CN101358182A - Recombinant baculovirus strain of porcine circovirus type 2 Cap protein expression, construction method and application thereof - Google Patents
Recombinant baculovirus strain of porcine circovirus type 2 Cap protein expression, construction method and application thereof Download PDFInfo
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Abstract
The present invention discloses a recombinant baculovirus strain rBac/PCV2Cap (microorganism preservation number: CGMCC NO.2083) efficiently expressing Porcine circovirus type 2 Cap protein and applications thereof. The recombinant baculovirus strain rBac/PCV2Cap constructed by the present invention can efficiently express recombinant PCV2-Cap protein in insect cells, and the expressed recombinant Cap protein, which has good immunocompetence and antigenicity, can serve as a subunit vaccine used to prevent the related plague caused by Porcine circovirus type 2 infection as well as a detecting and diagnostic antigen for Porcine circovirus type 2 serum antibody.
Description
Technical field
The present invention relates to a kind of recombinant baculovirus strain construction process, relate in particular to a kind of porcine circovirus 2 type recombinant C ap protein expression and application; The invention still further relates to the recombinant expression vector that is used for making up this porcine circovirus 2 type recombinant strain and this recombinant strain in the application of pig circular ring virus diagnosis, prevention, belong to the Vet Biotechnology field.
Background technology
Porcine circovirus 2 type (Porcine circovirus type 2, PCV2) be pmws (Postweaning multsystemic wasting syndrome, PMWS) cause of disease, cause symptom (Allan G M such as weanling pig generation progressive emaciation, cough, expiratory dyspnea, diarrhoea, ochrodermia or xanthochromia, lymphadenectasis, the greyish white oedema of kidney, Ellis J A.Porcine circoviruses:A review[J] .J Vet Diagn Invest, 2000,12:3-14).This disease was found in Canada first in 1991, had now confirmed to be worldwide popular, and some pig farms of China are this disease of ubiquity also.The PCV2 genome contains 2 main reading frames, i.e. ORF1 and ORF2.PCV2-ORF1 is by 945 based compositions, 314 amino acid of encoding, gene product relevant with rdrp virus (Rep); PCV2-ORF2 is by 702 based compositions, 233 amino acid of encoding, be constitute viral capsid proteins (Cap) composition (Cheung A K.Transcriptional analysis ofporcine circovirus type 2[J] .Virology, 2003,305:168-180; Hamel A L, LinL L, Nayar G P S.Nucleotide sequence of porcine circovirus associated withpostweaning multisystemic wasting syndrome in pigs[J] .J Viol, 1998,72:5262-5267.).(Nawagitgul P such as Nawagitgul, Morozov I, Bolin S R, et al.Openreading frame 2 of porcine circovirus type 2 encodes a major capsidprotein[J] .J Gen Virol, 2000,81:2281-2287; Nawagitgul P, Harms P A, MorozovI, et al.Modified indirect porcine circovirus (PCV) type 2-based andrecombinant capsid protein (ORF2)-based enzyme-linked immunosorbent assaysfor detection of antibodies to PCV[J] .Clin Diagn Lab Immunol, 2002,9:33-40.).With baculovirus expression the PCV2-ORF2 gene obtain the 30kDa protein product, observe the ability that this product has oneself's assembling pseudovirion under the Electronic Speculum.Because this virus infected cell do not present cytopathy, viral cultures poison valency is low, be difficult for purifying, brings certain difficulty to research work.Insect baculovirus expression system is expressed characteristics such as foreign protein output height, antigenicity are good, the easy purifying of product with it, extensively utilized in the research of multiple eqpidemic disease.
Summary of the invention
The object of the invention provides a kind of recombinant baculovirus strain, this recombinant strain can be expressed the Cap albumen of PCV2-ORF2 coding, expressed recombinant protein has good immunocompetence, can be used as the porcine circovirus 2 type subunit vaccine, also can be used as the porcine circovurus type 2 specific antibody diagnostic antigen.
The present invention seeks to be achieved through the following technical solutions:
A kind of recombinant baculovirus strain (rBac/PCV2Cap), its microbial preservation number are: CGMCC NO.2083; The classification name is: express porcine circovirus 2 type (Porcine circovirus type 2) the proteic recombinant baculovirus of Cap; The preservation time is: on June 13rd, 2007; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
The present invention's PCV2-ORF2 gene that at first increases, obtain 699bp goal gene segment, it is connected with baculovirus expression vector plasmid (being preferably pBlueBac4.5/V5-His-TOPO), obtains recombinant expression plasmid by endonuclease reaction evaluation and sequential analysis; Order-checking shows that amplification obtains the PCV2-ORF2 gene of 699bp, reaches more than 96.7% with the PCV2 sequence homology of having delivered, shows that this gene has very high conservative property.Resulting recombinant expression plasmid and linearizing baculovirus DNA are bathed altogether, add cell transfecting reagent, get commentaries on classics sense cells and supernatant and do plaque clone and shaker test, adopt the viral cultures supernatant, the inoculation insect cell, screening obtains a strain and stablizes, efficiently expresses the proteic recombinant baculovirus strain of PCV2-Cap (rBac/PCV2Cap), and virus titer reaches 1.28 * 10
8Pfu/ml.This recombinant strain can efficiently express reorganization PCV2-Cap albumen at insect cell, expressed reorganization PCV2-Cap albumen has good immune response activity, can be used as the porcine circovirus 2 type subunit vaccine, also can be used as the porcine circovurus type 2 specific antibody diagnostic antigen.
The expressed recombinant protein of recombinant strain of the present invention is removed the fusion part, its molecular weight and document (Nawagitgul P, Morozov I, Bolin S R, et al.Open reading frame 2 of porcinecircovirus type 2 encodes a major capsid protein[J] .J Gen Virol, 2000,81:2281-2287) Bao Dao 30kDa basically identical, and product is expressed output, is higher than the expression level of bibliographical information.Because the present invention has designed the His-tag sequence at the C-terminal of recombinant protein, for the purifying of next step recombinant protein is laid a good foundation.
(Mah é D according to the literature, Blanchard P, Truong C, et al.Differentialrecognition of ORF2 protein from type 1 and type 2 porcine circoviruses andidentification of immunorelevant epitopes[J] .J Gen Virol, 2000,81:1815-1824.), PCV1 and PCV2 list at genome sequence and have higher homology, but it is different to distribute, and two viruses are respectively 86% and 56% in the amino acid sequence homologous rate of ORF1 and ORF2 coding region.(Mah é D such as Mah é, Blanchard P, Truong C, et al.Differential recognition of ORF2 protein fromtype 1 and type 2 porcine circoviruses and identification of immunorelevantepitopes[J] .J Gen Virol, 2000,81:1815-1824.) ORF1 that expressed two kinds of viruses respectively, prove that there is significant antigenic cross-reaction in their gene product; But no remarkable antigenic cross-reaction between the ORF2 gene product of expressing.Infer that in view of the above the PCV2-ORF2 expression product that recombinant baculovirus of the present invention is expressed can be used as porcine circovurus type 2 specific antibody and detects target antigen, for further serology differential diagnosis provides the foundation.In addition, the albumen of PCV2-ORF2 genes encoding is the important component of forming virus structural protein, and it is pathogenic and induce the body protective immunne response to play an important role.Utilize expressed recombinant protein to can be used as the subunit vaccine immune animal, stimulate body to produce protective immune response, reach the purpose of anti-virus infection.
The present invention lays the foundation for further carrying out pig circular ring virus subunit vaccine, diagnostic antigen and molecular biology research.
Description of drawings
The result of the blue hickie screening of Fig. 1 recombinant baculovirus; A: wild-type baculovirus (hickie); B: recombinant type baculovirus (locus coeruleus).
Fig. 2 recombinant strain infects the viral inclusion body that forms behind the Sf-21 cell; A: wild-type baculovirus inclusion body; B: recombinant baculovirus inclusion body.
Fig. 3 expression of PCV2-Cap albumen in insect Sf-21 cell of recombinating; A:SDS-PAGE analytical results, 1-6 swimming lane are respectively 0,24,48,72, and the expression product of 96h time point, the 7th swimming lane are the wild-type baculovirus contrast, and the M swimming lane is a standard specimen albumen; B: the immunoblotting assay result, the application of sample order is with figure A; Arrow indication place is a recombinant protein among the figure.
Fig. 4 PCV2-Cap albumen Ni that recombinates
2+The affinity column purification result; The contrast of swimming lane 1:Sf-21 cell; Swimming lane 2: rough expression product; Swimming lane 3-6: the recombinant protein of purifying is partly collected sample.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Embodiment 1: the structure and the Recombinant Protein Expression of recombinant baculovirus strain of the present invention (rBac/PCV2Cap)
1 materials and methods
1.1 virus, cell and antiserum(antisera)
Porcine circovirus 2 type strain (PCV2/LG strain): be the sick pig internal organs from terrain pig farm clinical manifestation PMWS symptom, the pig kidney continuous cell line through not containing PCV1 separates acquisition, and passes for 29 generations continuously.Cell cultures adopts RPMI RPMI-1640 (Gibco), adds 10% foetal calf serum and 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates.Carry out cell cultures according to a conventional method, employing is with the footwork virus inoculation, when reaching 80% individual layer, press document (TischerI, Mields W, Wolff D, et al.Studies on epidemiology and pathogenicity ofporcine circovirus[J] .Arch Virol, 1986,91:271-276.) method is done the D-glucosamine processing.Sero-fast preparation: adopt the negative pig of 30 age in days antiviral antibodies, in tracheae, reach inguinal lymph nodes virus inoculation cell culture, in inoculation blood sampling after 42 days, preparation PCV2 positive serum.Insect cell line (Sf-21) is incubated at the GraceShi nutrient solution, adds 0.26% phosphoric acid Trypsin meat soup, and 10% foetal calf serum and 50 μ g/ml kantlex are used for DNA transfection, plaque clone and expression of recombinant proteins.
1.2 construction of recombinant plasmid
The amplification of PCV2-ORF2 gene order: get 100 μ l viral cultures, add 2 μ l Proteinase Ks (20mg/ml) in 37 ℃ of digestion 2h, boil through water-bath and boil 5min, the centrifugal 10min of 15000r/min gets supernatant and is used for DNA cloning.The design of primers reference, the PCV2 sequence (AF16652, AF027217, AF118097, AF264043 and AF201897) of GenBank login.Upstream primer: 5 '-ATGACGTATCCAGGAGGCG-3 '; Downstream primer: 5 '-GGGTTTAAGTGGGGGGTCT-3 ').Adopt Dalian Bao Bio-Engineering Company test kit to carry out pcr amplification reaction, the by specification operation.The DNA denaturation temperature is 94 ℃ of 2min; Amplified reaction carries out 35 circulations altogether: 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 1min; Extension is 72 ℃ of 7min.The PCR product is gone up (Invitrogen) through identifying rear clone to insect baculovirus transferring plasmid vector pBlueBac4.5/V5-His-TOPO, gene clone and analytical procedure reference (Sa nurse Brooker, D W Russell work (Huang Peitang etc. translate). molecular cloning experiment guide [M]. the 3rd plate, Science Press, 2002.).The recombinant plasmid dna of Qiagen column purification, 373A type sequence instrument carries out dna sequence analysis (Perkin-Elmer test kit), and DNASIS software carries out data processing.
1.3DNA transfection and plaque test
The linearizing baculovirus DNA of the plasmid DNA of purifying and suitable proportion is bathed altogether, and (Cellfectin, Invitrogen), by specification is operated to add an amount of cell transfecting reagent.Get commentaries on classics sense cells and supernatant and do plaque clone and shaker test.Plaque test: the viral cultures supernatant that adopts 10 times of serial dilutions, the inoculation insect cell, make 1h in 28 ℃ of senses, be that 1% agarose-GraceShi complete culture solution covers (including 150 μ g/ml X-gal) with final concentration after the sucking-off inoculation liquid, being inverted in 28 ℃ of incubators after solidifying cultivated 4, by day appearance of observation mazarine plaque, inhale Glass tubing picking locus coeruleus with hair, put in the 0.5ml serum-free GraceShi nutrient solution and blow and beat, inoculate the Sf-21 cell of new cultivation with this, carry out the plaque colony screening of 3 recombinant baculovirus repeatedly, the recombinant virus titre of acquisition is pressed plaque forming unit (pfu/ml) and is calculated.
1.4 recombinant protein expression analysis
The preparation of recombinant baculovirus basis seed poison: get the Sf-21 cell of new cultivation, dosage of inoculation is 0.1 infectious unit, inoculates results virocyte culture after 5～7 days.During expression of recombinant proteins, dosage of inoculation is 5 infectious units, collects cell culture by different time points (0,24,48,72,96 and 120h).The cell collected adds the SDS-PAGE sample buffer with PBS centrifuge washing 3 times, after boiling 5min through boiling and handling, and 12.5% separation gel electrophoresis, coomassie brilliant blue R250 dyeing, thin layer chromatography scanner is measured protein content.Western blot test is used to detect the immunocompetence reaction of recombinant protein, method see document (Sa nurse Brooker, D W Russell work (Huang Peitang etc. translate). molecular cloning experiment guide [M]. the 3rd plate, Science Press, 2002.).
2 experimental results
2.1PCV2-ORF2 the DNA product that gene amplification and clone PCR amplification obtain, size consistent with desired design (699bp), deletion viral protein terminator codon.With restriction enzyme reorganization plasmid analysis result is shown that the recombinant plasmid of structure contains the PCV2-ORF2 gene, closure is correct.
2.2DNA The sequencing results to the dna sequencing analytical results unanimity that 3 recombinant plasmids carry out, shows that mismatching phenomenon does not take place clone's PCR product.5 PCV2 strain gene order homologys of sequence data and GenBank login reach more than 96.7%.The PCV2-ORF2 full length gene is 702bp (containing 3 terminator codons), coding 233aa, and prediction has a potential glycosylation site.
2.3 the colony screening result of recombinant baculovirus clones through 3 circulation plaques, obtains a strain and stablizes, efficiently expresses the proteic recombinant baculovirus of PCV2-Cap (rBac/PCV2Cap), virus titer reaches 1.28 * 10
8Pfu/ml.The plaque that recombinant virus produces is mazarine reaction (Figure 1B), the plaque that wild nature type baculovirus the produces reaction (Figure 1A) that is creamy white.
As can be seen from Figure 2, behind the recombinant baculovirus inoculation Sf-21 cell, can cause occurring in the cell unique large-scale inclusion body (Fig. 2 B); And produce the small-sized inclusion body of dozens of (Fig. 2 A) during the wild-type baculovirus cells infected; The normal control cell does not have this phenomenon.
2.4 Recombinant Protein Expression recombinant baculovirus inoculation as a result Sf-21 cell is collected sample by different time and is carried out the evaluation of SDS-PAGE electrophoresis.See that Fig. 3 (A) result shows, destination gene expression albumen occurs at molecular weight 32.8kDa place, with the passing of sample time, expressing quantity increases gradually.Inoculation back 48h, recombinant protein begins to express, and 96h reaches peak value.Recombinant protein accounts for 17.2% of protein content after measured.Healthy cell and wild-type virus inoculation group do not have this protein band in same position and occur.
2.5 the immunocompetence qualification result of recombinant protein adopts western blot test that the immunocompetence of recombinant protein is identified, shown in Fig. 3 (B), the 32.8kDa albumen one of (Fig. 3 A) performance in the SDS-PAGE electrophorogram, the sample of gathering in the crops between 48h～120h all can produce specific immunity staining reaction, healthy cell and wild-type virus inoculation group dye-free with the PCV2 antiserum(antisera).The result confirms that the recombinant protein of acquisition belongs to PCV2-Cap albumen.
The PCV2-Cap protein Preparation subunit vaccine that the recombinant baculovirus strain (rBac/PCV2Cap) that the 1 usefulness the present invention of test example makes up is expressed
With the recombinant baculovirus strain (rBac/PCV2Cap) that the present invention makes up, the PCV2-Cap protein product of inoculation insect cell expression is an antigen, the preparation subunit vaccine.Research trial is optimized in the expression parameter of insect cell the preparation recombinant protein, with 5 infectious unit kinds poison inoculation insect cell, has carried out the development of subunit vaccine as antigen with 5 batches of recombinant proteins of expressing.Recombinant protein culture and immunological adjuvant emulsification are prepared subunit vaccine.Carried out the check of work in-process and finished product vaccine by the requirement of veterinary biologics rules, goods carry out clinical trial with being up to the standards.
Test-results: with 5 of PCV2-Cap subunit vaccine 2 multiple doses (4ml/ head) the intramuscular inoculation 25 age in days piglets of manufacturing experimently, clinical observation was not seen no abnormal reaction in 28 days, proved that vaccine product is safe to this animal.The vaccine immunity potency test is to adopt 0.5ml, 1ml, three kinds of dosage immunity of 2ml, 25 age in days piglets, 5 every group, establishes 3 of not vaccination contrast pigs.Sun is all changeed in antibody test in 28 days behind all vaccine immunities; The strong virus attack test-results shows that 1ml and 2ml immune group all obtain 100% protection, and the 0.5ml group obtains 80% (4/5) protection ratio.
The PCV2-Cap proteantigen that the recombinant baculovirus strain (rBac/PCV2Cap) that the 2 usefulness the present invention of test example make up is expressed is set up the ELISA diagnostic kit
With the recombinant baculovirus strain (rBac/PCV2Cap) that the present invention makes up, the inoculation insect cell prepares recombinant expressed PCV2-Cap albumen, utilizes terminal 6 the His-Tag sequence labels that insert of recombinant protein c, adopts commercialization Ni
2+Method of affinity column is carried out protein purification (Fig. 4)., set up and detect PCV2 specific antibody diagnostic kit by the ELISA Sptting plate with the recombinant protein antigen bag of purifying.
ELISA diagnostic kit schedule of operation: the detection reaction plate of getting method for preparing is put the room temperature preheating, embathe 1 time with PBS, serum sample to be checked dilutes by 1: 100 with PBS-T liquid (containing 0.05%Tween20+1%BSA), do reference with the PCV2 positive and negative serum, the serum of every part of dilution joins 2 antigen-reactive holes (100 μ L/ hole) respectively, puts 37 ℃ of wet boxes and hatches 1h; PBS-T washs 3 times, adds the SPA-HRP liquid (Zymed product) of 1: 3000 times of dilution, and every hole 100 μ L put 37 ℃ of wet boxes and hatch 1h; PBS-T washing 3 times adds 50 μ L/ hole substrate reactions liquid (ABTS, Sigma product), puts color development at room temperature 30min, adds 1%NaF solution termination reaction, measures the optical density value (OD of 405nm wavelength with enzyme mark determinator
405).Criterion as a result, P/N=[serum OD value to be checked-blank OD value]/[negative control sera OD value-blank OD value] 〉=2.1 are as the positive reaction threshold value; Result of determination was effective when the positive and negative control serum was all set up.
Test-results: is 1.46～2.39 μ g/mL with the recombinant protein bag by the optimal concentration of elisa plate; Serum diluting multiple to be checked is 1: 100, and sense is 1h as the time; Two antienzyme labeling antibody extension rates are 1: 3000, and sense is 1h as the time.The ELISA diagnostic kit of assembling is 98% to known PCV2 positive serum detection sensitivity; To PCV2 negative serum detection specificity is 100%.To Pestivirus suis (CSFV), pig parvoviral (PPV), PRV (Pseudorabies virus) (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastro-enteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), pig circular ring virus 1 type (PCV1) etc. with reference to the positive serum no cross reaction.Good with batch interior repeatability between diagnostic reagent is criticized, the variation coefficient is less than 15%.The coincidence rate that detects serum sample with conventional peroxidase monolayer cell stain test (IPMA) reaches 89%.Diagnostic kit can be put-20 ℃ and preserve 1 year effectively.
Claims (7)
1, recombinant baculovirus strain rBac/PCV2Cap, its microbial preservation number is: CGMCC NO.2083.
2, the purposes of the recombinant baculovirus strain rBac/PCV2Cap of claim 1 in preparation prevention porcine circovirus 2 type vaccine.
3, according to the purposes of claim 2, it is characterized in that: described vaccine is a subunit vaccine.
4, the recombinant baculovirus strain rBac/PCV2Cap of claim 1 is in the purposes of preparation diagnosis porcine circovirus 2 type Serum Antibody Detection diagnostic antigen.
5, the expressed reorganization porcine circovirus 2 type Cap albumen of the recombinant baculovirus strain rBac/PCV2Cap of claim 1.
6, the reorganization porcine circovirus 2 type Cap albumen of claim 5 is in the purposes of preparation diagnosis porcine circovirus 2 type Serum Antibody Detection diagnostic antigen.
7, a kind of ELISA diagnostic kit that detects porcine circovirus 2 type is characterized in that: contain the described reorganization porcine circovirus 2 type of claim 5 Cap albumen.
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| CN101875941A (en) * | 2010-03-09 | 2010-11-03 | 胡文锋 | Artificially modified and synthesized dORF2art gene and protein encoded by same |
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| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
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