CN102532281B - Classical swine fever virus recombinant E2 protein and IgM (immune globulin M) antibody ELISA (enzyme-linked immunosorbent assay) test kit thereof - Google Patents
Classical swine fever virus recombinant E2 protein and IgM (immune globulin M) antibody ELISA (enzyme-linked immunosorbent assay) test kit thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and relates to a classical swine fever virus recombinant E2 protein and an IgM (immune globulin M) antibody ELISA (enzyme-linked immunosorbent assay) test kit thereof. The classical swine fever virus E2 protein expressed by recombinant Escherichia coli is obtained by cloning the main antigen region of E2 protein into a pronucleus expression vector to obtain a recombinant expression vector, transforming the recombinant expression vector to Escherichia coli BL21 (DE3) and expressing and purifying with the recombinant Escherichia coli. A Westernblot test indicates that the protein has good antigenicity. According to the invention, an elisa plate is coated the protein; an ELISA method is established through optimization of antigen coating concentration, serum dilution and action time, secondary antibody concentration and action time as well as developing time for the purpose of detecting negative serum so as to determine a critical value and a judgment standard. According to the invention, the expression of a recombinant strain constructed by the recombinant E2 protein on a heterologous protein is stable, and the recombinant strain is good in antigenicity; and on the basis, the recombinant E2 protein disclosed by the invention is used for establishing a CSFV (classical swine fever virus) IgM antibody ELISA test kit for the first time.
Description
Technical field
The invention belongs to biology field, relate to a kind of Classical Swine Fever Virus recombinant E2 protein and Pestivirus suis IgM antibody ELISA detection kit.
Background technology
Escherichia expression system is the expression system of current comparative maturity, have simple to operate, safety non-toxic, exogenous protein expression amount advantages of higher, successfully for the production of multiple protein.E2 albumen is the main envelope protein of Pestivirus suis (CSFV), and mediation virus infection enters born of the same parents and protective immunological reaction, is the main immune protective antigen of CSFV.The detection method of hog cholera antibody is mainly set up indirectly or blocking-up ELISA and indirect hemagglutination test based on E2 albumen at present; but produce rule and the research that acts on not yet has report for this protein induced IgM antibody in immunoprotection; the present invention is intended to set up to detect the ELISA method of E2 protein I gM antibody, for clinical detection and correlative study lay the foundation.
Summary of the invention
Technical problem
The object of the invention is for the present situation there is no at present for CSFV IgM antibody detection method, a kind of IgM of CSFV accurately and reliably antibody ELISA detection kit is provided.
Another object of the present invention is to provide the preparation method for the restructuring E2 albumen of ELISA method.
Technical scheme
Object of the present invention is achieved through the following technical solutions:
A kind of CSFV E 2 protein of expression of recombinant e. coli, this albumen system enters prokaryotic expression carrier by the gene fragment clone that comprises E2 major antigen district and obtains recombinant expression vector, this recombinant expression vector is transformed to e. coli bl21 (DE3), this recombination bacillus coli BL21 (DE3) is cultured to that when OD600 reaches 0.6-0.8, to add final concentration be that the IPTG of 0.5mM carries out abduction delivering, and separating thallus obtains through Ni affinity chromatography column purification.
Described E2 aminoacid sequence is SEQ ID NO.1, i.e. the 791-969 amino acids residue of Pestivirus suis polyprotein.
LCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRDKPFPHRMDCVTTIVENEDLFYCKLGGNWTCVKGEPVVYTGGVVKQCRWCGFDFDGPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISTEGSHECLIGNTTVKVHASDERLGPMPCRPK
Its nucleotides sequence is classified SEQ ID NO.2 as.
CTGTGCCCGTTTGATACGAGTCCTGTTGTTAAGGGAAAGTACAATACGACCTTGTTGAACGGTAGTGCTTTCTATCTTGTCTGCCCAATAGGGTGGACGGGTGTCATAGAGTGCACAGCAGTGAGCCCAACAACTCTGAGGACAGAAGTGGTAAAGACCTTCAGGAGAGACAAGCCCTTTCCGCACAGAATGGATTGTGTGACCACCATAGTGGAAAATGAAGATTTATTCTATTGTAAGTTGGGGGGCAACTGGACATGTGTGAAAGGCGAGCCAGTGGTCTACACAGGGGGGGTAGTAAAACAATGTAGATGGTGTGGCTTCGACTTCGATGGGCCTGACGGACTCCCGCATTACCCCATAGGTAAGTGCATTTTGGCAAATGAGACAGGTTACAGAATAGTAGATTCAACGGACTGTAACAGAGATGGCGTTGTAATCAGCACAGAGGGGAGTCATGAGTGCTTGATCGGTAACACGACTGTCAAGGTGCATGCATCAGATGAAAGACTGGGCCCTATGCCATGCAGACCTAA
Described prokaryotic expression carrier is pET32a.
The preparation method of the CSFV E2 albumen of expression of recombinant e. coli of the present invention, comprises the steps:
(1) according to CSFV gene order design primers F: CCGGAATTCCGGCTGTGCCCGTTTGATACGAGTC(SEQ ID NO.3), R:CAAGCTTGTCTTTAGGTCTGCATGGCATAGG(SEQ ID NO.4), from hog cholera lapinised virus vaccine poison, extract RNA, through RT-PCR, increase and obtain encoding goal gene fragment (SEQ ID NO.2), by
ecorI and
hindtwo restriction enzyme sites of III, enter described gene fragment clone in prokaryotic expression carrier, and the evaluation of then cutting by enzyme and check order, obtains positive recombinant plasmid;
(2) by step (1) Suo Shu through the correct recombinant plasmid transformed e. coli bl21 (DE3) of sequencing, obtain recombinant strains BL21-△ E2;
(3) cultivate described recombinant strains BL21-△ E2, add IPTG to carry out abduction delivering, through affinity column purifying, obtain described recombinant protein.
ELISA detection kit prepared by the CSFV E 2 protein of described expression of recombinant e. coli, comprises the coated enzyme plate of described albumen, 0.5%BSA as best confining liquid, yin and yang attribute contrast, ELIAS secondary antibody IgM, TMB nitrite ion, stop buffer and washings.Its ELISA detection method for IgM antibody is, by above-mentioned recombinant protein coated elisa plate, ELISA method is set up in optimization by antigen coated concentration, serum dilution and action time, two anti-concentration and action time and developing time, detects negative serum and determines threshold value and criterion.
Beneficial effect:
At present the CSFV antibody detection method great majority of report be the E2 albumen of recombinating be basis, but be all to detect IgG antibody, about the detection method of IgM antibody report not also; In addition, also not about the change report of rule of IgM antibody after swine Fever Vaccine immunity and wild virus infection.The present invention has built the recombination bacillus coli of expressing swine fever E2 albumen, and use it for and set up CSFV IgM antibody ELISA detection method, Preliminary Applications result shows that this recombinant protein has good antigenicity, the ELISA test kit of setting up is special, accurate, responsive, reproducible after using, and this will lay the foundation for IgM antibody after systematic study hog cholera immune produces rule, the monitoring of clinical infection situation and the foundation of differential diagnostic method.
Accompanying drawing explanation
The pcr amplification of Fig. 1 goal gene.
1:PCR product; 2:DNA marker DL-2000
The enzyme of Fig. 2 recombinant plasmid is cut evaluation
1:DNA marker DL-2000; 2: the recombinant plasmid that enzyme is cut
Fig. 3 SDS-PAGE detects the expression and purification of recombinant protein
1: the albumen of purifying; 2: the supernatant after induction; 3: inclusion body; 4: empty plasmid induction contrast; M: albumen marker
The Western blot of Fig. 4 recombinant protein identifies (left figure: His monoclonal antibody is primary antibodie; Right figure: positive porcine blood serum is primary antibodie)
1: dye in advance albumen marker; 2: recombinant protein; 3: empty plasmid contrast
Embodiment
1.1 design of primers
According to designing primer according to CSFV gene order, each introduces upstream and downstream primer
ecorI and
hindiII site, sequence is as follows:
F: CCGGAATTCCGGCTGTGCCCGTTTGATACGAGT C（SEQ ID NO.3）；
R: CAAGCTTGTCTTTAGGTCTGCATGGCATAGG（SEQ ID NO.4）
Above primer is synthetic by Si Pujin bio tech ltd, Nanjing.
1.2 pcr amplification object fragments
Get 200 μ l hog cholera lapinised virus vaccine poison (purchased from Nanjing Tianbang Bio-industry Co., Ltd.), add 1ml Trizol reagent, concussion mixes, standing 10min, adds 200 μ l chloroforms, concuss, 4 ℃ of centrifugal 10min of 12000rpm, carefully draw supernatant, add equal-volume Virahol to mix, place 2h for-20 ℃, 4 ℃ of centrifugal 15min of 12000rpm, abandon supernatant, add 75% washing with alcohol, dry, add 10 μ l to dissolve RNA without the distilled water of Rnase.
According to reverse transcription test kit (Beijing Quanshijin Biotechnology Co., Ltd) Oligo for operation instructions (dT) 18, carry out reverse transcription, be specially: 2 * ES Reaction Mix, 10 μ l; EasyScript RT/RI Enzyme Mix 1 μ l; Oligo (dT) 18 1 μ l; RNA 5 μ l; Distilled water without Rnase complements to 20 μ l.42 ℃ of reaction 45min, 85 ℃ of heating 5min deactivation ThermoScript II, take reverse transcription product as template, carry out pcr amplification.PCR reaction system is: upstream, each 1 μ L of downstream primer (10pmol/L); DNTP s(2.5 mmol/L) 2 μ L; 10 * PCR buffer, 2.5 μ L; Template 4 μ L; Taq enzyme (5 U/μ L) 0.5 μ L; Sterilizing distilled water is mended to 25 μ L.Loop parameter: 94 ℃ of denaturation 5min; With 94 ℃ of 30 s, 52 ℃ of 30 s, 72 ℃ of 45 s carries out 35 circulations; Last 72 ℃ are extended 10 min.1% agarose gel electrophoresis is identified PCR product, can see the band (the results are shown in Figure 1) of about 560bp, reclaims PCR product, and-20 ℃ save backup.
The structure of 1.3 recombinant plasmids and evaluation
The goal gene PCR product reclaiming is used
ecorI and
hindiII double digestion, reclaims purifying and is cloned into the expression vector plasmid pET-32a(Novagen that same enzyme is cut processing afterwards) in, ligation is carried out at 16 ℃, will connect afterwards product and transform
e.
colidH5 α (purchased from Beijing Quanshijin Biotechnology Co., Ltd) competent cell, coating is dull and stereotyped containing the LB of penbritin, 37 ℃ of cultivations.Bacterium colony on picking flat board, containing the LB culture medium culturing of penbritin, alkaline lysis method of extracting plasmid, through PCR and
ecorI and
hindiII double digestion is identified (the results are shown in Figure 2), and positive plasmid pET32a-e2 obtains the object band of about 560bp.Positive plasmid send Si Pujin bio tech ltd, Nanjing to check order.
The structure of embodiment 2 recombinant strains BL21-△ E2
2.1 transformed competence colibacillus intestinal bacteria BL-21
In embodiment 1, through the correct recombinant plasmid pET32a-e2 of sequencing, transform e. coli bl21 (DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd), obtain recombinant strains BL21-△ E2, simultaneously by empty plasmid pET-32a with method transformed competence colibacillus intestinal bacteria BL-21.
The abduction delivering of 2.2 recombinant proteins
Respectively picking recombinant strains BL21-△ E2 and containing the e. coli bl21 list colony inoculation of empty plasmid in the LB liquid nutrient medium that contains penbritin, 37 ℃ of joltings of spending the night are cultivated.
(1) the bacterium liquid 10 μ L that get incubated overnight are inoculated in the LB liquid nutrient medium that 3 ml contain penbritin (50 μ g/ml), and 37 ℃ of 200rpm shaking culture 3h left and right, make OD
600reach 0.6～0.8, get 100 μ L samples and as induction is front, contrast in aseptic Eppendorf pipe;
(2) to adding final concentration in above-mentioned bacterium liquid, be the abduction delivering that the IPTG of 0.5mmol/L carries out recombinant protein, and 4h receive bacterium after adding IPTG; The bacterium of the 4 ℃ of 8000 centrifugal 5min collection of rpm abduction delivering, PBS(pH 7.2) resuspended bacterial sediment;
(3) by bacterium liquid multigelation 3 times, ultrasonic treatment bacterium, until bacterium liquid becomes limpid;
(4) 4 ℃ of 8000 centrifugal 10min of rpm, uses precipitation with the isopyknic PBS of supernatant resuspended, and respectively to get 100 μ L standby with precipitation suspension for supernatant.
2.3SDS-PAGE electrophoretic analysis
In supernatant and protein precipitation sample and empty carrier induced product, add 5 * protein sample sample-loading buffer (250mM Tris-HCl, pH6.8; 10%SDS; 0.5% tetrabromophenol sulfonphthalein; 50% glycerine; 5% 2 mercapto ethanol), fully mix latter 100 ℃ and boil 5min, instantaneous centrifugal before loading, draw supernatant and carry out SDS-PAGE, in precipitation, visible significantly 38kD object band, does not have in empty carrier induced product and supernatant (Fig. 3), shows that target protein is with the form correction of inclusion body.
The great expression of embodiment 3 recombinant proteins, purifying and antigenicity are identified
The great expression of 3.1 recombinant proteins, purifying
The positive bacterium colony of recombinant strains BL21-△ E2 in picking 2.2, in inoculation 200mlLB, press the abduction delivering condition abduction delivering of embodiment 2, centrifugal collection thalline, by every 100ml bacterium liquid, add the resuspended bacterial sediment of 5ml PBS, centrifugal after ultrasonic degradation, cleer and peaceful inclusion body in separation, inclusion body is resuspended with equal-volume Binding buffer, carries out the purifying (Fig. 3) of target protein according to the explanation of GE HisTrap HP affinity purification post.Spectrophotometric determination protein concentration is 1.2mg/mL ,-20 ℃ of preservations after packing.
The Western blot of 3.2 recombinant proteins identifies
（1）SDS-PAGE；
(2) transfer printing: after electrophoresis finishes, take off gel and transfer device (half-dried transfer printing) is installed in the following order:
+the utmost point-sponge-NC film-gel-sponge-
-the utmost point, transfer printing condition is 20V, 30min;
(3) NC film (Pall) is spent the night with the PBST confining liquid sealing containing 5% skimming milk;
(4) by His-Tag Mouse McAb(Abmart) and confining liquid 1:2000(or 1:500 for positive porcine blood serum (purchased from China Veterinery Drug Inspection Office)) after dilution, join on NC film room temperature jog 2h;
(5) with PBST washing 5 times, 5min/ time;
(6) add sheep anti-mouse igg-HRP or goat-anti pig IgG-HRP(Beijing Bo Aosen of 1:1000 dilution), room temperature jog 1.5h;
(7) with PBST washing 5 times, 5min/ time;
(10) with DAB colouring reagents box colour developing (Wuhan doctor's moral): add each of A, B, C liquid in 1ml deionized water, be added to after mixing on NC film, until there is obvious band to occur.The results are shown in Figure 4, recombinant protein swimming lane all has specific band (38kD) to occur, empty carrier contrast does not have.
Foundation and the application of embodiment 4 ELISA test kits
The selection of the best coated concentration of 4.1 antigens and serum optimum dilution degree
By matrix method, undertaken, the carbonate buffer solution of pH 9.6 of take is diluted to respectively final concentration as 2.0,1.0 by antigen protein, 0.5,0.25 μ g/mL, 4 ℃ of coated elisa plates that spend the night (Nunc).After washing, add swine fever positive serum and negative serum with 1:50,1:100,1:200,1:400,1:800,1:1600 dilution with PBST, each extent of dilution repeats once, get its mean value, calculate P/N value under each condition, select positive serum OD value approaches 1, P/N value is maximum reaction conditions as ELISA optimum reaction condition.Result is: the best coated concentration 0.5 μ g/mL of antigen; Serum optimum dilution degree 1:100(table 1).
The selection of the best coated concentration of table 1 antigen and serum optimum dilution degree
The selection of 4.2 confining liquids
With the best antigen concentration coated elisa plate of determining in 4.1, after washing, with 37 ℃, the PBST 200 μ L/ hole containing 5% skimming milk, 1%BSA, 0.5%BSA and 1% gelatin sealing 2 h, detect 5 parts of positive serums and 1 part of negative serum respectively.Calculate P/N value, finally select 0.5%BSA that P/N value is the highest as best confining liquid (table 2).
The selection of table 2 confining liquid
The selection of 4.3 serum to be checked action times
, sealase target coated by above-mentioned condition, detect 5 parts of positive serums and 1 part of negative serum, 37 ℃ act on respectively 0.5 h, 1.0 h, 1.5 h and 2.0 h, it are carried out to ELISA mensuration, calculate P/N value, finally select 1.5h that P/N value is the highest as the best use of time.
The selection of table 3 serum to be checked action time
The selection of 4.4 IgM bis-anti-working concentrations
By above-mentioned condition be coated with, sealing, application of sample, the anti-pig IgM-HRP(BETHYL of rabbit that adds respectively 1:10000,1:50000,1:100000 dilution after washing), 100 μ L/ holes, carry out ELISA mensuration, and the highest 1:50000 of the P/N value of usining is as best effort concentration.
The selection of table 4 two anti-concentration
The selection of 4.5 bis-anti-action times of IgM
IgM bis-is resisted and added after enzyme plate by the extension rate determining, and 37 ℃ act on respectively 0.5 h, 1 h, 1.5h, standard yin and yang attribute serum is blocked to ELISA and measure, and respectively organize the blocking-up rate of positive serum, to select suitable enzyme mark monoclonal antibody action time.
The selection of two anti-action times of table 5
The selection of 4.6 developing times
According to screening conditions above, carry out ELISA test, add develop the color respectively 5,10 after TMB, 15min, calculate P/N value.Choose 15min that P/N is the highest as best developing time.
The selection of table 6 developing time
Determining of 4.7 blocking-up ELISA threshold values
CSFV antibody ELISA test kit before IDEXX and Wuhan section is detected to 50 parts of all negative pig anteserum samples, and the ELISA setting up with the present invention detects.Calculating mean value (
x) and standard deviation (SD) be respectively, sample OD value>=X+3SD=0.14+0.21=0.35 person is judged to the positive ,≤X+2SD=0.14+0.14=0.28 person is judged to feminine gender, the person of falling between is judged to suspicious.
4.8 replica test
Use 5 batches of albumen coated elisa plates, select 5 parts of porcine blood serum criticize in and batch between replica test, calculate the variation coefficient, repeatability with checking present method, result shows variation within batch coefficient <5%, interassay coefficient of variation <10%, proves that present method has good repeatability.
4.9 susceptibility and specific test
By positive serum and the continuous doubling dilution of negative serum, calculate P/N value, until 1/1600 is still positive, prove that the method has very high susceptibility; With ELISA, porcine circovirus 2 type (PCV2) (positive control in the PCV2 ELISA of Tianjin Ruipu Biotechnology Co., Ltd test kit), porcine pseudorabies virus (PRV) (IDEXX ELISA test kit positive control), porcine reproductive and respiratory syndrome virus (PRRSV) (IDEXX ELISA test kit positive control), Bovine Viral Diarrhoea-Mucosal Disease (BVDV) (purchased from China Veterinery Drug Inspection Office) standard positive serum are detected, result is negative, proves that present method has good specificity.
The detection of 4.10 clinical serum samples
Apply the ELISA test kit that this research sets up 310 parts of clinical serum are detected, take in 4.7 definite criterion as according to detected result is judged.Positive rate is 76%.
4.11 ELISA test kit
ELISA test kit comprises that the contrast of the coated enzyme plate of described albumen (0.5%BSA is as best confining liquid), yin and yang attribute, enzyme mark IgM bis-are anti-, TMB nitrite ion, stop buffer, washings.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> Classical Swine Fever Virus recombinant E2 protein and IgM antibody ELISA detection kit thereof
<130> 0
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 179
<212> PRT
<213> hog cholera lapinised virus vaccine poison
<220>
<221> E2 albumen
<222> (1)..(179)
<223>
<400> 1
Leu Cys Pro Phe Asp Thr Ser Pro Val Val Lys Gly Lys Tyr Asn Thr
1 5 10 15
Thr Leu Leu Asn Gly Ser Ala Phe Tyr Leu Val Cys Pro Ile Gly Trp
20 25 30
Thr Gly Val Ile Glu Cys Thr Ala Val Ser Pro Thr Thr Leu Arg Thr
35 40 45
Glu Val Val Lys Thr Phe Arg Arg Asp Lys Pro Phe Pro His Arg Met
50 55 60
Asp Cys Val Thr Thr Ile Val Glu Asn Glu Asp Leu Phe Tyr Cys Lys
65 70 75 80
Leu Gly Gly Asn Trp Thr Cys Val Lys Gly Glu Pro Val Val Tyr Thr
85 90 95
Gly Gly Val Val Lys Gln Cys Arg Trp Cys Gly Phe Asp Phe Asp Gly
100 105 110
Pro Asp Gly Leu Pro His Tyr Pro Ile Gly Lys Cys Ile Leu Ala Asn
115 120 125
Glu Thr Gly Tyr Arg Ile Val Asp Ser Thr Asp Cys Asn Arg Asp Gly
130 135 140
Val Val Ile Ser Thr Glu Gly Ser His Glu Cys Leu Ile Gly Asn Thr
145 150 155 160
Thr Val Lys Val His Ala Ser Asp Glu Arg Leu Gly Pro Met Pro Cys
165 170 175
Arg Pro Lys
<210> 2
<211> 536
<212> DNA
<213> is artificial
<220>
<221> gene fragment
<222> (1)..(536)
<223>
<400> 2
ctgtgcccgt ttgatacgag tcctgttgtt aagggaaagt acaatacgac cttgttgaac 60
ggtagtgctt tctatcttgt ctgcccaata gggtggacgg gtgtcataga gtgcacagca 120
gtgagcccaa caactctgag gacagaagtg gtaaagacct tcaggagaga caagcccttt 180
ccgcacagaa tggattgtgt gaccaccata gtggaaaatg aagatttatt ctattgtaag 240
ttggggggca actggacatg tgtgaaaggc gagccagtgg tctacacagg gggggtagta 300
aaacaatgta gatggtgtgg cttcgacttc gatgggcctg acggactccc gcattacccc 360
ataggtaagt gcattttggc aaatgagaca ggttacagaa tagtagattc aacggactgt 420
aacagagatg gcgttgtaat cagcacagag gggagtcatg agtgcttgat cggtaacacg 480
actgtcaagg tgcatgcatc agatgaaaga ctgggcccta tgccatgcag acctaa 536
<210> 3
<211> 34
<212> DNA
<213> is artificial
<220>
<221> primers F
<222> (1)..(34)
<223>
<400> 3
ccggaattcc ggctgtgccc gtttgatacg agtc 34
<210> 4
<211> 31
<212> DNA
<213> is artificial
<220>
<221> primer R
<222> (1)..(31)
<223>
<400> 4
caagcttgtc tttaggtctg catggcatag g 31
Claims (2)
1. the CSFV E 2 protein of an expression of recombinant e. coli, this Argine Monohydrochloride sequence is SEQ ID NO.1, system enters prokaryotic expression carrier pET-32a by the gene fragment clone that comprises E2 major antigen district and obtains recombinant expression vector, this recombinant expression vector is transformed to e. coli bl21 (DE3) and obtain recombination bacillus coli BL21 (DE3), this recombination bacillus coli BL21 (DE3) is cultured to when OD600 reaches 0.6-0.8, to add final concentration be that the IPTG of 0.5mM carries out abduction delivering, separating thallus obtains through Ni affinity chromatography column purification.
2. the CSFV E 2 protein of expression of recombinant e. coli according to claim 1, the nucleotides sequence of the described E2 albumen that it is characterized in that encoding is classified SEQ ID NO.2 as.
3. the preparation method of the CSFV E 2 protein of the expression of recombinant e. coli described in claim 1 or 2, is characterized in that comprising the steps:
(1) according to CSFV gene order design primer
F: CCGGAATTCCGGCTGTGCCCGTTTGATACGAGTC、
R:CAAGCTTGTCTTTAGGTCTGCATGGCATAGG extracts RNA from hog cholera lapinised virus vaccine poison, and through RT-PCR, increase and obtain encoding goal gene fragment SEQ ID NO.2, by
ecorI and
hindtwo restriction enzyme sites of III, enter described gene fragment clone in prokaryotic expression carrier, and the evaluation of then cutting by enzyme and check order, obtains positive recombinant plasmid;
(2), by the positive recombinant plasmid transformed e. coli bl21 (DE3) step (1) Suo Shu, obtain recombinant strains BL21-△ E2;
(3) cultivate described recombinant strains BL21-△ E2, add IPTG to carry out abduction delivering, through affinity column purifying, obtain the CSFV E 2 protein of described expression of recombinant e. coli.
4. the ELISA detection kit of preparing with the CSFV E 2 protein of the expression of recombinant e. coli described in claim 1 or 2, comprises that the coated enzyme plate of the CSFV E 2 protein of described expression of recombinant e. coli, mass ratio 0.5%BSA are as confining liquid, yin and yang attribute contrast, the anti-pig IgM of ELIAS secondary antibody HRP mark rabbit, TMB nitrite ion, stop buffer and washings.
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| CN102967666B (en) * | 2012-11-16 | 2014-05-07 | 新疆天康畜牧生物技术股份有限公司 | Quantitative detection method for content of hog cholera virus E2 proteins |
| CN103091489B (en) * | 2013-01-10 | 2015-05-20 | 上海交通大学 | ELISA test kit of M protein antibody in animals infected with VSV and test method of ELISA test kit |
| CN103588864B (en) * | 2013-11-28 | 2015-01-14 | 华南农业大学 | Classic swine fever virus (CSFV) C strain E2 truncated protein and its preparation method and use |
| CN103951734A (en) * | 2014-04-21 | 2014-07-30 | 江苏省农业科学院 | Recombinant E2 protein of border disease virus and detection kit for IgG (immunoglobulin G) antibody of border disease virus |
| CN103983782A (en) * | 2014-06-06 | 2014-08-13 | 武汉中博生物股份有限公司 | ELISA kit for detecting hog cholera virus Erns IgM antibody |
| CN104730248A (en) * | 2015-03-17 | 2015-06-24 | 吉林吉和迅生物技术有限公司 | Rapid diagnosis and detection test paper card for detecting swine fever as well as preparation and application method of rapid diagnosis and detection test paper card |
| CN106749519B (en) * | 2016-11-15 | 2020-04-07 | 河南省农业科学院 | Peptide ligand sequence design and application based on computer simulation and used for CSFV E2 protein targeted combination |
| CN115433737A (en) * | 2022-08-23 | 2022-12-06 | 上海市农业科学院 | Indirect ELISA detection method for meishan swine enzootic pneumonia |
| CN115806595B (en) * | 2022-12-08 | 2023-07-28 | 江苏农牧科技职业学院 | Recombinant antigen protein for African swine fever virus detection, preparation method, detection kit and application thereof |
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