described in the previous embodiment, detect 300 parts and be defined as negative normal blood donation person's serum through indirect elisa method, get the normal distribution value and carry out statistical study.Getting average is 0.021, and SD is 0.004.Setting positive threshold value is 0.18+ negative control average.
Embodiment 7 dual-antigen sandwich methods detect syphilis helicoid antibody
On 96 hole micro reaction plates, add carbonate buffer solution (CB) dilution KTpp15, the KTpp17 use 0.02M pH9.6 in advance, KTpp47 coating buffer to optimal concentration (syphilis S-3 series antigen), 4 ℃ left standstill 24 hours, add the 0.02M pH7.4 PBS washings (PBS-T) that contains 0.1%TW-20 and fill it up with each hole, discard twice of repeated washing after leaving standstill the several seconds.Every hole adds the 0.02M pH7.4 phosphate buffered saline buffer (PBS) that 200 μ l go into 1% bovine serum albumin (BSA) and 1% skim-milk, and 4 ℃ were sealed 2 hours down.Discard confining liquid, dry under the room temperature, use the thin aluminum bag shrouding, 4 ℃ of preservations are stand-by.In the micro reaction plate hole, add and use enzyme-labelled antigen diluent (20% lowlenthal serum in advance, 1% casein, 0.02M PBS, pH7.4) to optimize the corresponding enzyme-labelled antigen of syphilis S-3 series antigen of good concentration dilution, every hole 50 μ l, add 15 again from difference experimenter's serum to be detected, 37 ℃ of incubations 60 minutes.Fill it up with each hole with the 0.02M PBS-T washings that contains 0.1%TW-20 then, discard repeated washing 5 times after leaving standstill the several seconds.Add the zymolyte B liquid that zymolyte A liquid that 50 μ l are made up of the acetate buffer of 0.06% urea peroxide (Sigma) 0.01MpH4.5 and 50 μ l are made up of the citrate buffer of 0.06%TMB (Sigma) 0.01M pH4.5 in every hole, 37 ℃ of incubations 15 minutes.The 2M H that adds 50 μ l in every hole 2SO 4Termination reaction.On microplate reader (Dynatech MR 4100), read the light absorption value (is reference wavelength with 630) at 450nm place.Wherein positive control and negative control are through TPHA, the human serum that RPR and FTA-ABS method are checked, and according to embodiment 6 determined threshold determination sample results, the sample that is higher than 0.18+0.024 is the treponema pallidum positive.Detect serum specimen from 49 parts of the patients with syphilis serum of BJ Medical University 1st Subsidiary Hospital Dermatology Department clinical definite, 11 parts of primary syphilis patients serums (I1-I11) wherein, (serum after 4 parts of treatments wherein is with II for secondary syphlilis patients serum 38 parts (II12----II49) *Expression).The RPR test kit that parallel laboratory test is adopted is that (lot number: 0349), the TPPA test kit is a Japanese fuji Biological Co., Ltd product (lot number: VN81204) to Organon company product.The gained result is as follows:
Table 3. dual-antigen sandwich method detects patients with syphilis serum result
The serum numbering ELISA (dual-antigen sandwich method) RPR TPPA
Absorbancy Differentiate Titre Differentiate
Positive control 2.231 + 1∶32 + +
Negative control 0.024 - / - -
I1 1.892 + 1∶16 + +
I2 0.544 + / - +
I3 1.766 + / - +
I4 1.869 + 1∶1 + +
I5 1.935 + 1∶2 + +
I6 1.436 + 1∶1 + +
I7 0.557 + / - +
I8 0.505 + / - +
I9 2.238 + 1∶1 + +
I10 0.658 + / - +
I11 1.154 + 1∶4 + +
II12 1.133 + 1∶8 + +
II13 * 2.159 + 1∶4 + +
II14 1.376 + 1∶8 + +
II15 0.530 + 1∶16 + +
II16 0.869 + 1∶16 + +
II17 0.901 + 1∶32 + +
II18 0.898 + 1∶8 + +
II19 0.760 + 1∶16 + +
II20 1.018 + 1∶32 + +
II21 0.964 + 1∶16 + +
II22 1.272 + 1∶8 + +
II23 1.932 + 1∶16 + +
II24 0.821 + 1∶8 + +
II25 0.741 + 1∶32 + +
II26 * 0.785 + 1∶4 + +
II27 0.682 + 1∶16 + +
II28 0.676 + 1∶32 + +
Table 3 (continuing)
II29 0.694 + 1∶32 + +
II30 1.457 + 1∶8 + +
II31 0.575 + 1∶16 + +
II32 1.164 + 1∶8 + +
II33 0.948 + 1∶16 + +
II34 1.453 + 1∶8 + +
II35 1.036 + 1∶16 + +
II36 1.136 + 1∶16 + +
II37 0.724 + 1∶16 + +
II38 0.690 + 1∶64 + +
II39 1.027 + 1∶64 + +
II40 1.271 + 1∶8 + +
II41 1.076 + 1∶128 + +
II42 0.660 + 1∶16 + +
II43 0.658 + 1∶128 + +
II44 1.034 + 1∶8 + +
II45 0.850 + 1∶8 + +
II46 * 1.284 + / - +
II47 * 1.878 + 1∶1 + +
II48 0.748 + 1∶8 + +
II49 1.728 + 1∶8 + +
The composition of embodiment 8 dual-antigen sandwich method treponema pallidum immunity detection reagents
The preferred dual-antigen sandwich method treponema pallidum of the present invention immunity detection reagent is made up of following components:
Envelope antigen: syphilis S-3 series antigen; That is: concentration is respectively KTpp15, KTpp17, the KTpp47 of 50,20,40 μ g/ml.
Antigen coated microplate: be homemade 96 hole detachable plates, by syphilis S-3 series antigen, vacuum-drying seals according to embodiment 4 described bags;
Enzyme-labelled antigen: the syphilis S-3 series antigen of horseradish peroxidase-labeled.That is: concentration is respectively enzyme-labelled antigen HRP-KTpp15, HRP-KTpp17, the HRP-KTpp47 of 20,40,20 μ g/ml.
Negative control and positive control serum: each 0.2 milliliter, contain 0.05% Thiomersalate, 0.01% sodium azide, every hole adds 50 μ l during use;
Enzyme mark S-3 antigen 6ml working fluid: with containing 20% sheep blood serum (the sharp blood products in Zhejiang three company produces), 0.05% Thiomersalate (Sigma), the enzyme-labelled antigen diluent of 0.02M pH7.4 PBS is marked extremely suitable the tiring of S-3 antigen diluent with enzyme.
0.01M pH4.5 citric acid (Sigma) damping fluid of substrate solution A:6ml 0.06% urea peroxide (Sigma);
Substrate solution B:6ml 0.06%TMB (Sigma) 0.01M pH4.5 citric acid (Sigma) damping fluid;
Stop buffer: 6ml 1M sulfuric acid
Washings: 20ml 50 * PBS contains 0.1% tween 20;
Anti-treponema pallidum positive serum OD is worthwhile more than 0.18+ negative control average.
Anti-treponema pallidum negative serum OD is worthwhile below 0.18.
Dual-antigen sandwich method immunodetection syphilis helicoid antibody test kit specification sheets portion.
The effect that embodiment 9 dual-antigen sandwich methods and indirect elisa method detect syphilis helicoid antibody compares
According to 96 orifice plates of previous embodiment condition with modified recombinant syphilis spirochete antigen protein KTpp bag quilt, adopt method that the antigen protein KTpp of embodiment 3 enzyme process marks abides by embodiment 7 to available from Inst. of Dermatology, Chinese Academy of Medical Sciences, BJ Clinic of Prevention ﹠ Cure of STD, the Beijing Red Cross Blood Center, clinical laboratory of China-Japan Friendship Hospital, the clinical serum of BJ Medical University 1st Subsidiary Hospital Dermatology Department, with dual-antigen sandwich method immunodetection syphilis helicoid antibody, gained result and conventional indirect elisa method compare.30 parts of positive serum evaluation results show that dual-antigen sandwich method and two kinds of methods of indirect elisa method detect weak positive, the ratio (S/CO) of comparative sample OD value and threshold value (cut off), as a result latent period syphilis (in the table with *The expression) and early stage syphilis (in the table with *Expression) value is higher than indirect elisa method.The results are shown in following table.
The comparison of table 4 dual-antigen sandwich method and indirect method (S/CO)
Confirm the positive serum numbering Dual-antigen sandwich method Indirect elisa method
The bag quilt KTpp15 KTpp17 KTpp47 KTpp15 KTpp17 KTpp47
The enzyme mark HRP-KTpp15、HRP-KTpp17、 HRP-KTpp47 HRR-McAb(αhuman IgG)
1 11.34 12.56 7.65 12.54 11.49 2.17
2 10.54 11.34 5.47 11.76 12.20 9.56
3 11.25 12.24 7.65 10.57 11.23 7.90
4 7.94 10.96 9.58 9.25 7.97 8.65
5 7.67 10.77 10.67 8.57 6.75 9.47
6 6.54 7.96 5.75 7.89 5.79 4.37
7 6.75 8.64 6.79 5.96 6.26 5.55
8 5.21 7.23 5.47 7.65 6.59 4.23
9 3.21 6.75 0.94 2.17 3.24 0.65
10 2.76 5.66 3.56 9.79 4.26 2.97
11 1.54 3.21 0.59 1.17 3.11 0.34
12 2.36 4.32 5.79 1.67 3.59 4.63
13 ** 4.27 5.24 7.76 156 3.25 5.39
14 2.17 3.26 8.54 1.47 3.14 6.75
15 1.56 2.54 7.67 1.22 2.34 5.39
16 ** 7.65 9.75 7.54 1.26 2.27 6.18
17 * 7.94 10.76 5.34 1.21 2.15 4.35
18 * 8.56 10.75 3.44 2.21 3.26 2.98
19 7.61 8.54 3.21 2.34 4.55 2.15
20 7.45 10.76 3.21 5.65 7.77 3.08
21 11.24 9.79 5.47 9.75 6.24 5.31
22 6.37 7.67 2.47 4.23 5.65 2.54
23 5.24 8.59 2.37 2.34 4.35 2.05
24 2.37 7.67 1.57 2.51 3.24 1.66
25 2.47 8.57 2.76 3.54 3.21 2.54
26 1.56 7.66 5.37 3.44 4.35 4.95
27 7.67 9.25 2.54 4.35 5.56 1.99
28 857 10.65 2.31 4.45 5.49 2.34
29 * 7.66 11.24 4.23 6.66 7.20 3.98
30 7.45 7.39 6.54 7.56 8.15 5.89
Above experimental result shows, dual-antigen sandwich method immunodetection treponema pallidum test kit is owing to can detect IgG simultaneously, IgM and IgA, compare with the indirect elisa method of main detection IgG antibody, both can early diagnosis syphilis in latent period, reduced the probability of omission again.
In addition, by mensuration to 400 parts of clinical negative serum samples, adopt the invention process dual-antigen sandwich method treponema pallidum test kit obviously to be better than indirect elisa method, OD value＜0.02 that dual-antigen sandwich method records, and indirect elisa method is about 0.04, the false positive rate of indirect elisa method is 5 ‰, and dual-antigen sandwich method is 0.
Between embodiment 10 reorganization KTpp antigens and not modified treponemal antigen Tpp
The effect that connects ELISA method mensuration syphilis helicoid antibody compares
In order relatively to adopt the antigenicity of the treponemal antigen that modified treponemal antigen KTpp15, KTpp17, KTpp47 and N-terminal that the present invention prepares and C-terminal do not add lysine residue, adopt indirect elisa method to compare.Promptly adopt two kinds of recombinant protein antigen Tpp15 of multiple concentration and KTpp15, Tpp17 and KTpp17, Tpp47 and KTpp47 to wrap respectively respectively by behind the solid enzyme target, pass through indirect elisa method, detect identical treponema pallidum positive serum sample, the situation that compares two kinds of Detection of antigen syphilis helicoid antibodies, concrete experimental implementation and result see table.
Use the reorganization KTpp antigen and the Tpp antigen of 0,2.5,5,10,15,20,25,30,40,50 (μ g/ml) concentration all to be made into bag by working fluid respectively, 4 ℃ of bags of 96 hole enzyme plates are spent the night with 0.05M pH9.6 carbonate buffer solution (CB).The 0.02M pH7.4 phosphate buffered saline buffer (PBS) that adds 1% bovine serum albumin (BSA) and 1% skim-milk then in the same manner, 4 ℃ of following sealings discarded after 2 hours.All add with the positive matter of a treponema pallidum and comment serum (being Tpp15, the positive serum of Tpp17 and Tpp47 band) 10 microlitres/hole through Western Blot conclusive evidence, the solution that contains the PBS of 1%BSA with 100 microlitres is sample diluting liquid, 37 ℃, after 30 minutes, discard.Wash twice with the 0.05M PBS washings (PBS-T) that contains 0.5%TW-20, add in the same manner with enzyme labelled antibody diluent (20% lowlenthal serum (the sharp blood products in Zhejiang three company produces), 1% casein (Sigma), 0.02M PBSpH7.4)) with 1: 5000 the dilution goat anti-human igg's (production of DAKO company) 100 microlitres/hole, 37 ℃, 30 minutes, wash twice with the 0.02MpH7.4 PBS washings (PBS-T) that contains 0.5% polysorbas20 (TW-20) again, add zymolyte A liquid that 50 μ l are made up of the acetate buffer of 0.06% urea peroxide (Sigma) 0.01M pH4.5 and 50 μ l in last every hole by 0.06%3,3`, 5,5`-tetramethyl benzidine (TMB) (Sigma), the zymolyte B that the citrate buffer of 0.01MpH4.5 is formed, 37 ℃, developed the color 10 minutes, and added 1M H at last 2SO 4Stop, read light absorption value (reference wavelength 630nm) at the 450nm place, the experimental result tabulation:
Table 5: indirect elisa method Tpp15 antigen and KTpp15 Detection of antigen syphilis spiral shell
Revolve the positive matter of body and comment the comparison (450nmOD value, reference wavelength 630nm) of serum
Bag is by concentration (μ g/ml) Tpp15 KTpp15
0 0 0
2.5 0.3 0.3
5.0 0.586 0.689
15 0.954 1.098
20 1.2 1.381
25 1.298 1.47
30 1.4 1.581
40 1.49 1.674
50 1.51 1.693
Table 6: indirect elisa method Tpp17 antigen and KTpp17 Detection of antigen syphilis spiral
The positive matter of body is commented the comparison (450nmOD value, reference wavelength 630nm) of serum
Bag is by concentration (μ g/ml) Tpp17 KTpp17
0 0 0
2.5 0.3 0.3
5.0 0.615 0.689
15 1.04 1.28
20 1.28 1.58
25 1.44 1.74
30 1.52 1.86
40 1.61 1.973
50 1.64 2
Table 7: the positive matter of indirect elisa method Tpp47 antigen and KTpp47 Detection of antigen treponema pallidum is commented the comparison (450nmOD value, reference wavelength 630nm) of serum
Bag is by concentration (μ g/ml) Tpp17 KTpp17
0 0 0
2.5 0.3 0.3
5.0 0.615 0.689
15 1.04 1.28
20 1.28 1.53
25 1.44 1.68
30 1.52 1.73
40 1.61 1.79
50 1.64 1.79
Experimental result shows that the antigen sensitivity level of KTpp15, KTpp17, KTpp47 is higher than Tpp15, Tpp17 and Tpp47.
Embodiment 11 enzyme indicated weight group KTpp antigens and the not modified syphilis of enzyme mark
The effect of former Tpp dual-antigen sandwich method for determining syphilis helicoid antibody relatively
Effect for the enzyme labelling mixture that relatively adopts the treponemal antigen that modified treponemal antigen KTpp15, KTpp17, KTpp47 and N-terminal that the present invention prepares and C-terminal do not add lysine residue adopts double antigens sandwich ELISA method to compare.Promptly the recombinant protein antigen Tpp15 of employing optimization concentration and KTpp15, Tpp17 and KTpp17, Tpp47 and KTpp47 wrap respectively by behind the solid enzyme target respectively, adopt the recombinant protein antigen Tpp15 of series concentration and the enzyme mark mixture of KTpp15, Tpp17 and KTpp17, Tpp47 and KTpp47 respectively, detect identical treponema pallidum positive serum sample, relatively two kinds of enzyme-labelled antigens are to detecting the situation of syphilis helicoid antibody influence.
Respectively with reorganization KTpp antigen (the concentration KTpp15 50 μ g/ml that indirect method is optimized, KTpp17 20 μ g/ml, KTpp47 40 μ g/ml) and Tpp antigen (the concentration Tpp15 50 μ g/ml that indirect method is optimized, Tpp17 30 μ g/ml, KTpp47 40 μ g/ml) all be made into bag by working fluid, 4 ℃ of bags of 96 hole enzyme plates are spent the night with 0.05MpH9.6 carbonate buffer solution (CB).The 0.02M pH7.4 phosphate buffered saline buffer (PBS) that adds 1% bovine serum albumin (BSA) and 1% skim-milk then in the same manner, 4 ℃ of following sealings discarded after 2 hours.Add then with enzyme-labelled antigen diluent (20% lowlenthal serum (the sharp blood products in Zhejiang three company produces), 1% casein (Sigma), 0.02M PBS pH7.4)) be diluted to 0,2.5,5,10,15,20,25, the enzyme-labelled antigen mixture HR-KTpp15 of 30,40,50 (μ g/ml) series concentration, HRP-KTpp17, HRP-KTpp47 and HRP-Tpp15, HRP-Tpp17, HRP-Tpp47,50 microlitres/hole, 37 ℃, 60 minutes, wash twice with the 0.02M pH7.4 PBS washings (PBS-T) that contains 0.5% polysorbas20 (TW-20), add zymolyte A liquid that 50 μ l are made up of the acetate buffer of 0.06% urea peroxide (Sigma) 0.01M pH4.5 and 50 μ l in last every hole by 0.06%3,3`, 5,5`-tetramethyl benzidine (TMB) is (Sigma), 0.01M the zymolyte B that the citrate buffer of pH4.5 is formed, 37 ℃, developed the color 10 minutes, add 1M H at last 2SO 4Stop, read light absorption value (reference wavelength 630nm) at the 450nm place, experimental result is seen Figure 10.
No matter experimental result shows that the reorganization KTpp15, the KTpp17 that adopt to modify, KTpp47's is antigen coated, still adopt reorganization Tpp15, Tpp17, Tpp47 antigen coated of unmodified, adopt the dual-antigen sandwich method of recombinant antigen HRP-KTpp15, HRP-KTpp17 that the enzyme mark modifies, HRP-KTpp47 to detect the positive matter of syphilis and comment serum always than recombinant antigen HRP-Tpp15, the HRP-Tpp17, the HRP-Tpp47 sensitivity level height that adopt enzyme mark unmodified.
Sequence table
(1) general information
(i) applicant:
(A) name: Wang builds golden red army
(B) street: heredity institute of Da Tun road, the Chaoyang District Chinese Academy of Sciences
(C) city: Beijing
(E) country: China
(F) postcode (ZIP): 100101
(G) phone: 010-64871664
(H) fax: 010-64889329
(ii) denomination of invention: modified treponema pallidum outer membrane protein, its test kit is used and contained to its immunodetection
(iii) sequence number: 15
(iv) computer-readable information:
(A) media type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: word 97
(v) current request for data:
(A) application number:
(vi) request for data formerly:
(A) application number: CN/99201335.6
(B) Date to Tender Notice of Readiness: on February 8th, 1999
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 149 amino acid
(B) type: amino acid
(D) topological classification: linearity
(ii) molecule type: protein
(xi) sequence description of SEQ ID NO:1:
Met Lys Lys Met Val Lys Arg Gly Arg Phe Ala Leu Cys Leu Ala Val Leu Leu Gly Ala Cys
1 10 20
Ser Phe Ser Ser Ile Pro Asn Gly Thr Tyr Arg Ala Thr Tyr Gln Asp Phe Asp Glu Asn Gly
30 40
Trp Lys Asp Phe Leu Glu Val Thr Phe Asp Gly Gly Lys Met Val Gln Val Val Tyr Asp Tyr
50 60
Gln His Lys Glu Gly Arg Phe Lys Ser Gln Asp Ala Asp Tyr His Arg Val Met Tyr Ala Ser
70 80
Ser Gly Ile Gly Pro Glu Lys Ala Phe Arg Glu Leu Ala Asp Ala Leu Leu Glu Lys Gly Asn
90 100
Pro Glu Met Val Asp Val Val Thr Gly Ala Thr Val Ser Ser Gln Ser Phe Arg Arg Leu Gly
110 120
Arg Ala Leu Leu Gln Ser Ala Arg Arg Aly Alu Lys Glu Ala Ile Ile Ser Arg Lys Lys Leu
130 140
Glu Lys
149
(2) information of SEQ ID NO:2:
(i) sequence signature:
(A) length: 164 amino acid
(B) type: amino acid
(D) topological classification: linearity
(ii) molecule type: protein
(xi) sequence description of SEQ ID NO:2:
Met Lys Lys Met Lys Gly Ser Val Arg Ala Leu Cys Ala Phe Leu Gly Val Gly Ala Leu Gly
1 10 20
Ser Ala Leu Cys Val Ser Cys Thr Thr Val Cys Pro His Ala Gly Lys Ala Lys Ala Glu Lys
30 40
Val Glu Cys Ala Leu Lys Gly Gly Ile Phe Arg Gly Thr Leu Pro Ala Ala Asp Cys Pro Gly
50 60
Ile Asp Thr Thr Val Thr Phe Asn Ala Asp Gly Thr Ala Gln Lys Val Glu Leu Ala Leu Glu
70 80
Lys Lys Ser Ala Pro Ser Pro Leu Thr Tyr Arg Gly Thr Trp Met Val Arg Glu Asp Gly Ile
90 100
Val Glu Leu Ser Leu Val Ser Ser Glu Gln Ser Lys Ala Pro His Glu Lys Glu Leu Tyr Glu
110 120
Leu Ile Asp Ser Asn Ser Val Arg Tyr Met Gly Ala Pro Gly Ala Gly Lys Pro Ser Lys Glu
130 140
Met Ala Pro Phe Tyr Val Leu Lys Lys Thr Lys Lys Lys Lys Leu Glu Lys
150 160
(2) information of SEQ ID NO:3:
(i) sequence signature:
(A) length: 374 amino acid
(B) type: amino acid
(D) topological classification: linearity
(ii) molecule type: protein
(xi) sequence description of SEQ ID NO:3:
Met Lys Lys Met Phe Asp Ala Val Ser Arg Ala Thr His Gly His Gly Ala Phe Arg Gln Phe
1 10 20
Phe Gln Tyr Ala Val Glu Val Leu Gly Glu Lys Val Leu Ser Lys Gln Glu Thr Glu Asp Ser
30 40
Arg Gly Arg Lys Lys Trp Glu Tyr Glu Thr Asp Pro Ser Val Thr Lys Met Val Arg Ala Ser
50 60
Ala Ser Phe Gln Asp Leu Gly Glu Asp Gly Glu Ile Lys Phe Glu Ala Val Glu Gly Ala Val
70 80
Ala Leu Ala Asp Arg Ala Ser Ser Phe Met Val Asp Ser Glu Glu Tyr Lys Ile Thr Asn Val
90 100
Lys Val His Gly Met Lys Phe Val Pro Val Ala Val Pro His Glu Leu Lys Gly Ile Ala Lys
110 120
Glu Lys Phe His Phe Val Glu Asp ser Arg Val Thr Glu Asn Thr Asn Gly Leu Lys Thr Met
130 140
Leu Thr Glu Asp Ser Phe Ser Ala Arg Lys Val Ser Ser Met Glu Ser Pro His Asp Leu Val
150 160
Val Asp Thr Val Gly Thr Val Tyr His Ser Arg Phe Gly Ser Asp Ala Glu Ala Ser Val Met
170 180
Leu Lys Arg Ala Asp Gly Ser Glu Leu Ser His Arg Glu Phe Ile Asp Tyr Val Met Asn Phe
190 200 210
Asn Thr Val Arg Tyr Asp Tyr Tyr Gly Asp Asp Ala Ser Tyr Thr Asn Leu Met Ala Ser Tyr
220 230
Gly Thr Lys His Ser Ala Asp Ser Trp Trp Lys Thr Gly Arg Val Pro Arg Ile Ser Cys Gly
240 250
Ile Asn Tyr Gly Phe Asp Arg Phe Lys Gly Ser Gly Pro Gly Tyr Tyr Leu Thr Leu Ile Ala
260 270
Asn Gly Tyr Arg Asp Val Val Ala Asp Val Arg Phe Leu Pro Lys Tyr Glu Gly Asn Ile Asp
280 290
Ile Gly Leu Lys Gly Lys Val Leu Thr Ile Gly Gly Ala Asp Ala Glu Thr Leu Met Asp Ala
300 310
Ala Val Asp Val Phe Ala Asp Gly Gln Pro Lys Leu Val Ser Asp Gln Ala Val Ser Leu Gly
320 330
Gln Asn Val Leu Ser Ala Asp Phe Thr Pro Gly Thr Glu Tyr Thr Val Glu Val Arg Phe Lys
340 350
Glu Phe Gly Ser Val Arg Ala Lys Val Val Ala Gln Lys Lys Leu Glu Lys
360 370
(2) information of SEQ ID NO:4:
(i) sequence signature:
(A) length: 34 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:4:
5’GGG TCGT CATATG AAG AAG ATG GTG AAA AGA GGT 3’
(2) information of SEQ ID NO:5:
(i) sequence signature:
(A) length: 31 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:5:
5’TTCG CTCGAG TTT TTT CCT GCT AAT AAT GGC 3’
(2) information of SEQ ID NO:6:
(i) sequence signature:
(A) length: 28 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:6:
5’GGG TCGT CATATG ATG GTG AAA AGA GGT 3’
(2) information of SEQ ID NO:7:
(i) sequence signature:
(A) length: 28 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:7:
5’TTCG CTCGAG CCT GCT AAT AAT GGC 3’
(2) information of SEQ ID NO:8:
(i) sequence signature:
(A) length: 34 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:8:
5’GATTGAG CATATG AAG AAG ATG AAA GGA TCT GTC3’
(2) information of SEQ ID NO:9:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:9:
5’GTCAG CTCGAG TTT TTT TTT CTT TGT TTT TTT 3’
(2) information of SEQ ID NO:10:
(i) sequence signature:
(A) length: 28 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:10:
5’GATTGAG CATATG ATG AAA GGA TCT GTC 3’
(2) information of SEQID NO:11:
(i) sequence signature:
(A) length: 26 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:11:
5’GTCAG CTCGAG TTT CTT TGT TTT TTT 3’
(2) information of SEQ ID NO:12:
(i) sequence signature:
(A) length: 34 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:12:
5’GTACTAG CATATG AAG AAG ATG TTC GAT GCA GTT 3’
(2) information of SEQ ID NO:13:
(i) sequence signature:
(A) length: 31 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:13:
5’CTGT CTCGAG TTT TTT CTG GGC GAC TAC CTT 3’
(2) information of SEQ ID NO:14:
(i) sequence signature:
(A) length: 28 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:14:
5’GTACTAG CATATG ATG TTC GAT GCA GTT 3’
(2) information of SEQ ID NO:15:
(i) sequence signature:
(A) length: 25 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological classification: linearity
(ii) molecule type: DNA
(xi) sequence description of SEQ ID NO:15:
5’CTGT CTCGAG CTG GGC GAC TAC CTT 3’

Claims (9)

1. protein with formula M etLysLys-Tpp-LysLysLeuGluLys, wherein Tpp is selected from treponema pallidum outer membrane protein Tpp15, Tpp17, Tpp47.
2. the protein of claim 1, it has the aminoacid sequence of SEQ ID No.1.
3. the protein of claim 1, it has the aminoacid sequence of SEQ ID No.2.
4. the protein of claim 1, it has the aminoacid sequence of SEQ ID No.3.
5. detection kit that is used for immunodetection people syphilis helicoid antibody, it comprises the check-out console with the protein bag quilt of the claim 1 of purifying.
6. the detection kit of claim 5, it also comprises the protein with the corresponding claim 1 through mark of the tested drafting board kind of bag.
7. the detection kit of claim 6, wherein the protein through the claim 1 of mark is that horseradish peroxidase, alkaline phosphatase, β--the enzyme of tilactase and acetylcholinesterase carries out mark with being selected from.
8. the detection kit of claim 5, it also comprises the second antibody at human IgG, IgM antibody through mark.
9. the detection kit of claim 8, wherein the second antibody at human IgG, IgM antibody is to carry out mark with the enzyme that is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and acetylcholinesterase.
CNB998154245A 1999-02-08 1999-08-06 Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit Expired - Lifetime CN1156491C (en)

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CN99201335.6 1999-02-08
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Cited By (1)

* Cited by examiner, † Cited by third party
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CN102095862A (en) * 2011-01-25 2011-06-15 厦门大学附属中山医院 Immune imprinting kit for syphilis specificity IgM antibody and preparation method thereof

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Publication number Priority date Publication date Assignee Title
CN101293919B (en) * 2008-01-22 2011-08-17 中国人民解放军第三军医大学 Syphilis spirochete membrane antigen with shortened expression and uses thereof
CN101738473B (en) * 2008-11-13 2013-06-05 威海威高生物科技有限公司 Treponema pallidum antibody diagnostic kit and preparation method thereof
CN105004858B (en) * 2015-06-25 2017-03-01 菲鹏生物股份有限公司 Conjugate and preparation method thereof, Lues Assay reagent and Lues Assay test kit
CN112684171B (en) * 2021-01-20 2022-09-30 季华实验室 Immunosensing carrier, kit and preparation method for syphilis antibody detection

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102095862A (en) * 2011-01-25 2011-06-15 厦门大学附属中山医院 Immune imprinting kit for syphilis specificity IgM antibody and preparation method thereof

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