CN102174109A - Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof - Google Patents

Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof Download PDF

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CN102174109A
CN102174109A CN2011100238124A CN201110023812A CN102174109A CN 102174109 A CN102174109 A CN 102174109A CN 2011100238124 A CN2011100238124 A CN 2011100238124A CN 201110023812 A CN201110023812 A CN 201110023812A CN 102174109 A CN102174109 A CN 102174109A
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hiv
genetically engineered
preparation
engineered recombinant
antigen
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杨怀宁
浦昀
徐卉
邵丽筠
李娟�
柴景春
邹红
王振国
谢兵
赵大力
王英超
刘洋
徐宏
寇玲
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JILIN BUREAU OF EMIGRATION AND INGRESSION EXAMINATION AND QUARATINE
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JILIN BUREAU OF EMIGRATION AND INGRESSION EXAMINATION AND QUARATINE
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Abstract

The invention provides acquired immune deficiency syndrome virus and treponema pallidum fusion protein which can be used for preparing GICA (Gold Immunochromatographic Assay) one-step double-combined acquired immune deficiency syndrome and syphilis antibody rapid detection test paper, simultaneously and jointly detecting acquired immune deficiency syndrome and syphilis viruses, simultaneously carrying out combined detection on two diseases and carrying out result reading by naked eyes without special instruments, thereby providing a convenient, rapid, accurate and cheap detecting method for the site rapid detection, such as blood center street compulsory blood donor screening, family self-help detection, disease screening for frontier port exit and entry people, and the like, site epidemiology investigation and other mechanisms needing rapid detection.

Description

Hiv virus and treponema pallidum fusion rotein and preparation method and application
Technical field
The present invention discloses a kind of hiv virus and treponema pallidum fusion rotein and preparation method thereof, GICA one step duplex rapid detection AIDS and syphilis antibody test strip and preparation method also further are provided, simultaneously rapid detection AIDS and syphilis antibody, be applicable to field quick detection and epidemiology survey, belong to the immunology detection field.
Background technology
AIDS and syphilis (hereinafter to be referred as: HIV and TP) be the transmissible disease of serious harm publilc health, two kinds of sick routes of transmission are and spread through sex intercourse, mother-to-baby transmission, blood propagation.In recent years, the sickness rate of acquired immune deficiency syndrome (AIDS) and syphilis all presents ascendant trend, case finding as early as possible, in time seek medical advice, control in time can play a positive role.
Colloidal gold immunochromatographimethod technology (Gold Immuno-Chromatographic Assay, GICA) be a kind of a kind of novel solid phase labelling immunoassay technology that several different methods such as colloidal gold-labeled method, immunoassay technology and chromatographic analysis technology are organically combined, obtained increasingly extensive application in biological, field of medical examination, have easy, fast, do not need advantages such as any plant and instrument, with low cost, shelf time be long, have huge development potentiality in the microorganism detection field.
Existing commercial colloidal gold strip is that AIDS or syphilis antibody are detected respectively now, during operational cost and inconvenient, and the overwhelming majority is an import reagent, adds up to the detection cost higher, does not still have the report of a step duplex rapid detection AIDS and syphilis antibody test strip at present.
Summary of the invention
The object of the invention provides a kind of hiv virus and treponema pallidum fusion rotein and preparation method, is used to prepare the reagent product that detects AIDS and syphilis simultaneously.
The invention also discloses a kind of GICA one step duplex rapid detection AIDS and syphilis antibody test paper, joint-detection AIDS and syphilis virus simultaneously.
Hiv virus provided by the present invention and treponema pallidum fusion rotein, the protein with one of following amino acid residue sequences:
HIV fusion rotein amino acid residue sequence: the SEQ ID № in the sequence table: 1;
TP fusion rotein amino acid residue sequence: the SEQ ID № in the sequence table: 2.
With SEQ ID № in the sequence table: 1 and SEQ ID №: the protein that 2 amino acid residue sequence obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
The preparation of hiv virus provided by the present invention and treponema pallidum fusion rotein comprises expression, evaluation and the purifying of main dominant antigen construction of prokaryotic expression vector, main dominant antigen;
The immune colloid gold detecting probe method of preparation HIV provided by the present invention and TP genetically engineered recombinant antigen comprises that the best pH of Preparation of Colloidal Gold, colloidal gold solution determines, the preparation of immune colloid gold probe and the purifying of definite and the HIV and the TP genetically engineered recombinant antigen of the suitableeest labelled antigen amount.
The GICA one step duplex rapid detection AIDS and the syphilis antibody test paper that are provided are made up of sample preparation pad, gold mark binding substances pad, nitrocellulose filter, suction paper washer and backing plate five parts.The sample preparation pad of the plain membrane prepare of glass fibre closely is connected in the gold mark binding substances pad of the immune colloid gold probe that contains HIV and TP genetically engineered recombinant antigen; Described gold mark pad closely is connected in nitrocellulose filter, nitrocellulose filter is provided with a calibration tape and a contrast band, contain HIV and TP genetically engineered recombinant antigen on the calibration tape, contrast with on contain the anti-antibody that contains HIV and TP genetically engineered recombinant antigen; Described nitrocellulose film close is connected in the suction paper washer of water-absorbing material preparation; Above-mentioned test paper closely is connected in a backing plate; The above-mentioned test paper of backing plate that has is by required size cutting, change plastics chromatography box over to, this chromatography box is provided with the point sample mouth corresponding to the sample pad place, position corresponding to calibration tape and contrast band is provided with observation window, be GICA one step duplex rapid detection AIDS and syphilis antibody test paper, add siccative after sealed packet deposit.
The method for preparing HIV and TP genetically engineered recombinant antigen provided by the present invention may further comprise the steps:
1) main dominant antigen construction of prokaryotic expression vector
Search pertinent literature, select the main dominant antigen epi-position of hiv virus and 2 kinds of pathogenic agent of treponema pallidum, and definite its nucleotide sequence, design specific PCR primer, by pcr amplification, obtain to contain the fragment of purpose antigen gene, the purpose fragment is inserted prokaryotic expression plasmid, and be transformed in the suitable competent cell, whether carry out the sequential analysis reorganization through ABI nucleic acid automatic sequencing analyser correct.
HIV antigen-4 fusion protein gene dna sequence dna: the SEQ ID № in the sequence table: 1;
TP fusion rotein nucleotide sequence: the SEQ ID № in the sequence table: 2.
2) expression and the evaluation of main dominant antigen
Select through the correct positive single bacterium colony of sequencing analysis reorganization, through expression condition is optimized (optimization that mainly is the abduction delivering time), being inoculated in the LB nutrient solution with resistance cultivates, express through the IPTG inducible protein, collect thalline is identified main dominant antigen with SDS-PAGE and Western blot expression.
3) purifying of main dominant antigen
According to INVITROGEN company nickel ion affinity purification operational manual target protein is carried out purifying, 4 ℃ of preservations are standby.
The immune colloid gold detecting probe method of preparation HIV provided by the present invention and TP genetically engineered recombinant antigen may further comprise the steps:
1) Preparation of Colloidal Gold by electron microscope observation, is determined the gold trichloride of preparation 40 nm colloid gold particles and the optimum proportion of trisodium citrate with reference to trisodium citrate reduction method and microwave technology.
The best pH of 2 colloidal gold solutions determines to utilize Gene Runner 3.00 analysis software that the iso-electric point of HIV and TP genetically engineered recombinant antigen is predicted, uses 25mM K 2CO 3The pH of colloidal gold solution is adjusted to respectively near the iso-electric point, adds excessive HIV and the effect of TP genetically engineered recombinant antigen, observing colloid gold color is determined the best pH of colloidal gold solution.
3) definite principle of the suitableeest labelled antigen amount is on the minimum basis of the protein add-on that do not change with the Radioactive colloidal gold color, increase by 30% protein content and be the suitableeest labelled amount, thereby the antigen concentration when preparing in a large number should be 1.3 times of measured value.By observing the colour-change of a certain proportion of Radioactive colloidal gold and HIV, TP recombinant antigen mixed solution, determine the suitableeest labelled antigen amount.
4) preparation of the immune colloid gold probe of HIV and TP genetically engineered recombinant antigen and purifying are got the colloidal gold solution of optimal ph, HIV and TP recombinant antigen are pressed optimal ph and the suitableeest labelled amount, be added dropwise in the colloidal gold solution, and adding stablizer PEG20000, centrifugal back will be precipitated resuspended with the PBS solution that contains PEG20000,4 ℃ of preservations are standby.
The method of the GICA of preparation provided by the present invention one step duplex rapid detection AIDS and syphilis antibody test paper may further comprise the steps:
1) the plain film of glass fibre is carried out pre-treatment with suitable phosphoric acid buffer, obtain the sample preparation pad;
2) with the immune colloid gold probe of HIV and TP genetically engineered recombinant antigen, bag is obtained gold mark binding substances pad by to the plain film of glass fibre, and it is sticked on the sample pad that step 1) obtains;
3) detection line and the control line of preparation nitrocellulose filter stick on 2 with it) on the gold that the obtains mark binding substances pad;
HIV and two kinds of genetically engineered recombinant antigens of TP concentration are respectively 500 μ g/mL, 200 μ g/mL on the described calibration tape; Described contrast is respectively 1.0mg/mL with the antibody concentration that goes up anti-HIV and two kinds of genetically engineered recombinant antigens of TP.
4) paper washer that will absorb water sticks on the end away from described calibration tape of the nitrocellulose filter that step 4) obtains;
5) test paper that step 5) is obtained closely is connected in a backing plate;
6) test paper that has backing plate that step 6) is obtained is by required size cutting, change plastics chromatography box over to, this chromatography box is provided with the point sample mouth corresponding to the sample pad place, position corresponding to calibration tape and contrast band is provided with observation window, be GICA one step duplex rapid detection AIDS and syphilis antibody test paper, add siccative after sealed packet deposit.
The present invention selects the main dominant antigen epi-position of HIV and TP respectively, the genetically engineered recombinant antigen of prokaryotic expression HIV and TP, and carry out colloid gold label; Preparation Radioactive colloidal gold-HIV antigen, TP antigen binding substances pad; With the genetically engineered recombinant antigen of HIV and TP by specking equipment respectively specking be fixed on the qualified nitrocellulose filter of pre-treatment; Adopt dual-antigen sandwich method, detect HIV antibody and TP antibody in the human blood respectively.
The present invention utilizes one step of immune colloidal gold technique preparation duplex rapid detection AIDS and syphilis antibody test paper, its principle is to wrap respectively by nitrocellulose filter with HIV and TP genetically engineered recombinant antigen to form two calibration tapes, in order to catch HIV or the TP antibody in the test sample, the immune colloid gold probe in detecting of used mark then HIV or TP genetically engineered recombinant antigen, if contain HIV or TP antibody in the test sample, through behind the of short duration chromatography corresponding visible chromatographic band can appear at the calibration tape place.In addition, another zone of described nitrocellulose filter is coated with the antibody of anti-HIV and TP genetically engineered recombinant antigen, form two rules contrast band, no matter whether contain HIV or TP antibody in the test sample, mark HIV or the immune colloid gold probe of TP genetically engineered recombinant antigen and the antibodies of anti-HIV or TP genetically engineered recombinant antigen, through two visible chromatographic bands occurring at contrast band place behind the of short duration chromatography,, prove that test paper lost efficacy if this band do not occur.
It is consistent with the ELISA method that immune chromatography test paper of the present invention detects the result, can reach specificity and the susceptibility same with the ELISA method, and more simple and efficient, and detect with low cost.
Positively effect of the present invention is: a kind of hiv virus and treponema pallidum fusion rotein are provided, can prepare GICA one step duplex rapid detection AIDS and syphilis antibody test paper, simultaneously joint-detection AIDS and syphilis virus, can carry out joint-detection simultaneously to two kinds of diseases, with the naked eye carry out interpretation as a result, do not need specific apparatus, be field quick detection such as the blood donation person's examination of Blood Center street corner, the self-service detection of family, frontier port inward and outward personnel's disorder in screening etc. and on-the-spot epidemiology survey, and some other mechanism of rapid detection that needs provides a kind of convenient and swift accurately cheap detection method.
Description of drawings
Fig. 1, Fig. 3 are test strip assembling synoptic diagram of the present invention;
1. sample preparation pad; 2. gold is marked the binding substances pad; 3. nitrocellulose filter; 4. suction paper washer; 5,6. backing plate; T 1, T 2. detection line; C. nature controlling line.
Fig. 2 is a test strip experimental result pattern of the present invention;
A AIDS positive test symbol; B syphilis positive test symbol; The equal positive test symbol of C AIDS and syphilis; The equal negative result of D AIDS and syphilis.
Embodiment
Method used in the following example is ordinary method if no special instructions.
Material: gold trichloride (HAuCl 4), trisodium citrate (NaC 6H 5O 7), polyvinylpyrrolidone (PVP-40) is all available from Sigma company; Bovine serum albumin (BSA) is available from Roch company; Nitrocellulose filter, the plain film of glass fibre, sample pad, absorbent pad are all available from Millipore company.
Bacterial strain plasmid: bacterial strain DH5 α, BL21(DE3), BL21(DE3) plysS is available from NOVAGEN company; Plasmid pRSET-B, pGEM-T are available from PROMEGA company.
Embodiment 1:
HIV and the antigenic preparation of TP genetically engineered recombination fusion protein
(1) acquisition of the main dominant antigen gene of HIV-1/2 and TP
The complete genome sequence of retrieval HIV-1 and 2 C-type virus Cs in the Genebank database, and determine its main dominant antigen gp41, gp120 and gp36, gp125 gene order, synthetic obtains the recombination template, and PCR obtains a large amount of genes.HIV gene template PCR primer is as follows:
Sense?primer：
GGATCCAGTTAAAATCGAACCGCTG
Anti-sense?primer：
AGCTTTCA CAGGTCTTCTTCAGAGATCAGTTTCTGTTCGCACGGCGCGTAGTTA。
TP gene template PCR primer is as follows:
Sense?primer：
GC GGATCCATGTGTTTCTTGCACCACTGT
Anti-sense?primer：
GC GAATTCTCA CAGGTCTTCTTCAGAGATCAGTTTCTGTTCCGCTTCTTTTTCACCACG
Wherein GGATCCFor The BamH IRestriction enzyme site, GAATTCFor The EcoR IRestriction enzyme site, AAGCTTFor The Hind IIIRestriction enzyme site, italic are the myc sequence label.
The reaction system of amplification is as follows: 10 * PCR buffer, 5 μ l, 10mM dNTP mixture 1 μ l, 50mM MgCL 21.5 μ l, template 1 μ l, Platinum Taq enzyme 0.2 μ l, aseptic double-distilled water 39.3 μ l, cumulative volume are 50 μ l.PCR reaction cycle parameter is: 95 ℃ of 5min, (95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 1min) 30 circulations, 72 ℃ of 10min.After PCR reaction was finished, product was with Gel Extraction kit(OMEGA company) carry out the purifying recovery.
(2) acquisition of the main dominant antigen expression vector of HIV-1/2 and TP pRSET-B
Fragment is reclaimed in isogeneity in elder generation and the big fragment gene of pRSET-B is transferred to suitable concn, by fragment and carrier mol ratio is that 3:1 is connected with the big fragment carrier of pRSET-B, and reaction system is as follows: 10 * Ligase Buffer, 1 μ l, the big fragment of pRSET-B (100ng) 1 μ l, insert fragment 1 μ l, T 4Dna ligase 1 μ l, aseptic double-distilled water 6 μ l.Behind the reactant mixing, 16 ℃ of water-bath 3h, 4 ℃ of reaction overnight then.To connect product conversion DH5 α competent cell then and carry out colony screening.After cell monoclonal to be transformed formed, the picking mono-clonal changes over to gathered in the crops thalline after 37 ℃ of 200rpm cultivate 12h in the 5ml LB substratum (Amp 100 μ g/ml), extracts test kit in a small amount with the OMEGA plasmid and extracts plasmid, and carry out BamH I/Hind IIIDouble digestion is identified and determined dna sequence.
(3) the main dominant antigen of HIV-1/2 and TP is expressed and is identified
Main operation is carried out according to the process specifications of the pRSET-B of INVITROGEN company expression vector, wherein distinct portions is described, with HIV recombination fusion protein protein expression is example: the expression strain of-70 ℃ of preservations is transferred by 1:100, and 37 ℃ of 200rpm cultivate 12h in the 5ml LB substratum (Amp 100 μ g/ml), transferring by 1:100 then, 37 ℃ of 200rpm continue to cultivate in the fresh LB substratum of 10ml (this moment microbiotic can add and can not add), work as OD 600During=0.4-0.6, take out the 1ml culture and gather in the crops thalline with 12000rpm ,-20 ℃ frozen, as inducing 0h to express contrast.In remaining culture, adding IPTG(final concentration 1mM then) 37 ℃ of 200rpm cultivate the expression with inducible protein.Take out the 1ml culture every 1h and gather in the crops thalline with 12000rpm, frozen standby in-20 ℃.Coinduction 6h.After waiting to induce end, the thalline of results is carried out the expression of SDS-PAGE or Western blot evaluation target protein.
(4) purifying of HIV-1/2 and the main dominant antigen of TP
Carry out the nickel ion affinity purification.Process specifications (the ProBond of the concrete operations main reference INVITROGEN company of nickel ion affinity purification TMPurification System:For purification of polyhistidine-containing recombinant proteins.Catalog no. K850-01, K851-01, K852-01, K853-01, K854-01, R801-01, R801-15 VersionK 2 September2004; 25-0006) carry out, mainly be described below:
Albumen 6ml(is dissolved in 8M urea, and adjust pH is 8.0) join in the nickel ion affinity column that balance finishes, make albumen and resin-bonded 30min, during turn upside down pillar for several times so that albumen fully combines with affine resin.Take down pillar lower end closed cap (snap-off cap) then, allow liquid rely on gravity independently to flow out; Add 6ml Denaturing Binding Buffer(pH8.0) wash pillar twice; 6mlDenaturing Wash Buffer(pH6.0) washes pillar twice; 6mlDenaturing Wash Buffer(pH5.3) washes pillar twice; 2-4ml Denaturing Elution Buffer(pH4.0) wash-out target protein.In purge process, each component fluids to be collected, follow-up SDS-PAGE analysis purposes albumen place component and purity thereof are prepared against in-20 ℃ of preservations, and carry out Western blot and identify.
Embodiment 2:
The immune colloid gold probe preparation of HIV and TP genetically engineered recombinant antigen:
1) Preparation of Colloidal Gold by electron microscope observation, is determined the gold trichloride of preparation 40 nm colloid gold particles and the optimum proportion of trisodium citrate with reference to trisodium citrate reduction method and microwave technology.Its operation steps is summarized as follows: the gold trichloride of 100 mL, 0.1 g/L is boiled in microwave oven, under the state of high-speed stirring, 10 g/L trisodium citrates of different volumes prepared fresh such as rapid adding 0.70,1.00,1.50,2.00,2.50 mL, after solution colour becomes mazarine, place and continue heating 5 min in the microwave oven, supply institute's dehydration branch, 25mM K after the room temperature cooling 2CO 3Adjust the pH of colloidal gold solution, 4 ℃ of preservations are standby behind the 0.22 μ m membrane filtration.Observe size and uniform particles degree down by electron microscope simultaneously.
2) the best pH of colloidal gold solution determines to utilize Gene Runner 3.00 analysis software that the iso-electric point of HIV and TP genetically engineered recombinant antigen is predicted, the PREDICTION FOR THE ISOELECTRIC POINT value of HIV genetically engineered recombinant antigen is 6.35, and the PREDICTION FOR THE ISOELECTRIC POINT value of TP genetically engineered recombinant antigen is 6.68.Use 25mM K 2CO 3The pH of colloidal gold solution is adjusted to respectively near the iso-electric point, the Radioactive colloidal gold of respectively getting the different pH of 1 mL adds excessive HIV and TP genetically engineered recombinant antigen respectively, add 100 μ L, 100 g/L NaCl after acting on 45 min, observing colloid gold color behind 4 ℃ of static 2 h.The minimum pH that the Radioactive colloidal gold color does not change is the best pH of colloidal gold solution.Measurement result shows, HIV genetically engineered recombinant antigen can keep color constant in the colloidal gold solution of pH7.0, pH7.4, TP genetically engineered recombinant antigen can keep color constant in the colloidal gold solution of pH7.3, pH7.7, therefore judge that HIV genetically engineered recombinant antigen and Radioactive colloidal gold bonded optimal ph are 7.0, TP genetically engineered recombinant antigen and Radioactive colloidal gold bonded optimal ph are 7.3.
3) the definite of the suitableeest labelled antigen amount gets the 5mL colloidal gold solution respectively, uses 25mM K 2CO 3The pH value is adjusted to 7.0 and 7.3 respectively.Simultaneously HIV genetically engineered recombinant antigen pH value of solution value is adjusted to 7.0, TP genetically engineered recombinant antigen pH value of solution value is adjusted to 7.3.The HIV and the TP genetically engineered reorganization solution that will mix up the pH value then respectively dilute 10 gradients with aseptic double-distilled water in 0-50 μ g/mL scope, getting 10,20,30,40,50,60,70,80,90,100 μ L in 96 orifice plates respectively adds in the 1 mL Radioactive colloidal gold, supply volume with ultrapure water, add 100 μ L, 100 g/L NaCl, 4 ℃ of static 2 h after acting on 45 min.Measurement result shows, HIV genetically engineered recombinant antigen all can make when 〉=25 μ g/mL and keep color constant in the colloidal gold solution, TP genetically engineered recombinant antigen all can make when 〉=15 μ g/mL and keep color constant in the colloidal gold solution, therefore judge that HIV genetically engineered recombinant antigen and Radioactive colloidal gold bonded Cmin are 25 μ g/mL, TP genetically engineered recombinant antigen and Radioactive colloidal gold bonded Cmin are 15 μ g/mL.When a large amount of preparation, principle is on the minimum basis of the protein add-on that do not change with the Radioactive colloidal gold color, increase by 30% protein content and be the suitableeest labelled amount, thereby the antigen concentration when preparing in a large number should be 1.3 times of measured value.
4) the immune colloid gold probe of HIV and TP genetically engineered recombinant antigen preparation and the purifying colloidal gold solution of getting the 30mL optimal ph places the clean beaker of silication, press optimal ph and the suitableeest labelled amount, under magnetic agitation, 975 μ g HIV genetically engineered recombinant antigens and 585 μ g TP genetically engineered recombinant antigen solution slowly are added dropwise in the colloidal gold solution respectively.After being added dropwise to complete, continuing middling speed and stir 20min, and add stablizer PEG20000, making its final concentration is 0.2%, prevents that albumen and Radioactive colloidal gold polymerization from precipitating, and continues to stir 30 min.After question response is complete, colloidal gold solution is placed new 50ml centrifuge tube 1000g, 4 ℃ of low-speed centrifugal 20min remove the precipitation of condensing in the reaction process; Careful sucking-off supernatant changes 14000g in the new 50ml centrifuge tube over to, 4 ℃ of centrifugal 40min(GS-15R type high speed freezing centrifuges, BECKMAN).Careful sucking-off supernatant, throw out (contains 0.02%NaN with the PBS solution that 3mL contains 0.2% PEG20000 3) will precipitate resuspendedly, 4 ℃ of preservations are standby.
Embodiment 3:
Preparation GICA one step duplex rapid detection AIDS and syphilis antibody test paper:
1) the plain film of glass fibre (300 * 20 mm) is soaked in 0.01 mol/L pH, 7.4 phosphoric acid buffers (20 g BSA, 25 g trehaloses, 3g PVP-40,0.2 g NaN 3,8 g NaCl, 0.2 g KCl, 2.9 g Na 2HPO 412H 2O, 0.2 g KH 2PO 4, be settled to 1000 mL with ultrapure water) in behind 30 min, in 37 ℃ of oven dry, obtain the sample preparation pad, Vacuum Package, put 4 ℃ standby.
2) HIV and two kinds of genetically engineered recombinant antigens of TP are carried out colloid gold label, Bio-Dot point film instrument is sprayed onto on the plain film of glass fibre (300 mm * 6 mm) with the amount of every centimetre 50 μ L, obtains gold mark binding substances pad, and it is standby that freeze drier is drained the back; It is sticked on the sample pad that step 1) obtains;
3) with the automatic micro-specking equipment of antibody utilization of HIV and two kinds of genetically engineered recombinant antigens of TP and anti-HIV and two kinds of genetically engineered recombinant antigens of TP, be fixed on the qualified nitrocellulose filter of pre-treatment, respectively as detection line and control line, two line midfeather 0.5CM; It is sticked on 2) on the gold that the obtains mark binding substances pad;
HIV and two kinds of genetically engineered recombinant antigens of TP concentration are respectively 500 μ g/mL, 200 μ g/mL on the described calibration tape; Described contrast is respectively 1.0mg/mL with the antibody concentration that goes up anti-HIV and two kinds of genetically engineered recombinant antigens of TP.
4) paper washer that will absorb water sticks on the end away from described calibration tape of the nitrocellulose filter that step 3) obtains;
5) above-mentioned test paper closely is connected in a backing plate, is combined into test strip;
6) the above-mentioned test paper of backing plate that has is by required size cutting, change plastics chromatography box over to, this chromatography box is provided with the point sample mouth corresponding to the sample pad place, position corresponding to calibration tape and contrast band is provided with observation window, be GICA one step duplex rapid detection AIDS and syphilis antibody test paper, add siccative after sealed packet deposit.See Fig. 1.
Test example 1:
GICA one step duplex rapid detection AIDS and syphilis antibody test paper effect assessment
1) collect each 10 parts of acquired immune deficiency syndrome (AIDS) positive serum and syphilis positive serums, 50 parts of negative control seras, standby as sample detection liquid;
2) get GICA one step duplex rapid detection AIDS and the syphilis antibody test paper that example 3 prepares, in sample well, add 3 of the sample detection liquid (about 100 μ L) in the step 1) respectively, begin observations behind the 5min, observe 15min.The result shows that 10 parts of positive serums all a red precipitate line occurs at calibration tape and contrast band place, and detected result is positive; 50 parts of negative control seras all do not develop the color at the calibration tape place, and a red precipitate line appears in the place at the contrast band, and detected result is negative.See Fig. 2, Fig. 3.
SEQ?ID?№：1
VKIEPLGVAPTKAKRRVVERGGGGRKSIRIGPGQAFYATGDIIGDIRQAHCGGGGRGPDRPEGIEEEGGEQDRDRSIRLVNGGGGNYTDIIYSLIEESQN QQEKNEQELLALDKWASLWNWFGGGGWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTNVPWNSSWGGGGAIEKYLKDQAQLNSWGCAFRQVCHT T VPWVNESLTPDWNNMTWQEWEKQVRYLEANISQSLEQAQIQQEKNMYELQKLNSWDVFGNGGGGSGLVFHSQPINKRPRQAWCWFKGNWTGGGGCQFN
SEQ?ID?№：2
CVSCTTVCPHAGKAKAEKVECALKGGIFRGTLPAADCPGIDTTVTFNADGTAQKVELALEKKSAPSPLTYRGTWMVREDGIVELSLVSSEQSKAPHEKELYELIDSNSVRYMGAPGAGKPSKEMAPFYVLKKTKKGGGGGSASGAKEEAEKKAAEQRALLGGGGVLSKQETEDSRGRKKWEYETDPSVGGGGVYDYQHKEGRFKSQDADYHRVGGGGARRGEKEA

Claims (5)

1. hiv virus and treponema pallidum fusion rotein, the protein with one of following amino acid residue sequences: 1) the SEQ ID № in the sequence table: 1 and SEQ ID №: 2;
2) with SEQ ID № in the sequence table: 1 and SEQ ID №: the protein that 2 amino acid residue sequence obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. the preparation method of the described fusion rotein of claim 1 comprises expression, evaluation and the purifying of main dominant antigen construction of prokaryotic expression vector, main dominant antigen.
3. the immune colloid gold probe preparation method of HIV and TP genetically engineered recombinant antigen comprises that the best pH of Preparation of Colloidal Gold, colloidal gold solution determines, the preparation of immune colloid gold probe and the purifying of definite and the HIV and the TP genetically engineered recombinant antigen of the suitableeest labelled antigen amount.
4. GICA one goes on foot duplex rapid detection AIDS and syphilis antibody test paper, comprise sample preparation pad, gold mark binding substances pad, nitrocellulose filter, suction paper washer and backing plate, it is characterized in that: gold mark binding substances pad contains the immune colloid gold probe of HIV and TP genetically engineered recombinant antigen; Contain HIV and TP genetically engineered recombinant antigen on the calibration tape; Contrast with on contain the anti-antibody that contains HIV and TP genetically engineered recombinant antigen.
5. the preparation method of the described test paper of claim 4 may further comprise the steps:
1) the plain film of glass fibre is carried out pre-treatment with suitable phosphoric acid buffer, obtain the sample preparation pad;
2) the immune colloid gold probe of HIV that step 3) is obtained and TP genetically engineered recombinant antigen, bag are obtained gold mark binding substances pad by to the plain film of glass fibre, and it is sticked on the sample pad that step 1) obtains;
3) detection line and the control line of preparation nitrocellulose filter stick on 2 with it) on the gold that the obtains mark binding substances pad;
HIV and two kinds of genetically engineered recombinant antigens of TP concentration are respectively 500 μ g/mL, 200 μ g/mL on the described calibration tape; Described contrast is respectively 1.0mg/mL with the antibody concentration that goes up anti-HIV and two kinds of genetically engineered recombinant antigens of TP;
4) paper washer that will absorb water sticks on the end away from described calibration tape of the nitrocellulose filter that step 3) obtains;
5) test paper that step 4) is obtained closely is connected in a backing plate;
6) test paper that has backing plate that step 5) is obtained is by required size cutting, change plastics chromatography box over to, this chromatography box is provided with the point sample mouth corresponding to the sample pad place, position corresponding to calibration tape and contrast band is provided with observation window, be GICA one step duplex rapid detection AIDS and syphilis antibody test paper, add siccative after sealed packet deposit.
CN2011100238124A 2011-01-21 2011-01-21 Acquired immune deficiency syndrome virus and treponema pallidum fusion protein as well as preparation method and application thereof Pending CN102174109A (en)

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CN108196050A (en) * 2018-02-02 2018-06-22 江苏维尔生物科技有限公司 For detecting the colloid gold test paper and its preparation and application of human immune defect virus antibody and syphilis helicoid antibody in saliva

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CN103163298B (en) * 2011-12-19 2015-04-08 天津中新科炬生物制药有限公司 Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof
CN103163298A (en) * 2011-12-19 2013-06-19 天津中新科炬生物制药有限公司 Rapid detective reagent strip of treponema pallidum immunoglobulin m (IgM) antibody and preparation method thereof
CN103172707A (en) * 2011-12-23 2013-06-26 上海市公共卫生临床中心 Diagnosis marker Talin 1 segment for human immunodeficiency virus infection and application thereof
CN102980997A (en) * 2012-06-23 2013-03-20 北京新兴四寰生物技术有限公司 EB virus capsid antigen IgM antibody colloidal gold method detection reagent and preparation method thereof
CN102980997B (en) * 2012-06-23 2014-11-05 北京新兴四寰生物技术有限公司 EB virus capsid antigen IgM antibody colloidal gold method detection reagent and preparation method thereof
CN103134935B (en) * 2013-02-28 2015-09-30 成都创宜生物科技有限公司 A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper
CN103134935A (en) * 2013-02-28 2013-06-05 成都创宜生物科技有限公司 Making method of immunochromatographic test paper for detecting premature rupture of fetal membranes
CN104749359A (en) * 2014-05-14 2015-07-01 陈岩松 Immunochromatographic test method and test paper for multi-item joint tests
CN104764877A (en) * 2014-05-14 2015-07-08 陈岩松 Immunity chromatography test method and test paper
CN104502586A (en) * 2014-06-11 2015-04-08 陈岩松 Immunochromatography detection method and test paper
CN104502586B (en) * 2014-06-11 2017-08-29 陈岩松 A kind of immunochromatography detection method and test paper
CN108181468A (en) * 2018-02-02 2018-06-19 江苏维尔生物科技有限公司 For detecting colloid gold test paper of syphilis helicoid antibody and preparation method thereof and application method in saliva
CN108196050A (en) * 2018-02-02 2018-06-22 江苏维尔生物科技有限公司 For detecting the colloid gold test paper and its preparation and application of human immune defect virus antibody and syphilis helicoid antibody in saliva

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Application publication date: 20110907