CN103235121B - A kind of indirect ELISA reagent kit detecting pig Transfusion transmitted virus 2 type antibody - Google Patents

Abstract

The present invention relates to a kind of for detecting the indirect ELISA reagent kit of pig Transfusion transmitted virus 2 type (TTV2) antibody, belonging to biological technical field.Comprise the application that the preparation of antigen recombinant protein, the foundation of indirect ELISA, criterion and clinical serum detect.Described antigen recombinant protein utilizes pcoldI prokaryotic expression carrier, the genetic engineering bacterium pcoldI-ORF1 of construction expression pig TTV2 ORF1 truncated protein, and the recombinant protein purification of expression is established indirect ELISA detection method as antigen.The method has reproducible, that specificity is high feature for detecting the antibody horizontal of TTV2 in Swine serum, can be used for the serosurvey of pig TTV2, for the control of this disease and the grasp of popularity provide a kind of quick, easy Serology test.Current the method is that the domestic pig TTV2 ORF1 recombinant protein that utilizes first sets up the ELISA detection method detecting TTV2 antibody in swinery.

Description

A kind of indirect ELISA reagent kit detecting pig Transfusion transmitted virus 2 type antibody

Technical field

The present invention relates to a kind of for detecting the indirect ELISA reagent kit of pig Transfusion transmitted virus 2 type (i.e. pig TTV2) antibody, belonging to technical field of veterinary biology.

Background technology

TTV(Torque Teno virus) have another name called Transfusion transmitted virus, belonging 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 without cyst membrane virus.Equaled to find for 1997 by Japanese scholars Nishizawa the earliest, obtain because this virus is separated in hepatitis body, therefore suspect that it is relevant with hepatitis, thus cause and show great attention to.Various countries have carried out epidemiology survey to TTV infection conditions in national different crowd subsequently, found that in crowd, TTV DNA positive rate is generally more than 10%.Verified at present, except the mankind can infect except 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 in succession detect the existence of TTV.Research shows that TTV extensively exists in world swinery, and may be relevant with causing of some disease, therefore draws attention in the whole world.

Segales J etc. are between 1985-2005 years, the 162 parts of blood serum samples picking up from Spain 99 pig farms carry out TTV detection, 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 is respectively 46.6% and 62.8%, TTV1 and TTV2 coinfection rate and is respectively 19.8% and 24.5%, and this result shows at least at pig TTV in 1985 just Already in Spain pig farm.Martinez etc. investigate the infection conditions of TTV in Spain 178 wild boars, result total positives rate is 84%, the positive rate of TTV1 and TTV2 be respectively 58% and the positive rate of 66%, TTV1 and TTV2 co-infection be 18%, but this result is considered to relevant with geographic position.King Meng Meng etc. detects domestic 258 parts of pig blood samples from 7 provinces such as Guangdong, Fujian and Jiangxi and tissue sample first, result shows that in swinery, TTV1 and TTV2 infection positive rate is respectively 37.6% and 82.6%, TTV2 positive rate is apparently higher than TTV1, the mixed infection rate of the two is 34.5%, confirm that there is TTV in China swinery infects, and comparatively popular with TTV2.This virus is often viral with other there is co-infection.The research such as McKeown N E shows, the mixed infection of this virus and known cause of disease can increase the seriousness of disease, and this virus have potential across kind of propagation to the harm of people, this also points out us can not ignore research to this virus.

The diagnosis sending out epidemic disease for new and anti-work processed are the keys that researcher primarily solves, only have at present and diagnosed by the nucleic acid of round pcr amplification TTV, set up the antibody detection method of this virus, extremely meaningful to the epidemiology survey of this disease, the evaluation of immune effect etc., and ELISA because of have detect flux large, result is definite, highly sensitive, be easy to the features such as basic unit's popularization and be favourably welcome, become current most widely used a kind of Serology test.Due to current pig TTV still can not cultivate in vitro in well bred, so set up in this, as envelope antigen the ELISA detecting anti-TTV antibody in Swine serum become optimal selection by the antigen protein of molecular biology method great expression TTV.

Pig TTV is divided into two hypotypes, i.e. TTV1 and TTV2, and the outstanding feature made a variation between genome is that compared with TTV2, TTV1 has a large amount of variant sites, and namely the variability of TTV1 is larger than TTV2.In TTV1, always have 1349 variant sites, average each variant sites has 1.27 substitutes, and in TTV2, variant sites is fewer, has 670.In addition, Catastrophe Model between ORFs is also different: ORF1 possibility encoding virus coat proteins (Cap) and replication-associated protein (Rep), more conservative, higher at the rate of change of the 3rd position of codon, 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, TTV2 infection rate is apparently higher than TTV1, and China TTV2 is comparatively popular.

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

Summary of the invention

technical matters

The object of this invention is to provide a kind of can fast, detect the indirect ELISA reagent kit of pig TTV2 serum antibody easily, namely pcoldI prokaryotic expression carrier is utilized, construct the carrier expressing pig TTV2 ORF1 portion gene, and wrap by elisa plate by after the recombinant protein purification of expression, to detect the antibody horizontal of TTV2 in Swine serum.

technical scheme

The present invention is realized by following steps:

Can fast, detect the indirect ELISA reagent kit of pig TTV2 serum antibody easily, be provided with antibody test plate in kit, confining liquid, sample diluting liquid, cleansing solution, enzyme conjugates working fluid, enzyme substrate solution, stop buffer, positive control and negative control; Wherein antibody test plate is that bag is by the removable 96 hole ELISA Plate of pig TTV2 restructuring ORF1 albumen, 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, wraps by pig TTV2 restructuring ORF1 protein sequence as follows:

1 DLTEPWLEGWGNAFYSVLGYEAIKDQGHWSNWAQIKYYWIYDTGVGNAVY

51 VVMLKKDIDDNPGRMATEFKTTPGQHPNAIDHIELINEGWPYWLYFFGKS

101 EQDIKKEAHSEEIAREYATNPKSKKLKIGIVGWASSNFTTPGSSQNVGGN

151 TAAIQGGYVAWAGGQGKLNLGAGSIGNLYQQGWPSNQNWPNTNRDETNFD

201 WGLRSLCMLRDNMQLGSQELDDECTMLSLFGPFVEKANPIFATTDPKYFK

251 PELKDYNLIMKYAFKFQWGGHGTERFKTTIGDPSTIPCPFEPGDRFHSGI

301 QDPSKVQNTVLNPWDYDCDGIVRKDTLKRLLELPTETEEEKAYPLLGQKT

351 EKEPLSDSDEESVISSTSSGSSQEEETQRRKHHKPSKRRLLKHLQRVVKR

401 MKT。

Described pig TTV2 restructuring ORF1 albumen is expressed by prokaryotic expression plasmid pcoldI-ORF1, and prokaryotic expression plasmid pcoldI-ORF1 is built by following methods and forms:

1) genetic engineering recombinant technique is utilized to build the prokaryotic expression plasmid of pig TTV2 ORF1 portion gene, called after pcoldI-ORF1; Built by following methods and form, devise a pair primer containing restriction enzyme site:

SFORF1:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG ( XhoI)

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA( HindIII)

Utilize above-mentioned Auele Specific Primer, amplification gene fragment from TTV2 positive pathological material of disease, obtain the amplified fragments of 1227bp, recycle the restriction enzyme on respective primer, carry out enzyme to pcr amplification product and pcoldI carrier respectively to cut, in turn the pcr amplified fragment that enzyme is cut is connected on pcoldI with T4 DNA ligase.Specifically, first by the fragment warp respectively of pcoldI carrier and SFORF1 and SRORF1 primer amplification xhoi and hindiII digestion, reclaims respective endonuclease bamhi, connects with T4 DNA ligase, builds pcoldI-ORF1 carrier, connects in product conversion bacillus coli DH 5 alpha competence, extracts plasmid, selects enzyme and cuts the correct clone of qualification and carry out order-checking and identify.

Picking warp xhoi and hindthe plasmid that III double digestion goes out 1215bp band checks order, and sequencing result shows that object fragment is correctly connected with pcoldI carrier, and the reading frame of expressing protein is correct, and result shows that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.

2) genetic engineering bacterium (called after BLpcoldI-ORF1) is become by plasmid pcoldI-ORF1 conversion Host Strains e. coli bl21, by BLpcoldI-ORF1 in LB nutrient culture media in 15 DEG C of cultivations, through 0.2mmol/L abduction delivering and through SDS-PAGE electrophoretic analysis display recombinant protein be present in thalline with insoluble inclusion bodies; Thus obtain the prokaryotic expression plasmid pcoldI-ORF1 of pig TTV2 recombinant protein.

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

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

(2) close: every hole adds confining liquid 200 μ l, acts on 1h, PBST buffer solution 5min × 3 time under 37 DEG C of conditions; Confining liquid is the PBS damping fluid containing mass ratio 2% skimmed milk power;

(3) serum to be checked is added: every hole adds the serum of 100 μ l after the PBS dilution containing mass ratio 0.5%BSA, acts on 1.0h, PBST buffer solution 5min × 3 time under 37 DEG C of conditions;

(4) add two to resist: every hole adds the goat-anti pig IgG of the HRP mark that 100 μ l dilute, and acts on 30 min, PBST buffer solution 5min × 3 time under 37 DEG C of conditions; The goat-anti pig IgG of the HRP mark of dilution is use the PBS of pH7.0 by 1:5000 dilution proportion;

(5) nitrite ion is added: the nitrite ion 100 μ l of every Kong Jiahan 1mg/ml TMB tetramethyl biphenyl diamines, 37 DEG C of lucifuge effect 5 min;

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

(7) result judges: be judged to be pig TTV2 antibody positive during serum OD450 value >=0.248 to be checked, OD450 value ∠ 0.248 is judged to be feminine gender.

beneficial effect

The inventive method utilizes molecular biology gene cloning and expression technology first, obtain pig TTV2 ORF1 recombinant protein, be that antigen sets up indirect ELISA with recombinant protein, the pig TTV2 that current China is popular effectively can be diagnosed, judge the situation of TTV2 antibody horizontal and natural infection in swinery, the anti-system for pig TTV2 virus provides a kind of quick, easy detection method.

Test proves, the indirect ELISA method that the recombinant protein (TTV2 ORF1) after the present invention utilizes expression and purification is first set up as antigen coated elisa plate, really can detect anti-pig TTV2 antibody in pig body, and have good specificity and repeatability.Detect clinical sample, result shows that this method can apply in production reality as a kind of SD method of pig TTV2.

Accompanying drawing explanation

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

1: ultrasonic treatment precipitates; 2: ultrasonic treatment supernatant; 3: induction afterproduct; 4: non-induced product; 5: albumen Marker

Fig. 2 western blot test display 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: empty carrier inducible protein; 2:pcoldI-ORF1 purifying protein; 3: albumen Marker

Embodiment

Main points of the present invention are: utilize pcoldI to build the prokaryotic expression carrier of part pig TTV2 ORF1 gene, and the recombinant protein after expressing is carried out purifying, set up indirect ELISA testing kit and detection method, and apply to clinical detection.

The preparation of envelope antigen:

1.1 specific primer design and synthesis: the pig TTV2 gene order (GenBank accession number is: EF514716) of including according to GenBank, choose coding pig TTV2 open reading frame ORF1 gene, utilize Primer Premier 5.0 Software for Design contain the primer of restriction enzyme site and submit to Shanghai Ying Jun company to synthesize.

SFORF1-:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG ( XhoI)

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA( HindIII)

The structure of 1.2 prokaryotic expression carriers: utilize above-mentioned Auele Specific Primer, amplification gene fragment from TTV2 positive pathological material of disease, obtain the amplified fragments of 1227bp, recycle the restriction enzyme on respective primer, carry out enzyme to pcr amplification product and pcoldI prokaryotic expression carrier (purchased from Takara) respectively to cut, in turn the pcr amplified fragment that enzyme is cut is connected on pcoldI with T4 DNA ligase (purchased from Takara).Specifically, first by the fragment warp respectively of pcoldI carrier and SFORF1 and SRORF1 primer amplification xhoi and hindiII digestion, reclaim respective endonuclease bamhi, connect with T4 DNA ligase, build pcoldI-ORF1 carrier, connect in product conversion bacillus coli DH 5 alpha (Beijing Quanshijin Biotechnology Co., Ltd) competence, extract plasmid, enzyme cuts qualification positive colony, and the Nanjing Jin Sirui company that send being accredited as positive colony carries out sequencing.

Picking warp xhoi and hindthe plasmid that III double digestion goes out 1215bp band checks order, and sequencing result shows that object fragment is correctly connected with pcoldI carrier, and the reading frame of expressing protein is correct, and result shows that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.Plasmid pcoldI-ORF1 is transformed in Host Strains e. coli bl21 and becomes genetic engineering bacterium, called after genetic engineering bacterium BLpcoldI-ORF1.

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

The abduction delivering of 1.3 recombinant proteins and analysis

The LB fluid nutrient medium that genetic engineering bacterium BLpcoldI-ORF1 is inoculated in containing Amp in 2% ratio (is added tryptone 10g in 950mL deionized water, yeast extract 5g, NaCl 10g, pH to 7.0 is adjusted with NaOH, 1L is settled to deionized water) in, 15 DEG C of shaken cultivation are to OD600 ≈ 0.4 ~ 0.6, adding IPTG to final concentration is 0.2mmol/L, carry out abduction delivering, the thalline that after collecting induction, different time is expressed, ultrasonication is carried out on ice bath, bacterium liquid after cracking is through the centrifugal 30min of 10000rpm, in separation, cleer and peaceful precipitation carries out SDS-PAGE electrophoresis (the discontinuous SDS-PAGE system of employing LaemimLi simultaneously, separation gel 12%, concentrated glue 4%), the expression of qualification destination protein and expression-form.

1.4 recombinant protein purifications:

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

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

Western Blotting analyzes: carry out SDS-PAGE according to the albumen of method purifying in 1.4, operate with reference to conventional Western Blotting method, finally adopts DAB substrate visualizingre agent (Wuhan doctor's moral company) to develop the color.There is specific band at 52KDa place in result, 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 the TTV2 ORF1 recombinant protein after purifying to do envelope antigen and set up indirect ELISA method, adopt square formation method to explore ELISA optimum protein bag by concentration and serum dilution, and ELISA optimum reaction condition.

The determination of 2.1 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 DEG C of bags are spent the night, closed 2h is carried out with 1%BSA after washing, add with the serum of optimum dilution degree dilution after washing, effect 1h, adds the ELIAS secondary antibody effect 1h of 1:5000 dilution after washing, add TMB room temperature lucifuge colour developing 10min after washing, determine most suitable coating buffer by OD450 value and P/N value.Result shows, and the bag of carbonate buffer solution is best by effect, therefore uses carbonate buffer solution as antigenic dilution coated elisa plate.

The best effort concentration of 2.2 antigen-antibodies

The carbonate buffer solution of the pcold-ORF1 recombinant protein 0.05mMpH9.6 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, and 100 μ l/ holes, 4 DEG C are spent the night.Taking-up next day PBST washs 3 times, each 5min.Bovine serum albumin(BSA) (BSA) with 1% is closed, 200 μ l/ holes, 37 DEG C 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, adds each hole successively from left to right, 100 μ l/ holes, 37 DEG C 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 DEG C of effect 1h, washing (the same); Every hole adds 100 μ l TMB nitrite ions, lucifuge color development at room temperature 10 min, finally adds 100 μ l stop buffer cessation reactions, uses microplate reader to measure its OD450 value immediately.The best effort concentration of antigen and antibody is determined according to OD450 value and P/N value.Result shows, and when the dilutability of antigen is 1:160, when the dilutability of antibody is 1:160, the OD450 value of positive serum is close to 1.0, and P/N value is maximum.Therefore, determine that recombinant protein the best bag is 1.65 μ g/mL by concentration, serum optimum dilution degree is 1:160.

2.2 best bags are by the selection of time

With best antigen concentration bag by elisa plate, be divided into three groups.First group: 37 DEG C of bags are by 2h; Second group: 37 DEG C hatch 1h after 4 DEG C spend the night; 3rd group: 4 DEG C bags are spent the night.Positive and negative serum respectively does 4 repetitions.Three kinds of different bags are wrapped rear under condition, washing, closed 2h is carried out with 1%BSA, add with the serum of optimum dilution degree dilution after washing, effect 1h, add the ELIAS secondary antibody effect 1h of 1:5000 dilution after washing, after washing, add TMB room temperature lucifuge colour developing 10min, determine that best bag is by condition by OD450 value and P/N value.Result determines that best bag is 4 DEG C by the time and spends the night.

The selection carbonate buffer solution of 2.3 best confining liquids by antigen diluent to optium concentration bag by elisa plate, 4 DEG C of bags are spent the night.After taking out washing next day, respectively using 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 respectively does 2 repetitions.Carry out closed 2h, add with the serum of optimum dilution degree dilution after washing, effect 1h, adds the ELIAS secondary antibody effect 1h of 1:5000 dilution after washing, adds TMB room temperature lucifuge colour developing 10min, determine best confining liquid by OD450 value and P/N value after washing.Result shows, and when carrying out closed with 2% skimmed milk power, P/N value is maximum, and sealing effect is best, therefore selects 2% skimmed milk power as best confining liquid.The selection carbonate buffer solution of 2.4 off-periods by antigen diluent to optium concentration bag by elisa plate, 4 DEG C of bags are spent the night.After taking out washing next day, close 30min, 60min, 90min, 120min respectively with 2% skimmed milk power, yin and yang attribute serum respectively does 4 repetitions.Add with the serum of optimum dilution degree dilution after washing, effect 1h, adds the ELIAS secondary antibody effect 1h of 1:5000 dilution after washing, adds TMB room temperature lucifuge colour developing 10min, determine best off-period by OD450 value and P/N value after washing.When being when closed 60min and 90min, P/N value is comparatively large and be more or less the same, and therefore in order to save time, this experiment selects off-period 60min as the best off-period.

The selection of 2.5 best serum dilutions

Closed composition is added in serum dilution, can reach and reduce 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 known with oneself carries out ELISA detection, observe the change of OD value and P/N change, and screen best primary antibodie dilution in contrast with PBS dilute serum.Result selects the PBS containing 0.5%BSA as best primary antibodie dilution.

The determination of 2.6 serum optimum reacting times

With carbonate buffer solution by antigen diluent to optium concentration bag by elisa plate, 4 DEG C of bags are spent the night.After taking out washing next day, close 60min with 2% skimmed milk power, after washing, add the serum being diluted to optium concentration with 1.5%BSA sample diluting liquid, act on 30min, 60min, 90min, 120min respectively, yin and yang attribute serum respectively does 4 repetitions.Add the ELIAS secondary antibody effect 1h of 1:5000 dilution after washing, add TMB room temperature lucifuge colour developing 10min after washing, determine serum optimum reacting time by OD450 value and P/N value.When serum action time is 60min, the value of P/N is obviously larger, and therefore the selection serum the best use of time is 60min.

The determination of 2.7 the suitableeest ELIAS secondary antibody working concentrations

With carbonate buffer solution by antigen diluent to optium concentration bag by elisa plate, 4 DEG C of bags are spent the night.After taking out washing next day, 60min is closed with 2% skimmed milk power, after washing, add the serum effect 60min being diluted to optium concentration with 1.5%BSA sample diluting liquid, after 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 respectively does 2 repetitions.Add TMB room temperature lucifuge colour developing 10min after washing, determined the optium concentration of ELIAS secondary antibody by OD450 value and P/N value.When ELIAS secondary antibody concentration is 1:5000, P/N value is maximum, and therefore this experiment selects 1:5000 as the best effort concentration of ELIAS secondary antibody.

The determination in 2.8 the suitableeest ELIAS secondary antibody reaction time

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

With carbonate buffer solution by antigen diluent to optium concentration bag by elisa plate, 4 DEG C of bags are spent the night.After taking out washing next day, 60min is closed with 2% skimmed milk power, after washing, add the serum effect 60min being diluted to optium concentration with 1.5%BSA sample diluting liquid, the ELIAS secondary antibody that concentration is 1:4000 is added after washing, effect 30min, add TMB room temperature lucifuge after washing and to develop the color respectively 3min, 5min, 8min, yin and yang attribute serum respectively does 4 repetitions.Substrate optimum reacting time is determined by OD450 value and P/N value.When being when reacted 5min, P/N value is maximum, therefore selects the substrate reactions time to be 5min.

The determination of 2.10 indirect ELISA method yin and yang attribute critical values

Negative Swine serum is detected with the indirect ELISA method set up, the mean value (X) calculating 40 parts of TTV negative serum OD450 is 0.191, standard deviation (SD) is 0.019, according to formula: X+3SD determines that the yin and yang attribute critical value of indirect ELISA detection method is 0.248, namely being the positive when OD450 value >=0.248 of sample, is then negative during OD450 value < 0.248.

The specificity of 2.11 indirect ELISAs and replica test

Known SS2(streptococcus suis 2-type is detected with the pig TTV2 indirect ELISA detection method set up) (see Zhang Xuehan, what Kong Wang, Zhou Junming etc. the structure [J] of streptococcus suis type 2 carnine acidohydrogenase deletion strain. Scientia Agricultura Sinica .2009.5 (42): 1789-1796.), HPS(haemophilus parasuis) (see Wang Haiyan, Liu Maojun, Feng Zhi is new. haemophilus parasuis cultural character and Study on Pathogenicity [J]. and Jinling School of Science and Technology journal. 2009.4 (25): 80-83), Mhp(mycoplasma hyopneumoniae) (see Wang Zhanwei, Liu Maojun, Feng Zhi is new. and osmotic pressure is on the impact [J] of mycoplasma hyopneumoniae 168 strain. agricultural Science & Technology. 2012.13 (10): 2051-2054), PCV2(porcine circovirus 2 type) (see Guo Rongli, what Kong Wang, Zhong Shulin etc. the separation qualification of two strain porcine circovirus 2 type viruses and sequential analysis [J] thereof. Jiangsu's agriculture journal. 2009.25 (5): 1063-1067.), PPV(pig parvoviral) (see 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 report. 2010.11 (32): 844-848), PRV(PRV) (see Zhang Xuehan, what Kong Wang, Miao Wenkai etc. Taqman probe real-time quantitative PCR detects PRV [J]. Jiangsu's agriculture journal. 2008.24 (4): 440-443.), TGEV(TGE) (see what Kong Wang, Lin Jihuang, also red China etc. transmissible gastro-enteritis virus weakening strain STC3 cell culture characteristic and Study on Pathogenicity [J]. Chinese Journal of Veterinary Science and Technology. 2001.31 (8): 8-9.), CSFV(CSFV) (see Zhang Xuehan, what Kong Wang, Guo Rongli etc. detect CSFV, JEV, the foundation [J] of PRRSV tri-kinds of RNA virus multiple RT-PCR methods. Chinese Preventive Veterinary Medicine report. 2007.29 (5): 385-388.), PRRSV(porcine reproductive and respiratory syndrome virus) (see Li Bin, Mao Li, He Kongwang etc. the clone of porcine reproductive and respiratory syndrome virus (PRRSV) Jiangsu separated strain NJGC ORF5 gene and Genetic Variation Analysis [J]. Jiangsu's agriculture journal. 2011.27 (4): 775-781.), FMDV(foot and mouth disease virus) (see Liu Yaofang, what Kong Wang, Zhang Dekun etc. utilize multiple RT-PCR to foot and mouth disease virus somatotype [J]. Jiangsu's agriculture science. 2007.5:136-139.) positive serum, set up TTV2 positive simultaneously, negative control, measure its OD450, determine whether cross reaction.Result OD value is all less than 0.248, is feminine gender, 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: the purified recombinant antigens bag prepared with same batch is by same elisa plate, 2 parts of positive serums and 2 parts of negative serum samples are detected, every part of serum sample repeats 4 holes, measure OD450 value, calculate the coefficient of variation (CV=SD/X), evaluate batch interior revision test effect.Result shows, the OD450 value coefficient of variation of 4 parts of serum is all less than 10%, shows that same sample degree of variation in same batch experiment is less, has good repeatability.

Repeat between batch: in the ELISA Plate of the series protein bag quilt prepared at different batches, often criticize and randomly draw 1, detect the serum that 6 parts of TTV2 antibody horizontals are different, every part of serum is parallel does 4 holes, calculates the coefficient of variation (CV=SD/X) of testing result.The OD450 value coefficient of variation of 6 parts of serum is all less than 5%, shows that same sample degree of variation in different batches test is less, has good repeatability.

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

3. the Preliminary Applications of indirect ELISA method

By the indirect ELISA method set up, 352 parts of Swine serum of province of Jiangsu and Anhui two censorship between 2010 ~ 2011 are detected.The serum of Jiangsu censorship totally 192 parts, wherein 82 parts of testing results are positive, and positive rate is 42.7%, the serum of Anhui censorship totally 160 parts, and wherein 12 parts of testing results are positive, and positive rate is the TTV2 total positives rate of 7.5%, two province's censorship serum is 26.7%.

SEQUENCE LISTING

 

 

<110> Jiangsu Province Agriculture Science Institute

 

 

<120> mono-kind detects the indirect ELISA reagent kit of pig Transfusion transmitted virus 2 type antibody

 

 

<130> 0

 

 

<160> 3

 

 

<170> PatentIn version 3.1

 

 

<210> 1

<211> 403

<212> PRT

<213> is 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

           

 

 

<210> 2

<211> 34

<212> DNA

<213> is artificial

 

 

<220>

<221> SFORF1-F

<222> (1)..(34)

<223>

 

 

 

<400> 2

ccgctcgagg acttaacgga accgtggcta gaag 34

 

 

<210> 3

<211> 38

<212> DNA

<213> is artificial

 

 

<220>

<221> SRORF1-R

<222> (1)..(38)

<223>

 

 

 

<400> 3

cccaagcttt gttttcatcc tctttaccac ccgctgga 38

 

Claims (3)

1. boar Transfusion transmitted virus 2 type (TTV2) antibody indirect ELISA diagnostic kit, is characterized in that: be provided with antibody test plate in kit, confining liquid, sample diluting liquid, cleansing solution, enzyme conjugates working fluid, enzyme substrate solution, stop buffer, positive control and negative control; Wherein antibody test plate is that bag is by the removable 96 hole ELISA Plate of pig TTV2 restructuring ORF1 albumen, 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, wraps by pig TTV2 restructuring ORF1 protein sequence as follows:

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 restructuring ORF1 albumen is expressed by prokaryotic expression plasmid pcoldI-ORF1, and prokaryotic expression plasmid pcoldI-ORF1 is built by following methods and forms:

Design a pair primer containing restriction enzyme site:

SFORF1:CCG CTCGAGGACTTAACGGAACCGTGGCTAGAAG XhoI

SRORF1:CCC AAGCTTTGTTTTCATCCTCTTTACCACCCGCTGGA HindIII

Utilize above-mentioned Auele Specific Primer, increase ORF1 genetic fragment from the positive pathological material of disease of TTV2, obtain the amplified fragments of 1227bp, recycle the restriction enzyme on respective primer, carry out enzyme to pcr amplification product and pcoldI prokaryotic expression carrier respectively to cut, with T4 DNA ligase, the digestion products of amplified fragments is connected on pcoldI digestion products, build pcoldI-ORF1 carrier, connect in product conversion bacillus coli DH 5 alpha competence, cut qualification and order-checking qualification through enzyme, show that recombinant prokaryotic expression vector plasmid pcoldI-ORF1 successfully constructs.

3. a boar TTV2 antibody indirect ELISA diagnostic kit according to claim 1 and 2, it for detecting the method for the antibody horizontal of pig TTV2 in Swine serum is:

(1) bag quilt: the final concentration after ORF1 protein purification of being recombinated by the boar TTV2 described in claim 1 or 2 with antigen coated liquid dilution being 1.65 μ g/mL, each ELISA hole adds 100 μ l, wraps and spent the night under 4 DEG C of conditions; Discard the coating buffer body in plate, PBST buffer solution 5min × 3 time, antigen coated liquid is the carbonate buffer solution of 0.05M, pH9.6; PBST damping fluid is the PBS damping fluid containing mass ratio 0.01%Tween-20;

(2) close: every hole adds confining liquid 200 μ l, acts on 1h, PBST buffer solution 5min × 3 time under 37 DEG C of conditions; Confining liquid is the PBS damping fluid containing mass ratio 2% skimmed milk power;

(3) serum to be checked is added: every hole adds the serum of 100 μ l after the PBS dilution containing mass ratio 0.5%BSA, acts on 1.0h, PBST buffer solution 5min × 3 time under 37 DEG C of conditions;

(4) add two to resist: every hole adds the anti-pig IgG of goat of the HRP mark that 100 μ l dilute, and acts on 30 min, PBST buffer solution 5min × 3 time under 37 DEG C of conditions; The anti-pig IgG of goat of the HRP mark of dilution is use the PBS of pH7.0 by 1:5000 dilution proportion;

(5) nitrite ion is added: the nitrite ion 100 μ l of every Kong Jiahan 1mg/ml TMB tetramethyl biphenyl diamines, 37 DEG C of lucifuge effect 5 min;

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