CN101293919B - Syphilis spirochete membrane antigen with shortened expression and uses thereof - Google Patents
Syphilis spirochete membrane antigen with shortened expression and uses thereof Download PDFInfo
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- CN101293919B CN101293919B CN2008100692793A CN200810069279A CN101293919B CN 101293919 B CN101293919 B CN 101293919B CN 2008100692793 A CN2008100692793 A CN 2008100692793A CN 200810069279 A CN200810069279 A CN 200810069279A CN 101293919 B CN101293919 B CN 101293919B
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Abstract
The invention discloses a DNA sequence expressing a truncated treponema pallidum membrane antigen and an amino acid sequence The membrane antigen is removed of the part having high homology with human fibronectin, so as to avoid false positive and improve the specificity of the serological test for diagnosis of treponema pallidum infection. The invention also discloses the application of the membrane antigen in preparing diagnostic reagents for detecting treponema pallidum infection.
Description
Technical field
The invention belongs to gene engineering technology field, concrete relate to a kind of antigene fragment and application thereof that is used to diagnose infection by Treponema pallidum.
Background technology
Syphilis is the sexually transmitted disease (STD) that is caused by treponema pallidum, and the ill back course of disease is very long, invades sexual organ and skin in early days, invades each organ of whole body late period, and pathology almost can be involved each internal organs of whole body, so very harmful.Syphilis can be divided into congenital syphilis and acquired syphilis by the route of transmission.This disease is imported China in 16th century, and syphilis was once once eliminated in China after the founding of the state.In recent years, frequent day by day along with foreign exchanges, this disease is revivable again in China, and sickness rate is ascendant trend year by year, should draw attention.Also not at the effective vaccine of syphilis,, can not prevent to infect again at present though penicillin has better curative effect to syphilis.Therefore detect patient in early days, treatment in time, the control contagium is the important measures that control disease is propagated.
The syphilis detection method has multiple, and serological reaction is the most frequently used detection method.Can be divided into two classes according to used antigen difference: the detection of specific antibody and non-specific antibody detect.The previous normal non-specific 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 method sensitivity are higher, but not special, are prone to false positive reaction, thereby often need do confirmatory test.With treponema pallidum is that antigenic method for detecting specificity comprises FTA-ABS test (FTA-ABS), the test of treponema pallidum hemagglutination (TPHA), treponema pallidum GAT (TPPA), these method susceptibilitys and specificity are all higher, but treponema pallidum is cultivated difficulty, and reagent cost is higher.
In recent years, detecting syphilis helicoid antibody with the ELISA method is popularized.This method can be used for each phase controller used in syphilis diagnosis, and is easy and simple to handle, once can carry out the detection originally of many increments, and the result judges objective and accurate, has avoided the influence of subjective factor to the result, is easy to carry out indoor Quality Control.It is the prefered method of present serodiagnosis of syphilis test.The ELISA test kit overwhelming majority utilizes corresponding antibody in four kinds of Detection of antigen serum such as the TpN15 of gene recombination (TP0171), TpN17 (TP0435), TpN47 (TP0574), TpN44.5 (TmpA TP0768).It is lower to utilize recombinant protein to do the general nonspecific reaction of antigen.But according to reports, use big pulsating recombinant antigen also nonspecific reaction can take place, as 4D antigen, can with most of yaws human serum generation cross reaction (Radolf JD, et al.Serodiagnosis of syphilis by enzyme-linked immunosorbentassay with purified recombinant Treponema pallidum antigen 4D.J infect Dis 1986Jun; 153 (6): 1023).Found to have among the TpN47 one section sequence (411PGTEYT416) and people's fibronectin that higher homology is arranged, may with some autoimmunity patients serum and relevant (the Baughn RE of syphilis detection reagent generation cross reaction, Jiang A, Abraham R, et al.Molecular mimicrybetween an immunodominant amino acid motif on the 47-kDa lipoprotein ofTreponema pallidum (Tpp47) and multiple repeats of analogous sequences infibronectin[J] .J Immunol, 1996,157 (2): 720-731) this cross reactivity can cause false-positive generation, cause clinical misdiagnosis, have serious consequences for patient's body and mind and even whole family.In case blood donor's sample syphilis detects the positive and promptly is eliminated, the blood waste that this must cause some amount brings certain stress to the blood donor simultaneously, and the specificity that therefore increases detection reagent is very important to avoiding false-positive generation.
Summary of the invention
Technical problem to be solved by this invention provides a kind of treponema pallidum TpN47 membrane antigen of shorten expression, and it is encoded by the described nucleotide sequence 5 ' end of SEQ ID NO:1 the 202nd～1230bp.The aminoacid sequence of fragment gene encoded protein is as described in the SEQ ID NO:2 thus.
The membrane antigen of this shorten expression has removed the part that higher homology is arranged with people's fibronectin, has avoided false-positive generation, has improved the specificity of the serological test of diagnosis infection by Treponema pallidum.
Another technical problem to be solved by this invention provides the expression vector of the treponema pallidum TpN47 membrane antigen that contains described shorten expression and utilizes this carrier transformed host cells.Expression vector can be selected pET serial carrier, pQE serial carrier, pTrc serial carrier, preferred expression plasmid pET-22b (+)-TP47 for use.Host cell can be selected e. coli bl21, intestinal bacteria M15, e. coli jm109 for use.
Another technical problem to be solved by this invention provides the application of treponema pallidum TpN47 membrane antigen in the diagnostic reagent of preparation detection infection by Treponema pallidum of described shorten expression.
The present invention utilizes prokaryotic expression system clonal expression treponema pallidum TpN47 membrane protein gene (gi:6626258) 202～1230bp, it is open reading frame 5 ' end 1029bp part, expressed albumen is 343 amino acid, removed the carboxyl terminal partial amino-acid that cross reaction takes place in the whole protein, and at expression in escherichia coli, the purified back of expression product is as the antigen of diagnosis infection by Treponema pallidum.
The reorganization membranin antigen of this brachymemma can be applicable to detect the antibody that produces behind the infection by Treponema pallidum, be applicable to enzyme linked immunosorbent assay (ELISA), dot-ELISA (Dot-ELISA), immunofluorescence antibody test (IP), colloidal gold test (colloidal gold), chemoluminescence test (chemoluminescence test) and western blot test (Western Blot).
Beneficial effect of the present invention is as follows:
1. this recombinant protein has significant antigenic response activity, genome with treponema pallidum type strain (Nichols strain) is a template, utilize prokaryotic expression carrier pET22b (+), express treponema pallidum TpN47 membrane antigen portion gene, and by with the enzyme linked immunosorbent assay of the syphilis helicoid antibody positive serum of TPHA method conclusive evidence, verified the antigenic response activity of recombinant protein.
2. through detecting with the treponema pallidum positive serum, the part TpN47 membranin of expressing has good antigen reactivity, can be used as diagnostic antigen and be applied to enzyme linked immunosorbent assay, dot-ELISA, the immunofluorescence antibody test, colloidal gold test, chemoluminescence test and western blot test detect the antibody that infection by Treponema pallidum produces.
3. present domestic first-selected enzyme linked immunosorbent assay is carried out the syphilis serology and is detected, corresponding antibodies in four kinds of Detection of antigen serum such as TpN15 (TP0171), the TpN17 (TP0435) of main applying gene reorganization, TpN47 (TP0574), TpN44.5 (TmpA TP0768), but still have false positive results.The treponema pallidum TpN47 membrane antigen of shorten expression then can be avoided the generation of cross reaction, prevents the appearance of false positive results.
The present invention is further detailed explanation below in conjunction with embodiment.
Description of drawings
Fig. 1. the PCR clonal expansion result of treponema pallidum TpN47 gene;
1 swimming lane is the nucleic acid molecular weight standard
2 swimming lanes are the amplified production of goal gene TpN47
Fig. 2. the enzyme of treponema pallidum TpN47 recombinant expression plasmid is cut qualification result;
1,2 swimming lanes are recombinant plasmid pET-22b (+)-TP47Nde I and Xho I double digestion product
3 swimming lanes are the nucleic acid molecular weight standard
The SDS-PAGE electrophoresis result that Fig. 3 .TpN47 gene recombination bacterium abduction delivering and expression-form are identified;
1 swimming lane is empty carrier pET-22b (+)/BL21 contrast
2 swimming lanes are induced preceding (0h) for the TpN47 gene recombination bacterium
3～5 swimming lanes induce 1,3,5h for the TpN47 gene recombination bacterium
6 swimming lanes are protein molecular weight standard
7 swimming lanes are induced the 3h supernatant for the TpN47 gene recombination bacterium and are expressed
8 swimming lanes be the TpN47 gene recombination bacterium ultrasonic after broken bacterium
9 swimming lanes are induced the 3h inclusion body for the TpN47 gene recombination bacterium and are expressed
Fig. 4. target protein TpN47 purification effect PAGE electrophorogram;
1 swimming lane is solubilization of inclusion bodies liquid (sample before the purifying)
2 swimming lanes are that stream is worn
3～10 swimming lanes are target protein elution peak sample
Fig. 5. detect the immunoreactivity of TpN47 and syphilis positive serum for Western blot.
A figure is TpN47 recombinant protein and the reaction of syphilis negative serum
B figure is TpN47 recombinant protein and the reaction of syphilitic's positive serum
1,2 swimming lanes are TpN47 recombinant protein and the reaction of syphilitic's positive serum
3 swimming lanes are blank
4 swimming lanes are protein molecular weight standard
Embodiment
The shorten expression of treponema pallidum TpN47 membrane antigen
The treponema pallidum TpN47 membranin antigen of shorten expression, utilize prokaryotic expression system clonal expression treponema pallidum type strain (Nichols strain), by prokaryotic expression system clonal expression TpN47 membrane protein gene open reading frame 5 ' end 1029bp sequence, the purified back of expressed products is as the antigen of diagnosis infection by Treponema pallidum, the TpN47 gene is by brachymemma, keep open reading frame 5 ' end 1029bp part, and at expression in escherichia coli.
The preparation of TP genomic dna: treponema pallidum Nichols strain is provided by Military Medical Science Institute, and the genomic extraction of TP is that Time Technology company limited bacterial genomes extraction test kit specification sheets carries out by sky, Beijing.
TpN47 gene order (gi:6626258) according to the GenBank announcement, 5 ' end 202bp with biosoftware PrimerPremier V5.0 design primer amplification TpN47 gene order (is an open reading frame 1～1029bp) (underscore is represented restriction enzyme site) and synthetic to 1230bp, carry out polymerase chain reaction (PCR) then, amplification purpose segment.
P1:5 '-CGC
CATATGTTCGATGCAGTTTCTC-3 ' wherein represents the NdeI restriction enzyme site with underscore;
P2:5 '-CCG
CTCGAGAGTGAAATCCGCAGAGAG-3 ' wherein represents Xho I restriction enzyme site with underscore.
DNA is a template with treponema pallidum Nichols pnca gene group, and P1, P2 are that primer carries out pcr amplification TpN47 gene fragment.PCR reaction system and program are as follows: add following reagent in the 200 μ l Eppendorf tubes: template DNA 2.5ng, each 1 μ L of upstream and downstream primer (0.025mmol/L), 10 * PCR damping fluid (not containing Mgcl2), 5 μ l, dNTP (10mmol/L) 4 μ L, MgCl2 (25mmol/L) 4 μ L, Ex Taq enzyme (5U/ μ L) 0.25 μ L adds deionized water to final volume 50 μ l.The PCR reaction parameter: 94 ℃ of pre-sex change are after 10 minutes, and 94 ℃, 45 seconds; 54 ℃, 45 seconds; 72 ℃, 1.5 minutes, 30 loop cycles, last 72 ℃ were extended 10 minutes.The gene amplification result as shown in Figure 1.
The structure of pMD18-T-TP47 plasmid: the PCR product is after reclaiming purifying, and (Dalian Takara company) is connected 4 hours at 16 ℃ with the pMD18-T-simple carrier.Reference literature (55-60 then, Sa nurse Brooker J. etc., " molecular cloning experiment guide ", the 2nd edition, Science Press) method described in, to connect product is converted in the bacillus coli DH 5 α competent cell, through the single bacterium colony of blue hickie screening picking white, extract plasmid DNA, identify through Nde I and Xho I double digestion, obtain recombinant plasmid pMD18-T-TP47, and carry out sequencing inserting segment.
The structure of expression plasmid pET-22b (+)-TP47: behind dna sequencing,, use T with Nde I and Xho I difference double digestion pMD18-T-TP47 and efficient prokaryotic expression carrier pET-22b (+) back recovery purpose fragment
4Dna ligase connects under 22 ℃ of conditions and spends the night.Then, will connect product and be converted in the e. coli bl21 competent cell, identify, obtain recombinant prokaryotic expression vector pET-22b (+)-TP47 by Amp+ resistance screening and digestion with restriction enzyme.Enzyme is cut the result as shown in Figure 2.
The abduction delivering of recombinant bacterial strain and expression-form are identified: the bacterium of will recombinating is in 37 ℃ of shaking table overnight incubation of Amp+ resistance LB nutrient solution.Next day, the ratio transferred species in 1% is in Amp+ resistance LB nutrient solution, and 37 ℃ are cultured to OD
600Add during ≈ 0.8 inductor IPTG to final concentration be the 1mmol/L abduction delivering, preceding respectively at inducing, induce back sampling in 1,3,5 hour and measure OD
600Value compares with the empty carrier bacterium simultaneously, and centrifugal collection thalline also boils the extraction whole bacterial protein, adopts the proteic expression of 12%SDS-PAGE testing goal.The relative content of the target protein that UVP gel scanner scanning analysis is expressed is 43%.The reorganization bacterium of abduction delivering is carried out the broken bacterium (300W of ice-bath ultrasonic, 9 seconds, 9 seconds at interval, repeat 98 times), after differential centrifugation (750g, 30 minutes) is removed residual cell debris, centrifugal 30 minutes of 12000g, collect respectively and go up cleer and peaceful precipitation (being inclusion body), carry out the proteic expression-form of 12%SDS-PAGE testing goal.Target protein is expressed and expression-form is identified as shown in Figure 3, mainly is with the inclusion body formal representation.
The fermentation culture of reorganization bacterium and the isolation and purification of target protein
Adopt German B.Braun 10L fermentor tank, ratio inoculation kind of the daughter bacteria in 10% keeps dissolved oxygen 70%, 37 ℃ of temperature, PH7.0.A
600Reach at 2 o'clock and add feed supplement, flow feeding-inferior final concentration of glucose, tryptone and 8% yeast extract that makes was respectively 0.5%, 0.2% and 0.2% in per afterwards 0.5 hour.Treat that after the 4th feed supplement glucose concn reduces at 0.1% o'clock and add IPTG500 μ mol/L and induce and received bacterium in 4 hours.Fermenting process is flow feeding on the batch culture basis of cascade dissolved oxygen control.Fermentation ends is collected bacterium liquid, and centrifugal 15 minutes of 4 ℃ of 8000g abandon supernatant, collect bacterium, and the back of weighing is frozen standby.The result: the 7LTpN47 zymocyte liquid obtains wet bacterium 290 grams.
The separation of target protein and purifying
Inclusion body extracts: the thalline 200-500 gram that efficiently expresses is suspended with 1: 10 (W/V) ratio of TE damping fluid, adopt the high speed dispersion device that it is mixed after 4 ℃ of precoolings.Adopting the high pressure homogenization machine is to break bacterium 5～8 times under the condition of 40-70Mpa at pressure.After broken bacterium finished, the bacterium liquid smear staining microscopically that takes a morsel was observed, and guaranteed fully broken.750g is centrifugal 30 minutes then, abandons precipitation, and centrifugal 40 minutes again with 15000g, collecting precipitation.Use washings A (20mmol/LTris, 1mmol/LEDTA, 1%TritonX-100, PH7.5 respectively with 1: 10 ratio; ) and B (20mmol/LTris, 2mol/LUrea PH7.5) respectively wash 2 times, and wash conditions is 4 ℃ and stirred 20 minutes, centrifugal 40 minutes of 15000g, and the collection inclusion body precipitates; At last with inclusion body with solubilization of inclusion bodies liquid (20mmol/LTris, 8mol/LUrea, 0.5mol/LNaCL, PH7.5).
Nickel ion column purification: select nickel ion post HiTrap Chelating to carry out purifying (the nickel ion column packing is selected from Q Sepharose HP, Q Sepharose FF, Q Sepharose), use 20mmol/LTris, 8mol/LUrea, 0.5mol/LNaCL, under the PH7.5 condition target protein is carried out purifying, the 0.5mol/L imidazoles carries out gradient elution.Purified target protein is carried out SDS-PAGE, examines and determine its purity.Purity＞95% meets and is used to detect the purity requirement that produces the diagnostic reagent of antibody behind the infection by Treponema pallidum.The results are shown in shown in Figure 4.
The immunologic competence analysis of recombinant protein
According to " molecular cloning experiment guide " (Sa nurse Brooker J., Flitch EF., Manny A Disi T., molecular cloning experiment guide the 2nd edition, Beijing: Science Press, 1999,888-897) the Western method described in, is two anti-with the patients with syphilis positive serum of confirming through the TPPA method of clinical collection, health examination person serum as a goat anti-human igg anti-, the HRP mark with the TpN47 recombinant protein electrotransfer of brachymemma respectively to the NC film, and the immunoblotting that carries out recombinant protein detects.The result shows: the recombinant syphilis antigen protein TpN47 of brachymemma of the present invention has good immunoreactivity with the early syphilis patient positive serum of confirming through the TPPA method, and do not react with health examination person serum, illustrate that the antigen of this shorten expression has immunoreactivity and specificity preferably.The result as shown in Figure 5.
The treponema pallidum TpN47 membranin of shorten expression is as the application of diagnostic antigen
The preparation of a enzyme-linked reaction plate
Behind the treponema pallidum TpN47 membranin purifying with shorten expression, be cushioned liquid (pH9.6 phosphate buffered saline buffer: NaCO with bag
31.95g, NaHCO
32.93g, add ddH
2O to 1000ml) be diluted to 1 μ g/ml, every hole 0.1ml adds in the microwell plate, and 4 ℃ of bags were by 24 hours.Take out microwell plate, pat dry.(Tris 2.42g, HCl transfers pH to 7.4, adds ddH with the 0.01MpH7.4TrisHCl washings
2O to 1000ml, polysorbas20 0.05%) every hole 0.4ml washing is 5 times, pats dry, with confining liquid (NaCl 8.0g, KH
2PO
40.2g, KCL 0.2g, Na
2HPO
41.15g, H
2O 1000ml, 1.0%BSA) sealing, every hole 0.3ml, 4 ℃ were sealed 24 hours, and the same washing pats dry, seals standby.
Wherein employed sample diluting liquid is for containing caseic phosphate buffered saline buffer; Enzyme-second antibody binding substances is the goat anti-human igg antibody with horseradish peroxidase-labeled; Employed Color Appearance System is the citrate buffer B that contains the citrate buffer A of hydrogen peroxide and contain tetramethyl benzidine (TMB); Employed stop buffer is 2M H
2SO4.
B ELISA indirect method
(1) application of sample: the pre-bag of getting TpN47 recombinant syphilis specific antigens bag quilt is established blank hole, negative serum control wells, positive serum control wells and sample well by elisa plate.Add sample diluent 50 μ l in each hole earlier, add negative serum contrast, positive serum contrast and each 50 μ l of serum to be checked then successively, behind the mixing, put 37 ℃ and hatched 30 minutes.
(2) wash plate: hatch end, discard the liquid in the plate hole, the same washing pats dry.
(3) add ELIAS secondary antibody: add anti-human IgG enzyme labelled antibody 100 μ l (the blank hole does not add), fully mixing is put 37 ℃ and was hatched 30 minutes.
(4) wash plate: method is the same.
(5) colour developing: add colour developing liquid A, each 50 μ l of B liquid successively in each hole, pat mixing, 37 ℃ of lucifuges developed the color 10 minutes.
(6) stop: add stop buffer 50 μ l successively in every hole, pat mixing.
(7) result judges: with microplate reader blank well is returned to zero, measure each hole absorbance value (OD value) at the 450nm wavelength, according to the ratio result of determination of measuring OD value and threshold value.If ratio is greater than 1, then positive reaction contains syphilis antibody in the interpret sample; If ratio is less than 1, then negative reaction, interpret sample does not contain syphilis antibody.
C detects syphilis positive serum 100 examples and negative serum 100 examples through the TPPA conclusive evidence with the Sptting plate of bag quilt, comprise diabetic serum 19 examples of having made a definite diagnosis in the negative serum, tuberculosis patient serum 7 examples, rheumatoid arthritis human serum 11 examples, systemic lupus erythematous patients serum's 2 examples, hepatitis patient serum's 10 examples, pregnancy serum 17 examples, all the other are the healthy blood donor.Wherein positive coincidence rate is 97%, and negative match-rate is 100%.Therefore, the TpN47 recombinant antigen of this shorten expression can be used for detecting the antibody that produces behind the infection by Treponema pallidum, has better specificity.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉syphilis spirochete membrane antigen of shorten expression and application thereof
<160>2
<210>1
<211>1305
<212>DNA
＜213〉treponema pallidum
<400>1
gtgaaagtga?aatacgcact?actttctgcc?ggagcgctgc?agttgttggt?tgtaggctgt 60
ggctcgtctc?atcatgagac?gcactatggc?tatgcgacgc?taagctatgc?ggactactgg 120
gccggggagt?tggggcagag?tagggacgtg?cttttggcgg?gtaatgccga?ggcggaccgc 180
gcgggggatc?tcgacgcagg?catgttcgat?gcagtttctc?gcgcaaccca?cgggcatggc 240
gcgttccgtc?agcaatttca?gtacgcggtt?gaggtattgg?gcgaaaaggt?tctctcgaag 300
caggagaccg?aagacagcag?gggaagaaaa?aagtgggagt?acgagactga?cccaagcgtt 360
actaagatgg?tgcgtgcctc?tgcgtcattt?caggatttgg?gagaggacgg?ggagattaag 420
tttgaagcag?tcgagggtgc?agtagcgttg?gcggatcgcg?cgagttcctt?catggttgac 480
agcgaggaat?acaagattac?gaacgtaaag?gttcacggta?tgaagtttgt?cccagttgcg 540
gttcctcatg?aattaaaagg?gattgcaaag?gagaagtttc?acttcgtgga?agactcccgc 600
gttacggaga?ataccaacgg?ccttaagaca?atgctcactg?aggatagttt?ttctgcacgt 660
aaggtaagca?gcatggagag?cccgcacgac?cttgtggtag?acacggtggg?taccggttac 720
cacagccgtt?ttggttcgga?cgcagaggct?tctgtgatgc?tgaaaagggc?tgatggctct 780
gagctgtcgc?accgtgagtt?catcgactat?gtgatgaact?tcaacacggt?ccgctacgac 840
tactacggtg?atgacgcgag?ctacaccaat?ctgatggcga?gttatggcac?caagcactct 900
gctgactcct?ggtggaagac?aggaagagtg?ccccgcattt?cgtgtggtat?caactatggg 960
ttcgatcggt?ttaaaggttc?agggccggga?tactacaggc?tgactttgat?tgcgaacggg 1020
tatagggacg?tagttgctga?tgtgcgcttc?cttcccaagt?acgaggggaa?catcgatatt 1080
gggttgaagg?ggaaggtgct?gaccataggg?ggcgcggacg?cggagactct?gatggatgct 1140
gcagttgacg?tgtttgccga?tggacagcct?aagcttgtca?gcgatcaagc?ggtgagcttg 1200
gggcagaatg?tcctctctgc?ggatttcact?cccggcactg?agtacacggt?tgaggttagg 1260
ttcaaggaat?tcggttctgt?gcgtgcgaag?gtagtggccc?agtag 1305
<210>2
<211>343
<212>PRT
＜213〉treponema pallidum
<400>2
Met?Phe?Asp?Ala?Val?Ser?Arg?Ala?Thr?His?Gly?His?Gly?Ala?Phe?Arg
5 10 15
Gln?Gln?Phe?Gln?Tyr?Ala?Val?Glu?Val?Leu?Gly?Glu?Lys?Val?Leu?Ser
20 25 30
Lys?Gln?Glu?Thr?Glu?Asp?Ser?Arg?Gly?Arg?Lys?Lys?Trp?Glu?Tyr?Glu
35 40 45
Thr?Asp?Pro?Ser?Val?Thr?Lys?Met?Val?Arg?Ala?Ser?Ala?Ser?Phe?Gln
50 55 60
Asp?Leu?Gly?Glu?Asp?Gly?Glu?Ile?Lys?Phe?Glu?Ala?Val?Glu?Gly?Ala
65 70 75 80
Val?Ala?Leu?Ala?Asp?Arg?Ala?Ser?Ser?Phe?Met?Val?Asp?Ser?Glu?Glu
85 90 95
Tyr?Lys?Ile?Thr?Asn?Val?Lys?Val?His?Gly?Met?Lys?Phe?Val?Pro?Val
100 105 110
Ala?Val?Pro?His?Glu?Leu?Lys?Gly?Ile?Ala?Lys?Glu?Lys?Phe?His?Phe
115 120 125
Val?Glu?Asp?Ser?Arg?Val?Thr?Glu?Asn?Thr?Asn?Gly?Leu?Lys?Thr?Met
130 135 140
Leu?Thr?Glu?Asp?Ser?Phe?Ser?Ala?Arg?Lys?Val?Ser?Ser?Met?Glu?Ser
145 150 155 160
Pro?His?Asp?Leu?Val?Val?Asp?Thr?Val?Gly?Thr?Gly?Tyr?His?Ser?Arg
165 170 175
Phe?Gly?Ser?Asp?Ala?Glu?Ala?Ser?Val?Met?Leu?Lys?Arg?Ala?Asp?Gly
180 185 190
Ser?Glu?Leu?Ser?His?Arg?Glu?Phe?Ile?Asp?Tyr?Val?Met?Asn?Phe?Asn
195 200 205
Thr?Val?rg?Tyr?Asp?Tyr?Tyr?Gly?Asp?Asp?Ala?Ser?Tyr?Thr?Asn?Leu
210 215 220
Met?Ala?Ser?Tyr?Gly?Thr?Lys?His?Ser?Ala?Asp?Ser?Trp?Trp?Lys?Thr
225 230 235 240
Gly?Arg?Val?Pro?Arg?Ile?Ser?Cys?Gly?Ile?Asn?Tyr?Gly?Phe?Asp?Arg
245 250 255
Phe?Lys?Gly?Ser?Gly?Pro?Gly?Tyr?Tyr?Arg?Leu?Thr?Leu?Ile?Ala?Asn
260 265 270
Gly?Tyr?Arg?Asp?Val?Val?Ala?Asp?Val?Arg?Phe?Leu?Pro?Lys?Tyr?Glu
275 280 285
Gly?Asn?Ile?Asp?Ile?Gly?Leu?Lys?Gly?Lys?Val?Leu?Thr?Ile?Gly?Gly
290 295 300
Ala?Asp?Ala?Glu?Thr?Leu?Met?Asp?Ala?Ala?Val?Asp?Val?Phe?Ala?Asp
305 310 315 320
Gly?Gln?Pro?Lys?Leu?Val?Ser?Asp?Gln?Ala?Val?Ser?Leu?Gly?Gln?Asn
325 330 335
Val?Leu?Ser?Ala?Asp?Phe?Thr
340
Claims (7)
1. the syphilis spirochete membrane antigen of a shorten expression, it is encoded by the described nucleotide sequence 5 ' end of SEQ ID NO:1 the 202nd～1230bp.
2. the syphilis spirochete membrane antigen of a shorten expression, its aminoacid sequence is as described in the SEQ ID NO:2.
3. expression vector, it comprises the described dna molecular of claim 1.
4. a kind of expression vector according to claim 3, it is characterized in that, it is expression plasmid pET-22b (+)-TP47, and described pET-22b (+)-TP47 inserts the described dna molecular of claim 1 between the multiple clone site Nde I of prokaryotic expression carrier pET-22b (+) and the Xho I and obtains.
5. host cell, it comprises claim 3 or 4 described expression vectors.
6. host cell according to claim 5 is characterized in that, described host cell is intestinal bacteria.
7. the application of the syphilis spirochete membrane antigen of claim 1 or 2 described shorten expressions in the diagnostic reagent of preparation detection infection by Treponema pallidum.
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| CN101293919B true CN101293919B (en) | 2011-08-17 |
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| CN101858914B (en) * | 2010-05-19 | 2013-03-27 | 厦门大学附属中山医院 | Reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and preparation method thereof |
| EP2617731A1 (en) * | 2012-01-19 | 2013-07-24 | Roche Diagnostics GmbH | Soluble immunoreactive Treponema pallidum TpN47 antigens |
| CN106397551A (en) * | 2016-09-29 | 2017-02-15 | 南华大学 | Treponema pallidum infection dependent antigens and kits and applications thereof |
| CN110981947B (en) * | 2019-12-16 | 2021-10-08 | 四川安可瑞新材料技术有限公司 | Preparation and application of treponema pallidum TP47 recombinant antigen |
| CN115975023B (en) * | 2022-12-16 | 2023-06-13 | 北京科跃中楷生物技术有限公司 | Preparation method of recombinant TP antigen and antibody detection reagent prepared by preparation method |
| CN118240037A (en) * | 2022-12-23 | 2024-06-25 | 菲鹏生物股份有限公司 | Recombinant antigen for detecting treponema pallidum antibodies and uses thereof |
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| US5681934A (en) * | 1986-09-30 | 1997-10-28 | Board Of Regents, The University Of Texas System | 47-kilodalton antigen of Treponema pallidum |
| CN1332750A (en) * | 1999-02-08 | 2002-01-23 | 汪建 | Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit |
| CN1370782A (en) * | 2001-02-26 | 2002-09-25 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant syphilis spirochete epitope antigen and multiple-epitope chimeric antigen |
| US7045363B2 (en) * | 1996-05-01 | 2006-05-16 | Fujirebio Inc. | Nucleic acid-bound polypeptide method of producing nucleic acid-bound polypeptide and immunoassay using the polypeptide |
| CN1916633A (en) * | 2006-09-08 | 2007-02-21 | 中国人民解放军第三军医大学 | Method and kit for detecting treponema pallidum |
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Patent Citations (5)
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| US5681934A (en) * | 1986-09-30 | 1997-10-28 | Board Of Regents, The University Of Texas System | 47-kilodalton antigen of Treponema pallidum |
| US7045363B2 (en) * | 1996-05-01 | 2006-05-16 | Fujirebio Inc. | Nucleic acid-bound polypeptide method of producing nucleic acid-bound polypeptide and immunoassay using the polypeptide |
| CN1332750A (en) * | 1999-02-08 | 2002-01-23 | 汪建 | Modified treponema pallidum outer membrane protein, its immunoassay use and immunoassay kit |
| CN1370782A (en) * | 2001-02-26 | 2002-09-25 | 中国人民解放军军事医学科学院基础医学研究所 | Recombinant syphilis spirochete epitope antigen and multiple-epitope chimeric antigen |
| CN1916633A (en) * | 2006-09-08 | 2007-02-21 | 中国人民解放军第三军医大学 | Method and kit for detecting treponema pallidum |
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| 肖洁等.梅毒螺旋体特异抗原TP0684的克隆和表达.《中国生物制品学杂志》.2007,第20卷(第4期),248-251. * |
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