CN102532281A - 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 CSFV reorganization E2 albumen and CSFV IgM antibody ELISA detection kit.
Background technology
Escherichia expression system is the expression system of present comparative maturity, have simple to operate, safety non-toxic, exogenous protein expression amount advantages of higher successfully has been used for the production of multiple protein.E2 albumen is the main envelope protein of CSFV (CSFV), and the mediation virus infection is gone into 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 in immunoprotection, acts on does not have report as yet for this protein induced IgM antibody; The present invention is intended to set up the ELISA method that detects E2 protein I gM antibody, for clinical detection and correlative study lay the foundation.
Summary of the invention
Technical problem
The objective of the invention is provides a kind of IgM of CSFV accurately and reliably antibody ELISA detection kit at present still useless in the present situation of CSFV IgM antibody detection method.
Another object of the present invention provides the proteic preparation method of reorganization E2 who is used for the ELISA method.
Technical scheme
The object of the invention is realized through following technical scheme:
A kind of CSFV E2 albumen of expression of recombinant e. coli; The gene fragment clone that this albumen system will comprise E2 major antigen district is gone into prokaryotic expression carrier and is obtained recombinant expression vector; With this recombinant expression vector transformed into escherichia coli BL21 (DE3); This recombination bacillus coli BL21 (DE3) adds final concentration when being cultured to OD600 and reaching 0.6-0.8 be that the IPTG of 0.5mM carries out abduction delivering, and separating thallus obtains through Ni affinity chromatography column purification.
Said E2 aminoacid sequence is SEQ ID NO.1, i.e. the 791-969 amino acids residue of CSFV polyprotein.
LCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRDKPFPHRMDCVTTIVENEDLFYCKLGGNWTCVKGEPVVYTGGVVKQCRWCGFDFDGPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISTEGSHECLIGNTTVKVHASDERLGPMPCRPK
Its nucleotides sequence is classified SEQ ID NO.2 as.
CTGTGCCCGTTTGATACGAGTCCTGTTGTTAAGGGAAAGTACAATACGACCTTGTTGAACGGTAGTGCTTTCTATCTTGTCTGCCCAATAGGGTGGACGGGTGTCATAGAGTGCACAGCAGTGAGCCCAACAACTCTGAGGACAGAAGTGGTAAAGACCTTCAGGAGAGACAAGCCCTTTCCGCACAGAATGGATTGTGTGACCACCATAGTGGAAAATGAAGATTTATTCTATTGTAAGTTGGGGGGCAACTGGACATGTGTGAAAGGCGAGCCAGTGGTCTACACAGGGGGGGTAGTAAAACAATGTAGATGGTGTGGCTTCGACTTCGATGGGCCTGACGGACTCCCGCATTACCCCATAGGTAAGTGCATTTTGGCAAATGAGACAGGTTACAGAATAGTAGATTCAACGGACTGTAACAGAGATGGCGTTGTAATCAGCACAGAGGGGAGTCATGAGTGCTTGATCGGTAACACGACTGTCAAGGTGCATGCATCAGATGAAAGACTGGGCCCTATGCCATGCAGACCTAA
Described prokaryotic expression carrier is pET32a.
The proteic preparation method of CSFV E2 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 the hog cholera lapinised virus vaccine poison, extract RNA; Through the RT-PCR amplification target gene fragment (SEQ ID NO.2) that obtains encoding, through
EcoRI with
HindTwo restriction enzyme sites of III are gone into described gene fragment clone in the prokaryotic expression carrier, and the evaluation of cutting through enzyme and check order then obtains positive recombinant plasmid;
(2) step (1) is described 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 and carry out abduction delivering, obtain described recombinant protein through the affinity column purifying.
The ELISA detection kit of the CSFV E2 protein Preparation of described expression of recombinant e. coli comprises enzyme plate that said albumen encapsulates, 0.5%BSA as best confining liquid, yin and yang attribute contrast, ELIAS secondary antibody IgM, TMB develop the color liquid, stop buffer and washings.Its ELISA detection method that is used for IgM antibody does; With above-mentioned recombinant protein coated elisa plate; The ELISA method is set up in optimization through antigen coated concentration, serum dilution and action time, two anti-concentration and action time and developing time, detects negative serum and confirms threshold value and criterion.
Beneficial effect:
The CSFV antibody detection method great majority of report are to be the basis with reorganization E2 albumen at present, but all are to detect IgG antibody, also do not have report about IgM detection of antibodies method; In addition, also not about the change report of rule of IgM antibody behind swine Fever Vaccine immunity and the wild virus infection.The present invention has made up the proteic recombination bacillus coli of expression swine fever E2; 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 being set up uses special, accurate, responsive, the good reproducibility in back, and this will lay the foundation for IgM antibody behind the systematic study hog cholera immune produces rule, the monitoring of clinical infection situation and the foundation of differential diagnosis method.
Description of drawings
The pcr amplification of Fig. 1 goal gene.
The 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 Recombinant Protein Expression and purifying
1: purified proteins; 2: the supernatant after inducing; 3: inclusion body; 4: empty plasmid is induced contrast; M: albumen marker
The Western blot of Fig. 4 recombinant protein identifies that (left figure: the His monoclonal antibody is one to resist; Right figure: positive porcine blood serum is one anti-)
1: dye albumen marker in advance; 2: recombinant protein; 3: the empty plasmid contrast
Embodiment
The structure of segmental pcr amplification of embodiment 1 raq gene and prokaryotic expression plasmid
1.1 design of primers
According to designing primer according to the CSFV gene order, each introduces the upstream and downstream primer
EcoRI with
HindThe III site, sequence is following:
F: CCGGAATTCCGGCTGTGCCCGTTTGATACGAGT C（SEQ ID NO.3）；
R: CAAGCTTGTCTTTAGGTCTGCATGGCATAGG（SEQ ID NO.4）
Above primer is synthetic by Nanjing Si Pujin bio tech ltd.
1.2 pcr amplification purpose fragment
Get 200 μ l hog cholera lapinised virus vaccines poison (available from Nanjing Tianbang Bio-industry Co., Ltd.), add 1ml Trizol reagent, the concussion mixing leaves standstill 10min; Add 200 μ l chloroforms, concuss, 4 ℃ of centrifugal 10min of 12000rpm carefully draw supernatant; Add equal-volume Virahol mixing, place 2h for-20 ℃, 4 ℃ of centrifugal 15min of 12000rpm abandon supernatant; Add 75% washing with alcohol, drying adds the distilled water dissolving RNA that 10 μ l do not have Rnase.
Carry out reverse transcription according to reverse transcription test kit (Beijing Quanshijin Biotechnology Co., Ltd) operation instructions with Oligo (dT) 18, 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; The distilled water of no Rnase complements to 20 μ l.42 ℃ of reaction 45min, 85 ℃ of heating 5min deactivation ThermoScript II are template with the reverse transcription product, carry out pcr amplification.The PCR reaction system is: the upper reaches, 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; The sterilization distilled water is mended to 25 μ L.Loop parameter: 94 ℃ of preparatory sex change 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 the PCR product, can see the band (result sees Fig. 1) of about 560bp, reclaims the PCR product, and-20 ℃ of preservations are subsequent use.
1.3 construction of recombinant plasmid and evaluation
The target gene PCR product that reclaims is used
EcoRI with
HindThe III double digestion is cloned into same enzyme after the recovery purifying and cuts among the expression vector plasmid pET-32a (Novagen) of processing, and ligation is carried out at 16 ℃, will connect product afterwards and transform
E.
ColiDH5 α (available from the Beijing Quanshijin Biotechnology Co., Ltd) competent cell, coating contain the LB flat board of penbritin, 37 ℃ of cultivations.Bacterium colony on the picking flat board is containing the LB culture medium culturing of penbritin, the alkaline lysis method of extracting plasmid, through PCR with
EcoRI with
HindThe III double digestion is identified (result sees Fig. 2), and positive plasmid pET32a-e2 obtains the purpose band of about 560bp.Positive plasmid send Nanjing Si Pujin bio tech ltd to check order.
The structure of embodiment 2 recombinant strains BL21-△ E2
2.1 transformed competence colibacillus intestinal bacteria BL-21
Among the embodiment 1 through the correct recombinant plasmid pET32a-e2 transformed into escherichia coli BL21 (DE3) (available from the Beijing Quanshijin Biotechnology Co., Ltd) of sequencing; Obtain recombinant strains BL21-△ E2, simultaneously with empty plasmid pET-32a with method transformed competence colibacillus intestinal bacteria BL-21.
2.2 the abduction delivering of recombinant protein
Picking recombinant strains BL21-△ E2 and the e. coli bl21 list colony inoculation that contains empty plasmid are in the LB liquid nutrient medium that contains penbritin respectively, and 37 ℃ of joltings of spending the night are cultivated.
(1) the bacterium liquid 10 μ L that get incubated overnight are inoculated in 3 ml and contain in the LB liquid nutrient medium of penbritin (50 μ g/ml), about 37 ℃ of 200rpm shaking culture 3h, make OD
600Reach 0.6～0.8, get the conduct in aseptic Eppendorf pipe of 100 μ L samples and induce preceding contrast;
(2) in above-mentioned bacterium liquid, adding final concentration is the abduction delivering that the IPTG of 0.5mmol/L carries out recombinant protein, and 4h receives bacterium after adding IPTG; The bacterium of the 4 ℃ of 8000 centrifugal 5min collection of rpm abduction delivering, the resuspended bacterial sediment of PBS (pH 7.2);
(3) with bacterium liquid multigelation 3 times, the ultrasonic treatment bacterium becomes limpid until bacterium liquid;
(4) 4 ℃ of centrifugal 10min of 8000 rpm will precipitate and use with the isopyknic PBS of supernatant resuspendedly, and respectively to get 100 μ L subsequent use with deposition suspension-s for supernatant.
2.3SDS-PAGE electrophoretic analysis
Add 5 * protein sample sample-loading buffer (250mM Tris-HCl, pH6.8 in supernatant and protein precipitation sample and the empty carrier induced product; 10%SDS; 0.5% tetrabromophenol sulfonphthalein; 50% glycerine; 5% 2 mercapto ethanol), 100 ℃ are boiled 5min behind the abundant mixing, instantaneous centrifugal before the last appearance; Draw supernatant and carry out SDS-PAGE; In the deposition there not being in empty carrier induced product and the supernatant (Fig. 3) visible significantly 38kD purpose band, shows that target protein correctly expresses with the form of inclusion body.
The great expression of embodiment 3 recombinant proteins, purifying and antigenicity are identified
3.1 the great expression of recombinant protein, purifying
The positive bacterium colony of recombinant strains BL21-△ E2 in the picking 2.2 among the inoculation 200mlLB, is pressed the abduction delivering condition abduction delivering of embodiment 2; Centrifugal collection thalline; Add the resuspended bacterial sediment of 5ml PBS by every 100ml bacterium liquid, centrifugal behind the ultrasonic degradation, cleer and peaceful inclusion body in the separation; Inclusion body is resuspended with equal-volume Binding buffer, according to the purifying (Fig. 3) of GE HisTrap HP affinity purification post explanation carrying out target protein.The spectrophotometric determination protein concentration is 1.2mg/mL ,-20 ℃ of preservations after the packing.
3.2 the Western blot of recombinant protein identifies
（1）SDS-PAGE；
(2) transfer printing: electrophoresis takes off gel and transfer device (half-dried transfer printing) is installed promptly by following order after finishing:
+The utmost point-sponge-NC film-gel-sponge-
-The utmost point, the transfer printing condition is 20V, 30min;
(3) NC film (Pall) is spent the night with the PBST confining liquid sealing that contains 5% skimming milk;
(4) with His-Tag Mouse McAb (Abmart) and positive porcine blood serum (available from China Veterinery Drug Inspection Office) with confining liquid 1:2000 (or 1:500) dilution after, join on the NC film room temperature jog 2h;
(5) with PBST washing 5 times, 5min/ time;
(6) add sheep anti-mouse igg-HRP or the goat-anti pig IgG-HRP (Beijing Bo Aosen) that 1:1000 dilutes, 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 respectively one of A, B, C liquid in the 1ml deionized water, be added to behind the mixing on the NC film, until there being obvious band to occur.The result sees Fig. 4, and the recombinant protein swimming lane all has specific band (38kD) to occur, and the empty carrier contrast does not then have.
The foundation and the application of embodiment 4 ELISA test kits
4.1 antigen the best encapsulates the selection of concentration and serum optimum dilution degree
Undertaken by matrix method, with the carbonate buffer solution of pH 9.6 antigen protein being diluted to final concentration respectively is 2.0,1.0,0.5,0.25 μ g/mL, 4 ℃ of coated elisa plates that spend the night (Nunc).The washing back adds swine fever positive serum and the 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 MV; Calculate P/N value under each condition, select positive serum OD value near 1, the reaction conditions of P/N value maximum is as the ELISA optimum reaction condition.The result is: antigen the best encapsulates concentration 0.5 μ g/mL; Serum optimum dilution degree 1:100 (table 1).
Table 1 antigen the best encapsulates the selection of concentration and serum optimum dilution degree
4.2 the selection of confining liquid
With the best antigen concentration coated elisa plate of confirming in 4.1, after the washing, with the 37 ℃ of sealings in the PBST 200 μ L/ holes that contain 5% skimming milk, 1%BSA, 0.5%BSA and 1% gelatin, 2 h, detect 5 parts of positive serums and 1 part of negative serum respectively.Calculate the P/N value, finally select the highest 0.5%BSA of P/N value as best confining liquid (table 2).
The selection of table 2 confining liquid
4.3 the selection of serum to be checked action time
By above-mentioned condition encapsulate, the sealase target; Detect 5 parts of positive serums and 1 part of negative serum, 37 ℃ act on 0.5 h, 1.0 h, 1.5 h and 2.0 h respectively, it is carried out ELISA measure; Calculate the P/N value, finally select the highest 1.5h of P/N value as the best use of time.
The selection of table 3 serum to be checked action time
4.4 the selection of IgM two anti-working concentrations
By above-mentioned condition encapsulate, sealing, application of sample; The anti-pig IgM-HRP of rabbit (BETHYL) that adds 1:10000,1:50000,1:100000 dilution after the washing respectively; 100 μ L/ holes are carried out ELISA and are measured, with the highest 1:50000 of P/N value as best effort concentration.
The selection of table 4 two anti-concentration
4.5 the selection of two anti-action times of IgM
IgM two is resisted by after confirming that good extension rate adds enzyme plate, and 37 ℃ act on 0.5 h, 1 h, 1.5h respectively, and standard yin and yang attribute serum is blocked ELISA mensuration, 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
4.6 the selection of developing time
Carry out ELISA test according to the front screening conditions, add develop the color 5,10 behind the TMB respectively, 15min, calculate the P/N value.Choose the highest 15min of P/N as best developing time.
The selection of table 6 developing time
4.7 confirming of blocking-up ELISA threshold value
To before IDEXX and Wuhan section, detect 50 parts of all negative pig anteserum samples by CSFV antibody ELISA test kit, the ELISA that sets 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 for use 5 parts of porcine blood serum criticize in and batch between replica test, calculate the variation coefficient; Repeatability with checking present method; The result show the variation within batch coefficient 5%, interassay coefficient of variation 10%, prove that present method has good repeatability.
4.9 susceptibility and specificity test
With positive serum and the continuous doubling dilution of negative serum, calculate the P/N value, up to 1/1600 still positive, prove that this method has very high susceptibility; With ELISA porcine circovirus 2 type (PCV2) (positive control in the PCV2 ELISA of the 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/bovine diarrhoea virus (BVDV) (available from China Veterinery Drug Inspection Office) standard positive serum are detected; The result is negative, proves that present method has good specificity.
4.10 the detection of clinical serum sample
Using the ELISA test kit that this research sets up 310 parts of clinical serum are detected, serves as according to detected result is judged with the criterion of confirming in 4.7.Positive rate is 76%.
4.11 ELISA test kit
The ELISA test kit comprises that enzyme plate (0.5%BSA is as best confining liquid), yin and yang attribute contrast, the enzyme mark IgM two that said albumen encapsulates resists, TMB develops the color liquid, stop buffer, washings.
SEQUENCE LISTING
< 110>Jiangsu Province Agriculture Science Institute
< 120>a kind of CSFV reorganization E2 albumen 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>manual work
<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>manual work
<220>
< 221>primers F
<222> (1)..(34)
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ccggaattcc ggctgtgccc gtttgatacg agtc 34
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<212> DNA
< 213>manual work
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Claims (5)
1. the CSFV E2 albumen of an expression of recombinant e. coli; The gene fragment clone that this albumen system will comprise E2 major antigen district is gone into prokaryotic expression carrier pET-32a and is obtained recombinant expression vector; This recombinant expression vector transformed into escherichia coli BL21 (DE3) is obtained recombination bacillus coli BL21 (DE3); The IPTG that adds final concentration when this recombination bacillus coli BL21 (DE3) is cultured to OD600 and reaches 0.6-0.8 and be 0.5mM carries out abduction delivering, and separating thallus obtains through Ni affinity chromatography column purification.
2. the CSFV E2 albumen of expression of recombinant e. coli according to claim 1 is characterized in that described E2 Argine Monohydrochloride sequence is SEQ ID NO.1.
3. the CSFV E2 albumen of expression of recombinant e. coli according to claim 1 and 2, the proteic nucleotides sequence of said E2 that it is characterized in that encoding is classified SEQ ID NO.2 as.
4. the proteic preparation method of CSFV E2 of the described expression of recombinant e. coli of one of claim 1-3 is characterized in that comprising the steps:
(1) according to CSFV gene order design primers F: CCGGAATTCCGGCTGTGCCCGTTTGATACG AGTC, R:CAAGCTTGTCTTTAGGTCTGCATGGCATAGG; From the hog cholera lapinised virus vaccine poison, extract RNA; Through the RT-PCR amplification target gene fragment SEQ ID NO.2 that obtains encoding, through
EcoRI with
HindTwo restriction enzyme sites of III are gone into described gene fragment clone in the prokaryotic expression carrier, and the evaluation of cutting through enzyme and check order then obtains positive recombinant plasmid;
(2) step (1) is described 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 and carry out abduction delivering, obtain described recombinant protein through the affinity column purifying.
5. with the ELISA detection kit of the CSFV E2 protein Preparation of one of claim 1-3 described expression of recombinant e. coli, comprise that enzyme plate that said recombinant protein encapsulates, mass ratio 0.5%BSA are as best confining liquid, yin and yang attribute contrast, the anti-pig IgM of ELIAS secondary antibody HRP mark rabbit, TMB develop the color liquid, stop buffer and washings.
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| CN102967666A (en) * | 2012-11-16 | 2013-03-13 | 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 |
| CN103091489A (en) * | 2013-01-10 | 2013-05-08 | 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 |
| CN103588864A (en) * | 2013-11-28 | 2014-02-19 | 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 |
| CN106749519A (en) * | 2016-11-15 | 2017-05-31 | 河南省农业科学院 | Peptide aglucon sequences Design and application that CSFVE2 targeting proteins based on computer simulation are combined |
| 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 |
| CN115806595A (en) * | 2022-12-08 | 2023-03-17 | 江苏农牧科技职业学院 | Recombinant antigen protein for detecting African swine fever virus, preparation method, detection kit and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101216489A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test paper for rapidly detecting hog cholera antibody and method for making same |
| CN101418304A (en) * | 2008-07-06 | 2009-04-29 | 中国农业科学院兰州兽医研究所 | Method for preparing soluble E2 antigen for detecting swine fever virus serum antibody |
| CN102175849A (en) * | 2010-12-22 | 2011-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for quickly detecting swine fever antibody and preparation method thereof |
-
2012
- 2012-01-17 CN CN201210013152.6A patent/CN102532281B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101216489A (en) * | 2008-01-10 | 2008-07-09 | 中国农业科学院哈尔滨兽医研究所 | Test paper for rapidly detecting hog cholera antibody and method for making same |
| CN101418304A (en) * | 2008-07-06 | 2009-04-29 | 中国农业科学院兰州兽医研究所 | Method for preparing soluble E2 antigen for detecting swine fever virus serum antibody |
| CN102175849A (en) * | 2010-12-22 | 2011-09-07 | 江苏出入境检验检疫局动植物与食品检测中心 | Kit for quickly detecting swine fever antibody and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李文良等: "猪瘟病毒Ｅ２蛋白主要抗原区的原核表达及其间接ＥＬＩＳＡ检测方法的建立与应用", 《动物医学进展》 * |
| 谢金文等: "猪瘟病毒E2 基因原核表达及间接ELISA 检测方法的初步建立", 《中国兽医杂志》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102967666A (en) * | 2012-11-16 | 2013-03-13 | 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 |
| 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 |
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| CN106749519A (en) * | 2016-11-15 | 2017-05-31 | 河南省农业科学院 | Peptide aglucon sequences Design and application that CSFVE2 targeting proteins based on computer simulation are combined |
| 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 |
| CN115806595A (en) * | 2022-12-08 | 2023-03-17 | 江苏农牧科技职业学院 | Recombinant antigen protein for detecting African swine fever virus, preparation method, detection kit and application thereof |
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