CN1151171C - Recombinant Treponema pallidum epitope antigen and multi-epitope chimeric antigen - Google Patents
Recombinant Treponema pallidum epitope antigen and multi-epitope chimeric antigenInfo
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Abstract
本发明涉及单片段重组梅毒螺旋体表位抗原，其氨基酸序列为ARRGEKEAVYDYQHKEGRFKSQDADYHRV或SVLSKQETEDSRGRKKWEYETDPSV。本发明还涉及包含上述表位抗原的多表位嵌合抗原。本发明的表位抗原活性高，多表位嵌合抗原特异性高、生产成本低。
The invention relates to a single-segment recombinant Treponema pallidum epitope antigen, the amino acid sequence of which is ARRGEKEAVYDYQHKEGRFKSQDADYHRV or SVLSKQETEDSRGRKKWEYETDPSV. The present invention also relates to multi-epitope chimeric antigens comprising the above-mentioned epitope antigens. The epitope antigen of the invention has high activity, high specificity of multi-epitope chimeric antigen and low production cost.
Description
The present invention relates to the recombinant syphilis spirochete epitope antigen, also relate to recombinant syphilis spirochete multi-epitope chimeric antigen.
Treponema pallidum is the pathogenic agent of venereal syphilis, the earliest the report be 14th century in Europe, import China into about 16th century greatly.After penicillin was heard generation, this disease had obtained control to a certain degree, but still eliminated far away.And closely during the last ten years, along with spreading of AIDS, syphilis has the gesture of new line again, should draw attention.Also not at the effective vaccine of syphilis, therefore, detect patient in early days at present, treatment in time, control blood source is the important measures that effective control disease is propagated.
Detecting infection by Treponema pallidum has several different methods, as can directly detecting pathogenic agent with lesion tissue; Detect the spirochete gene with the PCR method report (Pietravalle M, et al.Diagnostic relevance ofpolymerase chain reaction technology for T.pallidum in subjects with syphilis indifferent phases of infection.New Microbiol 1999 Apr are also arranged; 22 (2): 99-104) but also not general.Serological reaction is present the most frequently used detection method.The previous normal non-special lipoids antigen (Val) that adopts detects anticardiolipin antibody, as Venereal Disease Research Laboratory (VDRL), do not heat plain test of sero-reaction (USR) and rapid plasma reagin ring test (RPR) etc.These methods are sensitive, but not special, false positive reaction is arranged, thereby often need do validation test.With treponema pallidum is antigenic method for detecting specificity, and syphilis fluorescence helicoid antibody adsorption test (FTA-ABS) and treponemal hemagglutination test (TPHA) are arranged, and its specificity and sensitivity are all higher.But because treponema pallidum is cultivated difficulty, the cost of reagent is higher, because the antigenicity of treponema pallidum is complicated and other pathogenic agent, especially similarly spirochetosis (as refined department) still can have certain nonspecific reaction again.In view of above situation, people are seeking to make antigenic approach with recombinant protein.In recent years, the syphilis whole genome sequence has all obtained determining: genome total length about 1,138,006bp, has 1041 encoder block (FraserCM et al according to inferring, Complete Genome Sequence of Treponema pallidum, the SyphilisSpirochete.Science, 1998; 281:375).Development along with gene clone technology, with the syphilis recombinant protein is the existing many researchs of antigenic method of immunity, captures EIA (ICE) assay method (Young H.et al.Novel recombinant-antigen enzyme immunoassay forserological diagnosis of sphilis.J Clin Microbiol 1998 Apr as immunity; 36 (4): 913).It is reported aspect specificity and sensitivity, all with the FTA-ABS method quite or better.Show that this fermentoid immunologic function test reagent has vast potential for future development.In order to obtain good enzyme immunologic function test reagent, antigenic research is extremely important.Be used for SD recombinant syphilis antigen in early days 4D antigen (Radolf JD, et al.Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purifiedrecombinant Treponema pallidum antigen 4D.J Infect Dis 1986 Jun are arranged; 1023) and TmpA (Ijsselmuiden OE, et al.Sensitivity and specificity of an enzyme-linkedimmunosorbent assay using the recombinant DNA-derived Treponema pallidumprotein TmpA for serodiagnosis of syphilis and the potential use of TmpA forassessing the effect of antibiotic therapy.J Clin Microbiol 1989 Jan 153 (6):; 27 (1): 152-7), it is reported that the latter is 76% to the verification and measurement ratio of primary syphilis, the second phase is 100%, also can reach 98% to asymptomatic syphilis, and is suitable with the blood clotting method.But according to recent studies have shown that, in numerous antigens of syphilis genes encoding, except that TmpA (also claiming TpN42), also has TpN17, TpN15, TpN47 has antigens with higher, wherein again with antigenic activity the highest (Fujimura K, et al.Reactivity ofrecombinant Treponema pallidum (r-Tp) the antigens with anti-Tp antibodies inhuman syphilitic sera evaluated by ELISA.J Clin Lab Anal 1997 of TpN17; 11 (6): 315. and Sato NS, et al.Analysis of Treponema pallidum recombinant antigens fordiagnosis of syphilis by Western blotting technique.Rev Inst Med Trop Sao Paulo1999 Mar-Apr; 41 (2): 115).But there is report to think TpN17, TpN15, three kinds of antigen couplings of TpN47, the more any again antigen list of detection sensitivity is with being high (Gerber A, RecombinantTreponema pallidum antigens in syphilis serology.Immunobiology1996-97; 196 (5): 535).It is lower that recombinant protein is made the general non-specific responding of antigenic reagent, but use big segmental recombinant antigen, also there is report that certain non-specific responding can be arranged, as 4D antigen, can with most of yaws human serum generation cross reaction (Radolf JD, et al.Serodiagnosis ofsyphilis by enzyme-linked immunosorbent assay with purified recombinantTreponema pallidum antigen 4D.J Infect Dis 1986 Jun; 153 (6): 1023).Also having been found that has one section sequence (411PGTEYT416) and people's fiber adhesion albumen (fibronectin) to exist higher homology in TpN47, this may with some autoimmune disorder, can relevant (Baughn RE, et al.Molecular mimicry between animmunodominant amino acid motif on the 47-kDa lipoprotein of Treponemapallidum (Tpp47) and multiple repeats of analogous sequences in fibronectin.JImmunol 1996 Jul 15 with syphilis detection reagent generation cross reactivity; 157 (2): 720), this homology and cross reactivity also may exist in other syphilis antigen.Therefore, if can use the epitope antigen of small segment, and need not big segmental recombinant protein, should can further improve the specificity of detection.But at present on the document, study (Antoni G, et al.Detection of antigenicdeterminants in the Treponema pallidum membrane protein TmpA usingoverlapping synthetic peptides.J Immunol Methods 1996 Jan 16 report of its major antigen epi-position with overlapping peptide except that TpN42 is existing; 189 (1): 137-40), yet there are no other report.
An object of the present invention is to provide the recombinant syphilis epitope antigen of some single slice.
Another object of the present invention provides a multi-epitope chimeric antigen that merges each epitope.
According to an aspect of the present invention, the aminoacid sequence of single slice recombinant syphilis spirochete epitope antigen is:
ARRGEKEAVYDYQHKEGRFKSQDADYHRV or
SVLSKQETEDSRGRKKWEYETDPSV。
According to another aspect of the present invention, recombinant syphilis spirochete multi-epitope chimeric antigen comprises above-mentioned single slice epitope antigen.
Recombinant syphilis spirochete multi-epitope chimeric antigen of the present invention is preferably the fusion of the 23rd to 41 amino acids of the 22nd to 156 amino acids of above-mentioned single slice epitope antigen and TpN17 and TpN42, and its aminoacid sequence is:
ARRGEKEAVYDYQHKEGRFKSQDADYHRVSGGGSSGGGSVLSKQETEDSRGRKKWEYETDPSGGGSSGGGSASGAKEEAEKKAAEQRALLSGGGSSGGGSCVSCTTVCPHAGKAKAEKVECALKGGIFRGTLPAADCPGIDTIVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKK。
Above-mentioned epitope of the present invention and multi-epitope chimeric antigen can obtain by the following technical programs.
In the protein of numerous syphilis genes encodings, the present known protein that can be used for diagnostic reagent mainly contains four kinds of TpN17, TpN15, TpN42 and TpN47.In order to select each proteinic major antigen epi-position, (Gene-Bank) obtains above four kinds of proteinic amino acid complete sequences at first from network; Analyze each antigenic epitope (also claiming antigenic determinant) with computer software Gold-Key, and it is recombinant expressed therefrom to select main epitope to be added on, and further is added on to be connected.
The synthetic available PCR method of antigen gene, amplification obtains from the syphilis gene, also available chemical synthesis, the former is comparatively favourable to obtaining of big gene fragment, and it is segmental synthetic that the latter is fit to minigene.Consider that we want the synthetic gene fragment not long, intend adopting chemical synthesis.The chemosynthesis gene can save the operation of extracting templet gene, and is fast convenient; Another advantage is to be chosen in colibacillary bias codon, can help like this obtaining to express efficiently in E.coli.So we select chemical synthesis for use, also directly do not select the codon of the syphilis gene order of bibliographical information for use, but, infer that nucleotide sequence is synthesized according to the bias codon of high expression level in the peptide sequence of epitope and the intestinal bacteria.The chemical method synthetic gene once can the synthetic limited length, generally is no more than 60 bases.To TpN15, TpN47 and TpN42, can synthesize two 3 ' ends earlier has one section complementary eclipsed gene fragment, and polymerase extension is used in the two annealing back, obtains positive and negative double-stranded DNA gene.But synthesize total length 135 amino acid TpN17 genes, synthetic two gene fragments are far from being enough, so synthesized nine altogether partial sequence eclipsed gene fragment are arranged, and progressively extend synthetic from the middle to both ends.
Interconnect for the ease of intergenic, and can obtain antigenic activity preferably after connection, between epitope one connecting arm need be arranged, we add G-G-G-S and S-G-G-G respectively in the upstream and downstream of epi-position, so should add corresponding gene when synthetic gene.Arrive expression vector for the ease of the synthetic gene clone at last, also synthesized two universal primers that match with connecting arm, introduce two restriction enzyme site XhoI and XbaI, to be fit to expression vector pBVIL1.
Carrier pBVIL1 is the interleukin-11 expression plasmid pBV220/IL-1 β (Guo Fukun that made up before us, Ling Shigan etc., IL-1 β and mutant clone thereof, expression and activity research, institute of Military Medical Science Institute periodical, 1999,23 (3): basis 238-9) last structure form.That is: select g-ring place, IL-1 beta molecule surface, the restriction enzyme site of Gong the gene clone of insertion is XhoI and XbaI, finally is built into expression vector pBVIL1.This carrier has carried out enough preservations on January 27th, 2000, preservation mechanism and be numbered CGMCC NO0437.In on January 28th, 2000 application Chinese patent, number of patent application is 00100695.9 to its construction process, one is listed as bibliography of the present invention at this.
The above-mentioned two ends of having mentioned the synthetic gene have been introduced restriction enzyme site XhoI and XbaI, and this conforms to carrier pBVIL1.So each gene fragment all can be inserted among the carrier pBVIL1 with the double digestion method easily, is built into corresponding expression plasmid.The expression plasmid that makes up need be identified with the PCR method, inserts to prove that each gene fragment has obtained correctly.
Multi-epitope chimeric antigen expression plasmid is to make up to form on the basis of each syphilis epitope antigen expression plasmid.One of them plasmid as carrier; Another plasmid that contains gene TpN15 is as template, with the universal primer that contains SpeI and contain former IL-1 gene 3 ' the end primer of BamHI, amplify the gene fragment that contains TpN15 and part IL-1, insert back, obtain to contain the plasmid of TpN47 and two genes of TpN15 with XhoI and the two TpN47 genes of cutting of BamHI.Because in the novel plasmid, the intergenic connection of TpN47 and TpN15 is by linking to each other between complementary enzyme XbaI and the SpeI, connecting the back restriction enzyme site has not existed, therefore, novel plasmid can become new carrier again, and another contains the TpN42 plasmid, can be used as template, amplify the gene fragment that contains TpN42, be connected the back of TpN47-15, obtain plasmid pBVIL1/TpN47-15-42.In like manner, further can obtain plasmid pBVIL1/TpN47-15-42-17.The multi-epitope chimeric plasmid also will be identified through PCR, proves to amplify corresponding fusion gene fragment.
Each epitope antigen and multi-epitope chimeric antigen expression plasmid behind the Transformed E .coli HB101, are cultivated by thermal induction, get full bacterium liquid and carry out the SDS-PAGE evaluation, prove that each expression plasmid has all obtained to express efficiently.Through ion exchange column and gel-filtration purifying, all can obtain the pure product of electrophoretically pure syphilis epitope antigen and multi-epitope chimeric antigen again.
Experiment showed, that different epitope antigen of the present invention all has certain activity, wherein the inspection rate with TpN17 is the highest, and the activity of multi-epitope chimeric antigen is higher, and our antigen only needs a chimeric antigen, and production cost is low, be epitope antigen again, higher specificity should be arranged.
Below in conjunction with accompanying drawing the present invention is done to describe in further detail, these embodiment only are used for explaining and do not limit the present invention in any way.
Fig. 1 is TpN17 molecule and the Characterization of antigenic epitopes figure (aminoacid sequence of a.TpN17 molecule; B. Characterization of antigenic epitopes illustrates, and abscissa is an amino acid position, and ordinate is an antigenicity).
Fig. 2 is the TpN15 molecular antigen epitope analysis (aminoacid sequence of a.TpN15 molecule; B. Characterization of antigenic epitopes figure; C. the epitope of Xuan Zeing position and aminoacid sequence in molecule).
Fig. 3 is the TpN47 molecular antigen epitope analysis figure (aminoacid sequence of a.TpN47 molecule; B. Characterization of antigenic epitopes figure; C. the epitope of Xuan Zeing position and aminoacid sequence in molecule).
Fig. 4 is the TpN42 molecular antigen epitope analysis figure (aminoacid sequence of a.TpN42 molecule; B. Characterization of antigenic epitopes figure; C. the epitope of Xuan Zeing position and aminoacid sequence in molecule).
Fig. 5 is the aminoacid sequence of four kinds of main epitope antigens and the gene order of deduction thereof.
Fig. 6 is for synthetic four kinds of syphilis epitope antigen genes, needs synthetic gene fragment and universal primer.
Fig. 7 is a TpN17 gene stepwise synthesis synoptic diagram.
Fig. 8 is the structure synoptic diagram of syphilis epitope antigen expression plasmid, the arbitrary epitope antigen of TpNx representative among the figure.
Fig. 9 connects between epitope antigen and multi-epitope antigen expression plasmid structure synoptic diagram.
Figure 10 is the SDS-PAGE qualification result figure (1.TpN47,2.TpN42,3.TpN17,4.TpN15,5.TpN47+15,6.TpN47+15+42,7.TpN47+15+42+17,8. protein standard) of the pure product of various recombinant syphilis antigens.
Embodiment 1: the selection of recombinant syphilis epitope
1.TpN17 the selection of epitope
The complete sequence of TpN17 has 156 amino acid as shown in Figure 1a, and with Gold-Key software analysis antigenic determinant, shown in Fig. 1 b, wherein the N end is the hydrophobic signal peptide sequence, does not have clear and definite epitope, exists a plurality of epitopes in the other parts of molecule.Because TpN17 activity in each antigen is the highest, so we have selected the other parts except that 21 amino acid of N-end, i.e. 22～156 (135 amino acid).
2.TpN15 the selection of epitope
The complete sequence of TpN15 is shown in Fig. 2 a, and complete sequence has 124 amino acid, with Gold-Key software analysis epitope, shown in Fig. 3 b, 2 main epitopes are arranged in TpN15, the one, in the stage casing (the 41st～60 amino acid), be again at C-end (113～120 amino acid).So we have selected this two epi-positions, and then are connected, so the epitope combined amino acid sequence that fits to is shown in Fig. 2 c.
3.TpN47 the selection of epitope:
The complete sequence of TpN47 is shown in Fig. 3 a, total order is shown 415 amino acid, with Gold-Key software analysis antigenic determinant, shown in Fig. 3 b, though several epitopes are arranged in the TpN47 molecule, but stronger epitope has only 1, mainly is 82～97 at molecule, and we also only select this epi-position, sequence may be to the influence of epi-position three-dimensional structure before and after having considered simultaneously, suitably be added on prolongation, select the 78th～101 amino acid at last, shown in Fig. 3 c.
4.TpN42 the selection of epitope:
The complete sequence of TpN42 is shown in Fig. 4 a, total order is shown 405 amino acid, with Gold-Key software analysis antigenic determinant, shown in Fig. 4 b, though several epitopes are arranged in the TpN42 molecule, stronger epitope also has only 1, mainly be at molecule N end 25-37 amino acid, and suitably extend forwards, backwards, selecting sequence at last is 23～41, the epitope of studying with overlapping peptide on this and the document is consistent.Also avoided simultaneously the section that bibliographical information and fibronectin have cross-immunoreactivity.
The cloning and expression of embodiment 2. syphilis epitope antigens and multi-epitope chimeric antigen
1. the structure that contains epitope antigen and multi-epitope chimeric antigen expression plasmid
1.1. the segmental design of synthetic gene is with synthetic:
According to the needs of each synthetic gene, designed the gene fragment of having mentioned as Fig. 6.All entrusting Shanghai to give birth to worker's biotechnology Services Co., Ltd after the gene fragment order design synthesizes.
1.2.PCR method obtains the TpNs gene:
TpN15, the method for synthesizing gene of Tpn47 and TpN42, except that used synthetic gene fragment difference, all use the two-step pcr synthesis method:
The first step: in the pcr amplification pipe, add successively: H
2O 41 μ l, 10 * PCR Buffer, 5 μ l, add each 1 μ l of a pair of gene fragment of synthetic of gene (20pmole/ μ l) R, F (Fig. 6) separately respectively, 10mmol/L 4 dNTP 1 μ l, the Taq archaeal dna polymerase 1 μ l of 2U/ μ l, mixing, 1000rpm centrifugal 30 ", put into pcr amplification instrument (GeneAmp PCR System 2400) amplification then.The PCR reaction conditions be 94 ℃ 1 minute, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, 5 circulations.
Second step: the PCR product with the first step is that template increases.Promptly in the pcr amplification pipe, add successively: H
2O 40 μ l, 10 * PCR Buffer, 5 μ l, each 1 μ l (Fig. 6) of 20pmole/ μ l universal primer R1, F1,1/10 the first step PCR product 1 μ l, 10mmol/L 4 dNTP 1 μ l, the Taq archaeal dna polymerase 1 μ l of 2U/ μ l, mixing, 1000rpm centrifugal 30 ", put into the pcr amplification instrument then and increase.The PCR reaction conditions is 94 ℃ after 3 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, after 30 circulations again 72 ℃ extended 5 fens.
Six steps of branch for TpN17 are synthesized (Fig. 7): 1) earlier with TpN17 primers F 5, R6 increases with above-mentioned the first step method.2) is template all after with last amplified production, respectively with F4 and R7, F3 and R8, F2 and R9, F1 and R9, and universal primer F1 and R1 be primer, by above-mentioned second one step process amplification.
The purifying of PCR product: " PCR product purification in a small amount " test kit of producing with Shanghai China Shun biotechnology company limited carries out purifying; promptly in the PCR product, add PB 0.25ml; full dose moves into adsorption column, and the by specification method is cleaned and wash-out then, and electrophoresis is identified then.
1.3 the structure of expression plasmid
1.3.1PCR product and expression vector pBVIL1 double digestion
PCR product and each 30ul of pBVIL1 expression vector of getting above purifying are put in respectively in the Eeppendorf centrifuge tube, each adds 10 * buffer (H) 4ul, XbaI (10u/ul) and each 1ul of XhoI (12u/ul), add sterile purified water to 40ul, put 37 ℃ of water-bath enzymes and cut and spend the night.
Enzyme is cut the agarose gel electrophoresis purifying and the recovery of product: PCR product and carrier pBVIL1 carry out purifying with 1.2% sepharose behind double digestion, and concrete grammar is undertaken by the method for " molecular cloning " (Science Press, second edition).The a small amount of glue that purified genes is produced with Shanghai China Shun biotechnology company limited again reclaims test kit and reclaims: promptly downcut the agarose that contains plasmid and goal gene under ultraviolet lamp respectively; be put in the Eeppendorf centrifuge tube; each adds S1 liquid; putting 55 ℃ of water-baths separated peptization in 10 minutes; add the equivalent Virahol, mixing, 55 ℃ of temperature were bathed 1 minute; after moving into adsorption column respectively then, carry out purifying by the test kit specification sheets.
1.3.2 connect: the carrier after the above-mentioned enzyme of adding is cut in sterilization Eeppendorf centrifuge tube and each 1ul of goal gene, 10 * T4 DNA Ligase buffer 1ul, T4 DNA Ligase (12u/ul) 1ul, add sterile purified water to 10ul, put 16 ℃ and spend the night.
1.3.3 transform: in Bechtop, get 100 μ l competent cells (competent cell is undertaken by the method for " molecular cloning " (Science Press, second edition)) suspension with aseptic suction nozzle and in Eppendorfl, add above-mentioned connector 5 μ l, rotate mixing gently, ice bath 30 minutes.Transfer to immediately in 42 ℃ of water-baths and placed 2 minutes, every pipe adds 0.5ml LB substratum (not added with antibiotic), and 30 ℃ of shaking baths were cultivated after 60 minutes, respectively gets 0.2ml and is applied to respectively on the LB agar culture plate and (contains microbiotic), after room temperature is dried, put 37 ℃ of thermostat containers and be inverted overnight incubation.Select several bacterium colonies, be inoculated in respectively (5ml/ pipe) among the LB, overnight incubation is respectively got 0.1ml next day and is transferred to (2ml/ pipe) among the LB, cultivated 3 hours for 32 ℃, inducing culture 4h in 42 ℃ of shaking baths receives bacterium, identify with SDS-PAGE, select goal gene to obtain the high expression level bacterial strain, do further to identify.
1.3.4 plasmid extracts and identifies
Get the bacterial strain that above-mentioned SDS-PAGE assay certificate goal gene obtains high expression level, extract plasmid by " molecular cloning " (Science Press, second edition) method.With the plasmid is template, carries out pcr amplification (method is with above-mentioned second step) with the F1 primer of each gene and the general R2 of drawing, and identifies with agarose gel electrophoresis then.
2. the expression and purification of epitope antigen and multi-epitope chimeric antigen
2.1 the cultivation of expression strain: the expression strain 20ul that gets-70 ℃ of preservations is inoculated in (100ml LB/500ml triangular flask) in the LB substratum, 30 ℃ of air shaking table overnight incubation, next day in 5% ratio transferred species in LB substratum (the same), 30 ℃ of air shaking tables were cultivated about 3 hours, when the OD600 value reaches 0.7, immediately culturing bottle is forwarded in 42 ℃ of shaking baths to inducing culture 4 hours.Bacterium liquid is merged, and centrifugal 20 minutes of 6000rpm abandons supernatant, the collecting precipitation part.
2.2 extraction inclusion body: will precipitate and claim weight in wet base, will precipitate with the 20mmol/L pH8.0 TE damping fluid of 10 times of volumes and hang, adding N,O-Diacetylmuramidase (1mg/ml suspension), at room temperature magnetic agitation is 10 minutes.Ultrasonic disruption bacterium in ice bath surpassed for 30 seconds at every turn, at interval 30 seconds, surpassed altogether 10 times.8 ℃, 12000rpm, centrifugal 20 minutes, abandon supernatant, precipitation is washed once with 1mol/L NaCl (preparing with TE), washes 2 times collecting precipitation again with TE.Precipitation adds 1% beta-mercaptoethanol with 8M urea (preparing with PH8.0TE) dissolving.In 20 ℃, 12000rpm centrifugal 10 minutes, goes precipitation to get supernatant again.
2.3 purifying: above-mentioned dissolved inclusion body solution is crossed Q-Sepharose FF anion-exchange column, with balance liquid (pH8.0,20mmol/L TE contains the 6mol/L urea, 0.1% beta-mercaptoethanol) wash, collect not bound fraction, after S-Sepharose FF cationic exchange coloum, after the balance liquid cleaning,, collect each elution peak with NaCl (preparing) wash-out of different concns with balance liquid, identify through SDS-PAGE, collect 0.1mol/L NaCl elution peak.After Sephardex G-50 gel-filtration column, collect first elution peak.Various purification of Recombinant epitope antigens and multi-epitope chimeric antigen are all identified (Figure 10) with SDS-PAGE.
3. syphilis epitope antigen activity identification
With the syphilis epitope antigen and the MEFA7.1 of purifying, be diluted to 2.0ug/ml with the carbonate buffer solution of pH9.5, bag, to 122 parts of syphilis positive serums that blood station, Red Cross Society of China Beijing provides, is measured after sealing by the ELISA assay plate.And and Japanese TPHA test kit and the test kit assembled with the syphilis antigen of import (reorganization TpN17, TpN15, three kinds of antigens combinations of pN47) compare mensuration, the result is as shown in table 1.
Table 1. epitope antigen, multi-epitope chimeric antigen are to 122 parts of syphilis positive serum measurement results
The negative umber of the positive umber of test kit (antigen title)
15 70 52
17 91 31
42 88 34
47 64 58
Merge 94 28
Japan (TPHA) 91 31
WK (import antigen) 92 30
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