CN106397603B - A kind of special fused antigen of SMCY gender and its antibody and application - Google Patents
A kind of special fused antigen of SMCY gender and its antibody and application Download PDFInfo
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- CN106397603B CN106397603B CN201610726961.XA CN201610726961A CN106397603B CN 106397603 B CN106397603 B CN 106397603B CN 201610726961 A CN201610726961 A CN 201610726961A CN 106397603 B CN106397603 B CN 106397603B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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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
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Abstract
The present invention provides a kind of recombinant antigens, contain amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.The present invention also provides a kind of colloidal gold fast detecting test paper strips, contain above-mentioned recombinant antigen and the immune polyclonal antibody generated of above-mentioned above-mentioned recombinant antigen.The present invention is directed to the amino acid sequence of mankind SMCY antigen, screening has the segment of larger difference with SMCX, it is connected together with the method for molecular biology, form a new SMCY genetic recombinants, it is significantly reduced with the homology of SMCX, to improve the male specificity of the special fused antigen of SMCY gender.The present invention establishes double-antibody sandwich elisa detection technique on the basis of obtaining specific antibody, and establishes a kind of field fast detection method by detecting the SMCY antigen of human biological's sample and carrying out sex determination.
Description
Technical field
The invention belongs to bioengineering fields, are related to a kind of antigen, and specifically a kind of SMCY gender specifically merges anti-
Former and its antibody and application.
Background technique
Criminal case is badly in need of us by quickly identifying evidence at the scene feature, provides technology branch for quickly detection criminal case
Support.The method that sex identification mainly uses detection of nucleic acids at present, but the detection method is time-consuming and laborious, in addition can also miss solve a case it is good
Machine, it is therefore desirable to the quick sex appraisal method in scene.
H y antigen be encoded by Y chromosome, the distinctive histocompatibility antigen of buck, be by Eichwald and
What Silmser had found for the first time when carrying out heterogenous skin transplant experiment with mouse inbred lines.H y antigen belongs to a kind of secondary tissue phase
Capacitive compound, is different from major histocompatibility complex, and h y antigen has in the transplanting of homogenic type male and female individual asked
Rejection.Shapiro M etc. proved by antiserum adsorption test, the antigenic determinant for the h y antigen that Serologic detection arrives
It is a kind of carbohydrate.It is found simultaneously using indirect immunofluorescence, H-Y specificity fluorescent appears on cell membrane, illustrates h y antigen
For a kind of membranous antigen, H-Y is prompted to be present in all male sex cell surfaces containing Y chromosome in mammals.Different hairs
Bright person has found the candidate gene of a series of h y antigen when the T lymph poison cell to patient's body is invented.1995
When year Scott etc. in the murine genes of h y antigen feminine gender to analyzing, in Y chromosome one conservative gene of long-armed discovery,
With extensive tissue expression, it is named as SMCY.SMCX on the gene and X chromosome has very high homology, male
The SMCY product of property can induce repulsion of the female cell to h y antigen.But in some amino acid sections of SMCY and phase in SMCX
Answering section, there are larger differences, carry out going deep into invention in sex identification with very important meaning for these otherness peptide fragments
Justice.
H y antigen (histocompatibility antigen Y) is encoded by Y chromosome, is distinctive group of buck
Compatibility antigen is knitted, the glycoprotein of cell surface is incorporated in, is present in all males containing Y chromosome in mammals
Cell surface.Inorganization and organ specificity in male.H y antigen belongs to weak histocompatibility antigen, participates in organ
Specific immune response in transplanting, blood transplant and pregnancy reaction.There is inventive result to show the patient from transplanting versus-host disease
In the h y antigen peptide separated can be in conjunction with MHC molecule, cytotoxic T lymphocyte technology is reflected using this characteristic
Not different h y antigen peptides.Domestic and international many laboratories have carried out the gender of a large amount of body early embryo using H-Y antiserum or monoclonal antibody
The work of identification, and achieve preliminary effect.SMCY (selected mouse cDNA on Y) is first determining H-Y
Antigen candidate gene only all expresses in male tissue, encodes 1500 amino acid sequences.Sequence alignment analysis is as the result is shown
SMCX on SMCY and X chromosome has very high homology, but still more than on 200 amino acid and X chromosome
SMCX is different, it is possible to produce sex-specific peptide fragment.
Summary of the invention
For above-mentioned technical problem in the prior art, the present invention provides a kind of special fused antigen of SMCY gender and its
Antibody and application, a kind of special fused antigen of described this SMCY gender and its antibody and application will solve to make in the prior art
With the time-consuming and laborious technical problem of detection of nucleic acids method for distinguishing.
The present invention provides a kind of recombinant antigen (SMCY), contain SEQ ID NO.1, SEQ ID NO.2 and SEQ ID
Amino acid sequence shown in NO.3.
Further, the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 forms,
Connectionless segment between amino acid sequence shown in SEQ ID NO.1, the SEQ ID NO.2, the EQ ID NO.2
There is a junction fragment between amino acid sequence shown in SEQ ID NO.3, the junction fragment is by two or three
A serine composition.
The present invention also provides the nucleotide sequences for encoding above-mentioned recombinant antigen.
The present invention also provides a kind of inclusion bodys, contain above-mentioned recombinant antigen.
The present invention also provides a kind of carriers, contain the nucleotide sequence for encoding above-mentioned recombinant antigen.
The present invention also provides a kind of polyclonal antibodies, are generated by above-mentioned recombinant antigen is immune.
The present invention also provides the test strips that a kind of colloidal gold quickly detects gender, contain above-mentioned recombinant antigen and above-mentioned
Polyclonal antibody.
Further, the colloidal gold quickly detect gender test strips include a backboard, the backboard it is upper
One end of side is provided with a sample pad, and the other end of the upside of backboard is provided with a water absorption pad, in the sample pad
A cellulose membrane is provided between water absorption pad, water absorption pad and the cellulose membrane closely connect, in sample pad and fiber
It is provided with a gold-labelled pad between plain film, is closely connected between the sample pad, gold-labelled pad and cellulose membrane, in cellulose membrane
Nature controlling line is arranged in upper one end close to water absorption pad, above-mentioned recombinant antigen is coated on the nature controlling line, in the Quality Control
Detection line is set on the cellulose membrane between line and gold-labelled pad, above-mentioned polyclonal antibody, institute are provided in the detection line
The probe of colloid gold label is provided in the gold-labelled pad stated, one end of the cellulose membrane is arranged under the gold-labelled pad
The downside of the sample pad, the colloid gold label probe in the gold-labelled pad is arranged in side, one end of the gold-labelled pad
It is the polyclonal antibody that rabbit is immunized using above-mentioned recombinant antigen as antigen and obtains.
Further, the cellulose membrane is selected from nitrocellulose filter or cellulose acetate film, the support colloid
The gold-labelled pad of golden label probe is selected from glass fibre membrane or polymer PET, and the sample pad is selected from blood filter membrane, glass fibre or water suction
Paper, the water absorption pad are blotting paper.
The present invention also provides a kind of purposes of the above-mentioned recombinant antigen in identification mankind's gender.
The present invention will be directed to the amino acid sequence of mankind SMCY antigen, and screening has the segment of larger difference with SMCX,
It is connected together with the method for molecular biology, forms a new SMCY genetic recombinants, the homology with SMCX
It significantly reduces, to improve the male specificity of the special fused antigen of SMCY gender.SMCY genetic recombinants carry out prokaryotic expression,
And antibody is prepared with the antigen-immunized animal of purifying, double-antibody sandwich elisa inspection is established on the basis of obtaining specific antibody
Survey technology, and tentatively establish it is a kind of by detect human biological's sample SMCY antigen and carry out sex determination scene quickly inspection
Survey method.
The present invention is in order to establish the quick sex appraisal method in scene, for the amino acid sequence of mankind's SMCY antigen, screening
Three segments with SMCX with larger difference out, i.e. segment 1:256-293aa, segment 2:814-845aa, segment 3:
1467-1560aa.It is connected together with the method for molecular biology, forms one and new only encode these three segments
SMCY genetic recombinants are significantly reduced with the homology of SMCX, to substantially increase the hero of the special fused antigen of SMCY gender
Property specificity.Then prokaryotic expression is carried out, the SMCY gender expressed in e. coli bl21 (DE3) with inclusion bodies is obtained
Special fused antigen is 90% or more by the destination protein purity that affinity chromatography purifies, and is resisted with what this was purified
Original is immunized purebred White Rabbit and is successfully prepared polyclonal antibody, and the fusion protein of Western marking testing result display expression can
It is specifically bound with how anti-, it is 10 that ELISA, which detects 500 times of diluted potency of enzyme,6, antigen lowest detection line is 1ng；1000
Times diluted potency of enzyme is 2 × 106, antigen lowest detection line is 2ng.
The present invention is compared with prior art, and technological progress is significant.The present invention is established based on the special SMCY of gender
The colloidal gold fast detecting test paper strip of antigen is marked using the double-antibody sandwich detection method of colloidal gold immunochromatographimethod technology in gold
The SMCY rabbit that colloid gold label is coated on pad is mostly anti-, for the SMCY albumen in qualitative detection serum or plasma sample.By pair
The detection of random men and women's serum sample, this colloidal gold fast detecting test paper strip can distinguish male and female serum sample substantially；
In addition, by being detected to female male mice serum sample, it is as a result reactionless, illustrate that the special fused antigen of SMCY gender has
Species specificity, this reagent strip are suitable for the identification of human sample gender.
Detailed description of the invention
Fig. 1 shows SMCY and SMCX amino acid sequence differences section.
Fig. 2 shows the special fused antigen B cell epitope analysis of gender.
Fig. 3 shows that pET-28a/SMCY expression plasmid constructs schematic diagram.
Fig. 4 is pcr amplification product electrophoretic identification, wherein M.DL2000DNA marker；The coding of 1.SMCY fusion section
Gene (516bp).
Fig. 5 is SMCY fused antigen SDS-PAGE electrophoretic identification, 1. full bacterium；2. ultrasonication supernatant；3. ultrasonication
Precipitating；4.Ni column purification antigen；M. albumen relative molecular mass marker.
Fig. 6 shows Anti-TNF-α antibody preparation procedures.
Fig. 7 shows polyclonal antibody after purification, M. albumen relative molecular mass marker；1. mostly anti-after purification.
Fig. 8 is Western Blotting experimental result, 1.0.1mg/ml SMCY antigen；2.0.05mg/ml SMCY is anti-
It is former；3.0.02mg/ml SMCY antigen.
Fig. 9 is double-antibody sandwich detection method schematic diagram.
Figure 10 shows testing result.
Figure 11 is 30 male's serum sample testing results.
Figure 12 is male's serum sample positive test symbol.
Figure 13 is 20 women serum sample testing results.
Figure 14 is mice serum sample testing result.
Specific embodiment
1 SMCY sequence alignment of embodiment and analysis
As shown in Figure 1, the amino acid sequence of tri- variants of SMCY and SMCX is inquired and downloaded from NCBI, sequence is utilized
Software is analyzed by SCMY (1539aa) and SMCX variant 1 (1560aa), variant2 (1379aa), variant 3
(1559aa) carries out sequence alignment respectively, it was found that three segment difference heteroleptics:
Segment 1:256-293aa (DKTVHKKVTCPPTVTVKDEQSGGGNVSSTLLKQHLSL)；
Segment 2:814-845aa (RLKNCLSEVEACIAQVLGLVSGQVARMDTPQL)；
Segment 3:1467-1560aa (QKVDQGRNVENLVQQELQSKRARSSGIMSQVGREEEHYQEKADRENMFLT
PSTDHSPFLKGNQNSLQHKDSGSSAACPSMPLLQLSYSDEQQL)。
B cell epitope analysis (Fig. 2) is carried out to different regions fused antigen using BIOSUN software, fusion is anti-as the result is shown
Original still maintains original B cell epitope, and has preferable antigenicity.
The synthesis of 2 SMCY gene difference section of embodiment
It is counter to push away nucleic acid sequence according to the amino acid sequence of three segment difference heteroleptics, and codon optimization is carried out, according to nucleic acid sequence
Then column design primer is connected the primer of synthesis by taking turns PCR with the bypass method of molecular biology more step by step,
Form the new SMCY genetic recombinants for only encoding these three segments:
The amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 forms, the SEQ
Connectionless segment between amino acid sequence shown in ID NO.1, SEQ ID NO.2, EQ ID NO.2 and the SEQ ID
There is a junction fragment, the junction fragment is by two or three serine groups between amino acid sequence shown in NO.3
At.
It is significantly reduced with the homology of SMCX, so that the male for substantially increasing the special fused antigen of SMCY gender is special
Property.The gene is sequenced, choosing the right-on gene of sequence, gene carries out subsequent experimental as a purpose.
Shown in the primer sequence of design following (such as NO.4~23 SEQ ID))
1F3:GCGGATCCGATAAAACCGTGCATAAGAAAGTTACGTGCCCGCCAACTGT
1F2:CGTGCCCGCCAACTGTGACCGTCAAAGACGAACAGAGCGGCGGTGGCAACG
1F1:AGCGGCGGTGGCAACGTTTCTTCCACTCTGTTGAAGCAACACCTGTCGTTA
1R1:CACGCTTCTACCTCGCTAAGACAATTTTTCAGACGTAACGACAGGTGTTGC
1R2:CCTGGCCACTAACGAGACCCAGCACCTGGGCAATGCACGCTTCTACCTCGC
1R3:GCTCTAGACAGTTGCGGCGTATCCATGCGCGCGACCTGGCCACTAACGAG
2F4:GCACTAGTCAGAAAGTGGATCAAGGCCGTAACGTTGAAAATCTGGTGCA
2F3:TTGAAAATCTGGTGCAGCAGGAGTTGCAAAGCAAGCGCGCGCGTTCTTCCGG
2F2:GCGCGCGTTCTTCCGGTATTATGTCGCAGGTCGGCCGCGAAGAAGAGCATTA
2F1:GCGAAGAAGAGCATTATCAGGAAAAAGCCGACCGTGAAAACATGTTTCTGAC
2R1:TTACCTTTTAAGAATGGACTGTGATCCGTGCTCGGGGTCAGAAACATGTTTT
2R2:CTGCTGCCCGAATCCTTATGCTGCAGAGAGTTTTGATTACCTTTTAAGAATG
2R3:AGCTGGAGCAGTGGCATAAGAGACGGGCATGCCGCGCTGCTGCCCGAATCCT
2R4:GCGAATTCCAGCTGTTGCTCGTCCGAGTAGGACAGCTGGAGCAGTGGCA
F1:AGCGCGCAGGATCTGCGTGGATCCGATAAAACCGT
F2:AGCGCGCAGGATCTGCGT
R1:GC TCTAGACAGCTGTTGCTCGTCCGAGTA
F3:GCACTAGTGATAAAACCGTGCATAAGAAAGT
R2:AAGGCAACAATCACAGAGGAATTCCAGCTGTTGCTCGT
R3:AAGGCAACAATCACAGAG
The construction and expression of 3 SMCY fused antigen carrier of embodiment
The right-on fusion section antigen gene of sequence is chosen after once repeating with BamH1, EcoR1 double digestion, is returned
The target fragment (about 516bp) cut is received, then is connect with the pET-28a carrier through BamH1, EcoR1 double digestion with T4 ligase,
It constructs recombinant expression plasmid pET-28a/SMCY (Fig. 3, Fig. 4).Then it is transformed into competent escherichia coli cell BL21 (DE3)
In, picking positive colony bacterium colony is inoculated with the LB culture solution containing kanamycins, and it is to be grown to after logarithmic phase, IPTG is added, 37 DEG C lure
It leads overnight, collects bacterium solution, SDS-PAGE electroresis appraisal protein expression situation.The special fused antigen of SMCY gender is as the result is shown to wrap
Contain body mode to express, relative molecular weight is about 43KD (Fig. 5).
The extraction and purification of 4 SMCY fused antigen of embodiment
Bacterium is expressed after full-page proof ferments, collects bacterium solution, ultrasonic fragmentation extracts inclusion body from expression bacterium, and with dissolving
Liquid (25mmol/LTE, 1% beta -mercaptoethanol, 8mol/L urea, pH8.5) is dissolved, and is collected sample and is carried out Ni column purification, with washing
Buffer (25mmol/L TE, 1% beta -mercaptoethanol, 6mol/L urea, pH8.5) washing cylinder is washed, SMCY sample loading crosses column.
Foreign protein is eluted with the washing buffer of the imidazoles containing 25mmol/L after end of the sample, it is finally slow with imidazole wash containing 250mmol/L
Fliud flushing elutes and collects destination protein, each eluting peak collected by last SDS-PAGE electrophoretic analysis and the (figure of the sample before loading
5)。
5 polyclonal antibody of embodiment is prepared and purified
New zealand white rabbit is immunized with SMCY fused antigen after purification, takes blood as negative control, skin before first immunisation
Lower multi-point injection method is immunized, and by just exempting from, two exempts from and booster immunization, each immunization interval one month, it is anti-to prepare anti-SMCY
Former polyclonal antibody (Fig. 6).Heart extracting blood after third time is immune, is collected by centrifugation serum, utilizesAntibody
purification- G kit kit purifies it, and Guo Zhuhou obtains SMCY polyclonal antibody (figure after purification
7) it is, 3.1mg/ml through detectable concentration, is successfully prepared polyclonal antibody, and subsequent experimental can be carried out.
6 polyclonal antibody specificity of embodiment and reactivity identification
By the SMCY antigen (1.0mg/ml) of purifying according to 10 ×, 20 ×, 50 × be diluted, make the final concentration be respectively
0.1mg/ml, 0.05mg/ml, 0.02mg/ml are added 3 × sample diluting liquid, boil 5min, applied sample amount 10ul, concentration is
15%, voltage 160V carry out SDS-PAGE electrophoresis.It takes out gel after electrophoresis, membrane-transferring device is installed, with filter paper, NC film,
Gel, the sequence of filter paper stack alignment one by one, avoid generating bubble.Transferring film condition is 4 DEG C, 300mA electrotransfer 2h.Take out NC
Film is cut to suitable size, closes 1h at room temperature with the TBST containing 5% skimmed milk power；H-Y polyclonal antibody (1:6000 is added
Dilution), 4 DEG C of oscillation incubations are stayed overnight；TBST washes film 3 times, each 8min.Be added corresponding HRP label goat anti-rabbit igg (1:
2000 dilutions), oscillation incubation 1h at room temperature.TBST is washed film 3 times, and TBS washes film 1 time, each 8min, chemiluminescence A, B liquid (ECL)
After the mixing of 1:1 ratio, film is immersed into developing solution, develops the color and takes pictures in the colour developing instrument that shines after 1min.It is prepared as the result is shown
Polyclonal antibody can with specific recognition be immunized antigen (Fig. 8).
The measurement of 7 Anti-TNF-α precursor reactant potency of embodiment
Mostly anti-label HRP: take the SMCY antibody 2mg of purifying in bag filter, pH9.6 carbonate buffer solution dialysed overnight.It takes
2mg HRP is dissolved in 0.2mL distilled water, and the 0.06M NaIO newly configured is added4Aqueous solution 0.2mL is protected from light stirring after mixing
20min.Glycol water 0.2mL, the room temperature avoid light place 30min of 0.16M is added, the SMCY antibody that 2mg has dialysed is added
Aqueous solution, dialyse 6h after mixing.Take out the NaBH that 5mg/mL is added4Solution 0.2mL, 4 DEG C of placement 2h, after glycerol is added by 1:1
It saves.
The foundation (Fig. 9) of double-antibodies sandwich ELISA: SMCY antibody is diluted to the concentration packet of 5 μ g/mL with coating buffer
By elisa plate, 4 DEG C of coatings are overnight.SMCY plate board-washing is patted dry afterwards twice, adds 110 μ L confining liquids to close 5h, pats dry rear room temperature wind
It is dry.The SMCY antigen of sample-adding product diluted 1mg/ml, extension rate are as follows: 100,500,1000,2000,4000,8000,
16000,32000,64000,1.282 × 105, 2.56 × 105, 5.12 × 105, 1 × 106, 2 × 106, 4 × 106, 8 × 106,
1.6×107, 3.2 × 107, 6.4 × 107, 1.28 × 108, 2.56 × 108, 5.12 × 108, 1 × 109, after sample-adding plus 37 DEG C anti-
Answer 30min.It is patted dry after board-washing 5 times, respectively enzyme labeling antibody, 37 DEG C of reaction 20min.It is patted dry after board-washing 5 times, color developing agent A liquid B liquid
Each 50 μ L, mixes well, and 37 DEG C are protected from light 10 minutes.50 μ L of terminate liquid is added, OD value is measured in 10 minutes, detects dual wavelength
For 450nm/630nm.Testing result is that be respectively as follows: 500 times of diluted potency of enzyme be 10 to SMCY antibody titer6, the minimum inspection of antigen
Survey line is 1ng；1000 times of diluted potency of enzyme are 2 × 106, antigen lowest detection line is 2ng.
The development of colloidal gold fast detecting test paper strip of the embodiment 8 based on the special SMCY antigen of gender
Double-antibody sandwich detection method uses colloidal gold immunochromatographimethod technology, and colloid gold label is coated in gold-labelled pad
SMCY rabbit is mostly anti-, for the SMCY albumen in qualitative detection serum or plasma sample.SMCY when detecting male sample, in sample
The more anti-bindings of the SMCY rabbit of albumen and colloid gold label form compound, since chromatography acts on compound and sample in nitric acid fibre
Tie up flow forward inside plain film.When compound passes through T line with the coated more anti-bindings of SMCY rabbit, formed " Au-rabbit is mostly anti--
SMCY albumen-rabbit is mostly anti-" and be aggregated colour developing, the mostly anti-SMCY protein binding with C line of remaining colloid gold label SMCY rabbit and
Agglutination colour developing.When detecting women sample, SMCY albumen is free of in sample, therefore cannot form compound, then only have at C line aobvious
Color.
It prepares colloidal gold: 0.8g trisodium citrate being taken to be dissolved in the bis- pure water of 5mL；The bis- pure water of 1.2L are taken to be added in round-bottomed flask
5min is boiled with electric jacket heating；It takes 2.4mL gold chloride to be added in the above-mentioned double pure water boiled, boils 5min；By what is dissolved
Citric acid three sodium solution is added at one time in chlorauric acid solution, boils 10min；Cooled to room temperature.
Gold mark SMCY rabbit is mostly anti-: the how anti-CB dialysis 48h by PH9.6 of the SMCY rabbit of purifying；Take the colloidal gold prepared
15,20,25,30 μ L 0.1mol/L K are added in solution 1mL thereto respectively2CO3Solution adjusts PH；Respectively into above-mentioned solution
The how anti-solution of SMCY rabbit of 15 μ L 1.0mg/mL is added, mixes well and stands 30min；It continues up to state and 100 μ L is added in solution
10%BSA solution stands 20min after mixing well；The gold solution of different PH is separately added into 100 μ L, 10% sodium chloride solution, sees
It examines and whether becomes blue and precipitate, as a result select 25 μ L 0.1mol/L K of optimal set2CO3Solution adjusts PH；Take the colloidal gold prepared
250 μ L0.1mol/L K are added in solution 10mL thereto respectively2CO3Solution adjusts PH；150 μ L are separately added into above-mentioned solution
The how anti-solution of purifying SMCY rabbit of 1mg/mL mixes well and stands 30min；It continues up to state and 200 μ L 10% is added in solution
BSA solution stands 20min after mixing well；4 DEG C of above-mentioned solution are centrifuged, 14,000r/min centrifugation 30min；Discard supernatant liquid
6.0mL re-suspension liquid is added afterwards, ultrasonic 2min is mixed；In Fusion5 colloidal gold pad after oil gidling, 50 DEG C of drying overnight.
It is mostly anti-to be coated with SMCY rabbit: how anti-CB the dialysis 48h, the 50mM that coating condition is PH9.6 by PH9.6 of SMCY rabbit
It is 1mg/mL that CB, which dilutes the how anti-peridium concentration of rabbit,；The speed of NC film superior synovial membrane instrument is 1 μ L/cm, is T line under scribing line slightly.
Be coated with nature controlling line: coating SMCY albumen, for albumen by the CB dialysis 48h of PH9.6, coating condition is PH9.6's
50mM CB diluted protein peridium concentration is 1mg/mL；The speed of NC film superior synovial membrane instrument is 1 μ L/cm, is crossed on slightly, is C line.
Detecting step:
Prepare before examining: 100 μ L micropipettors and mating pipette tips are several.
Checkout procedure: will test and be placed on dry horizontal table, the well blocked with micropipettor to detection
Then 50 μ L sample dilutions (about two drops) is added in 50 μ L test serums of interior addition or plasma sample, the dilution is one
Kind of buffer, such as phosphate buffer (sodium dihydrogen phosphate or potassium dihydrogen phosphate), are added timing after sample, and 15 to 20 minutes
Interior observation result.
Detection process points for attention: 1. this kit and sample to be tested are placed in room temperature environment can just make after balance
For detecting；2. detection card uses in 30 minutes behind Kaifeng；3. observation result is invalid after twenty minutes.
The explanation (Figure 10) of testing result: C line, T line develop the color: positive；Only C line develops the color: negative；Only T line develops the color: nothing
Effect；C line, T line do not develop the color: invalid.
9 pattern detection result of embodiment
One, human serum pattern detection result
50, human serum sample are randomly selected, wherein male 30, women 20, is examined according to above-mentioned detecting step
It surveys, as the result is shown such as table 1, Figure 11, Figure 12 and Figure 13.Male's serum sample reactivity is significantly stronger than women serum sample；Male
There are 27 presentation positive reactions, positive rate 90.0% in 30 serum samples；Only 3 presentations can in 20 serum samples of women
It doubts as a result, remaining is negative reaction.
1. serum sample testing result of table
Two, mice serum sample testing result
Mature BALB/c mouse serum sample 4 is randomly selected, wherein male 2, female 2 are walked according to above-mentioned detection
Suddenly it is detected, as the result is shown such as Figure 14.Male and female mice serum sample are reactionless, illustrate that this kit is suitable for mirror
The gender for determining human sample, with mouse no cross reaction.
Claims (8)
1. a kind of recombinant antigen, it is characterised in that: the ammonia as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3
Base acid sequence forms, connectionless segment between amino acid sequence shown in the SEQ ID NO.1, SEQ ID NO.2, described
EQ ID NO.2 and SEQ ID NO.3 shown in there is between amino acid sequence a junction fragment, the junction fragment
It is made of two or three serines.
2. encoding the nucleotide sequence of recombinant antigen described in claim 1.
3. a kind of inclusion body, it is characterised in that: contain recombinant antigen described in claim 1.
4. a kind of carrier contains the nucleotide sequence for encoding recombinant antigen described in claim 1.
5. a kind of polyclonal antibody is generated by recombinant antigen described in claim 1 is immune.
6. a kind of colloidal gold quickly detects the test strips of gender, contain recombinant antigen described in claim 1 and claim 5 institute
The polyclonal antibody stated.
7. the test strips that a kind of colloidal gold according to claim 6 quickly detects gender, it is characterised in that: carried on the back including one
Plate, one end of the upside of the backboard are provided with a sample pad, and the other end of the upside of backboard is provided with a water suction
Pad, is provided with a cellulose membrane, water absorption pad and the cellulose membrane closely connect between the sample pad and water absorption pad
It connects, a gold-labelled pad is provided between sample pad and cellulose membrane, it is tight between the sample pad, gold-labelled pad and cellulose membrane
Close connection is arranged nature controlling line close to one end of water absorption pad on cellulose membrane, is coated with claim 1 institute on the nature controlling line
The recombinant antigen stated, is arranged detection line on the cellulose membrane between the nature controlling line and gold-labelled pad, in the detection line
It is provided with polyclonal antibody described in claim 5, the probe of colloid gold label, the fibre are provided in the gold-labelled pad
The downside of the gold-labelled pad is arranged in the one end for tieing up plain film, and one end of the gold-labelled pad is arranged under the sample pad
Side, the colloid gold label probe in the gold-labelled pad are more grams for being immunized that rabbit obtains with recombinant antigen described in claim 1
Grand antibody.
8. the test strips that a kind of colloidal gold according to claim 7 quickly detects gender, it is characterised in that: the fiber
Plain film is selected from nitrocellulose filter or cellulose acetate film, and the gold-labelled pad is selected from glass fibre membrane or polymer PET, the sample
Product pad is selected from blood filter membrane, glass fibre or blotting paper, and the water absorption pad is blotting paper.
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