CN102998453A - Indirect enzyme-linked immuno sorbent assay (ELISA) kit of torque teno virus 1 (TTSuV1) antibody and preparation method thereof - Google Patents
Indirect enzyme-linked immuno sorbent assay (ELISA) kit of torque teno virus 1 (TTSuV1) antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an indirect enzyme-linked immuno sorbent assay (ELISA) kit of a torque teno virus 1 (TTSuV1) antibody and a preparation method thereof. The indirect ELISA kit of the TTSuV1 antibody for detecting the level of the TTSuV1 antibody in a porcine serum is built by the steps of preparing TTSuV1 ORF1a recombinant protein envelope antigen expressed by yeast, screening standard positive serum and standard negative serum, determining antigen envelope concentration and serum dilutability, and assembling the kit; and the TTSuV1 ORF1a recombinant protein can be used for competitively restraining combination of the antibody in the serum and a solid-phase envelope antigen. The kit disclosed by the invention has good specificity on the TTSuV1 antibody; and the maximal variable coefficients (CV) inside batches and between batches of the kit respectively are 6.87% and 11.4%, and lower than 15% industrial standard, so that the kit has good repeatability and stability.
Description
Technical field
The present invention relates to animal doctor's technical field, be specifically related to the thin circovirus virus TTSuV1 of boar I type antibody indirect ELISA diagnostic kit and preparation method thereof, set up TTSuV1 antibody indirect ELISA diagnostic kit for detection of the antibody horizontal of TTSuV1 in the pig serum.
Background technology
Transfusion transmitted virus (Transufsion Transmitted Viurs, TTV) be a class without cyst membrane, icosahedron, sub-thread minus strand cyclic DNA virus, electron microscopic observation virion diameter is about 30 32nm.TTV is found from the non-first of a routine unknown cause of disease-non-penta type post-transfusion hepatitis patient's serum by Japanese scholars first, people are non-human primate and pig, ox, sheep, dog subsequently, have also found the infection of TTV in the multiple domestic and wild animal body such as cat, poultry.2009, international virus taxis association (ICTV) was divided into the thin Circovirus (Iotatorquevirus) of finger ring Viraceae (Anelloviridae) with the TTV of pig, and thin Circovirus Further Division is two kinds; The thin circovirus virus of pig I type (Torque teno sus virus I, TTSuV1) and the thin circovirus virus of pig I type (Torque teno sus virus II, TTSuV2).As a kind of comparatively novel virus, the pathogenesis of TTSuV2 is known little about it.2004, McKeown was to China, the Iowa, and Thailand, Ontario, Korea S, Spain, 154 parts of pig serum in these six country variant areas of Quebec and Saskatchewan detect, and the TTSuV2 positive rate can be up to 66.2% (102/154).Aramouni etc. have confirmed between TTSuV1 and pmws (PMWS) and the scorching nephrotic syndrome of pigskin (PDNS) certain contact is arranged and find that TTSuV2 and other known porcine pathogen coinfection can cause disease to increase the weight of.In recent years, increase gradually about the report of TTSuV1 and TTSuV2, the infection of TTSuV2 has popularity and ubiquity, and these characteristics have caused the concern of many researchers.Therefore, strengthen the epidemiology survey to TTSuV2, determine its antigenicity point, the control of TTSuV2 is of great significance.
At present, both at home and abroad in the epidemiology report to TTSuV1 and TTSuV2, comparatively pay attention to the infection conditions of TTSuV2, as if the infection of TTSuV2 plays a part aspect PMWS and the PDNS illness even more importantly strengthening, and has had report to set up " the blocking-up ELISA method " of TTSuV2 antibody diagnosis at present.Yet in nearly all report, TTSuV1 recall rate (PCR method) above 30% in pig serum is enough to cause that people are to the attention of TTSuV1 harm.Set up gradually the method for TTSuV1 viral nucleic acid diagnosis (PCR) both at home and abroad, there is no the report about TTSuV1 antibody diagnosis (ELISA) kit.
Summary of the invention
For overcoming the defective of prior art, the object of the present invention is to provide the thin circovirus virus TTSuV1 of boar I type antibody indirect ELISA diagnostic kit, the TTSuV1 antibody indirect ELISA diagnostic kit of setting up has good repeatability and stable, can detect the antibody horizontal of TTSuV1 in the pig serum.
Another object of the present invention is to provide the preparation method of the thin circovirus virus TTSuV1 of boar I type antibody indirect ELISA diagnostic kit.
The technical solution adopted in the present invention is as follows for achieving the above object:
The thin circovirus virus TTSuV1 of one boar I type antibody indirect ELISA diagnostic kit, it comprises with the coated ELISA Plate of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression, and the standard positive serum and the standard female serum that go out as the immunodiagnosis antigen selection with the unique structural proteins ORF1 of TTSuV1, wherein the sequence with the TTSuV1 ORF1a recombinant protein of Yeast expression is SEQ ID NO:1.
Among the present invention, the thin circovirus virus TTSuV1 of described pig I type antibody indirect ELISA diagnostic kit, it also comprises
1) the anti-pig two anti-liquid of the rabbit of HRP mark: it is the dilution proportion of 1 ﹕ 2000-3000 according to volume ratio that the anti-pig two of rabbit resists with HRP protection liquid, and consumption is 100 μ l;
2) antibody diluent: 0.1%BSA1.0g adds PBS to 1000ml, and consumption is 100 μ l;
3) nitrite ion: 50mg TMB, 10mL DMSO, 9.8g citric acid and 1.2ml 30%H
2O
2Adding distil water is settled to 1000ml, and consumption is 100 μ l;
4) stop buffer: distilled water 178.3mL and 98% concentrated sulphuric acid 21.7mL mix, and consumption is 100 μ l.
In the preferred scheme, the thin circovirus virus TTSuV1 of above-mentioned pig I type antibody indirect ELISA diagnostic kit comprises that also the pH value is 7.4,0.2g KH
2PO
4, 2.9g Na
2HPO
412H
2O, 8.0g NaCl, 0.2g KCl, 0.5mL 0.05%(V/V) Tween-20, add the eluent that distilled water is settled to 100ml.
In a preferred scheme, in the thin circovirus virus TTSuV1 of the described pig I type antibody indirect ELISA diagnostic kit, be 0.25～1 μ g/ml with the coated concentration of the coated ELISA Plate of the TTSuV1 ORF1a of Yeast expression restructuring egg envelope antigen.
The preparation method of the thin circovirus virus TTSuV1 of one boar I type antibody indirect ELISA diagnostic kit, it may further comprise the steps:
1) preparation of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression
A.TTSuV1 ORF1a retrieval: in conjunction with TTSuV1 sequence information in the Genebank database, at virus genome sequence conserved regions design diagnostic primers SEQ ID NO:2 and primer SEQ ID NO:3; Use rTaq archaeal dna polymerase pcr amplification purpose fragment, pcr amplification product carries out electrophoresis, and purifying, order-checking obtain to have checked order row; Then at the row basis reverse amplimer SEQ ID NO:4 of design and the primer SEQ ID NO:5 of checking order, amplification comprises the virus gene sequence of TTSuV1 ORF1 complete sequence; Use the LA TaqDNA polymerase PCR purpose fragment that oppositely increases, pcr amplification product is carried out electrophoresis, purifying, obtain TTSuV1 ORF1a albumen;
The Yeast expression of b.TTSuV1 ORF1a: use Primer Premier5.0 design and express primer SEQ ID NO:6 and primer SEQ ID NO:7, take the positive nucleic acid of TTSuV1 as template, use the PCR reaction of Kod plus high-fidelity enzyme, behind electrophoresis, the purifying purpose band, double digestion PCR product and carrier connect Transformed E .coli:DH5 α, extract recombinant plasmid pPICZ alpha-TTSuV1 ORF1a, the linearization of Sac I single endonuclease digestion; Saccharomycete X-33 is prepared into competent cell; getting 80 μ L competent cells mixes with the linearizing recombinant expression plasmid of 5～10 μ g; change 0.2cm electricity revolving cup over to; electricity transforms; after bacterium liquid after electricity transforms is used 10 times of dilutions of 1ml ice bath sorbierite; coating basic dextrose culture-medium selects on the flat board; place 30 ℃ to cultivate 2～3d; have high Zeocin resistance and well-grown bacterial strain as expressing strain in the screening of the YPD flat board of 200-1000 μ g/ml concentration Zeocin; inoculate single bacterium colony in 25ml BMGY nutrient culture media, be cultured to OD in 30 ℃
600nm≈ 4.0; Centrifugal, collection thalline, with the resuspended cultivation of 100ml BMMY nutrient culture media, 1ml takes a sample every day, and to add pure methyl alcohol be 0.5% in nutrient culture media to final concentration, abduction delivering 6d, the centrifugal collection supernatant of sample thief is made SDS-PAGE and is analyzed, and take Anti-HIS-antibody as first antibody, Western Blotting filters out college entrance examination shellfish of step 1) TTSuV1 ORF1a albumen;
C.TTSuV1 ORF1a purifying: get step 2) college entrance examination shellfish that filters out in, with albumen in the 50% ammonium sulfate precipitation supernatant, dissolving, after the restructuring TTSuV1 ORF1a of the method purifying yeast expression system expression of application affinity chromatography dialyses in PBS, using the BCA method carries out quantitatively, obtain the TTSuV1 ORF1a recombinant protein of Yeast expression, sequence is SEQ ID NO:1;
2) screening of standard positive serum and standard female serum
A. make up the TTSuV1 ORF1 prokaryotic expression carrier that inserts the pET-21 empty carrier: use PrimerPremier5.0 design primer SEQ ID NO:8 and SEQ ID NO:9, take the positive nucleic acid of TTSuV1 as template, pcr amplification, reclaim purifying PCR purpose fragment, double digestion PCR product and pET-21 empty carrier, connect, picking monoclonal bacterium colony behind the Transformed E .coli:DH5 α, extract plasmid, with Plasmid Transformation E.coli:Rosetta (DE3) the pLysS competent cell that correctly makes up, identify positive colony, positive colony inoculation 2ml LA fluid nutrient medium, 37 ℃, 180rpm reaches at 0.6 o'clock to bacterium liquid OD value and adds 1mM IPTG, induced 6 hours, centrifugal collection bacterium mud adds Loading buffer and boils sample, and the SDS-PAGE analyzing proteins is expressed, use Western blotting checking, obtain TTSuV1 ORF1 recombinant protein;
The screening of b.TTSuV1 standard positive serum and standard female serum: preparation TTSuV1 ORF1 recombinant protein, cut after glue purification, the emulsification with dosage 2 monthly ages of immunity of 5mg/ head piglet without TTSuV1 cause of disease (PCR detection), repeat immunity with same dose after 1 month, after 15 days, Western Blotting and ELISA experiment are carried out in blood sampling, will experimental results show that positive serum is that TTSuV1 antibody is with reference to positive serum; Utilize the TTSuV1 ORF1a recombinant protein of Yeast expression to be the solid-phase coating thing, 50 parts of serum are carried out ELISA detect, filtering out the serum consistent with reference positive serum Western blotting and ELISA experiment reaction signal is standard positive serum; The immunological experiment no signal, and the serum of PCR detection TTSuV1 cause of disease feminine gender is standard female serum;
3) the coated concentration of defined antigen and two anti-dilutabilitys
Adopt the chessboard titrimetry, get first positive OD
450nmValue and then is therefrom found out positive serum OD about 1
450nmValue and negative serum OD
450nmThe antigen concentration of the reacting hole that the ratio of value is the highest and two anti-dilutabilitys are best antigen coated concentration and two anti-dilutabilitys;
4) assembling of kit
According to the coated concentration that determines, with the TTSuV1 ORF1a recombinant protein coated elisa plate of Yeast expression, 1 of ELISA Plate, each 1 pipe of standard yin and yang attribute serum, 1 bottle of antibody diluent, 1 bottle of ELIAS secondary antibody, 1 bottle of substrate nitrite ion, 1 bottle of stop buffer, operational manual portion are assembled into the ELISA kit.
Among the above-mentioned preparation method, step 1) is used rTaq archaeal dna polymerase pcr amplification purpose fragment specific procedure and is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 30s, 52 ℃ of annealing 30s, 72 ℃ are extended 30s, and 35 circulations are set, and 72 ℃ are extended 8min; The Ago-Gel of usefulness 1.0% carried out electrophoresis to pcr amplification product after amplification was finished.
Among the above-mentioned preparation method, it is 94 ℃ of denaturation 5min that step 1) is used the LA Taq archaeal dna polymerase PCR purpose fragment specific procedure that oppositely increases, 94 ℃ of sex change 30s, and 52 ℃ of annealing 30s, 72 ℃ are extended 120s, and 30 circulations are set, and 72 ℃ are extended 8min; The Ago-Gel of usefulness 0.8% carried out electrophoresis to pcr amplification product after amplification was finished.
Among the above-mentioned preparation method, the specific procedure of the PCR of step 1) Kod plus high-fidelity enzyme reaction is 94 ℃ of denaturation 5min, 94 ℃ of sex change 30s, and 52 ℃ of annealing 30s, 68 ℃ are extended 45s, and 30 circulations are set, and 68 ℃ are extended 8min.
Among the above-mentioned preparation method, step 2) condition of pcr amplification is 94 ℃ of 5min; 94 ℃ of 30S, 52 ℃ of 30S, 68 ℃ of 120S, 30 circulations; 68 ℃ are extended 10min.
Among the above-mentioned preparation method, the concrete steps of step 3) chessboard titrimetry are: with the TTSuV1ORF1a recombinant protein of Yeast expression on the ELISA Plate from left to right take initial concentration as 4 μ g/ml according to after hole concentration be 1/2 gradient concentration dilution coated elisa plate of hole concentration formerly, last row are as blank, every hole is coated with 100 μ l, placed 1 hour for 37 ℃, wash plate 1 time with PBST, every hole adds 10% lamb serum, 37 ℃ were sealed 1 hour, and positive control serum and negative control sera carry out respectively gradient dilution, join in the ELISA Plate from top to bottom, every hole adds 100 μ l, last column is the serum-free contrast, and 37 ℃ were reacted 1 hour, PBST washing 3 times, the anti-pig two of HRP mark rabbit that every hole adds 100 μ l resists, 37 ℃ were reacted 1 hour, PBST washing 3 times, and every hole adds substrate nitrite ion 100 μ l, lucifuge colour developing 10 minutes, every hole adds 100 μ L stop buffers, and readout instrument reads the OD value in the 450nm place, and 620nm is as the reference wavelength.
Compared to existing technology, beneficial effect of the present invention is: by the thin circovirus virus TTSuV1 of the pig I type antibody indirect ELISA diagnostic kit of preparation method's preparation of the present invention, the TTSuV1 ORF1a recombinant protein that exists in the TTSuV1 Positive Sera can the competitive combination that suppresses Serum Antibody and solid-phase coating antigen, kit of the present invention is better to the specificity of TTSuV1 antibody, kit of the present invention criticize interior and batch between the maximum coefficient of variation (CV) be respectively 6.87% and 11.4%, all be lower than 15% of industry standard, therefore, kit of the present invention has good repeatability and stability.
Below in conjunction with concrete embodiment the present invention is described in further detail.
Embodiment
It below is the concrete preferred embodiment of the present invention.
Embodiment 1
The thin circovirus virus TTSuV1 of pig I type antibody indirect ELISA diagnostic kit, it comprises with the coated ELISA Plate of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression, and the standard positive serum and the standard female serum that go out as the immunodiagnosis antigen selection with TTSuV1 ORF1, wherein the sequence with the TTSuV1 ORF1a recombinant protein of Yeast expression is SEQ ID NO:1, and the coated concentration of ELISA Plate is 0.25 μ g/ml for coated concentration; The anti-pig two anti-liquid 12ml of the rabbit of HRP mark: i.e. the anti-pig two anti-lgM of rabbit and HRP protection liquid is the dilution proportion of 1 ﹕ 2000 according to volume ratio; Antibody diluent 12ml; Nitrite ion 12ml; Stop buffer 12ml; And eluent 12ml.
Embodiment 2
The thin circovirus virus TTSuV1 of pig I type antibody indirect ELISA diagnostic kit, it comprises with the coated ELISA Plate of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression, and the standard positive serum and the standard female serum that go out as the immunodiagnosis antigen selection with TTSuV1 ORF1, wherein the sequence with the TTSuV1 ORF1a recombinant protein of Yeast expression is SEQ ID NO:1, and the coated concentration of ELISA Plate is 0.5 μ g/ml for coated concentration; The anti-pig two anti-liquid 12ml of the rabbit of HRP mark: i.e. the anti-pig two anti-lgM of rabbit and HRP protection liquid is the dilution proportion of 1 ﹕ 2500 according to volume ratio; Antibody diluent 12ml; Nitrite ion 12ml; Stop buffer 12ml; And eluent 12ml.
Embodiment 3
The thin circovirus virus TTSuV1 of pig I type antibody indirect ELISA diagnostic kit, it comprises with the coated ELISA Plate of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression, and the standard positive serum and the standard female serum that go out as the immunodiagnosis antigen selection with TTSuV1 ORF1, wherein the sequence with the TTSuV1 ORF1a recombinant protein of Yeast expression is SEQ ID NO:1, and the coated concentration of ELISA Plate is 1 μ g/ml for coated concentration; The anti-pig two anti-liquid 12ml of the rabbit of HRP mark: namely the anti-pig of rabbit two anti-with HRP protection liquid be the dilution proportion of 1 ﹕ 3000 according to volume ratio; Antibody diluent 12ml; Nitrite ion 12ml; Stop buffer 12ml; And eluent 12ml.
Application Example 1
Use the thin circovirus virus TTSuV1 of pig I type of the present invention antibody indirect ELISA diagnostic kit and detect 40 parts of sick pig serum (picking up from pig farm, Zengcheng, Guangdong); Operation steps is as follows:
(1) the TTSuV1 ORF1a recombinant protein with purifying is diluted to 0.5 μ g/ml with coating buffer, is added on the ELISA Plate, and every hole 100 μ l placed 1 hour for 37 ℃.Wash plate 1 time with PBST, every hole adds the PBST that contains 10% lamb serum, and 37 ℃ were sealed 1 hour;
(2) blood serum sample to be checked is diluted 40 times with PBST, be added on the ELISA Plate, every hole 100 μ l arrange feminine gender and positive control, and 37 ℃ were reacted 1 hour;
(3) the PBST washing is 3 times, and every hole adds the anti-pig two of HRP mark rabbit anti-(3000 times of dilutions) of 100 μ l, and 37 ℃ were reacted 1 hour, PBST washing 3 times, every hole add substrate nitrite ion 100 μ l, lucifuge colour developing 10 minutes, every hole adds 100 μ l stop buffers, and readout instrument reads the OD value in the 450nm place; Result such as following table 1:
The OD value of table 1:40 part pig serum
The data based kit criterion of above table has the OD450nm of 22 parts of blood serum samples〉0.35.Antibody positive rate is 55%.
Performance Detection
1. specific test
Cross matching: collect PRV, PCV2, PRRSV, HCV antibody positive pig serum, after WestBlotting and ELISA check TTSuV1 antibody are negative, with the indirect ELISA method of foundation above-mentioned serum is detected, judge whether to have cross reaction.
Competitive inhibition test: standard positive and negative serum are carried out 2 times of gradient dilutions with dilution, and positive serum dilutes 2 groups, and negative serum dilutes 1 group.Get 1 group the dilution after positive serum and the negative serum of dilution mix with isopyknic TTSuV1 ORF1a recombinant protein (1 μ g/ml), be designated as respectively the 1st and the 2nd group; Get 1 group of positive serum after the dilution and mix with the equal-volume serum dilution, as the unrestraint control group, be designated as the 3rd group.Place 37 ℃ of effects after 2 hours 3 groups of serum, adopt the indirect ELISA method that establishes to detect, respectively organize the OD value of serum.
Table 2: cross-over experiment and the competitive experimental result that suppresses
* organize the mixed group of 1:TTSuV1 positive serum and TTSuV1 ORF1a recombinant protein.
The mixed group of * group 2:TTSuV1 negative serum and TTSuV1 ORF1a recombinant protein.
The mixed group of * * group 3:TTSuV1 positive serum and diluted protein solution.
2. repeated experiment
Get the coated ELISA Plate of 3 different batches, detect 10 parts of known serum, every block of plate of each sample arranges 3 repetitions, calculates interassay coefficient of variation CV=(SD/OD
450nmMean value) * 100%; Repeat in the same ELISA Plate is criticized, detect 10 parts of known positive serums, each sample arranges 3 repetitions, and record mean value calculates variation within batch coefficient CV=(SD/OD
450nmMean value) * 100%.
Table 3: repeated experiments in batch
Table 4: repeated experiments between batch
According to principle of statistics, as the OD of sample
450nmValue is greater than negative sample OD
450nmDuring value mean value+3 standard deviations (SD), can be judged to be the positive in 99.9% level; By statistics, the critical value of kit male/female antibody is OD
450Nm=0.35, OD
450nmBe higher than 0.35 and be judged to be the positive, OD
450nmBe lower than 0.35 and be judged to be feminine gender.
The TTSuV1 ORF1a recombinant protein that exists in the TTSuV1 Positive Sera can the competitive combination that suppresses Serum Antibody and solid-phase coating antigen, illustrates that this TTSuV1 ORF1a recombinant protein is better to the specificity of TTSuV1 antibody.Can find out in the data of table 2 and table 3 that the result of repeated experiment shows, kit criticize interior and batch between the maximum coefficient of variation (CV) be respectively 6.87% and 11.4%, all be lower than 15% of industry standard, illustrate that the repeatability of this ELISA method and stability are fine.
Above-mentioned embodiment only is preferred implementation of the present invention; can not limit the scope of protection of the invention with this, the variation of any unsubstantiality that those skilled in the art does on basis of the present invention and replacement all belong to the present invention's scope required for protection.
＜110〉research institute of Guangdong modern agriculture group company limited
＜120〉one boar I type thin circovirus virus TTSuV1 antibody indirect ELISA diagnostic kit and preparation sides thereof
〈130〉
〈160〉 9
〈170〉 PatentIn version 3.5
〈210〉 1
〈211〉 193
〈212〉 PRT
〈213〉 TTSuV1 ORF1a
〈400〉 1
met asp trp asp ala glu arg gln trp val trp lys glu gly glu
1 5 10 15
pro pro asp gln tyr gly tyr leu val gln tyr gly gly gly trp
20 25 30
gly ser gly glu ile ser leu ala gly leu tyr arg glu his leu
35 40 45
leu trp arg asn ser trp ser lys gly asn asp gly met asp leu
50 55 60
val arg tyr phe gly cys ser ile lys leu tyr pro thr gln asn
65 70 75
met asp tyr tyr phe trp trp asp thr asp phe lys pro gly tyr
80 85 90
glu asp lys val lys glu tyr ser gln pro ser val met met met
95 100 105
ala lys asn ser arg ile val ile ala arg asp arg ala pro asn
110 115 120
arg arg arg ile arg lys leu phe ile pro pro pro ser arg asp
125 130 135
thr thr gln trp gln phe gln thr asp phe cys asn arg pro leu
140 145 150
phe thr trp ala ala gly leu ile asp leu gln lys pro phe asp
155 160 165
ser lys gly ser phe arg asn ala trp trp met glu asp arg thr
170 175 180
glu asp gly asn met glu tyr ile glu lys trp gly arg
185 190
〈210〉 2
〈211〉 18
〈212〉 DNA
〈213〉 TTSuV1-F
〈400〉 2
TACACTTCCG GGTTCAGG 18
〈210〉 3
〈211〉 18
〈212〉 DNA
〈213〉 TTSuV1-R
〈400〉 3
AACGCCGCCA TCTCCTTA 18
〈210〉 4
〈211〉 18
〈212〉 DNA
〈213〉 TTSuV1cycle-F
〈400〉 4
TAAGGAGATG GCGGCGTT 18
〈210〉 5
〈211〉 18
〈212〉 DNA
〈213〉 TTSuV1cycle-R
〈400〉 5
CCTGAACCCG GAAGTGTA 18
〈210〉 6
〈211〉 29
〈212〉 DNA
〈213〉 TTSuV1-ORF1a-EcorF
〈400〉 6
CGGAATTCAT GGACTGGGAC GCAGAAAGA 29
〈210〉 7
〈211〉 29
〈212〉 DNA
〈213〉 TTSuV1-ORF1a-XbaR
〈400〉 7
GCTCTAGATC TTCCCCACTT TTCTATGTA 29
〈210〉 8
〈211〉 28
〈212〉 DNA
〈213〉 TTSuV1 ORF1 Hind III F
〈400〉 8
CCCAAGCTTC GCTTTAGACG ACGCAGAT 28
〈210〉 9
〈211〉 29
〈212〉 DNA
〈213〉 TTSuV1 ORF1 Xho I R
〈400〉 9
CCGCTCGAGC TCTGAAAATC TTCGTCGCT 29
Claims (10)
1. the thin circovirus virus TTSuV1 of boar I type antibody indirect ELISA diagnostic kit, it is characterized in that: it comprises with the coated ELISA Plate of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression, and the standard positive serum and the standard female serum that go out as the immunodiagnosis antigen selection with TTSuV1 ORF1, wherein the sequence with the TTSuV1 ORF1a recombinant protein of Yeast expression is SEQ ID NO:1.
2. the thin circovirus virus TTSuV1 of pig I type according to claim 1 antibody indirect ELISA diagnostic kit is characterized in that it also comprises
1) the anti-pig two anti-liquid of the rabbit of HRP mark: it is the dilution proportion of 1 ﹕ 2000-3000 according to volume ratio that the anti-pig two of rabbit resists with HRP protection liquid, and consumption is 100 μ l;
2) antibody diluent: 0.1%BSA1.0g adds PBS to 1000ml, and consumption is 100 μ l;
3) nitrite ion: 50mg TMB, 10mL DMSO, 9.8g citric acid and 1.2ml 30% H
2O
2Adding distil water is settled to 1000ml, and consumption is 100 μ l;
4) stop buffer: distilled water 178.3mL and 98% concentrated sulphuric acid 21.7mL mix, and consumption is 100 μ l.
3. the thin circovirus virus TTSuV1 of pig I type according to claim 2 antibody indirect ELISA diagnostic kit, it is characterized in that: it comprises that also the pH value is 7.4,0.2g KH
2PO
4, 2.9g Na
2HPO
412H
2O, 8.0g NaCl, 0.2g KCl, 0.5mL 0.05%(V/V) Tween-20, add the eluent that distilled water is settled to 100ml.
4. the thin circovirus virus TTSuV1 of each described pig I type antibody indirect ELISA diagnostic kit according to claim 1-3, it is characterized in that: in the coated ELISA Plate of the TTSuV1 ORF1a restructuring egg envelope antigen of Yeast expression, coated concentration is 0.25～1 μ g/ml.
5. the preparation method of the thin circovirus virus TTSuV1 of boar I type antibody indirect ELISA diagnostic kit is characterized in that it may further comprise the steps:
1) preparation of the TTSuV1 ORF1a recombinant protein envelope antigen of Yeast expression
A. TTSuV1 ORF1a retrieval: in conjunction with TTSuV1 sequence information in the Genebank database, at virus genome sequence conserved regions design diagnostic primers SEQ ID NO:2 and primer SEQ ID NO:3; Use rTaq DNA polymerase pcr amplification purpose fragment, the PCR amplified production carries out electrophoresis, and purifying, order-checking obtain to have checked order row; Then at the row basis reverse amplimer SEQ ID NO:4 of design and the primer SEQ ID NO:5 of checking order, amplification comprises the virus gene sequence of TTSuV1 ORF1 complete sequence; Use the LA Taq archaeal dna polymerase PCR purpose fragment that oppositely increases, pcr amplification product is carried out electrophoresis, purifying, obtain TTSuV1 ORF1a albumen;
B. the Yeast expression of TTSuV1 ORF1a: use Primer Premier5.0 design and express primer SEQ ID NO:6 and primer SEQ ID NO:7, take the positive nucleic acid of TTSuV1 as template, use the PCR reaction of Kod plus high-fidelity enzyme, behind electrophoresis, the purifying purpose band, double digestion PCR product and carrier connect Transformed E. coli:DH5a, extract recombinant plasmid pPICZ a-TTSuV1 ORF1a, the linearization of Sac I single endonuclease digestion; Saccharomycete X-33 is prepared into competent cell; getting the 80mL competent cell mixes with the linearizing recombinant expression plasmid of 5～10mg; change 0.2cm electricity revolving cup over to; electricity transforms; bacterium liquid after electricity transforms with 10 times of dilutions of 1 ml ice bath sorbierite after; coating basic dextrose culture-medium selects on the flat board; place 30 ℃ to cultivate 2～3 d; have high Zeocin resistance and well-grown bacterial strain as expressing strain in the screening of the YPD flat board of 200-1000mg/ml concentration Zeocin; inoculate single bacterium colony in 25 ml BMGY nutrient culture media, be cultured to OD in 30 ℃
600nm4.0; Centrifugal, collection thalline, with the resuspended cultivation of 100 ml BMMY nutrient culture media, 1ml takes a sample every day, and to add pure methyl alcohol be 0.5% in nutrient culture media to final concentration, abduction delivering 6 d, the centrifugal collection supernatant of sample thief is made SDS-PAGE and is analyzed, and take Anti-HIS-antibody as first antibody, Western Blotting filters out height copy of step 1) TTSuV1 ORF1a albumen;
C. college entrance examination shellfish that filters out TTSuV1 ORF1a purifying: get step 2), with albumen in the 50% ammonium sulfate precipitation supernatant, dissolving, after the restructuring TTSuV1 ORF1a of the method purifying yeast expression system expression of application affinity chromatography dialyses in PBS, using the BCA method carries out quantitatively, obtain the TTSuV1 ORF1a recombinant protein of Yeast expression, sequence is SEQ ID NO:1;
2) screening of standard positive serum and standard female serum
A. make up the TTSuV1 ORF1 prokaryotic expression carrier that inserts the pET-21 empty carrier: use Primer Premier5.0 design primer SEQ ID NO:8 and SEQ ID NO:9, take the positive nucleic acid of TTSuV1 as template, pcr amplification, reclaim purifying PCR purpose fragment, double digestion PCR product and pET-21 empty carrier, connect, Transformed E. picking monoclonal bacterium colony behind the coli:DH5a, extract plasmid, with Plasmid Transformation E. coli:Rosetta (DE3) the pLysS competent cell that correctly makes up, identify positive colony, positive colony is inoculated 2 ml LA fluid nutrient mediums, 37 ℃, 180 rpm reach at 0.6 o'clock to bacterium liquid OD value and add 1mM IPTG, induced 6 hours, centrifugal collection bacterium mud adds Loading buffer and boils sample, and the SDS-PAGE analyzing proteins is expressed, use Western blotting checking, obtain TTSuV1 ORF1 recombinant protein;
B. the screening of TTSuV1 standard positive serum and standard female serum: preparation TTSuV1 ORF1 recombinant protein, cut after glue purification, the emulsification with dosage 2 monthly ages of immunity of 5 mg/ heads piglet without TTSuV1 cause of disease (PCR detection), repeat immunity with same dose after 1 month, after 15 days, Western Blotting and ELISA experiment are carried out in blood sampling, will experimental results show that positive serum is that TTSuV1 antibody is with reference to positive serum; Utilize the TTSuV1 ORF1a recombinant protein of Yeast expression to be the solid-phase coating thing, 50 parts of serum are carried out ELISA detect, filtering out the serum consistent with reference positive serum Western blotting and ELISA experiment reaction signal is standard positive serum; The immunological experiment no signal, and the serum of PCR detection TTSuV1 cause of disease feminine gender is standard female serum;
3) the coated concentration of defined antigen and two anti-dilutabilitys
Adopt the chessboard titrimetry, get first positive OD
450nmValue and then is therefrom found out positive serum OD about 1
450nmValue and negative serum OD
450nmThe antigen concentration of the reacting hole that the ratio of value is the highest and two anti-dilutabilitys are best antigen coated concentration and two anti-dilutabilitys;
4) assembling of kit
According to the coated concentration that determines, with the TTSuV1 ORF1a recombinant protein coated elisa plate of Yeast expression, 1 of ELISA Plate, each 1 pipe of standard yin and yang attribute serum, 1 bottle of antibody diluent, 1 bottle of ELIAS secondary antibody, 1 bottle of substrate nitrite ion, 1 bottle of stop buffer, operational manual portion are assembled into the ELISA kit.
6. the preparation method of the thin circovirus virus TTSuV1 of pig I type according to claim 5 antibody indirect ELISA diagnostic kit, it is characterized in that: using rTaq DNA polymerase pcr amplification purpose fragment specific procedure in the step 1) is: 94 ℃ of denaturation 5min, 94 ℃ of sex change 30 s, 52 ℃ of annealing 30 s, 72 ℃ are extended 30s, 35 circulations are set, and 72 ℃ are extended 8min; The Ago-Gel of usefulness 1.0% carried out electrophoresis to the PCR amplified production after amplification was finished.
7. the preparation method of the thin circovirus virus TTSuV1 of pig I type according to claim 5 antibody indirect ELISA diagnostic kit, it is characterized in that: using the LA Taq archaeal dna polymerase PCR purpose fragment specific procedure that oppositely increases in the step 1) is 94 ℃ of denaturation 5min, 94 ℃ of sex change 30 s, 52 ℃ of annealing 30 s, 72 ℃ are extended 120s, 30 circulations are set, and 72 ℃ are extended 8 min; The Ago-Gel of usefulness 0.8% carried out electrophoresis to pcr amplification product after amplification was finished.
8. the preparation method of the thin circovirus virus TTSuV1 of pig I type according to claim 5 antibody indirect ELISA diagnostic kit, it is characterized in that: the specific procedure of the PCR of Kod plus high-fidelity enzyme reaction is 94 ℃ of denaturation 5min in the step 1), 94 ℃ of sex change 30 s, 52 ℃ of annealing 30 s, 68 ℃ are extended 45s, 30 circulations are set, and 68 ℃ are extended 8 min.
9. the preparation method of the thin circovirus virus TTSuV1 of pig I type according to claim 5 antibody indirect ELISA diagnostic kit is characterized in that: step 2) in the condition of pcr amplification be 94 ℃ of 5min; 94 ℃ of 30 S, 52 ℃ of 30 S, 68 ℃ of 120 S, 30 circulations; 68 ℃ are extended 10 min.
10. the preparation method of the thin circovirus virus TTSuV1 of pig I type according to claim 5 antibody indirect ELISA diagnostic kit, it is characterized in that: the concrete steps of chessboard titrimetry are in the step 3): with the TTSuV1 ORF1a recombinant protein of Yeast expression on the ELISA Plate from left to right take initial concentration as 4 mg/ml according to after hole concentration be 1/2 gradient concentration dilution coated elisa plate of hole concentration formerly, last row are as blank, every hole is coated with 100 ml, placed 1 hour for 37 ℃, wash plate 1 time with PBST, every hole adds 10% lamb serum, 37 ℃ were sealed 1 hour, positive control serum and negative control sera carry out respectively gradient dilution, join in the ELISA Plate from top to bottom, every hole adds 100ml, last column is the serum-free contrast, 37 ℃ were reacted 1 hour, PBST washing 3 times, the anti-pig two of HRP mark rabbit that every hole adds 100 ml resists, 37 ℃ were reacted 1 hour, PBST washing 3 times, every hole adds substrate nitrite ion 100 ml, lucifuge colour developing 10 minutes, every hole adds 100 ml stop buffers, and readout instrument reads the OD value in the 450nm place, and 620nm is as the reference wavelength.
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