CN102998453B - 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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- CN102998453B CN102998453B CN201210485102.8A CN201210485102A CN102998453B CN 102998453 B CN102998453 B CN 102998453B CN 201210485102 A CN201210485102 A CN 201210485102A CN 102998453 B CN102998453 B CN 102998453B
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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 the antibody horizontal of TTSuV1 antibody indirect ELISA diagnostic kit for detection of TTSuV1 in 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-non-penta type post-transfusion hepatitis patient's of a routine unknown cause of disease 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, the TTV of pig is divided into the thin Circovirus (Iotatorquevirus) of finger ring Viraceae (Anelloviridae) by international virus taxis association (ICTV), 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, Iowa, and Thailand, Ontario, Korea S, Spain, 154 parts of pig serum in these six country variant areas of Quebec and Saskatchewan detect, and TTSuV2 positive rate can be up to 66.2% (102/154).Aramouni etc. have confirmed there is certain contact between TTSuV1 and pmws (PMWS) and the scorching nephrotic syndrome of pigskin (PDNS) and have found TTSuV2 and porcine pathogen coinfection that other are known 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 this feature has 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 of TTSuV1 and TTSuV2, comparatively focus on the infection conditions of TTSuV2, the infection of TTSuV2 seems to play a part aspect PMWS and PDNS illness even more importantly strengthening, and has had report to set up " the blocking-up ELISA method " of TTSuV2 antibody diagnosis at present.But in nearly all report, TTSuV1 exceedes 30% recall rate (PCR method) and is enough to cause the attention of people to TTSuV1 harm in pig serum.The method of having set up gradually 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 defect 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 stability, can detect the antibody horizontal of TTSuV1 in 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 the coated ELISA Plate of TTSuV1 ORF1a recombinant protein envelope antigen with Yeast expression, and the standard positive serum and the standard female serum that go out as immunodiagnosis antigen selection with the unique structural proteins ORF1 of TTSuV1, be wherein SEQ ID NO:1 by the sequence of the TTSuV1 ORF1a recombinant protein of Yeast expression.
In the present invention, the described thin circovirus virus TTSuV1 of pig I type antibody indirect ELISA diagnostic kit, it also comprises
1) the anti-liquid of the anti-pig two 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, 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 preferred scheme, the thin circovirus virus TTSuV1 of above-mentioned pig I type antibody indirect ELISA diagnostic kit also comprises that 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 distilled water and be settled to the eluent of 100ml.
In a preferred scheme, in the described thin circovirus virus TTSuV1 of pig I type antibody indirect ELISA diagnostic kit, with the coated concentration of the coated ELISA Plate of the TTSuV1 ORF1a restructuring egg envelope antigen of Yeast expression be 0.25～1 μ g/ml.
The preparation method of the thin circovirus virus TTSuV1 of one boar I type antibody indirect ELISA diagnostic kit, it comprises the following 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 Genebank database, at virus genome sequence conserved regions design diagnostic primers SEQ ID NO:2 and primer SEQ ID NO:3; Application rTaq archaeal dna polymerase pcr amplification object fragment, pcr amplification product carries out electrophoresis, and purifying, order-checking obtain the row that checked order; Then in sequence basis, design reverse amplimer SEQ ID NO:4 and primer SEQ ID NO:5 surveying, amplification comprises the virus gene sequence of TTSuV1 ORF1 complete sequence; The application LA TaqDNA polymerase PCR object fragment that oppositely increases, carries out electrophoresis to pcr amplification product, purifying, obtains TTSuV1 ORF1a albumen;
The Yeast expression of b.TTSuV1 ORF1a: primer SEQ ID NO:6 and primer SEQ ID NO:7 are expressed in application Primer Premier5.0 design, taking the positive nucleic acid of TTSuV1 as template, the PCR reaction of application Kod plus high-fidelity enzyme, after electrophoresis, purifying object 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; proceed to 0.2cm electricity revolving cup; electricity transforms; bacterium liquid after electricity transforms is with after 10 times of dilutions of 1ml ice bath sorbierite; coating basic dextrose culture-medium selects on flat board; be placed in 30 DEG C and cultivate 2～3d; on the YPD flat board of 200-1000 μ g/ml concentration Zeocin, screening has high Zeocin resistance and well-grown bacterial strain as expressing strain; inoculate single bacterium colony in 25ml BMGY nutrient culture media, be cultured to OD in 30 DEG C
600nm≈ 4.0; Centrifugal, collection thalline, with the resuspended cultivation of 100ml BMMY nutrient culture media, sample 1ml 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 taking 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) in college entrance examination shellfish filtering out, with albumen in 50% ammonium sulfate precipitation supernatant, dissolve, after the restructuring TTSuV1 ORF1a of the method purifying yeast expression system expression of application affinity chromatography dialyses in PBS, application BCA method is carried out quantitatively, the TTSuV1 ORF1a recombinant protein that obtains Yeast expression, sequence is SEQ ID NO:1;
2) screening of standard positive serum and standard female serum
A. build the TTSuV1 ORF1 prokaryotic expression carrier that inserts pET-21 empty carrier: application PrimerPremier5.0 design primer SEQ ID NO:8 and SEQ ID NO:9, taking the positive nucleic acid of TTSuV1 as template, pcr amplification, reclaim purifying PCR object fragment, double digestion PCR product and pET-21 empty carrier, connect, picking monoclonal bacterium colony after Transformed E .coli:DH5 α, extract plasmid, by plasmid Transformed E .coli:Rosetta (DE3) the pLysS competent cell correctly building, identify positive colony, positive colony inoculation 2ml LA fluid nutrient medium, 37 DEG C, 180rpm reaches at 0.6 o'clock to bacterium liquid OD value and adds 1mM IPTG, induce 6 hours, centrifugal collection bacterium mud, add Loading buffer to boil sample, SDS-PAGE analyzing proteins is expressed, application 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, emulsification with dosage 2 monthly ages of immunity of 5mg/ head piglet without TTSuV1 cause of disease (PCR detection), after 1 month, repeat immunity with same dose, 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; The TTSuV1 ORF1a recombinant protein that utilizes Yeast expression is solid-phase coating thing, and 50 parts of serum are carried out to ELISA detection, filters out and be standard positive serum with reference to the consistent serum of positive serum Western blotting and ELISA experiment reaction signal; Immunological experiment no signal, and the serum of PCR detection TTSuV1 cause of disease feminine gender is standard female serum;
3) determine antigen coated concentration and two anti-dilutabilitys
Adopt chessboard titrimetry, first get positive OD
450nmvalue is in 1 left and right, and then therefrom finds out positive serum OD
450nmvalue and negative serum OD
450nmantigen concentration and the two anti-dilutabilitys of the reacting hole that the ratio of value is the highest are best antigen coated concentration and two anti-dilutabilitys;
4) assembling of kit
According to the coated concentration determining, 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 ELISA kit.
In above-mentioned preparation method, step 1) application rTaq archaeal dna polymerase pcr amplification object fragment specific procedure is: 94 DEG C of denaturation 5min, and 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 72 DEG C are extended 30s, and 35 circulations are set, and 72 DEG C are extended 8min; The Ago-Gel of the rear use 1.0% that increased carries out electrophoresis to pcr amplification product.
In above-mentioned preparation method, the step 1) application LA Taq archaeal dna polymerase PCR object fragment specific procedure that oppositely increases is 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, and 52 DEG C of annealing 30s, 72 DEG C are extended 120s, and 30 circulations are set, and 72 DEG C are extended 8min; The Ago-Gel of the rear use 0.8% that increased carries out electrophoresis to pcr amplification product.
In above-mentioned preparation method, the specific procedure of the PCR reaction of step 1) Kod plus high-fidelity enzyme is 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, and 52 DEG C of annealing 30s, 68 DEG C are extended 45s, and 30 circulations are set, and 68 DEG C are extended 8min.
In above-mentioned preparation method, step 2) condition of pcr amplification is 94 DEG C of 5min; 94 DEG C of 30S, 52 DEG C of 30S, 68 DEG C of 120S, 30 circulations; 68 DEG C are extended 10min.
In above-mentioned preparation method, the concrete steps of step 3) chessboard titrimetry are: by the TTSuV1ORF1a recombinant protein of Yeast expression in ELISA Plate from left to right taking initial concentration as 4 μ g/ml according to posterior hole concentration 1/2 the gradient concentration dilution coated elisa plate as hole concentration formerly, last is listed as blank, every hole is coated with 100 μ l, place 1 hour for 37 DEG C, wash plate 1 time with PBST, every hole adds 10% lamb serum, 37 DEG C are sealed 1 hour, positive control serum and negative control sera carry out respectively gradient dilution, join from top to bottom in ELISA Plate, every hole adds 100 μ l, last column is serum-free contrast, 37 DEG C are reacted 1 hour, PBST washing 3 times, every hole adds the anti-pig two of the HRP mark rabbit of 100 μ l anti-, 37 DEG C are reacted 1 hour, PBST washing 3 times, every hole adds substrate nitrite ion 100 μ l, lucifuge colour developing 10 minutes, every hole adds 100 μ L stop buffers, in 450nm place, readout instrument reads OD value, 620nm is as with reference to wavelength.
Compared to existing technology, beneficial effect of the present invention is: the thin circovirus virus TTSuV1 of the pig I type antibody indirect ELISA diagnostic kit of preparing by preparation method of the present invention, the TTSuV1 ORF1a recombinant protein existing in 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 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 is below 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 the coated ELISA Plate of TTSuV1 ORF1a recombinant protein envelope antigen with Yeast expression, and the standard positive serum going out as immunodiagnosis antigen selection with TTSuV1 ORF1 and standard female serum, be wherein SEQ ID NO:1 by the sequence of the TTSuV1 ORF1a recombinant protein of Yeast expression, the coated concentration of ELISA Plate for coated concentration be 0.25 μ g/ml; The anti-liquid 12ml of the anti-pig two of rabbit of HRP mark: the rabbit anti-lgM of anti-pig two and HRP protection liquid are 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 the coated ELISA Plate of TTSuV1 ORF1a recombinant protein envelope antigen with Yeast expression, and the standard positive serum going out as immunodiagnosis antigen selection with TTSuV1 ORF1 and standard female serum, be wherein SEQ ID NO:1 by the sequence of the TTSuV1 ORF1a recombinant protein of Yeast expression, the coated concentration of ELISA Plate for coated concentration be 0.5 μ g/ml; The anti-liquid 12ml of the anti-pig two of rabbit of HRP mark: the rabbit anti-lgM of anti-pig two and HRP protection liquid are 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 the coated ELISA Plate of TTSuV1 ORF1a recombinant protein envelope antigen with Yeast expression, and the standard positive serum going out as immunodiagnosis antigen selection with TTSuV1 ORF1 and standard female serum, be wherein SEQ ID NO:1 by the sequence of the TTSuV1 ORF1a recombinant protein of Yeast expression, the coated concentration of ELISA Plate for coated concentration be 1 μ g/ml; The anti-liquid 12ml of the anti-pig two of rabbit of HRP mark: 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
Apply 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 of purifying is diluted to 0.5 μ g/ml with coating buffer, is added in ELISA Plate, every hole 100 μ l, place 1 hour for 37 DEG C.Wash plate 1 time with PBST, every hole adds the PBST containing 10% lamb serum, 37 DEG C of sealings 1 hour;
(2) by 40 times of PBST dilutions for blood serum sample to be checked, be added in ELISA Plate, every hole 100 μ l, arrange feminine gender and positive control, 37 DEG C of reactions 1 hour;
(3) PBST washing 3 times, every hole adds the anti-pig two of the HRP mark rabbit of 100 μ l anti-(3000 times of dilutions), and 37 DEG C are reacted 1 hour, PBST washing 3 times, every hole adds substrate nitrite ion 100 μ l, lucifuge colour developing 10 minutes, every hole adds 100 μ l stop buffers, and in 450nm place, readout instrument reads OD value; Result is as following table 1:
The OD value of table 1:40 part pig serum
The data of above table, according to kit criterion, have the OD450nm>0.35 of 22 parts of blood serum samples.Antibody positive rate is 55%.
Performance Detection
1. specific test
Cross matching: collect PRV, PCV2, PRRSV, HCV antibody positive pig serum, through WestBlotting and ELISA inspection TTSuV1 antibody negative after, above-mentioned serum is detected with the indirect ELISA method of foundation, judge whether to there is cross reaction.
Competitive inhibition test: standard positive and negative serum are carried out to 2 times of gradient dilutions with dilution, and positive serum dilutes 2 groups, and negative serum dilutes 1 group.Get 1 group 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 dilution and mix with equal-volume serum dilution, as unrestraint control group, be designated as the 3rd group.3 groups of serum were placed in to 37 DEG C of effects after 2 hours, adopt the indirect ELISA method establishing 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 3 ELISA Plate that different batches is coated, 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%; In criticizing in same ELISA Plate, repeat, detect 10 parts of known positive serums, each sample arranges 3 repetitions, records mean value, calculates variation within batch coefficient CV=(SD/OD
450nmmean value) × 100%.
Table 3: repeat experiment in batch
Table 4: repeat experiment between batch
According to principle of statistics, as the OD of sample
450nmvalue is greater than negative sample OD
450nmwhen value mean value+3 standard deviations (SD), can in 99.9% level, be judged to be the positive; By statistics, the critical value of kit male/female antibody is OD
450nm=0.35, OD
450nmbe judged to be the positive, OD higher than 0.35
450nmbe judged to be feminine gender lower than 0.35.
The TTSuV1 ORF1a recombinant protein existing in 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.In the data of table 2 and table 3, can find out, 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 lower than 15% of industry standard, illustrate that the repeatability of this ELISA method and stability are fine.
Above-mentioned embodiment is only the preferred embodiment 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 > Guangdong Institute of Modern Agricultural Group Co., Ltd.
< 120 > mono-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 the coated ELISA Plate of TTSuV1ORF1a recombinant protein envelope antigen with Yeast expression, and the standard positive serum going out as immunodiagnosis antigen selection with TTSuV1ORF1 and standard female serum, be wherein SEQ ID NO:1 by the sequence of the TTSuV1ORF1a recombinant protein of Yeast expression.
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-liquid of the anti-pig two 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, consumption is 100 μ l;
3) nitrite ion: 50mg TMB, 10mL DMSO, 9.8g citric acid and 1.2ml30%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, is characterized in that: it also comprises that pH value is 7.4,0.2g KH
2pO
4, 2.9g Na
2hPO
412H
2o, 8.0g NaCl, 0.2g KCl, 0.5mL0.05% (V/V) Tween-20, adds distilled water and is settled to the eluent of 100ml.
4. according to the thin circovirus virus TTSuV1 of the pig I type antibody indirect ELISA diagnostic kit described in claim 1-3 any one, it is characterized in that: with in the coated ELISA Plate of the TTSuV1ORF1a recombinant protein 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 comprises the following steps:
1) preparation of the TTSuV1ORF1a recombinant protein envelope antigen of Yeast expression
A.TTSuV1ORF1a retrieval: in conjunction with TTSuV1 sequence information in Genebank database, at virus genome sequence conserved regions design diagnostic primers SEQ ID NO:2 and primer SEQ ID NO:3; Application rTaq archaeal dna polymerase pcr amplification object fragment, pcr amplification product carries out electrophoresis, and purifying, order-checking obtain the row that checked order; Then in sequence basis, design reverse amplimer SEQ ID NO:4 and primer SEQ ID NO:5 surveying, amplification comprises the virus gene sequence of TTSuV1ORF1 complete sequence; The application LA Taq archaeal dna polymerase PCR object fragment that oppositely increases, carries out electrophoresis to pcr amplification product, purifying, obtains TTSuV1ORF1a protein gene;
The Yeast expression of b.TTSuV1ORF1a: primer SEQ ID NO:6 and primer SEQ ID NO:7 are expressed in application Primer Premier5.0 design, taking above-mentioned TTSuV1ORF1a protein gene as template, the PCR reaction of application Kod plus high-fidelity enzyme, after electrophoresis, purifying object band, double digestion PCR product and carrier, connect Transformed E .coli:DH5a, extract recombinant plasmid pPICZ a-TTSuV1ORF1a, the linearization of Sac I single endonuclease digestion; Saccharomycete X-33 is prepared into competent cell; getting 80mL competent cell mixes with the linearizing recombinant expression plasmid of 5～10mg; proceed to 0.2cm electricity revolving cup; electricity transforms; bacterium liquid after electricity transforms is with after 10 times of dilutions of 1ml ice bath sorbierite; coating basic dextrose culture-medium selects on flat board; be placed in 30 DEG C and cultivate 2～3d; on the YPD flat board of 200-1000mg/ml concentration Zeocin, screening has high Zeocin resistance and well-grown bacterial strain as expressing strain; inoculate single bacterium colony in 25ml BMGY nutrient culture media, be cultured to OD in 30 DEG C
600nm≈ 4.0; Centrifugal, collection thalline, with the resuspended cultivation of 100ml BMMY nutrient culture media, sample 1ml 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 taking Anti-HIS-antibody as first antibody, Western Blotting filters out step 1) height of TTSuV1ORF1a albumen copy;
C.TTSuV1ORF1a purifying: get the height filtering out copy in step b, with albumen in 50% ammonium sulfate precipitation supernatant, dissolve, after the restructuring TTSuV1ORF1a of the method purifying yeast expression system expression of application affinity chromatography dialyses in PBS, application BCA method is carried out quantitatively, the TTSuV1ORF1a recombinant protein that obtains Yeast expression, sequence is SEQ ID NO:1;
2) screening of standard positive serum and standard female serum
A. build the TTSuV1ORF1 prokaryotic expression carrier that inserts pET-21 empty carrier: application Primer Premier5.0 design primer SEQ ID NO:8 and SEQ ID NO:9, taking the positive nucleic acid of TTSuV1 as template, pcr amplification, reclaim purifying PCR object fragment, double digestion PCR product and pET-21 empty carrier, connect, picking monoclonal bacterium colony after Transformed E .coli:DH5a, extract plasmid, by plasmid Transformed E .coli:Rosetta (DE3) the pLysS competent cell correctly building, identify positive colony, positive colony inoculation 2ml LA fluid nutrient medium, 37 DEG C, 180rpm reaches at 0.6 o'clock to bacterium liquid OD value and adds 1mM IPTG, induce 6 hours, centrifugal collection bacterium mud, add Loading buffer to boil sample, SDS-PAGE analyzing proteins is expressed, application Western blotting checking, obtain TTSuV1ORF1 recombinant protein,
The screening of b.TTSuV1 standard positive serum and standard female serum: preparation TTSuV1ORF1 recombinant protein, cut after glue purification, emulsification with dosage 2 monthly ages of immunity of 5mg/ head piglet without TTSuV1 cause of disease, after 1 month, repeat immunity with same dose, 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; The TTSuV1ORF1a recombinant protein that utilizes Yeast expression is solid-phase coating thing, and 50 parts of serum are carried out to ELISA detection, filters out and be standard positive serum with reference to the consistent serum of positive serum Western blotting and ELISA experiment reaction signal; Immunological experiment no signal, and the serum of PCR detection TTSuV1 cause of disease feminine gender is standard female serum;
3) determine antigen coated concentration and two anti-dilutabilitys
Adopt chessboard titrimetry, first get positive OD
450nmvalue is in 1 left and right, and then therefrom finds out positive serum OD
450nmvalue and negative serum OD
450nmantigen concentration and the two anti-dilutabilitys of the reacting hole that the ratio of value is the highest are best antigen coated concentration and two anti-dilutabilitys;
4) assembling of kit
According to the coated concentration determining, with the TTSuV1ORF1a 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 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: step 1) in application rTaq archaeal dna polymerase pcr amplification object fragment specific procedure be: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 72 DEG C are extended 30s, 35 circulations are set, and 72 DEG C are extended 8min; The Ago-Gel of the rear use 1.0% that increased carries out electrophoresis to pcr amplification product.
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: step 1) in the application LA Taq archaeal dna polymerase PCR object fragment specific procedure that oppositely increases be 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 72 DEG C are extended 120s, 30 circulations are set, and 72 DEG C are extended 8min; The Ago-Gel of the rear use 0.8% that increased carries out electrophoresis to pcr amplification product.
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: step 1) in the specific procedure of PCR reaction of Kod plus high-fidelity enzyme be 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 52 DEG C of annealing 30s, 68 DEG C are extended 45s, 30 circulations are set, and 68 DEG C are extended 8min.
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 DEG C of 5min; 94 DEG C of 30S, 52 DEG C of 30S, 68 DEG C of 120S, 30 circulations; 68 DEG C are extended 10min.
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: step 3) in the concrete steps of chessboard titrimetry be: by the TTSuV1ORF1a recombinant protein of Yeast expression in ELISA Plate from left to right taking initial concentration as 4 μ g/ml according to posterior hole concentration 1/2 the gradient concentration dilution coated elisa plate as hole concentration formerly, last is listed as blank, every hole is coated with 100 μ l, place 1 hour for 37 DEG C, wash plate 1 time with PBST, every hole adds 10% lamb serum, 37 DEG C are sealed 1 hour, positive control serum and negative control sera carry out respectively gradient dilution, join from top to bottom in ELISA Plate, every hole adds 100 μ l, last column is serum-free contrast, 37 DEG C are reacted 1 hour, PBST washing 3 times, every hole adds the anti-pig two of the HRP mark rabbit of 100 μ l anti-, 37 DEG C are reacted 1 hour, PBST washing 3 times, every hole adds substrate nitrite ion 100 μ l, lucifuge colour developing 10 minutes, every hole adds 100 μ l stop buffers, in 450nm place, readout instrument reads OD value, 620nm is as with reference to wavelength.
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