CN103995121A - Preparation method for colloidal-gold test paper based on single-chain antibody - Google Patents

Preparation method for colloidal-gold test paper based on single-chain antibody Download PDF

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CN103995121A
CN103995121A CN201410059162.2A CN201410059162A CN103995121A CN 103995121 A CN103995121 A CN 103995121A CN 201410059162 A CN201410059162 A CN 201410059162A CN 103995121 A CN103995121 A CN 103995121A
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chain antibody
gold
small peptide
label
gene
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CN103995121B (en
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胡学军
曹际娟
赵昕
杨春光
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Dalian Jinrong Bande Biotechnology Co.,Ltd.
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Dalian University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

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Abstract

The invention discloses a preparation method for colloidal-gold test paper based on a single-chain antibody, which belongs to the field of biotechnology. The method extensively applies single-chain antibodies in preparation of the colloidal-gold test paper. The method comprises a step of preparing a single-chain antibody chained through neutral amino acid short peptide and having an electropositive short peptide label in Escherichia coli, wherein the single-chain antibody can stably bind with a colloidal gold particle having negative spots, so a stable immunocolloidal gold particle is prepared. The invention has the following beneficial effects: the single-chain antibody can be easily, rapidly and highly efficiently applied to preparation of the colloidal-gold test paper, and a basis is laid for wide application of the single-chain antibody in immunocolloidal gold technology.

Description

The preparation method of the gold test strip based on single-chain antibody
Technical field
The invention belongs to biological technical field, relate to a kind of method that single-chain antibody merging by neutral amino acid small peptide with positive electricity amino acid short peptide label is prepared gold test strip, single-chain antibody can be widely used in to gold test strip preparation.
Background technology
Single-chain antibody is one of minimum recombinant antibody fragment, only has 1/8 of whole antibody.Single-chain antibody has only retained whole antibody and antigen-binding site, is conventionally connected with 15 amino acid short peptide chains, keeps and the very close structure in whole antibody antigen binding domain, keeps and antigen binding capacity.Single-chain antibody is compared with whole antibody, and application single-chain antibody can improve the quantity of unit area sessile antibody, significantly improve immune detection susceptibility.
The fixing instability of single-chain antibody is its major obstacle that is applied to field of immunodetection.Because its molecule is little, be difficult to be stably fixed at solid surface, or fixed rear portion divides antibody to lose activity.
At present, the method of fixing single-chain antibody is mainly to introduce the fixing single-chain antibody of specific label by gene engineering research, as introduce histidine, the soft chain label of arginine at single-chain antibody C end or soft interchain or merge can and the small peptide label of material surface specific molecular combination, if be combined with the streptomysin of biotin specific bond protein fragments, can be combined with streptomysin protein bound small peptide, can with polystyrene specific bond peptide or the labels such as albumen of being combined with cellulose.But in the time of the fixing single-chain antibody of these labels of application, single-chain antibody often loses or part and antigen binding capacity, greatly reduces immune detection susceptibility.
Collaurum is the gold grain that gold chloride can be grouped to a certain size under reductive agent effect, forms electronegative hydrophobic sol solution.With after colloid gold label monoclonal antibody, can be used for qualitative or Quasi-quantitative measurement.Be usually used in chromatography and carry out fast detecting.The monoclonal antibody of anti-certain antigen of gold mark is because chromatography effect compound moves forward along nitrocellulose membrane, and in the time running into coated specific antigen, compound is enriched on coated line, forms red precipitation line.If the monoclonal antibody of golden mark is prior to specific antigen combination to be detected, compound just can not with the antibody of the local Concentration of Gold mark of envelope antigen, just can't see red precipitate line.On coated film, also have a nature controlling line contrast, thus in the time having two red lines, be judged to feminine gender, positive in the time only having a red line of nature controlling line.
Preparing at present colloid gold test paper is both at home and abroad all to use colloid gold label whole antibody, there is no the report that utilizes single-chain antibody to prepare colloid gold test paper.
Summary of the invention
The invention provides a kind of gold test strip preparation method based on single-chain antibody, is that a kind of positively charged amino acid short peptide label merges by neutral amino acid small peptide the method that single-chain antibody is prepared colloid gold test paper, is applied to gold test strip development.This set of tags becomes: by the neutral asparagine residue of poly, (8 to 20 asparagine residues, are expressed as N 8-20) with positively charged arginine (formed by 5-12 arginine, be expressed as R5-12), or lysine (formed by 5-12 lysine, be expressed as K5-12), or arginine and lysine (6-12 amino acid, is expressed as (RK) 3-6) the small peptide label of positively charged of the common composition of polyamino acid small peptide of composition.
The preparation method of the gold test strip based on single-chain antibody, comprises the following steps:
1. the positively charged label single-chain antibody gene expression vector establishment connecting by neutral amino acid small peptide: (1) introduces coding-belt positive electricity small peptide label at 5 of single-chain antibody gene ' end by genetic recombination, fusion forms successively by the small peptide of coding-belt positive electricity, neutral small peptide and single-chain antibody gene.Or introduce coding-belt positive electricity small peptide label at 3 of single-chain antibody gene ' end by genetic recombination, fusion composition is made up of the small peptide label of coding single-chain antibody gene, neutral small peptide and positively charged successively.Positively charged small peptide label composition: 5-12 arginine residues or 5-12 lysine residue or 6-12 the two kilnitamin residue small peptide label of composition jointly, represent with R5-12, K5-12, (RK) 3-6 respectively, R arginine residues, K represents lysine residue; Neutral small peptide is made up of 8 to 20 asparagine residues, N8-20, and N represents asparagine residue.At the other end of gene, introduce 6 histidine residues base sequences of coding by genetic recombination, (2), by above Fusion gene construction to the expression vector of all chambeies of Escherichia coli matter, the single-chain antibody of fusion will be expressed in all chambeies of Escherichia coli matter.
2. the single-chain antibody gene matter week chamber expression vector of structure is transformed into colibacillus engineering strain, the positive transformant of identifying is carried out to self-induction expression, abduction delivering 3-6 hour, adds and the corresponding microbiotic of resistant gene that expression vector is with in nutrient culture media.
3. collect thalline, extract matter week chamber soluble protein, use affinity chromatography method, the single-chain antibody of positively charged label is carried in separation and purification.
4. preparation gold mark single-chain antibody.0.5 milliliter of (300 nanograms/milliliter) single-chain antibody is dropwise added to (collaurum of 40 nanometers is purchased an outstanding Bioisystech Co., Ltd in Shanghai) in the colloidal gold solution of 8 milliliters, mixes gently.Mixed liquor was room temperature reaction 1 hour.Then, seal 30 minutes with 10% bovine serum albumin(BSA) (BSA).Mixed liquor, at 4 degrees Celsius, leaves the heart 30 minutes with per minute 14000, discards supernatant, and gold grain precipitation is hanged again with the BSA of the Tris buffering of 20 mM pH8.0 of 450 microlitres 1%, adds 0.1 the sodium nitride that falls.Before using, golden target single-chain antibody is stored in 4 degrees Celsius of refrigerators.
5. the preparation of immunity test strip.The monoclonal antibody of the determined antigen of 1 microlitre and anti-6 histidines is fixed on to detection zone and the Quality Control district on nitrocellulose filter in room temperature respectively.The colloid gold label single-chain antibody of purifying is done after dilution in various degree, and evenly equivalent is dipped in onesize glass fibre element film and makes gold mark pad.In the situation that other conditions are constant, according to reaction result, determine the dilutability of the suitableeest colloid gold label single-chain antibody that reaches the requirement of test strips susceptibility, or be called working concentration.Getting the golden labeling antibody of suitable working concentration, to be added to gold mark pad with 10 microlitres/bar upper, then vacuum freeze drying, and sealing is preserved.
The invention has the beneficial effects as follows, single-chain antibody can be applied to gold test strip preparation simply, fast and efficiently, lays the foundation for single-chain antibody is widely used in immune-gold labeled technology.
Brief description of the drawings
Fig. 1 is 2H12-R4K2 gene structure figure of the present invention;
Fig. 2 is the carrier structure figure of pIG6 table of the present invention;
Fig. 3 is 2H12-R4K2 single chain antibody protein matter separation and purification figure of the present invention,
In figure: 1. the 2H12-R4K2 of separation and purification, M. standard protein;
Fig. 4 is 2H12-R5K1 gene structure figure of the present invention;
Fig. 5 is 2H12-R5K1 single chain antibody protein matter separation and purification figure of the present invention,
In figure: 1. the 2H12-R4K2 single-chain antibody of separation and purification, M. standard protein;
Fig. 6 is R4K2-2H12 gene structure figure of the present invention;
Fig. 7 is R4K2-2H12 single chain antibody protein matter separation and purification figure of the present invention,
In figure: 1. the R4K2-2H12 single-chain antibody of separation and purification, M. standard protein.
Embodiment
Below in conjunction with the drawings and the specific embodiments, the present invention is further illustrated, but do not affect protection scope of the present invention.
embodiment mono-
1. to adopt anti-domoic acid single-chain antibody be pattern gene (EMBL logins number AJ867814 and AJ867813) to the present embodiment, this gene is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.Introduce respectively 6 histidine residues of coding at 5 ' end of gene and 3 ' end by genetic recombination and by 10 poly asparagine residues and positively charged 4 arginine residues and 2 small peptide label (arginine-arginine-lysine-arginine-lysine-arginine that lysine residue forms, be expressed as R-R-K-R-K-R) base sequence, fusion sequence is shown in gene order 1, and fusion structure is shown in Fig. 1.Gene is building up to pIG6 by genetic recombination, carrier sequence is shown in gene order table [002], carrier structure is shown in Fig. 2, and this carrier is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.This carrier has coding ompA signal peptide sequence in genes of interest upstream, can guide recombinant protein to enter in Escherichia coli matter week chamber.
2. by synthetic the anti-domoic acid single-chain antibody 2H12 fusion expression cassette sequence shown in sequence table [001] rear (Nanjing Genscript Biotechnology Co., Ltd.), use ecor V and hinit is upper that d III is building up to coli expression carrier pIG6, obtains recombinant vector pIG6-2H12-R4K2.
3. the expression vector electric shock building is transformed to JM109 coli strain (purchasing in precious bioengineering (Dalian) company limited), then transformant is inoculated in 10 milliliters of LB nutrient culture media that contain ampicillin (100 every milliliter of microgram) to incubated overnight.Next day, is inoculated in 500 milliliters of 2 liters of triangular flasks that contain ampicillin (100 every milliliter of microgram) LB nutrient culture media with 1:100, and 200rpm, 37 DEG C of cultivations, until OD 600reach 0.6.Then, under 25 DEG C, 200rpm condition, add 1 mM isopropylthiogalactoside (IPTG) carry out abduction delivering 3 hours, in this process, Escherichia coli matter week chamber express single-chain antibody.The formula of LB nutrient culture media is as follows: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L.Prepare soluble protein by conventional method, with the single-chain antibody of ni-sepharose purification His-Tag, the single-chain antibody 2H12-RK of purifying as shown in Figure 3.
4. single-chain antibody 2H12-R4K2 step 3 being obtained is for the preparation of golden target single-chain antibody.0.5 milliliter of (300 nanograms/milliliter) single-chain antibody is dropwise added to (collaurum of 40 nanometers is purchased an outstanding Bioisystech Co., Ltd in Shanghai) in the colloidal gold solution of 8 milliliters, mixes gently.Mixed liquor was room temperature reaction 1 hour.Then, seal 30 minutes with 10% BSA.Mixed liquor leaves the heart 30 minutes at 4 degrees Celsius with per minute 14000, discards supernatant, and 1% the BSA that gold grain precipitation cushions with the Tris of 450 microlitre 20 mM pH8.0 hangs again, adds 0.1% the sodium nitride that falls.Before using, golden target single-chain antibody is stored in 4 degrees Celsius of refrigerators.
5. the preparation of immunity test strip.The monoclonal antibody (purchasing the company in Sigma-Aldrich) of the antigen 1 microlitre of the domoic acid of preparing by conventional method and BSA coupling (concentration is 0.8 mg/ml) and anti-6 histidines is fixed on to detection zone and the Quality Control district on nitrocellulose filter in room temperature respectively.Suitably, after dilution, evenly equivalent is dipped in onesize glass fibre element film and makes gold mark pad.The golden labeling antibody of working concentration is added to gold mark pad with 10 microlitres/bar upper, then vacuum freeze drying, sealing is preserved.
6. 1 milligram of standard domoic acid (purchasing the company in Sigma-Aldrich) is dissolved in 50 microlitre dimethyl sulfoxide (DMSO)s, then, be diluted to the domoic acid of 20 nanograms/milliliter, 10 nanograms/milliliter, 1 nanograms/milliliter, 0.5 nanograms/milliliter, 0.1 nanograms/milliliter with the PBS of pH7.4.The standard items of getting the above serial dilution of 90 μ l drip the sample application zone in test strips, observe and record experimental result in 5-10 minute, and result is as shown in table 1.
embodiment bis-
1. to adopt anti-domoic acid single-chain antibody be pattern gene (EMBL logins number AJ867814 and AJ867813) to the present embodiment, this gene is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.Introduce respectively 6 histidine residues of coding at 5 ' end of gene and 3 ' end by genetic recombination and by 8 poly asparagine residues and positively charged 5 arginine residues and 1 small peptide label (arginine-arginine-lysine-arginine-lysine-arginine that lysine residue forms, be expressed as R-R-K-R-R-R) base sequence, fusion sequence is shown in gene order table [003], and fusion structure is shown in Fig. 4.Gene is building up to pIG6 by genetic recombination, carrier sequence is shown in gene order table [002], carrier structure is shown in Fig. 2, and this carrier is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.This carrier has coding ompA signal peptide sequence in genes of interest upstream, can guide recombinant protein to enter in Escherichia coli matter week chamber.
2. by synthetic the anti-domoic acid single-chain antibody 2H12 fusion expression cassette sequence shown in sequence table [003] rear (Nanjing Genscript Biotechnology Co., Ltd.), use ecor V and hinit is upper that d III is building up to coli expression carrier pIG6, obtains recombinant vector pIG6-2H12-R5K1.
3. the expression vector electric shock building is transformed to JM109 coli strain (purchasing in precious bioengineering (Dalian) company limited), then transformant is inoculated in 10 milliliters of LB nutrient culture media that contain ampicillin (100 every milliliter of microgram) to incubated overnight.Next day, is inoculated in 500 milliliters of 2 liters of triangular flasks that contain ampicillin (100 every milliliter of microgram) LB nutrient culture media with 1:100, and 200 rpm, 37 DEG C of cultivations, until OD 600reach 0.6.Then, under 25 DEG C, 200 rpm conditions, add 1 mM isopropylthiogalactoside (IPTG) carry out abduction delivering 3 hours, in this process, Escherichia coli matter week chamber express single-chain antibody.The formula of LB nutrient culture media is as follows: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L.Prepare soluble protein by conventional method, with the single-chain antibody of ni-sepharose purification His-Tag, the single-chain antibody 2H12-R5K1 of purifying as shown in Figure 5.
4. single-chain antibody 2H12-R5K1 step 3 being obtained is for the preparation of golden target single-chain antibody.0.5 milliliter of (300 nanograms/milliliter) single-chain antibody is dropwise added to (collaurum of 40 nanometers is purchased an outstanding Bioisystech Co., Ltd in Shanghai) in the colloidal gold solution of 8 milliliters, mixes gently.Mixed liquor was room temperature reaction 1 hour.Then, seal 30 minutes with 10% BSA.Mixed liquor leaves the heart 30 minutes at 4 degrees Celsius with per minute 14000, discards supernatant, and 1% the BSA that gold grain precipitation cushions with the Tris of 450 microlitre 20 mM pH8.0 hangs again, adds 0.1% the sodium nitride that falls.Before using, golden target single-chain antibody is stored in 4 degrees Celsius of refrigerators.
5. the preparation of immunity test strip.The monoclonal antibody (purchasing the company in Sigma-Aldrich) of the antigen 1 microlitre of the domoic acid of preparing by conventional method and BSA coupling (concentration is 0.8 mg/ml) and anti-6 histidines is fixed on to detection zone and the Quality Control district on nitrocellulose filter in room temperature respectively.Suitably, after dilution, evenly equivalent is dipped in onesize glass fibre element film and makes gold mark pad.The golden labeling antibody of working concentration is added to gold mark pad with 10 microlitres/bar upper, then vacuum freeze drying, sealing is preserved.
6. 1 milligram of standard domoic acid (purchasing the company in Sigma-Aldrich) is dissolved in 50 microlitre dimethyl sulfoxide (DMSO)s, then, be diluted to the domoic acid of 20 nanograms/milliliter, 10 nanograms/milliliter, 1 nanograms/milliliter, 0.5 nanograms/milliliter, 0.1 nanograms/milliliter with the PBS of pH7.4.The standard items of getting the above serial dilution of 90 μ l drip the sample application zone in test strips, observe and record experimental result in 5-10 minute, and result is as shown in table 2.
embodiment tri-
1. to adopt anti-domoic acid single-chain antibody be pattern gene (EMBL logins number AJ867814 and AJ867813) to the present embodiment, this gene is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.5 ' end and 3 ' end at gene are introduced respectively coding positively charged 4 arginine residues and 2 lysine residue (arginine-arginine-lysine-arginine-lysine-arginine by genetic recombination, be expressed as R-R-K-R-K-R) and 10 small peptide label and 6 histidine residues base sequences that poly asparagine residue forms, fusion sequence is shown in gene order table [004], and fusion structure is shown in Fig. 6.Gene is building up to pIG6 by genetic recombination, carrier sequence is shown in gene order table [002], carrier structure is shown in Fig. 2, and this carrier is at Journal of Biotecnology (ISSN:0168-1656), 2005 120 (1): on p. 38-45, have a detailed description.This carrier has coding ompA signal peptide sequence in genes of interest upstream, can guide recombinant protein to enter in Escherichia coli matter week chamber.
2. by synthetic the anti-domoic acid single-chain antibody 2H12 fusion expression cassette sequence shown in sequence table [004] rear (Nanjing Genscript Biotechnology Co., Ltd.), use ecor V and hinit is upper that d III is building up to coli expression carrier pIG6, obtains recombinant vector pIG6-R4K2-2H12.
3. the expression vector electric shock building is transformed to JM109 coli strain (purchasing in precious bioengineering (Dalian) company limited), then transformant is inoculated in 10 milliliters of LB nutrient culture media that contain ampicillin (100 every milliliter of microgram) to incubated overnight.Next day, is inoculated in 500 milliliters of 2 liters of triangular flasks that contain ampicillin (100 every milliliter of microgram) LB nutrient culture media with 1:100, and 200rpm, 37 DEG C of cultivations, until OD 600reach 0.6.Then, under 25 DEG C, 200rpm condition, add 1 mM isopropylthiogalactoside (IPTG) carry out abduction delivering 3 hours, in this process, Escherichia coli matter week chamber express single-chain antibody.The formula of LB nutrient culture media is as follows: tryptone (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium chloride (NaCl) 10g/L.Prepare soluble protein by conventional method, with the single-chain antibody of ni-sepharose purification His-Tag, the single-chain antibody R4K2-2H12 of purifying as shown in Figure 7.
4. single-chain antibody R4K2-2H12 step 3 being obtained is for the preparation of golden target single-chain antibody.0.5 milliliter of (300 nanograms/milliliter) single-chain antibody is dropwise added to (collaurum of 40 nanometers is purchased an outstanding Bioisystech Co., Ltd in Shanghai) in the colloidal gold solution of 8 milliliters, mixes gently.Mixed liquor was room temperature reaction 1 hour.Then, seal 30 minutes with 10% BSA.Mixed liquor leaves the heart 30 minutes at 4 degrees Celsius with per minute 14000, discards supernatant, and 1% the BSA that gold grain precipitation cushions with the Tris of 450 microlitre 20 mM pH8.0 hangs again, adds 0.1% the sodium nitride that falls.Before using, golden target single-chain antibody is stored in 4 degrees Celsius of refrigerators.
5. the preparation of immunity test strip.The monoclonal antibody (purchasing the company in Sigma-Aldrich) of the antigen 1 microlitre of the domoic acid of preparing by conventional method and BSA coupling (concentration is 0.8 mg/ml) and anti-6 histidines is fixed on to detection zone and the Quality Control district on nitrocellulose filter in room temperature respectively.Suitably, after dilution, evenly equivalent is dipped in onesize glass fibre element film and makes gold mark pad.The golden labeling antibody of working concentration is added to gold mark pad with 10 microlitres/bar upper, then vacuum freeze drying, sealing is preserved.
6. 1 milligram of standard domoic acid (purchasing the company in Sigma-Aldrich) is dissolved in 50 microlitre dimethyl sulfoxide (DMSO)s, then, be diluted to the domoic acid of 20 nanograms/milliliter, 10 nanograms/milliliter, 1 nanograms/milliliter, 0.5 nanograms/milliliter, 0.1 nanograms/milliliter with the PBS of pH7.4.The standard items of getting the above serial dilution of 90 μ l drip the sample application zone in test strips, observe and record experimental result in 5-10 minute, and result is as shown in table 3.
Embodiment of the present invention is not limit this, according to foregoing of the present invention, according to ordinary skill knowledge and the universal method of this area, is not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, and the present invention can also have other embodiment.As expressed with all chambeies of other Escherichia coli matter expression vector etc. the single-chain antibody of the type.Therefore, the present invention can also make amendment, replacement or the change of other various ways, within all dropping on rights protection scope of the present invention.
The above; it is only preferably embodiment of the present invention; but protection scope of the present invention is not limited to this; any be familiar with those skilled in the art the present invention disclose technical scope in; be equal to replacement or changed according to technical scheme of the present invention and inventive concept thereof, within all should being encompassed in protection scope of the present invention.
Sequence table
<110> University Of Dalian
The preparation method of the gold test strip of <120> based on single-chain antibody
<160> 4
<170> PatentIn version 3.3
<210> 1
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cctatttgca gatcaacaac ctcaaaaatg aggacacggc tacatatttc tgtgtaagag 360
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gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa 1080
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 1140
ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 1200
aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 1260
gataacaatt tcacacagga aacagctatg accatgatta cgaatttcta gataacgagg 1320
gcaaaaaatg aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt 1380
agcgcaggcc gactacaaag atatcgaaca gaaactgatc tctgaagaag acctgaacca 1440
ccaccaccac caccactgat aagcttgacc tgtgaagtga aaaatggcgc acattgtgcg 1500
acattttttt tgtctgccgt ttaccgctac tgcgtcacgg atccccacgc gccctgtagc 1560
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 1620
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 1680
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 1740
ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 1800
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 1860
actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg 1920
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 1980
aaaatattaa cgcttacaat ttcaggtggc acttttcggg gaaatgtgcg cggaacccct 2040
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2100
taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2160
cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 2220
aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 2280
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 2340
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 2400
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 2460
catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 2520
aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 2580
ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 2640
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 2700
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 2760
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 2820
gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 2880
gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 2940
gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3000
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3060
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3120
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3180
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3240
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3300
ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3360
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3420
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3480
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3540
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3600
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3660
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3720
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 3780
ttcctggcct tttgctggcc ttttgctcac atg 3813
<210> 3
<211> 854
<212> DNA
<213> is manually synthetic
<400> 3
ggccgatatc tctggttctc accaccacca ccaccacggt tctggttctt ctggttcctc 60
cggcatccag atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt 120
caagatctcc tgcaaggctt ctggatatac cttcacaaac tatggaatga actgggtgaa 180
acaggctcca ggaaagggtt taaagtggat gggctggata aacaccaaca ctggagagcc 240
aacatatgct gaagagttca agggacggtt tgccttctct ttggaaacct ctgccaacac 300
tgcctatttg cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgtaag 360
agggaggaat gggtttgctt actggggcca agggactctg gtcactgtcg gtggtggtgg 420
ttctggcggc ggcggctccg gtggtggtgg ttctagtatt gtgatgaccc agactcccaa 480
attcctgctt gtatcagcag gagacagggt taccataacc tgcaaggcca gtcagagtgt 540
gagtaatgat gtagcttggt accaacagaa gccagggcag tctcctaaac tgctgatata 600
ctatgcatac aatcgctata agggagtccc tgaccgcttc actggcagtg gatatgggac 660
ggatttcact ttcaccatca gcactgtgca ggctgaagac ctggcagttt atttctgtca 720
gcaggattat agctctccgt acacgttcgg aggggggacc aagttggaaa taaaacggag 780
ctcgaacaac aacaacaata acaataacaa caacagtggt tctcgccgca agcgccgccg 840
ctgaaagctt cgcg 854
<210> 4
<211> 845
<212> DNA
<213> is manually synthetic
<400> 4
ggccgatatc tctcgccgca agcgccgccg cagctcgaac aacaacaaca ataacaataa 60
caacaacagt ggttctcaga tccagttggt gcagtctgga cctgagctga agaagcctgg 120
agagacagtc aagatctcct gcaaggcttc tggatatacc ttcacaaact atggaatgaa 180
ctgggtgaaa caggctccag gaaagggttt aaagtggatg ggctggataa acaccaacac 240
tggagagcca acatatgctg aagagttcaa gggacggttt gccttctctt tggaaacctc 300
tgccaacact gcctatttgc agatcaacaa cctcaaaaat gaggacacgg ctacatattt 360
ctgtgtaaga gggaggaatg ggtttgctta ctggggccaa gggactctgg tcactgtcgg 420
tggtggtggt tctggcggcg gcggctccgg tggtggtggt tctagtattg tgatgaccca 480
gactcccaaa ttcctgcttg tatcagcagg agacagggtt accataacct gcaaggccag 540
tcagagtgtg agtaatgatg tagcttggta ccaacagaag ccagggcagt ctcctaaact 600
gctgatatac tatgcataca atcgctataa gggagtccct gaccgcttca ctggcagtgg 660
atatgggacg gatttcactt tcaccatcag cactgtgcag gctgaagacc tggcagttta 720
tttctgtcag caggattata gctctccgta cacgttcgga ggggggacca agttggaaat 780
aaaacggggt tctggttctt ctggttcctc cggccaccac caccaccacc actgaaagct 840
tcgcg 845

Claims (1)

1. the preparation method of the gold test strip based on single-chain antibody, is characterized in that, comprises the following steps:
One, the positively charged label single-chain antibody gene expression vector establishment connecting by neutral amino acid small peptide:
(1) introduce coding-belt positive electricity small peptide label at 5 of single-chain antibody gene ' end by genetic recombination, fusion composition is successively by the small peptide of coding-belt positive electricity, neutral small peptide and single-chain antibody gene, or introduce coding-belt positive electricity small peptide label at 3 of single-chain antibody gene ' end by genetic recombination, fusion composition is successively by coding single-chain antibody gene, the small peptide label composition of neutral small peptide and positively charged, positively charged small peptide label composition: 5-12 arginine residues or 5-12 lysine residue or 6-12 the two kilnitamin residue small peptide label of composition jointly, respectively with R5-12, K5-12, (RK) 3-6 represents, R arginine residues, K represents lysine residue, neutral small peptide is made up of 8 to 20 asparagine residues, N8-20, and N represents asparagine residue, at the other end of gene, introduces 6 histidine residues base sequences of coding by genetic recombination,
(2) by above Fusion gene construction to Escherichia coli matter week chamber expression vector, the single-chain antibody of fusion will be expressed in Escherichia coli matter week chamber;
Two, the single-chain antibody gene matter week chamber expression vector of structure is transformed into colibacillus engineering strain, the positive transformant of identifying is carried out to self-induction expression, abduction delivering 3-6 hour, adds and the corresponding microbiotic of resistant gene that expression vector is with in nutrient culture media;
Three, collect thalline, extract matter week chamber soluble protein, use affinity chromatography method, the single-chain antibody of positively charged label is carried in separation and purification;
Four, preparation gold mark single-chain antibody, the 300 nanograms/milliliter single-chain antibodies of 0.5 milliliter are dropwise added in the colloidal gold solution of 8 milliliters, mix gently, mixed liquor was room temperature reaction 1 hour, then, bovine serum albumin(BSA) (BSA) sealing with 10% 30 minutes, mixed liquor is at 4 degrees Celsius, leave the heart 30 minutes with per minute 14000, discard supernatant, gold grain precipitation is hanged again with the BSA of the Tris buffering of 20 mM pH8.0 of 450 microlitres 1%, adds 0.1 the sodium nitride that falls, and before using, golden target single-chain antibody is stored in 4 degrees Celsius of refrigerators;
Five, the preparation of immunity test strip, the monoclonal antibody of the determined antigen of 1 microlitre and anti-6 histidines is fixed on to detection zone and the Quality Control district on nitrocellulose filter in room temperature respectively, the colloid gold label single-chain antibody of purifying is done after dilution in various degree, evenly equivalent is dipped in onesize glass fibre element film and makes gold mark pad, in the situation that other conditions are constant, according to reaction result, determine the dilutability of the suitableeest colloid gold label single-chain antibody that reaches the requirement of test strips susceptibility, or be called working concentration, getting the golden labeling antibody of suitable working concentration is added on gold mark pad with 10 microlitres/bar, then vacuum freeze drying, sealing is preserved.
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