CN103235121A - Indirect ELISA kit for detecting porcine transfusion transmitted virus 2 (TTV2) antibody - Google Patents

Abstract

The invention relates to an indirect ELISA kit for detecting a porcine transfusion transmitted virus 2 (TTV2) antibody and belongs to the field of biotechnology. The invention comprises antigen recombinant protein preparation, indirect ELISA establishing, and use of determination standard and clinical serological test. Through pcoldI prokaryotic expression vectors, a gene engineering bacterium pcoldI-ORF1 for expression of a porcine TTV2ORF1 truncated protein is constructed and the expressed antigen recombinant protein is purified and is used as an antigen so that an indirect ELISA detection method is established. The indirect ELISA detection method is used for detecting a TTV2 antibody level of porcine serum, has good repeatability and high singularity, can be used for porcine TTV2 serology investigation and is a fast and simple serological test method for prevention, treatment and prevalence state control of porcine transfusion transmitted diseases. The indirect ELISA detection method utilizes the porcine TTV2ORF1 recombinant protein to detect the porcine TTV2 antibody first in China.

Description

A kind of indirect ELISA reagent kit that detects pig blood transfusion transmitted virus 2 type antibody

Technical field

The present invention relates to a kind of indirect ELISA reagent kit for detection of pig blood transfusion transmitted virus 2 types (being pig TTV2) antibody, belong to the Vet Biotechnology field.

Background technology

TTV(Torque Teno virus) have another name called the blood transfusion transmitted virus, belong to finger ring Viraceae (Anelloviridae), the thin Circovirus of type in the ninth of the ten Heavenly Stems (Iotatorquevirus) is a kind of sub-thread ring-type minus-strand dna virus of not having cyst membrane.Equaled to find in 1997 by Japanese scholar Nishizawa the earliest, obtain because this virus is separated in the hepatitis body, therefore suspect that it is relevant with hepatitis, thereby caused and show great attention to.Various countries have carried out epidemiology survey to TTV infection conditions in the national different crowd subsequently, found that TTV DNA positive rate is generally more than 10% among the crowd.Verified at present, except the mankind can infect TTV, in primate (chimpanzee, anthropoid cape and monkey), domestic animal (pig, ox, sheep, dog, cat, chicken) and other animal (mouse, tree shrew and camel) body, all detected the existence of TTV in succession.Studies show that TTV extensively exists in world swinery, and may be relevant with causing of some disease, therefore draw attention in the whole world.

Segales J etc. are in the period of 1985-2005,162 parts of blood serum samples that pick up from 99 pig farms of Spain carry out TTV and detect, the result all detects pig TTV in all times, in sow and pork pig, the positive rate of TTV1 is respectively 34.2% and 30.9%, the positive rate of TTV2 be respectively 46.6% and 62.8%, TTV1 and TTV2 coinfection rate be respectively 19.8% and 24.5%, this result shows at least at pig TTV in 1985 just Already in the Spain pig farm.Martinez etc. investigate the infection conditions of TTV in 178 wild boars of Spain, the total positives rate is 84% as a result, the positive rate of TTV1 and TTV2 be respectively 58% and the common positive rate that infects of 66%, TTV1 and TTV2 be 18%, but this result is considered to relevant with the geographic position.King Meng Meng etc. detect domestic 258 parts of pig blood samples from 7 provinces such as Guangdong, Fujian and Jiangxi and tissue sample first, the result shows that TTV1 and TTV2 infect positive rate in the swinery and is respectively 37.6% and 82.6%, the TTV2 positive rate is apparently higher than TTV1, the mixed infection rate of the two is 34.5%, confirm to exist in China swinery TTV to infect, and comparatively popular with TTV2.Frequent and the common infection of other virus generations of this virus.McKeown N E etc. studies show that the mixed infection of this virus and known cause of disease can increase severity of disease, and this virus has the potential harm that the people is given in kind of propagation of striding, and this also points out us can not ignore research to this virus.

Diagnosis and anti-work processed for the New Development eqpidemic disease are the primary keys that solves of researcher, have only at present by the nucleic acid of round pcr amplification TTV and diagnose, set up this viral antibody detection method, extremely meaningful to evaluation of this sick epidemiology survey, immune effect etc., and ELISA detect because having that flux is big, the result is definite, highly sensitive, be easy to characteristics such as basic unit's popularizations and be favourably welcome, become present most widely used a kind of serology detection method.Because pig TTV still can not well be bred in vitro culture at present, so become optimal selection by the antigen protein of molecular biology method great expression TTV and with this ELISA that sets up anti-TTV antibody in the detection pig serum as envelope antigen.

Pig TTV is divided into two hypotypes, i.e. TTV1 and TTV2, and an outstanding feature that makes a variation between the genome is, compares with TTV2, TTV1 has a large amount of variant sites, i.e. the variability of TTV1 big than TTV2.In TTV1, always have 1349 variant sites, on average each variant sites has 1.27 substitutes, and variant sites is fewer in TTV2, has 670.In addition, sudden change pattern between the ORFs also is different: may encode viral capsid proteins (Cap) and copy associated protein (Rep) of ORF1, more conservative, at the rate of change of the 3rd position of codon than higher, although reported some conservative regions of ORF2 and ORF3, but comparatively speaking, the conservative property of the two is lower.And the popularity of China TTV shows that the TTV2 infection rate is apparently higher than TTV1, and China TTV2 is comparatively popular.

Therefore, the present invention is template with the relative conservative gene ORF1 gene of the popular hypotype TTV2 of pig TTV, carry out prokaryotic expression, purifying, utilize the recombinant protein of purifying to set up indirect ELISA detection method as antigen, for this viral early diagnosis and large-scale detection provide technological means.

Summary of the invention

Technical matters

The purpose of this invention is to provide a kind of indirect ELISA reagent kit that can detect pig TTV2 serum antibody fast, easily, namely utilize the pcoldI prokaryotic expression carrier, made up the carrier that to express pig TTV2 ORF1 portion gene, and will wrap by elisa plate behind the recombinant protein purification of expressing, to detect the antibody horizontal of TTV2 in the pig serum.

Technical scheme

The present invention realizes by following steps:

A kind of indirect ELISA reagent kit that can detect pig TTV2 serum antibody fast, easily is provided with the antibody test plate, confining liquid, sample diluting liquid, cleansing solution, enzyme conjugates working fluid, enzyme substrate solution, stop buffer, positive control and negative control in the kit; Wherein the antibody test plate is for wrapping by the removable 96 hole ELISA Plate of pig TTV2 reorganization ORF1 albumen, the enzyme conjugates working fluid is the anti-pig IgG polyclonal antibody of horseradish peroxidase-labeled goat, positive control is pig TTV2 standard positive serum, negative control is pig standard female serum, and bag is as follows by pig TTV2 reorganization ORF1 protein sequence:

1 DLTEPWLEGWGNAFYSVLGYEAIKDQGHWSNWAQIKYYWIYDTGVGNAVY

51 VVMLKKDIDDNPGRMATEFKTTPGQHPNAIDHIELINEGWPYWLYFFGKS

101 EQDIKKEAHSEEIAREYATNPKSKKLKIGIVGWASSNFTTPGSSQNVGGN

151 TAAIQGGYVAWAGGQGKLNLGAGSIGNLYQQGWPSNQNWPNTNRDETNFD

201 WGLRSLCMLRDNMQLGSQELDDECTMLSLFGPFVEKANPIFATTDPKYFK

251 PELKDYNLIMKYAFKFQWGGHGTERFKTTIGDPSTIPCPFEPGDRFHSGI

301 QDPSKVQNTVLNPWDYDCDGIVRKDTLKRLLELPTETEEEKAYPLLGQKT

351 EKEPLSDSDEESVISSTSSGSSQEEETQRRKHHKPSKRRLLKHLQRVVKR

401 MKT。

Described pig TTV2 reorganization ORF1 albumen is expressed by prokaryotic expression plasmid pcoldI-ORF1, and prokaryotic expression plasmid pcoldI-ORF1 is made up by following method and forms:

1) utilize the genetic engineering recombinant technique to make up the prokaryotic expression plasmid of pig TTV2 ORF1 portion gene, called after pcoldI-ORF1; Formed by following method structure, designed a pair of primer that contains restriction enzyme site:

SFORF1:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG ( XhoI)

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA( HindIII)

Utilize above-mentioned Auele Specific Primer, amplification gene fragment from the positive pathological material of disease of TTV2, obtain the amplified fragments of 1227bp, recycling is the restriction enzyme on the primer separately, respectively pcr amplification product and pcoldI carrier are carried out enzyme and cut, the pcr amplified fragment of in turn enzyme being cut with the T4 dna ligase is connected on the pcoldI.Specifically, at first with the fragment of pcoldI carrier and SFORF1 and SRORF1 primer amplification warp respectively XhoI and HindThe III enzyme is cut, and reclaims endonuclease bamhi separately, connects with the T4 dna ligase, makes up the pcoldI-ORF1 carrier, connects in the product transformed into escherichia coli DH5 α competence, extracts plasmid, selects enzyme and cuts and identify the correct clone evaluation of checking order.

The picking warp XhoI and HindThe plasmid that the III double digestion goes out the 1215bp band checks order, and sequencing result shows that the purpose fragment correctly links to each other with the pcoldI carrier, and the reading frame of expressing protein is correct, and the result shows that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.

2) will become genetic engineering bacterium (called after BLpcoldI-ORF1) in the plasmid pcoldI-ORF1 conversion host bacterium e. coli bl21, with BLpcoldI-ORF1 in the LB nutrient culture media in 15 ℃ of cultivations, show that through the 0.2mmol/L abduction delivering and through the SDS-PAGE electrophoretic analysis recombinant protein is present in the thalline with insoluble inclusion body form; Thereby obtain the prokaryotic expression plasmid pcoldI-ORF1 of pig TTV2 recombinant protein.

3) be that envelope antigen prepares the ELISA check-out console with the recombinant protein, detect the antibody horizontal of pig TTV2 in the pig serum;

(1) bag quilt: diluting with antigen coated liquid behind the recombinant protein purification that the prokaryotic expression plasmid pcoldI-ORF1 of claim 1 or 2 described boar TTV2 ORF1 truncated proteins is expressed is the final concentration of 1.65 μ g/mL, each ELISA hole adds 100 μ l, and bag is spent the night under 4 ℃ of conditions.Discard the coating buffer body in the plate, PBST damping fluid washing 5min * 3 time.Antigen coated liquid is 0.05M, the carbonate buffer solution of pH9.6; The PBST damping fluid is the PBS damping fluid that contains mass ratio 0.01%Tween-20;

(2) sealing: every hole adds confining liquid 200 μ l, acts on 1h under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time; Confining liquid is the PBS damping fluid that contains mass ratio 2% skimmed milk power;

(3) add serum to be checked: every hole adds the serum of 100 μ l after with the PBS dilution that contains mass ratio 0.5%BSA, acts on 1.0h under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time;

(4) add two anti-: every hole adds the goat-anti pig IgG of the HRP mark of 100 μ l dilution, effect 30 min under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time; The goat-anti pig IgG of the HRP mark of dilution is pressed the 1:5000 dilution proportion for the PBS with pH7.0;

(5) add colour developing liquid: every hole adds the colour developing liquid 100 μ l that contain 1mg/ml TMB tetramethyl biphenyl diamines, 37 ℃ of lucifuge effect 5 min;

(6) add stop buffer: every hole adds 100 μ l stop buffers, and microplate reader is measured 450 nm light absorption value OD450; Stop buffer is 2M H ₂SO ₄

(7) result judges: serum OD450 value to be checked 〉=be judged to be pig TTV2 antibody positive at 0.248 o'clock, OD450 value ∠ 0.248 is judged to be feminine gender.

Beneficial effect

The inventive method is utilized molecular biology gene cloning and expression technology first, obtain pig TTV2 ORF1 recombinant protein, be that antigen is set up indirect ELISA with the recombinant protein, can carry out effective diagnosis to the popular pig TTV2 of present China, judge the situation of TTV2 antibody horizontal and natural infection in the swinery, for the anti-system of pig TTV2 virus provides a kind of quick, easy detection method.

The indirect ELISA method that evidence, the present invention utilize the recombinant protein (TTV2 ORF1) behind expression and the purifying to set up as antigen coated elisa plate first can detect anti-pig TTV2 antibody in the pig body really, and have good specificity and repeatability.Clinical sample is detected, and the result shows that this method can be used as the SD a kind of method of pig TTV2 and applies in the production reality.

Description of drawings

SDS-PAGE behind Fig. 1 expression of recombinant proteins and the purifying analyzes

1: the ultrasonic treatment precipitation; 2: the ultrasonic treatment supernatant; 3: induce afterproduct; 4: induced product not; 5: albumen Marker

Fig. 2 western blot test shows pig TTV2 ORF1 recombinant expression protein

1: albumen Marker; The purified product of 2:pcoldI-ORF1 expressing protein

The Western blotting of Fig. 3 purifying protein detects

1: the empty carrier inducible protein; The 2:pcoldI-ORF1 purifying protein; 3: albumen Marker

Embodiment

Main points of the present invention are: utilize pcoldI to make up part pig TTV2 ORF1 Prokaryotic Expression carrier, and the recombinant protein after will expressing carries out purifying, set up indirect ELISA testing kit and detection method, and apply to clinical detection.

The preparation of envelope antigen:

1.1 the Auele Specific Primer design is with synthetic: (the GenBank accession number is the pig TTV2 gene order of including according to GenBank: EF514716), choose coding pig TTV2 open reading frame ORF1 gene, utilize Primer Premier 5.0 softwares to design the primer that contains restriction enzyme site and submit to Shanghai Ying Jun company to synthesize.

SFORF1-:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG ( XhoI)

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA( HindIII)

1.2 construction of prokaryotic expression vector: utilize above-mentioned Auele Specific Primer, amplification gene fragment from the positive pathological material of disease of TTV2, obtain the amplified fragments of 1227bp, recycling is the restriction enzyme on the primer separately, respectively pcr amplification product and pcoldI prokaryotic expression carrier (available from Takara) are carried out enzyme and cut, the pcr amplified fragment of in turn enzyme being cut with T4 dna ligase (available from Takara) is connected on the pcoldI.Specifically, at first with the fragment of pcoldI carrier and SFORF1 and SRORF1 primer amplification warp respectively XhoI and HindThe III enzyme is cut, reclaim endonuclease bamhi separately, connect with the T4 dna ligase, make up the pcoldI-ORF1 carrier, connect in product transformed into escherichia coli DH5 α (Beijing Quanshijin Biotechnology Co., Ltd) competence, extract plasmid, enzyme is cut the evaluation positive colony, and the Nanjing Jin Sirui company that send that is accredited as positive colony carries out sequencing.

The picking warp XhoI and HindThe plasmid that the III double digestion goes out the 1215bp band checks order, and sequencing result shows that the purpose fragment correctly links to each other with the pcoldI carrier, and the reading frame of expressing protein is correct, and the result shows that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.To become genetic engineering bacterium, called after genetic engineering bacterium BLpcoldI-ORF1 in the plasmid pcoldI-ORF1 conversion host bacterium e. coli bl21.

The recombinant protein sequence that described recombinant prokaryotic expression vector plasmid pcoldI-ORF1 expresses is as follows:

1 DLTEPWLEGWGNAFYSVLGYEAIKDQGHWSNWAQIKYYWIYDTGVGNAVY

51 VVMLKKDIDDNPGRMATEFKTTPGQHPNAIDHIELINEGWPYWLYFFGKS

101 EQDIKKEAHSEEIAREYATNPKSKKLKIGIVGWASSNFTTPGSSQNVGGN

151 TAAIQGGYVAWAGGQGKLNLGAGSIGNLYQQGWPSNQNWPNTNRDETNFD

201 WGLRSLCMLRDNMQLGSQELDDECTMLSLFGPFVEKANPIFATTDPKYFK

251 PELKDYNLIMKYAFKFQWGGHGTERFKTTIGDPSTIPCPFEPGDRFHSGI

301 QDPSKVQNTVLNPWDYDCDGIVRKDTLKRLLELPTETEEEKAYPLLGQKT

351 EKEPLSDSDEESVISSTSSGSSQEEETQRRKHHKPSKRRLLKHLQRVVKR

401 MKT

1.3 the abduction delivering of recombinant protein and analysis

Genetic engineering bacterium BLpcoldI-ORF1 is inoculated in the LB fluid nutrient medium that contains Amp in 2% ratio (in the 950mL deionized water, adds tryptone 10g, yeast extract 5g, NaCl 10g, transfer pH to 7.0 with NaOH, be settled to 1L with deionized water) in, 15 ℃ of shaken cultivation are to OD600 ≈ 0.4～0.6, add IPTG to final concentration be 0.2mmol/L, carry out abduction delivering, the thalline that collection induces the back different time to express, on ice bath, carry out ultrasonication, bacterium liquid after the cracking is through the centrifugal 30min of 10000rpm, cleer and peaceful precipitation is carried out SDS-PAGE electrophoresis (the discontinuous SDS-PAGE of employing LaemimLi system simultaneously in the separation, separation gel 12%, concentrate glue 4%), expression and the expression-form of evaluation destination protein.

1.4 recombinant protein purification:

Get 100ml LB fluid nutrient medium, induce cultivation in a large number according to protein induced expression condition in 1.3, get inclusion body and carry out protein purification according to the Protino Ni-TED Resin of Macherey-Nagel company kit instructions, fetch receive protein sample and add the 2xSDS gel loading buffer and boil 5 min after, carry out SDS-PAGE and identify purity of protein.Reclaim protein sample and adopt the spectrophotometric determination protein concentration.

SDS-PAGE analyzes demonstration, recombinant protein is successful expression in e. coli bl21 (Beijing Quanshijin Biotechnology Co., Ltd) cell, be present in the thalline with insoluble inclusion body form, the molecular weight of albumen size is 52kDa(Fig. 1), recombinant protein size basically identical with prediction, the nickel post reclaims the back protein example and carries out SDS-PAGE, and visible single purpose band (Fig. 2) shows with this expressing protein effect of ni-sepharose purification better.

Western Blotting analyzes: the albumen according to method purifying in 1.4 carries out SDS-PAGE, operates with reference to conventional Western Blotting method, adopts DAB substrate visualizingre agent (Wuhan doctor's moral company) to develop the color at last.The result specific band occurs at the 52KDa place, shows that the albumen of expression and purification and pig TTV2 positive serum have good reactionogenicity (Fig. 3).

2. set up indirect ELISA with recombinant protein:

Get TTV2 ORF1 recombinant protein behind the purifying and do envelope antigen and set up indirect ELISA method, adopt the square formation method to explore ELISA optimum protein bag by concentration and serum dilution, and the ELISA optimum reaction condition.

2.1 determining of antigen coated liquid

Respectively with phosphate buffer (0.01M, pH7.4, PBS), Tris-HCl damping fluid (0.05 M, pH8.5), carbonate buffer solution (0.05 M, pH9.6), distilled water is as coating buffer, with optimum dilution degree dilution antigen, 4 ℃ of bags are spent the night, 2h is sealed with 1%BSA in the washing back, the washing back adds the serum with the optimum dilution degree dilution, effect 1h, and the washing back adds the ELIAS secondary antibody effect 1h of 1:5000 dilution, the washing back adds TMB room temperature lucifuge colour developing 10min, determines only coating buffer by OD450 value and P/N value.The result shows that the bag of carbonate buffer solution is best by effect, therefore uses carbonate buffer solution as the antigenic dilution coated elisa plate.

2.2 the best effort concentration of antigen-antibody

The pcold-ORF1 recombinant protein of purifying is taken turns doing 1:10,1:20,1:40,1:80,1:160,1:320,1:640,1:1280 gradient dilution from top to bottom with the carbonate buffer solution of 0.05mMpH9.6,100 μ l/ holes, 4 ℃ are spent the night.Take out with PBST washing 3 times each 5min next day.Bovine serum albumin(BSA) with 1% (BSA) seals, 200 μ l/ holes, 37 ℃ of effect 2h.Standard yin and yang attribute serum is made 1:10,1:20,1:40,1:80,1:160,1:320 gradient dilution, add each hole from left to right successively, 100 μ l/ holes, 37 ℃ of effect 1h, washing (the same); Every hole adds goat-anti pig IgG-HRP(Wuhan doctor's moral company that 100 μ l are diluted to working concentration (1:5000) subsequently); 37 ℃ of effect 1h, washing (the same); Every hole adds 100 μ l TMB colour developing liquid, and lucifuge color development at room temperature 10 min add 100 μ l stop buffer cessation reactions at last, use microplate reader to measure its OD450 value immediately.Best effort concentration according to OD450 value and the definite antigen of P/N value and antibody.The result shows, when the dilutability of antigen is 1:160, when the dilutability of antibody was 1:160, the OD450 value of positive serum was close to 1.0, and P/N value maximum.Therefore, determine that the best bag of recombinant protein is 1.65 μ g/mL by concentration, the serum optimum dilution degree is 1:160.

2.2 best bag is by the selection of time

By elisa plate, be divided into three groups with best antigen concentration bag.First group: 37 ℃ of bags are by 2h; Second group: hatch behind the 1h 4 ℃ for 37 ℃ and spend the night; The 3rd group: 4 ℃ of bags are spent the night.Positive and negative serum is respectively done 4 repetitions.After three kinds of different bags are wrapped quilt under the condition, washing, seal 2h with 1%BSA, the washing back adds the serum with the optimum dilution degree dilution, effect 1h, the washing back adds the ELIAS secondary antibody effect 1h of 1:5000 dilution, and the washing back adds TMB room temperature lucifuge colour developing 10min, determines that by OD450 value and P/N value best bag is by condition.The result determines that best bag is 4 ℃ by the time and spends the night.

2.3 the selection of best confining liquid with carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.After taking out washing next day, respectively with 1%BSA, 2%BSA, 2% skimmed milk power, 5% skimmed milk power, 1% gelatin, 2% gelatin, 5% calf serum, 10% calf serum as confining liquid, yin and yang attribute serum is respectively done 2 repetitions.Seal 2h, the washing back adds the serum with the optimum dilution degree dilution, effect 1h, and the washing back adds the ELIAS secondary antibody effect 1h of 1:5000 dilution, and the washing back adds TMB room temperature lucifuge colour developing 10min, determines best confining liquid by OD450 value and P/N value.The result shows that when sealing with 2% skimmed milk power, the P/N value is maximum, and sealing effect is best, therefore selects for use 2% skimmed milk power as best confining liquid.2.4 the selection of off-period with carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.With 2% skimmed milk power respectively seal 30min, 60min, 90min, 120min after taking out washing next day, and yin and yang attribute serum is respectively done 4 repetitions.The washing back adds the serum with the optimum dilution degree dilution, effect 1h, and the washing back adds the ELIAS secondary antibody effect 1h of 1:5000 dilution, and the washing back adds TMB room temperature lucifuge colour developing 10min, determines best off-period by OD450 value and P/N value.When be 60min and 90min off-period, the P/N value was big and be more or less the same, and therefore in order to save time, this experiment selects for use off-period 60min as best off-period.

2.5 the selection of best serum dilution

In serum dilution, add the sealing composition, can reach and reduce the non-specific adsorption effect, in PBS, add 0.5%BSA, 2% skimmed milk power, 5% skimmed milk power and 1% gelatin for this reason, the positive and negative serum of knowing with oneself carries out the ELISA detection, observe the variation of OD value and P/N and change, and screen best primary antibodie dilution in contrast with the PBS dilute serum.The result selects to contain the PBS of 0.5%BSA as best primary antibodie dilution.

2.6 determining of serum optimum reacting time

With carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.After taking out washing next day, with 2% skimmed milk power sealing 60min, after the washing, add the serum that is diluted to optium concentration with the 1.5%BSA sample diluting liquid, act on 30min, 60min, 90min, 120min respectively, yin and yang attribute serum is respectively done 4 repetitions.The washing back adds the ELIAS secondary antibody effect 1h of 1:5000 dilution, and the washing back adds TMB room temperature lucifuge colour developing 10min, determines the serum optimum reacting time by OD450 value and P/N value.When serum was 60min action time, the value of P/N was obviously bigger, so to select the serum the best use of time be 60min.

2.7 determining of the suitableeest ELIAS secondary antibody working concentration

With carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.After taking out washing next day, with 2% skimmed milk power sealing 60min, after the washing, add the serum effect 60min that is diluted to optium concentration with the 1.5%BSA sample diluting liquid, after the washing, add the ELIAS secondary antibody that concentration is 1:2000,1:3000,1:4000,1:5000 respectively, effect 1h, yin and yang attribute serum is respectively done 2 repetitions.The washing back adds TMB room temperature lucifuge colour developing 10min, determines the optium concentration of ELIAS secondary antibody by OD450 value and P/N value.When ELIAS secondary antibody concentration was 1:5000, the P/N value was maximum, so this experiment selects for use 1:5000 as the best effort concentration of ELIAS secondary antibody.

2.8 determining of the suitableeest ELIAS secondary antibody reaction time

With carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.After taking out washing next day, with 2% skimmed milk power sealing 60min, after the washing, add the serum effect 60min that is diluted to optium concentration with the 1.5%BSA sample diluting liquid, it is the ELIAS secondary antibody of 1:4000 that the washing back adds concentration, act on 30min, 60min, 90min, 120min respectively, yin and yang attribute serum is respectively done 4 repetitions.The washing back adds TMB room temperature lucifuge colour developing 10min, determines the ELIAS secondary antibody optimum reacting time by OD450 value and P/N value.When be 30min two anti-action times, the P/N value was maximum, therefore selected 30min as the ELIAS secondary antibody optimum reacting time.2.9 determining of the suitableeest substrate reactions time

With carbonate buffer solution with antigen diluent to the optium concentration bag by elisa plate, 4 ℃ of bags are spent the night.After taking out washing next day, with 2% skimmed milk power sealing 60min, after the washing, add the serum effect 60min that is diluted to optium concentration with the 1.5%BSA sample diluting liquid, it is the ELIAS secondary antibody of 1:4000 that the washing back adds concentration, effect 30min, washing back adds TMB room temperature lucifuge develop the color respectively 3min, 5min, 8min, and yin and yang attribute serum is respectively done 4 repetitions.Determine the substrate optimum reacting time by OD450 value and P/N value.When the reaction time was 5min, the P/N value was maximum, and therefore selecting the substrate reactions time is 5min.

2.10 determining of indirect ELISA method yin and yang attribute critical value

Detect negative pig serum with the indirect ELISA method of having set up, the mean value (X) that calculates 40 parts of TTV negative serum OD450 is 0.191, standard deviation (SD) is 0.019, determine that according to formula: X+3SD the yin and yang attribute critical value of indirect ELISA detection method is 0.248, namely positive when the OD450 of sample value 〉=0.248, OD450 value＜0.248 is o'clock then negative.

2.11 the specificity of indirect ELISA and replica test

Detect known SS2(streptococcus suis 2-type with the pig TTV2 indirect ELISA detection method of setting up) (referring to Zhang Xuehan, what Kong Wang, Zhou Junming etc. the structure of streptococcus suis type 2 carnine acidohydrogenase deletion strain [J]. Scientia Agricultura Sinica .2009.5 (42): 1789-1796.), the HPS(haemophilus parasuis) (referring to Wang Haiyan, Liu Maojun, Feng Zhi is new etc. haemophilus parasuis cultural character and Study on Pathogenicity [J]. and the Jinling School of Science and Technology journal. 2009.4 (25): 80-83), the Mhp(mycoplasma hyopneumoniae) (referring to Wang Zhanwei, Liu Maojun, Feng Zhixin etc. osmotic pressure is to the influence [J] of mycoplasma hyopneumoniae 168 strains. Agricultural Science ﹠amp; Technology. 2012.13 (10): 2051-2054), the PCV2(porcine circovirus 2 type) (referring to Guo Rongli, what Kong Wang, Zhong Shulin etc. the separation evaluation of two strain porcine circovirus 2 type viruses and sequential analysis [J] thereof. Jiangsu agricultural journal. 2009.25 (5): 1063-1067.), the PPV(pig parvoviral) (referring to Pan Qunxing, Wang Yongshan, He Kongwang etc. the preparation [J] of recombinant expressed foot and mouth disease virus t cell epitope pig parvoviral virus sample particle. Chinese Preventive Veterinary Medicine newspaper. 2010.11 (32): 844-848), the PRV(PRV) (referring to Zhang Xuehan, what Kong Wang, Miu Wenkai etc. Taqman probe real-time quantitative PCR detects PRV [J]. Jiangsu agricultural journal. 2008.24 (4): 440-443.), the TGEV(TGE) (referring to what Kong Wang, Lin Jihuang, red China etc. also. transmissible gastro-enteritis virus weakening strain STC3 cell cultural character and Study on Pathogenicity [J]. Chinese Journal of Veterinary Science and Technology. 2001.31 (8): 8-9.), the CSFV(CSFV) (referring to Zhang Xuehan, what Kong Wang, Guo Rongli etc. detect CSFV, JEV, the foundation [J] of three kinds of RNA virus multiple RT-PCRs of PRRSV method. Chinese Preventive Veterinary Medicine newspaper. 2007.29 (5): 385-388.), the PRRSV(porcine reproductive and respiratory syndrome virus) (referring to Li Bin, hair power, He Kongwang etc. clone and the Genetic Variation Analysis [J] of porcine reproductive and respiratory syndrome virus (PRRSV) Jiangsu separated strain NJGC ORF5 gene. Jiangsu agricultural journal. 2011.27 (4): 775-781.), the FMDV(foot and mouth disease virus) (referring to Liu Yaofang, what Kong Wang, Zhang Dekun etc. utilize multiple RT-PCR to foot and mouth disease virus somatotype [J]. the Jiangsu agricultural sciences. 2007.5:136-139.) positive serum, set up the TTV2 positive simultaneously, negative control, measure its OD450, determined whether cross reaction.The OD value is all less than 0.248 as a result, and is negative, shows the positive serum no cross reaction of this recombinant protein and above-mentioned virus, and this indirect ELISA method high specificity is described.

Repeat in batch: with the antigen coated same elisa plate of the purification of Recombinant of same batch of preparation, 2 parts of positive serums and 2 parts of negative serum samples are detected, and every part of serum sample repeats 4 holes, measures the OD450 value, calculate the coefficient of variation (CV=SD/X), estimate batch interior revision test effect.The result shows that the OD450 value coefficient of variation of 4 parts of serum shows that all less than 10% same sample degree of variation in same batch of test is less, has good repeatability.

Repeat between batch: in the ELISA Plate of the series protein bag quilt that different batches prepares, randomly draw 1 for every batch, detect 6 parts of serum that the TTV2 antibody horizontal is different, every part of parallel 4 holes of doing of serum, the coefficient of variation (CV=SD/X) of calculating testing result.The OD450 value coefficient of variation of 6 parts of serum shows that all less than 5% same sample degree of variation in the different batches test is less, has better repeatability.

In the result shows batch and batch between the coefficient of variation all less than 5%, show that this method has better repeatability.

3. the Preliminary Applications of indirect ELISA method

Detect by the indirect ELISA method set up 352 parts of pig serum to Jiangsu between 2010～2011 and the censorship of Anhui two provinces.Totally 192 parts of the serum of Jiangsu censorship, wherein 82 parts of testing results are positive, and positive rate is 42.7%, totally 160 parts of the serum of Anhui censorship, wherein 12 parts of testing results are positive, and positive rate is that the TTV2 total positives rate of 7.5%, two province censorship serum is 26.7%.

SEQUENCE LISTING

＜110〉Jiangsu Province Agriculture Science Institute

＜120〉a kind of indirect ELISA reagent kit that detects pig blood transfusion transmitted virus 2 type antibody

<130> 0

<160> 3

<170> PatentIn version 3.1

<210> 1

<211> 403

<212> PRT

＜213〉artificial

<220>

＜221〉pig TTV2 recombinant capsid protein sequence

<222> (1)..(403)

<223>

<400> 1

Asp Leu Thr Glu Pro Trp Leu Glu Gly Trp Gly Asn Ala Phe Tyr Ser

1 5 10 15

Val Leu Gly Tyr Glu Ala Ile Lys Asp Gln Gly His Trp Ser Asn Trp

20 25 30

Ala Gln Ile Lys Tyr Tyr Trp Ile Tyr Asp Thr Gly Val Gly Asn Ala

35 40 45

Val Tyr Val Val Met Leu Lys Lys Asp Ile Asp Asp Asn Pro Gly Arg

50 55 60

Met Ala Thr Glu Phe Lys Thr Thr Pro Gly Gln His Pro Asn Ala Ile

65 70 75 80

Asp His Ile Glu Leu Ile Asn Glu Gly Trp Pro Tyr Trp Leu Tyr Phe

85 90 95

Phe Gly Lys Ser Glu Gln Asp Ile Lys Lys Glu Ala His Ser Glu Glu

100 105 110

Ile Ala Arg Glu Tyr Ala Thr Asn Pro Lys Ser Lys Lys Leu Lys Ile

115 120 125

Gly Ile Val Gly Trp Ala Ser Ser Asn Phe Thr Thr Pro Gly Ser Ser

130 135 140

Gln Asn Val Gly Gly Asn Thr Ala Ala Ile Gln Gly Gly Tyr Val Ala

145 150 155 160

Trp Ala Gly Gly Gln Gly Lys Leu Asn Leu Gly Ala Gly Ser Ile Gly

165 170 175

Asn Leu Tyr Gln Gln Gly Trp Pro Ser Asn Gln Asn Trp Pro Asn Thr

180 185 190

Asn Arg Asp Glu Thr Asn Phe Asp Trp Gly Leu Arg Ser Leu Cys Met

195 200 205

Leu Arg Asp Asn Met Gln Leu Gly Ser Gln Glu Leu Asp Asp Glu Cys

210 215 220

Thr Met Leu Ser Leu Phe Gly Pro Phe Val Glu Lys Ala Asn Pro Ile

225 230 235 240

Phe Ala Thr Thr Asp Pro Lys Tyr Phe Lys Pro Glu Leu Lys Asp Tyr

245 250 255

Asn Leu Ile Met Lys Tyr Ala Phe Lys Phe Gln Trp Gly Gly His Gly

260 265 270

Thr Glu Arg Phe Lys Thr Thr Ile Gly Asp Pro Ser Thr Ile Pro Cys

275 280 285

Pro Phe Glu Pro Gly Asp Arg Phe His Ser Gly Ile Gln Asp Pro Ser

290 295 300

Lys Val Gln Asn Thr Val Leu Asn Pro Trp Asp Tyr Asp Cys Asp Gly

305 310 315 320

Ile Val Arg Lys Asp Thr Leu Lys Arg Leu Leu Glu Leu Pro Thr Glu

325 330 335

Thr Glu Glu Glu Lys Ala Tyr Pro Leu Leu Gly Gln Lys Thr Glu Lys

340 345 350

Glu Pro Leu Ser Asp Ser Asp Glu Glu Ser Val Ile Ser Ser Thr Ser

355 360 365

Ser Gly Ser Ser Gln Glu Glu Glu Thr Gln Arg Arg Lys His His Lys

370 375 380

Pro Ser Lys Arg Arg Leu Leu Lys His Leu Gln Arg Val Val Lys Arg

385 390 395 400

Met Lys Thr

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<211> 34

<212> DNA

＜213〉artificial

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<221> SFORF1-F

<222> (1)..(34)

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ccgctcgagg acttaacgga accgtggcta gaag 34

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<211> 38

<212> DNA

＜213〉artificial

<220>

<221> SRORF1-R

<222> (1)..(38)

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cccaagcttt gttttcatcc tctttaccac ccgctgga 38

Claims (3)

1. boar blood transfusion transmitted virus 2 types (TTV2) antibody indirect ELISA diagnostic kit is characterized in that: be provided with the antibody test plate in the kit, confining liquid, sample diluting liquid, cleansing solution, enzyme conjugates working fluid, enzyme substrate solution, stop buffer, positive control and negative control; Wherein the antibody test plate is for wrapping by the removable 96 hole ELISA Plate of pig TTV2 reorganization ORF1 albumen, the enzyme conjugates working fluid is the anti-pig IgG polyclonal antibody of horseradish peroxidase-labeled goat, positive control is pig TTV2 standard positive serum, negative control is pig standard female serum, and bag is as follows by pig TTV2 reorganization ORF1 protein sequence:

1 DLTEPWLEGWGNAFYSVLGYEAIKDQGHWSNWAQIKYYWIYDTGVGNAVY

51 VVMLKKDIDDNPGRMATEFKTTPGQHPNAIDHIELINEGWPYWLYFFGKS

101 EQDIKKEAHSEEIAREYATNPKSKKLKIGIVGWASSNFTTPGSSQNVGGN

151 TAAIQGGYVAWAGGQGKLNLGAGSIGNLYQQGWPSNQNWPNTNRDETNFD

201 WGLRSLCMLRDNMQLGSQELDDECTMLSLFGPFVEKANPIFATTDPKYFK

251 PELKDYNLIMKYAFKFQWGGHGTERFKTTIGDPSTIPCPFEPGDRFHSGI

301 QDPSKVQNTVLNPWDYDCDGIVRKDTLKRLLELPTETEEEKAYPLLGQKT

351 EKEPLSDSDEESVISSTSSGSSQEEETQRRKHHKPSKRRLLKHLQRVVKR

401 MKT。

2. a boar TTV2 antibody indirect ELISA diagnostic kit according to claim 1, it is characterized in that: described pig TTV2 reorganization ORF1 albumen is expressed by prokaryotic expression plasmid pcoldI-ORF1, and prokaryotic expression plasmid pcoldI-ORF1 is made up by following method and forms:

Design a pair of primer that contains restriction enzyme site:

SFORF1:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG XhoI

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA HindIII

Utilize above-mentioned Auele Specific Primer, amplification ORF1 genetic fragment from the positive pathological material of disease of TTV2, obtain the amplified fragments of 1227bp, recycling is the restriction enzyme on the primer separately, respectively pcr amplification product and pcoldI prokaryotic expression carrier being carried out enzyme cuts, with the T4 dna ligase enzyme of amplified fragments being cut product is connected to the pcoldI enzyme and cuts on the product, make up the pcoldI-ORF1 carrier, connect in the product transformed into escherichia coli DH5 α competence, cut the evaluation of identifying and check order through enzyme, show that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.

3. ELISA kit according to claim 1 and 2, its method for detection of the antibody horizontal of pig TTV2 in the pig serum is:

(1) bag quilt: will be the final concentration of 1.65 μ g/mL with antigen coated liquid dilution behind claim 1 or the 2 described boar TTV2 reorganization ORF1 protein purifications, each ELISA hole adds 100 μ l, wraps under 4 ℃ of conditions and is spent the night; Discard the coating buffer body in the plate, PBST damping fluid washing 5min * 3 time, antigen coated liquid is 0.05M, the carbonate buffer solution of pH9.6; The PBST damping fluid is the PBS damping fluid that contains mass ratio 0.01%Tween-20;

(2) sealing: every hole adds confining liquid 200 μ l, acts on 1h under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time; Confining liquid is the PBS damping fluid that contains mass ratio 2% skimmed milk power;

(3) add serum to be checked: every hole adds the serum of 100 μ l after with the PBS dilution that contains mass ratio 0.5%BSA, acts on 1.0h under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time;

(4) add two anti-: every hole adds the goat-anti pig IgG of the HRP mark of 100 μ l dilution, effect 30 min under 37 ℃ of conditions, PBST damping fluid washing 5min * 3 time; The goat-anti pig IgG of the HRP mark of dilution is pressed the 1:5000 dilution proportion for the PBS with pH7.0;

(5) add colour developing liquid: every hole adds the colour developing liquid 100 μ l that contain 1mg/ml TMB tetramethyl biphenyl diamines, 37 ℃ of lucifuge effect 5 min;

(6) add stop buffer: every hole adds 100 μ l stop buffers, and microplate reader is measured 450 nm light absorption value OD450; Stop buffer is 2M H ₂SO ₄

(7) result judges: serum OD450 value to be checked 〉=be judged to be pig TTV2 antibody positive at 0.248 o'clock, OD450 value ∠ 0.248 is judged to be feminine gender.

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