CN102517260A - Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof - Google Patents
Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion protein carrying an NS3 preponderant area of a non-structural protein of a bovine viral diarrhea virus (BVDV) as well as a recombinant expression method and application thereof. The fusion protein disclosed by the invention is obtained after the amino (N) end of the NS3 preponderant area of the non-structural protein of the BVDV is connected with foldase DsbA. The activated high expression of the intracellular solubility of an NS3 gene can be realized by the fusion of the DsbA after being mutated and the NS3 preponderant area of the non-structural protein of the BVDV, and the expression quantity is greater than 30 percent. In the expression method of the fusion protein, which is disclosed by the invention, a pET-DsbA expression vector carrying a T7 promoter and a DsbA gene is adopted, the cleavage site of thrombin is adopted as the insertion site of a gene of the NS3 preponderant area of the non-structural protein of the BVDV, and the expressed NS3 immunodominance protein of the bovine viral diarrhea virus has very good antigenicity. The fusion protein carrying the NS3 preponderant area of the non-structural protein of the BVDV can be used for the ELISA (Enzyme-Linked Immuno Sorbent Assay) detection of the bovine viral diarrhea virus.
Description
Technical field
The invention belongs to fusion rotein and recombinant expression method thereof and application; Particularly relate to a kind of carry bovine viral diarrhea virus (Bovine viral diarrhea virus, the application during BVDV) fusion rotein in Nonstructural Protein NS3 advantage district and recombinant expression method thereof and its ELISA at bovine viral diarrhea virus detect.
Background technology
Bovine viral diarrhea virus (Bovine viral diarrhea virus; BVDV) belong to flaviviridae (Flaviviridae) pestivirus (Pestivirus), be the sub-thread positive chain RNA virus, with sheep border disease virus (Border diseasevirus; BDV) and CSFV (Classical swine fever virus; CSFV) on serology, have cross reaction (Yin Zhen, Liu Jinghua. animal virology [M]. the 2nd edition. Science Press, 1997.631-645).That BVDV infects subclinical often property or cause slight and non-specific clinical symptom.Yet; But the persistent infection calf of diaplacental infection output immunological tolerance, these persistent infection animals are with poison throughout one's life, continue toxin expelling; Become the important contagium in the cows; Increase the weight of infection (Weiland E.Ahl R.Stark et al.A second envelope lycoproteinmediate neutralization of a pestivirus hog cholera virus [J] Virol, 1992, (66): 3677-3682) of cows.
The infection of BVDV distributes global; Though infection rate is different; But infect often popularly in many countries, the ox of persistent infection (PI) can reach 1-2%, the Niu Keda 60-85% of antibody positive (Houe H.Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections.Vet Microbiol; 1999,64 (2-3): 89-107).For a long time, this disease has a strong impact on Developing of Animal Industry always, and simultaneously BVDV still be the source of pollution of being everlasting of ox source biological products (serum, freeze essence, frozen embryo and vaccine etc.), has caused enormous economic loss to livestock industry and relative commercial field.
" gold standard " that BVDV detects is virus neutralization tests (Edwards S.The diagnosis of bovine virus diarrhoea-mucosal disease in cattle.Rev Sci Tech; 1990; 9 (1): 115-30); This method is responsive and special, but need carry out cell cultures and labor capacity big.And ELISA method detection BVDV is easy and simple to handle, and expense is low, can be used for the quarantine of sample in enormous quantities, is highly suitable for clinical diagnosis and quarantine.The cell that traditional ELISA test kit infects based on BVDV; But the protein content that BVDV produces in tissue is lower; The true protein that is difficult to the acquisition sufficient amount is used for diagnosis (the Justewicz DM of BVDV; Magar R, Marsolais G, et al.Bovine viral diarrhea virus-infected MDBK monolayer as antigen in enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies in bovine sera.Vet.Immunol.Immunopathol; 1987,14 (4): 377-384).
In viral protein, structural protein E0 and E2, Nonstructural Protein NS3 have been confirmed as main immunogens (Bolin SR.Immunogens of bovine viral diarrhea virus.Vet.Microbiol, 1993,37 (3-4): 263-271) of BVDV.Nonstructural Protein NS3 is conservative in pestivirus, exercises similar function, and in the virus multiplication process, NS3 albumen appears in the endoplasmic reticulum, participates in the course of processing of viral protein directly.This albumen has protease activity (Protease) and helicase activity (Helicase).NS3 albumen is a kind of immunodominance albumen; Can produce in the animal after natural infection or attenuated live vaccines immunity to the proteic antibody of NS3 (Brown LM, Papa RA, Frost MJ; Et al.A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3protein.Virus Res; 2002,84 (1-2): 111-124), have important diagnostic value.Simultaneously, because the unstructuredness matter of NS3 albumen itself, animal is behind the traditional inactivated vaccine of inoculation, and the NS3 detection of antibodies can be distinguished the animal of vaccine inoculation or natural infection.A lot of researchists advise by NS3 as diagnostic detect reagent; Be convenient to vaccine inoculation or NS3 serology monitoring natural infection (Sandvik T.Laboratory diagnostic investigations for bovine viral diarrhoea virus infections in cattle.Vet Microbiol; 1999,64 (2-3): 123-34; Reddy JR; Kwang J; Okwumabua O; Et al.Application of recombinant bovine viral diarrhea virus proteins in the diagnosis of bovine viral diarrhea infection in cattle.Vet Microbiol, 1997,57 (2-3): 119-33; Deregt D; Masri SA; Cho HJ; Et al.Monoclonal antibodies to the p80/125gp53proteins of bovine viral diarrhea virus:their potential use as diagnostic reagents.Can J Vet Res, 1990,54 (3): 343-348).Closely during the last ten years, external existing expression NS3 albumen or its truncated segment are set up the report that the ELISA diagnostic method is used for detecting cows BVDV antibody as diagnostic antigen.And Zoth etc. was at confirming relatively that NS3 antigen can detect by virus neutralization experiment and be judged to be anti-BVDV antibody (the Chimeno Zoth S in the negative sample with other structural protein ELISA method through multiple reorganization ELISA method in 2006; Taboga O.Multiple recombinant ELISA for the detection of bovine viral diarrhoea virus antibodies in cattle sera.J Virol Methods; 2006,138 (1-2): 99-108).
Summary of the invention
The purpose of this invention is to provide a kind of reorganization diagnostic antigen, relate to a kind of fusion rotein that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
The fusion rotein that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district provided by the present invention is the fusion rotein that is connected with folding enzymes DsbA at amino (N) end in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
Wherein, Two halfcystines (Cys) relevant with the redox function are mutated into Serine (Ser) to said DsbA from aminoterminal (N end) position; Purpose is that the DsbA after making goal gene and suddenling change merges, and realizes the activated high expression level of solubility (expression amount is greater than 30%) in the born of the same parents.
Specifically, the said fusion rotein that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district, called after DsbA/BVDV NS3 is one of following amino acid residue sequences:
1) the SEQ ID No:1 in the sequence table;
2) with the amino acid residue sequence of SEQ ID No:1 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and protein with serine protease, rna helicase enzymic activity.
SEQ IDNo:1 in the sequence table is made up of 563 amino-acid residues; From amino (N) end 1-215 position is the amino acid residue sequence of folding enzymes DsbA, is the amino acid residue sequence in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district from aminoterminal 218-561 position.
The above-mentioned gene (DsbA/BVDVNS3) that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district fusion rotein of encoding also belongs to protection scope of the present invention, and it is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID No:2 in the sequence table;
2) dna sequence dna of SEQ ID No:1 in the code sequence tabulation;
3) with sequence table in the nucleotide sequence that limits of the SEQ ID No:2 nucleotide sequence that has 90% above homology and have serine protease, rna helicase enzymic activity;
The nucleotide sequence of the dna sequence dna hybridization that 4) under the rigorous condition of height, can limit with SEQ ID No:2 in the sequence table.
The rigorous condition of said height for hybridization back with contain 0.1 * SSPE (or 0.1 * SSC), the solution of 0.1%SDS washes film under 65 ℃.
SEQ ID No:2 in the sequence table is by 1689 based compositions; Its encoding sequence is from 5 ' end 1-1689 bit base; Coding has the protein of the amino acid residue sequence of SEQ ID No:1 in the sequence table; Wherein, from 5 ' end 1-645 bit base coding folding enzymes DsbA, from 5 ' end 652-1683 bit base coding bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
Increase arbitrary segmental primer in the above-mentioned fusion rotein encoding sox to also within protection scope of the present invention.
Another object of the present invention provides a kind of above-mentioned recombinant expression method that carries the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
The above-mentioned recombinant expression method that carries the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district provided by the present invention; Be to contain the above-mentioned recombinant expression vector that carries the fusion rotein encoding sox in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district to import among the host, obtain carrying the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district through expression, purifying.
In the recombinant expression method of the above-mentioned fusion rotein that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district; The said fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district can adopt transcribes polymerase chain reaction (RT-PCR) amplification and obtains, and the upstream and downstream primer sequence of this gene that is used for increasing is respectively shown in sequence table SEQ ID No:3 and SEQ ID No:4.
SEQ ID No:1 is by 33 based compositions in the sequence table; SEQ ID No:2 is by 31 based compositions; Wherein SEQ ID No:1 from 5 ' end the 1st to the 6th bit base be the BagII restriction enzyme site, SEQ ID No:2 from 5 ' end the 1st to the 6th bit base be the NheI restriction enzyme site.
The carrier that sets out that is used for making up the recombinant expression vector contain the above-mentioned fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district can be any one and carries folding enzymes DsbA Prokaryotic Expression carrier pET-DsbA.
Wherein, be that the recombinant expression vector that containing of vector construction carry the fusion rotein encoding sox in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district that sets out is pET-DsbA-NS3 with pET-DsbA.
The said fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district is between the BagII and Nhe I restriction enzyme site at prokaryotic expression carrier pET-DsbA MCS place.
Said host can be intestinal bacteria, yeast, mammalian cell, insect cell or Bacillus subtilus etc., is preferably intestinal bacteria.
Said intestinal bacteria specifically can be BL21 (Rosetta), BL21 (DE3), BL21 (DE3, plysS), E.coli JM109, E.coli HB101 or E.coli Topl0 etc. are preferably BL21 (Rosetta).
Wherein, be that the engineering bacteria of the recombinant expression vector pET-DsbA-NS3 that contains the fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district that makes up of starting strain is BL21 (Rosetta)/DsbA-NS3 with e. coli bl21 (Rosetta).
Above-mentioned recombinant expression vector and engineering bacteria all can make up according to ordinary method.
Cultivation contains the substratum and the culture condition of host cell that the present invention carries the fusion rotein encoding sox in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district, all can be substratum and the culture condition of cultivating the host that sets out.
When said host was intestinal bacteria, needing to add final concentration was glucose and 0.8mM-1.2mM (being preferably 1mM) the IPTG abduction delivering of 0.1-0.3% (being preferably 0.2%), and inducing temperature is 30 ℃, and induction time is 1-5 hour.
The method that said target protein to abduction delivering carries out purifying is preferably nickel sepharose affinitive layer purification.
The invention provides a kind of fusion rotein and encoding sox thereof that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.Fusion rotein of the present invention is the fusion rotein that obtains behind amino (N) the end connection folding enzymes DsbA with bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.Said DsbA has removed signal peptide; Simultaneously that DsbA is relevant with the redox function two halfcystines (Cys) are mutated into Serine (Ser), merge the activated high expression level of solubility (expression amount is greater than 30%) in the born of the same parents that can realize the NS3 gene through DsbA after the sudden change and bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.Adopt the pET-DsbA expression vector that carries T7 promotor and DsbA gene in the Expression of Fusion Protein method of the present invention; Select the insertion site of zymoplasm cleavage site simultaneously for use as bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district gene; And because the proteins encoded of this gene is a non-glycosylated protein; Thereby the bovine viral diarrhea virus NS3 immunodominance albumen of expressing has good antigenicity, and the ELISA that can be used for bovine viral diarrhea virus detects, and has a extensive future.
Below in conjunction with specific embodiment the present invention is explained further details.
Description of drawings
Fig. 1 is a bovine viral diarrhea virus NS3 protein immunization advantage district Gene RT-PCR amplification
Fig. 2 is the PCR qualification result of cloning vector pMD18-T-NS3
Fig. 3 is the PCR qualification result of recombinant expression vector pET-DsbA-NS3
The sequencing result of Fig. 4 recombinant expression vector pET-DsbA-NS3
Fig. 5 is the 12%SDS-PAGE electrophoresis detection result who carries the fusion protein expression products in bovine viral diarrhea virus Nonstructural Protein NS3 advantage district
Fig. 6 is the 12%SDS-PAGE electrophoresis detection result after carrying that the expressing fusion protein supernatant in bovine viral diarrhea virus Nonstructural Protein NS3 advantage district is purified and concentrating
Fig. 7 measures the result of bovine viral diarrhea virus antibody for indirect ELISA
Embodiment
Embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
Method therefor is ordinary method if no special instructions among the following embodiment.
One, carries the amplification of the fusion rotein encoding sox in bovine viral diarrhea virus Nonstructural Protein NS3 advantage district
1, design of primers
MRNA sequence according to bovine viral diarrhea virus Nonstructural Protein NS3; Be designed for the upstream and downstream primer P1 and the P2 of amplification bovine viral diarrhea virus Nonstructural Protein NS3 advantage district gene; Wherein in upstream primer P1, add the BagII restriction enzyme site; Add the NheI restriction enzyme site among the downstream primer P2, concrete primer sequence is P1:
AGATCTATGGTCAAGAAGATAACCAGCATGAAC (base sequence of band underscore is the BagII restriction enzyme site) (SEQ ID No:3 in the sequence table), P2:
GCTAGCCTCCCTAAAGGATTTCGTCACGTTG (base sequence of band underscore is the NheI restriction enzyme site) (SEQ ID No:4 in the sequence table).
2, the amplification of bovine viral diarrhea virus Nonstructural Protein NS3 immunodominance district gene
With the method amplification bovine viral diarrhea virus Nonstructural Protein NS3 immunodominance district gene of reverse transcription-polymerase chain reaction (RT-PCR), concrete grammar may further comprise the steps:
1) the viral suspension multigelation of MDBK cell (bovine kidney cells) being cultivated 3 times, 4 ℃, the centrifugal 20min of 8000r get supernatant, add final concentration respectively and be 10% and 3% PEG20000 and NaCl, and 4 ℃ of magnetic agitation are spent the night.Next day, 4 ℃, the centrifugal 30min of 8000r abandon supernatant, precipitate resuspended with PBS.Dispose 30%, 35%, 40%, 45% CsCl solution respectively, preparation CsCl concentration gradient, add viral liquid concentrator after, 4 ℃, the centrifugal 16h of 22000r/min carry out purifying to spissated viral liquid.
2) get viral liquid concentrator 10 μ l and add 200 μ l PBS, add 400 μ l Trizol, gently mix 1min, the static 10min of room temperature; Add 400 μ l chloroforms again, gently mix 1min, the static 10min of room temperature; In 4 ℃, the centrifugal 15min of 12000g, draw upper strata liquid, add the long-pending above Virahol of monoploid;-30 ℃ of deposition 30min are in 4 ℃, the centrifugal 10min of 14000g, with the resuspended deposition of sterilization 9 μ l DEPC water.
3) obtain the cDNA of bovine viral diarrhea virus Nonstructural Protein NS3 immunodominance district gene with reverse transcription-polymerase chain reaction (RT-PCR), reaction system is: 5 * RT-PCR buffer, 4 μ l, downstream primer P22 μ l; Mouse source ThermoScript II 1 μ l, DEPC water 4 μ l, 42 ℃ of reverse transcription 1h; Be that template is carried out pcr amplification again with cDNA, reaction system is: cDNA 3 μ l, 2 * PCR buffer, 12.5 μ l; Upstream primer P1 1 μ l, downstream primer P2 1 μ l, ddH
2O 8.5 μ l, reaction conditions is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 1min, totally 32 circulations; Last 72 ℃ are extended 10min, 4 ℃ of termination reactions.
4) dna fragmentation that step 3) is obtained through pcr amplification carries out purifying with the dna fragmentation purification kit of sky, Beijing root company; Concrete steps are: the DNA cloning fragment is carried out 0.8% agarose gel electrophoresis separate separating resulting (M:DL2000Marker as shown in Figure 1; A: negative control; B:DsbA/BVDV NS3 Gene RT-PCR product), obtained the purpose fragment of about 1032bp, conformed to, after the EB dyeing with expected results through pcr amplification; Under long-wave ultra violet lamp, cut and contain the segmental sepharose of this purpose, put into an aseptic 1.5mL EP pipe, add 400 μ l sol solutionses, in 50 ℃ of water-bath 5min; Flick the EP pipe during this time, gel dissolves fully, and the liquid that will dissolve then goes to one and reclaims post, leaves standstill 5min in room temperature; The centrifugal 1min of 8000r/min outwells the waste liquid in the collection tube, uses the washings washed twice; The last centrifugal 1min of 12000r/min reclaims post and goes in the clean aseptic 1.5mL EP pipe, adds 10 μ l elutriant wash-outs; Place 5min in room temperature, the centrifugal 1min of 12000r/min is the dna fragmentation of recovery in the collection tube.DNA behind the wash-out is stored in 4 ℃ or-20 ℃.
Two, carry fusion rotein recombinant expressed in bovine viral diarrhea virus Nonstructural Protein NS3 advantage district
1, the structure of cloning vector pMD18-T-NS3
The goal gene of step 1 purifying is connected with cloning vector pMD18-T (available from the precious biotechnology in Dalian ltd), and 20 μ l linked systems comprise 0.5 μ l pMD18-T carrier, 4.5 μ l goal gene purified products and 5 μ l Solution I, and 16 ℃ are spent the night.Reaction will connect product and be converted in the E.coli TOP10F competent cell after finishing, and extract DNA and carry out PCR evaluation, result (M:DL2000Marker as shown in Figure 2; A: negative control; The b:pMD18-T-NS3PCR qualification result); Show the positive colony that has obtained to carry bovine viral diarrhea virus Nonstructural Protein NS3 advantage district gene, this is carried the recombinant clone plasmid called after pMD 18-T-NS3 of bovine viral diarrhea virus Nonstructural Protein NS3 advantage district gene.
2, the structure of recombinant expression plasmid pET-DsbA-NS3 and conversion BL21 (Rosetta) competence bacteria
The pMD18-T-NS3 plasmid is carried out BagII, NheI double digestion; Expression vector pET-DsbA (available from crystalline substance U.S. biotech firm) carries out BamHI, NheI double digestion; Double digestion product with two carriers spends the night with 16 ℃ of connections of T4DNA ligase enzyme again, will connect product transformed into escherichia coli competence bacteria BL21 (Rosetta), extracts DNA; With P1, P2 is that primer carries out pcr amplification, result (M:DL2000Marker as shown in Figure 3; The a:pET-DsbA-NS3PCR qualification result; B: negative control), obtained the dna fragmentation of 1032bp, conformed to expected results through increasing; Select positive colony again; The evaluation of checking order, sequencing result is as shown in Figure 4, and the gene (called after DsbA/BVDV NS3) that result's coding carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district fusion rotein has the dna sequence dna of SEQ ID No:2 in the sequence table; SEQ ID No:2 in the sequence table is by 1689 based compositions; Its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID No:1 in the sequence table, wherein from 5 ' end 1-1689 bit base; From 5 ' end 1-645 bit base coding folding enzymes DsbA, from 5 ' end 652-1683 bit base coding bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.Above-mentioned sequencing result shows and has obtained the recombinant expression vector that the antigen-4 fusion protein gene in bovine viral diarrhea virus Nonstructural Protein NS3 advantage district is carried in all correct the containing of insertion sequence and position; Called after pET-DsbA-NS3 is with recombination bacillus coli engineering bacteria called after BL21 (the Rosetta)/DsbA-NS3 that carries pET-DsbA-NS3.
3, carry the abduction delivering of bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district fusion rotein
Recombination bacillus coli engineering bacteria BL21 (Rosetta)/DsbA-NS3 of the single clone of picking; Be inoculated in 3mL and contain the LB substratum of penbritin (100mg/L), 37 ℃ of jolting overnight cultures, next day is in 1% ratio enlarged culturing; Behind 37 ℃ of joltings cultivation 2h (absorption photometric is 0.6-0.8); Add IPTG (final concentration is 1.0mmol/L), glucose (final concentration is 0.2%), continue at 30 ℃ of joltings cultivations and induce 4h-6h, collect thalline.
4, the SDS-PAGE protein electrophoresis detects
With the recombinant expressed albumen of 12%SDS-PAGE gel separation step 3, concentrated gum concentration is 5%, and resolving gel concentration is 12%, the result as shown in Figure 5 (a, b, c, d, e is respectively pET-DsbA-NS3 and induces 5h, 4h, 3h, 2h, 1h product; F is that empty carrier is induced 3h; M is a protein relative molecular weight standard: 250kDa, 150kDa, 100kDa, 80kDa; 60kDa, 50kDa, 40kDa, 30kDa; 25kDa, 20kDa, 15kDa, 10kDa); Through expressing the recombinant protein that has obtained 62.9kDa, conform to expected results, show that bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district fusion rotein has obtained to express.
5, the separation and purification of expression product
Get and induce bacterium liquid 200mL to collect thalline, with the about 5mL suspension of the PBS damping fluid thalline of 50mmol/L pH7.4, the broken thalline of ultrasonic method; Ultrasound condition is: power 200W, and ultrasonic 3s, to stop 2s be a circulation, totally 180 cyclic ultrasonic breakings; The centrifugal 12000r/min 15min of ultrasonic liquid; Get supernatant, with the nickel sepharose column purification to the His label, concrete operations are: sample is added through damping fluid (0.5mol/L NaH
2PO
419mL, 0.5mol/LNa
2HPO
481mL, NaCl 29.3g, the pH7.4) affinity column of washing carries out stepwise elution with the damping fluid that contains the 100mmol/L imidazoles, the dialysis tubing of packing into of the albumen behind the purifying, in dialyzate (pH7.4PBS), 4 ℃ of dialysis 20h.Purifying protein is carried out 12%SDS-PAGE gel branch; From protein, the result is as shown in Figure 6 (after a is pET-DsbA-NS3 induced product purifying; Before b is pET-DsbA-NS3 induced product purifying; M is protein relative molecular weight standard: 250kDa, 150kDa, and 100kDa, 80kDa, 60kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa 10kDa), shows that bovine viral diarrhea virus NS3 protein immunization advantage district fusion rotein has obtained purifying.
Embodiment 2, indirect elisa method detect bovine viral diarrhea virus antibody
1, the selection of indirect ELISA optimum reaction condition
BVDV antigen the best encapsulates the selection (adopt square formation test carry out the screening of working concentration) of concentration and negative and positive serum optimum dilution degree: get the reorganization NS3 fusion rotein that the embodiment 1 of purifying obtains, with coating buffer (0.05mol/L yellow soda ash-sodium bicarbonate buffer liquid, pH9.6) diluting is 8 μ g/mL, 4 μ g/mL, 2 μ g/mL, 1 μ g/mL; Add enzyme plate; 100 μ L/ holes, each concentration is done delegation, and 4 ℃ are spent the night; Take out with PBST (NaCl 8.0g, KH
2PO
40.2g, Na
2HPO4 1.15g, KCl 0.2g, Tween-20 0.5mL adds deionized water to 1000mL, transfers pH to 7.4) wash 3 times; Every then hole adds 250 μ L confining liquids (PBST+1% gelatin), 37 ℃ of sealing 2h, PBST washing 3 times; With PBST ox positive serum and negative serum (standard positive serum and standard female serum) are done dilution in 1: 20,1: 40,1: 80,1: 160,1: 320 respectively; Add in the enzyme plate; Each extent of dilution is made row; With PBST is blank, and wash 3 times in 37 ℃ of reaction 1h in 100 μ L/ holes; Add 1: the 1000 anti-ox IgG-HRP of rabbit (thing scientific and technological development Ltd of the rich Hang Seng of Beijing longitude and latitude) 100 μ L/ hole, 37 ℃ of reaction 1h add tmb substrate 100 μ L after washing, and room temperature lucifuge effect 10-15min adds 50 μ L 2mol/L H
2SO
4Termination reaction is measured OD
450nmNear 1.0, the antigen of P/N >=2.1, serum greatest dilution are as the righttest working concentration of antigen and test serum with the OD value in positive serum hole.Experiment confirms that envelope antigen concentration is 2 μ g/mL), the serum antibody optimum dilution degree is 1: 40.
2, indirect elisa method is measured the bovine viral diarrhea virus serum antibody
With the determined antigen coated concentration of square formation titration (2 μ g/mL) as the envelope antigen working concentration; 6 parts of Ox blood serum samples (through bovine viral diarrhea virus antibody assay kit (INGEZIM Company products) test positive) to collecting carry out antibody test, establish blank hole (PBST) and negative control hole (standard female serum) simultaneously.
Concrete operations: get the reorganization NS3 fusion rotein that the embodiment 1 of purifying obtains, with 2 μ g/mL coated elisa plates, with in the Ox blood serum sample of dilution in 1: 40 and the negative control sera adding enzyme plate; 100 μ L/ holes, in 37 ℃ of reaction 1h, the washing back adds 1: 1000 anti-ox IgG-HRP of rabbit (thing scientific and technological development Ltd of the rich Hang Seng of Beijing longitude and latitude); 100 μ L/ holes, 37 ℃ of reaction 1h, the washing back adds tmb substrate 100 μ L; Room temperature lucifuge effect 10-15min adds 50 μ L2mol/L H
2SO
4Termination reaction is measured OD
450nm
Result's (1-6 is 6 parts of serum) as shown in Figure 7 shows that reorganization NS3 albumen can produce positive reaction with the ox antiserum(antisera), and gram is used for detecting the bovine viral diarrhea virus serum antibody.
Claims (10)
1. carrying the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district, is the fusion rotein that is connected with folding enzymes DsbA at amino (N) end in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
2. fusion rotein according to claim 1 is characterized in that, said DsbA is mutated into Serine (Ser) from aminoterminal two halfcystines (Cys) relevant with the redox function.
3. fusion rotein according to claim 1 is characterized in that, the said fusion rotein that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district is one of following amino acid residue sequences:
1) the SEQ ID No:1 in the sequence table;
2) with the amino acid residue sequence of SEQ ID No:1 in the sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and have serine protease and the protein of rna helicase enzymic activity.
4. coding claim 1 or 2 or 3 said genes that carry the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
5. gene according to claim 4 is characterized in that, the said gene that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district fusion rotein of coding claim 3 is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID No:2 in the sequence table;
2) dna sequence dna of SEQ ID No:1 in the code sequence tabulation;
3) with sequence table in the nucleotide sequence that limits of SEQ ID No:2 have 90% above homology and have Tryase and the nucleotide sequence of rna helicase enzymic activity;
The nucleotide sequence of the dna sequence dna hybridization that 4) under the rigorous condition of height, can limit with SEQ ID No:2 in the sequence table.
6. contain claim 4 or 5 said expression carrier, transgenic cell line or host bacterium.
7. recombinant expressed claim 1 or 2 or 3 said methods of carrying the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district; Be that the recombinant expression vectors that will contain claim 4 or the 5 or 6 said fusion rotein encoding soxs that carry bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district import among the host, through express, purifying obtains carrying the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district;
The said fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district adopts transcribes polymerase chain reaction (RT-PCR) amplification and obtains, and the nucleotide sequence of the upstream and downstream primer of this gene that is used for increasing is respectively shown in sequence table SEQ ID No:3 and SEQ ID No:4.
8. according to the said recombinant expression method that carries the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district of claim 7; It is characterized in that the carrier that sets out that is used for making up the recombinant expression vector contain the above-mentioned fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district carries folding enzymes DsbA Prokaryotic Expression carrier pET-DsbA for any one; With pET-DsbA is that the recombinant expression vector that containing of vector construction carry the fusion rotein encoding sox in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district that sets out is pET-DsbA-NS3; The said fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district is between the BagII and Nhe I restriction enzyme site at prokaryotic expression carrier pET-DsbA MCS place.
9. according to claim 7 or the 8 said recombinant expression methods that carry the fusion rotein in bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district, it is characterized in that said host is intestinal bacteria; Said intestinal bacteria are specially BL21 (Rosetta), BL21 (DE3), BL21, and (DE3 plysS), E.coli JM109, E.coli HB101 or E.coli Topl0, is preferably BL21 (Rosetta); With e. coli bl21 (Rosetta) is that the engineering bacteria of the recombinant expression vector pET-DsbA-NS3 that contains the fusion rotein encoding sox that carries bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district that makes up of starting strain is BL21 (Rosetta)/DsbA-NS3.
10. claim 1 or the 2 or 3 said application of fusion rotein in the ELISA of bovine viral diarrhea virus detects of carrying bovine viral diarrhea virus (BVDV) Nonstructural Protein NS3 advantage district.
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Application publication date: 20120627 |