CN102839195A - Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus - Google Patents
Method for expression of PCV 2 Cap protein by pFast Bac Dual baculovirus Download PDFInfo
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Abstract
The invention discloses a method for expression of a PCV 2 (Porcine circovirus type2) Cap protein by a pFast Bac Dual baculovirus. The method comprises the following steps of: amplifying a gene fragment of an encoded PCV 2 Cap protein with a His tag; connecting the gene fragment to a pFast Bac Dual plasmid so as to obtain a recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2; transforming Escherichia coli DH10Bac with the recombinant transfer plasmid, and carrying out blue-white selection to obtain a recombinant shuttle vector Bac-p10-ORF2-pH-ORF2; transfecting an insect cell with the recombinant shuttle vector so as to obtain a recombinant baculovirus Ac.Dual-Cap; and poisoning the insect cell with the recombinant baculovirus, performing cultivation, then collecting the insect cell, and purifying an expression product so as to obtain the recombinant Cap protein. The method disclosed in the invention solves the problem of low expression level of the Cap protein in eukaryotic cells. The recombinant Cap protein in the invention is designed with a His tag, thus being beneficial to the follow-up purification. And the recombinant Cap protein has biological activity superior to that of a Cap protein expressed by a prokaryotic expression system, thus being applicable to establishment of epidemiological diagnosis methods and reseach as well as development of PCV2 subunit vaccines.
Description
Technical field
The invention belongs to the genetically engineered field, be specifically related to the proteic method of a kind of pFast of utilization Bac Dual baculovirus expression porcine circovirus 2 type (PCV2) Cap.
Background technology
PMWS came to light in Canada from 1991 first, had popular (the Kennedy and Allan1998) of this disease at present in a plurality of countries and regions.It is also very serious in the popularity of China that PCV2 infects the relative disease that causes, caused enormous economic loss (Lang Hongwu .2000 such as Zhang Guangchuan to pig industry; Zhang Chaoxia .2008 such as Liu Changming).
Still do not have at present control and eliminate the effective measure that PCV2 infects, though the vaccine that can prevent PCV2 to infect, its expensive, the unable use in most of pig farm are arranged on the market.So, strengthen the detection of PCV2 is seemed particularly important.Detect the PCV2 effective means PCR, immunohistochemical methods, in-situ nucleic acid hybridization and elisa technique are arranged.Elisa technique has high susceptibility and specificity with respect to other technologies, and simple, convenient easy row is convenient to the detection of a large amount of samples.
The PCV2 genome is a sub-thread ring-type minus-strand dna, and total length is 1767bp or 1768bp.The genome of PCV2 encode altogether 11 open reading frames (open reading frames, ORFs), wherein importantly ORF1 and ORF2 on the function.The Rep albumen (Mankertz, Mankertz et al.1998) that the ORF1 coding is relevant with the viral DNA rolling-circle replication.ORF2 has 702 Nucleotide, is positioned on the genome complementation chain nucleocapsid protein of coding virus, i.e. Cap albumen.Cap albumen is the unique structural protein of PCV2 virus particle, and the about 32kDa of molecular weight of albumen size also is the main immunogenic protein of PCV2 simultaneously.
The proteic N end of Cap 65 ~ 87aa, 113 ~ 147aa, 157 ~ 183aa zone and 4 amino acid of C end are the major antigen site of virus.Wherein N end 69 ~ 83aa and two antigen sites of 117 ~ 131aa have the type specificity to PCV2, can be by many anti-identifications of PCV2, and N end 157 ~ 183aa antigen site is that PCV1 and PCV2 have jointly.Though there is a common antigen site in the Cap albumen of PCV1 and PCV2, the serology experimental result shows, does not have antigenic cross property between the Cap albumen of two kinds of serotypes, and this possibly be because whole C ap albumen has been covered its common antigen site.
Cap albumen is behind vivoexpression; Can the oneself be assembled into viroid appearance particle; Immunological characteristic with PCV2 virus particle in the research of epidemiology diagnosis and vaccine, have very critical role, so Cap albumen is the research focus of PCV2 in external efficiently expressing.
The proteic N end of Cap is by one section nuclear localization signal peptide of being made up of 41 amino acid (nuclear localization signal; NLS) (Liu; Tikoo et al.2001); This NLS nucleotide sequence contains 30% the escherichia coli rare codon (Trundova and Celer2007) of having an appointment, so Cap albumen is difficult to obtain great expression (Lou, Li et al.2011) in the prokaryotic expression system of escherichia coli.
In recent years, the many investigators Cap albumen (Fan Huiying, Chen Huanchun, the yellow loud and clear .2005 that waits that attempt utilizing baculovirus to express and have its natural radioactivity; Liu Changming, Lu Yuehua .2005 such as Zhang Chaofan), but expression amount is all lower.
Summary of the invention
In order to overcome low, the active defect of insufficient of PCV2Cap expressing quantity in the existing method; The object of the present invention is to provide the proteic method of a kind of pFast of utilization Bac Dual baculovirus expression porcine circovirus 2 type (PCV2) Cap; This method utilizes the pFast Bac Dual baculovirus transfer vector in the Bac-to-Bac baculovirus efficient expression system to come the proteic recombinant baculovirus of construction expression Cap; Add the His label; Utilize NI-NTA purifying resin purification Cap albumen, resulting porcine circovirus 2 type Cap albumen has kept its natural radioactivity, and is to efficiently express.
The object of the invention is realized through following technical proposals:
A kind of proteic method of pFast Bac Dual baculovirus expression PCV2Cap of utilizing may further comprise the steps:
(1) DNA of extracting PCV2 virus; According to the sequences Design primer of PCV2ORF2, amplification obtains having the proteic gene fragment of coding PCV2Cap of His label;
(2) gene fragment that step (1) amplification is obtained is connected on the pFast Bac Dual plasmid, obtains recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2;
(3) with recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2 transformed into escherichia coli DH10Bac, transposition takes place, after blue hickie screening, obtain containing the recombinant shuttle vector Bac-p10-ORF2-pH-ORF2 of ORF2 gene;
(4) with liposome-mediated method with recombinant shuttle vector Bac-p10-ORF2-pH-ORF2 transfection insect cell, obtain recombinant baculovirus Ac.Dual-Cap;
(5) recombinant baculovirus Ac.Dual-Cap is connect poison in insect cell, cultivate the back and collect insect cell, recombinant C ap albumen obtains expression in the nucleus of insect cell and tenuigenin; With obtaining recombinant C ap albumen behind the expression product purifying;
The sequence of the said primer of step (1) is:
P10F：5’–
CCATGGATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
P10R：5’-TGCA
GGTACCTTAAGGGTTAAGTGGGGGGTCT-3’；
PHF：5’-
GGATCCATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
PHR：5’-CGC
AAGCTTTTAAGGGTTAAGTGGGGGGTCTT-3’；
The described gene fragment that step (1) amplification is obtained of step (2) is connected on the pFast Bac Dual plasmid, is that the gene fragment that step (1) amplification obtains is inserted BamHI, the HindIII cloning site in NcoI, KpnI cloning site and the pH promotor downstream in pFast Bac Dual plasmid p10 promotor downstream respectively;
The described recombinant baculovirus Ac.Dual-Cap of step (4) has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on May 4th, 2012; Preserving number is CGMCC 6069, and the classification name is the false type baculovirus of reorganization.
Step (4) and the preferred Sf9 insect cell of (5) described insect cell;
The described purifying of step (5) is to use the NI-NTA resin purification.
The present invention has following advantage and effect with respect to prior art:
1, the present invention utilizes pFast Bac Dual baculovirus expression system to express PCV2Cap albumen, can solve the low difficult problem of Cap albumen expression amount in eukaryotic cell, makes that Cap albumen successfully obtains to efficiently express in eukaryotic expression system.
2, the Cap albumen of the inventive method reorganization is designed with the His label, is beneficial to its follow-up purifying.
3, the proteic biological activity of Cap of baculovirus expression system expression of the present invention is superior to the expressed Cap albumen of prokaryotic expression system, can be applied to the foundation of epidemiology diagnostic method and the research and development of PCV2 subunit vaccine.
Description of drawings
Fig. 1 is the pcr amplification gel electrophoresis figure of PCV2ORF2; Wherein, M-DNA Marker DL2000; 1-PCR amplified production ORF2-1 contains NcoI, KpnI site, about 720bp; 2-PCR amplified production ORF2-2 contains BamHI, HindIII site, about 720bp.
Fig. 2 is that the double digestion of recombinant transfer plasmid is identified gel electrophoresis figure; Wherein, M1-DNA MarkerDL15000; M2-DNA Marker DL2000; M3-DNA Marker1kb ladder; 1-NcoI, KpnI double digestion identify the product that is connected of ORF2-1 gene and pFast Bac Dual plasmid; 2-BamHI, HindIII double digestion are identified recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2.
Fig. 3 is the indirect immunofluorescence assay figure as a result of recombinant baculovirus Ac.Dual-Cap; Wherein, A-does not connect the normal Sf9 insect cell (* 200) of poison; B-recombinate shape virus infection Sf9 insect cell (* 200).
Fig. 4 is the proteic SDS-PAGE analysis of recombinant C ap; Wherein, M-albumen relative molecular mass standard; 1-does not connect the Sf9 insect cell contrast of poison; 2,3-infects the Sf9 insect cell of recombinant baculovirus.
Fig. 5 is the proteic Western Blot of recombinant C ap qualification result figure; Wherein, M-albumen relative molecular mass standard; 1-does not connect the Sf9 insect cell contrast of poison; 2-infects the Sf9 insect cell of recombinant baculovirus; Cap albumen behind the 3-purifying.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
Embodiment
A kind of pFast Bac Dual baculovirus expression system that utilizes is expressed the proteic method of PCV2Cap, may further comprise the steps:
One, the structure of recombinant shuttle vector Bac p10-ORF2-pH-ORF2
1, the extraction of the DNA of PCV2
PCV2 strain (Fan, Ye et al.2012) is inoculated in the PK15 cell that no PCV pollutes, in 37 ℃ CO
2Incubator is cultivated, and collects and handle cell then, according to the DNA of the Blood DNAKit test kit specification sheets extracting PCV2 of OMEGA company, as template DNA.
2, PCR design of primers
2 pairs of Auele Specific Primers of sequences Design according to PCV2ORF2:
P10F：5’-
CCATGGATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
P10R：5’-TGCA
GGTACCTTAAGGGTTAAGTGGGGGGTCT-3’；
PHF：5’-
GGATCCATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
PHR：5’-CGC
AAGCTTTTAAGGGTTAAGTGGGGGGTCTT-3’；
Amplification contains the ORF2 gene (about 720bp) of His label.Article four, primer comprises NcoI, KpnI, BamHI, HindIII site respectively.Primer is synthetic by Invitrogen company.
3, the amplification of ORF2 gene
The PCR reaction system is 50 μ L, template DNA 1.5 μ L wherein, each 1 μ L of upstream and downstream primer (20pmol/L), dNTP s (2.5mmol/L) 4 μ L, 10 * Ex Taq
TMBuffer5 μ L, Ex Taq
TMArchaeal dna polymerase (5U/ μ L) 0.5 μ L, deionized water 37 μ L.The PCR reaction conditions is: 94 ℃ of preparatory sex change 3min; Get into the PCR circulation, 94 ℃ of sex change 30s, 58 ℃ of annealing 45s, 72 ℃ are extended 40s, carry out 30 circulations after, 72 ℃ are extended 10min.Reaction detects amplification (Fig. 1) with agarose gel electrophoresis after finishing, and the PCR product is carried out glue reclaim purifying.
4, recombinant transfer vector pFast Bac-p10-ORF2-pH-ORF2 makes up
PCR product ORF2-1 and pFast Bac Dual plasmid to purifying all carry out NcoI, KpnI double digestion, and recovery and purifying enzyme are cut product.Connect ORF2-1 gene and pFast Bac Dual plasmid with T4DNA Ligase enzyme, will connect product and transform DH5 α competent cell, identify (Fig. 2 A), the positive recombinant plasmid of dna sequencing evaluation and screening through double digestion.
PCR product ORF2-2 and the recombinant plasmid followed purifying then all carry out BamHI, HindIII double digestion; Recovery and purifying enzyme are cut product; ORF2-2 and recombinant plasmid after T4DNA Ligase enzyme ligase enzyme is cut; To connect product and transform DH5 α competent cell, identify that through double digestion (Fig. 2 B), dna sequencing evaluation obtain positive recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2.
5, the acquisition of recombinant baculovirus shuttle vectors
Get 2 μ L recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2 and transform the DH10Bac competent escherichia coli cell that 100 μ L contain shuttle vectors Bacmid; Behind the ice bath 30min; Carry out heat shock in 42 ℃ of water-bath 45s, ice bath 2min adds 900 μ L LB liquid nutrient mediums (high salt) then; 4 ~ 5h is cultivated in 37 ℃ of joltings, by 10
-1, 10
-2, 10
-3Behind the diluting cells, respectively get 100 μ L and coat that three high salt tolerance LB are dull and stereotyped (to contain 50 μ g/ml Kan, 7 μ g/ml Gen; 10 μ g/ml Tet), cultivate 36 ~ 48h, select white colony for 37 ℃; Continue inoculation on three kinds of antibiotic LB flat boards in same containing and go down to posterity once, the positive bacterium colony of purifying, the picking hickie (contains 50 μ g/ml Kan in high salt LB liquid nutrient medium; 7 μ g/ml Gen, 10 μ g/ml Tet), when 37 ℃ of joltings are cultured to the bacterium logarithmic phase; Carry out bacterium liquid PCR with general pUC M13 upstream and downstream primer and identify that positive bacteria liquid extracts shuttle plasmid, thereby obtain recombinant baculovirus shuttle vectors Bac-p10-ORF2-pH-ORF2.
Two, the proteic expression of the acquisition of recombinant baculovirus Ac.Dual-Cap and Cap
1, transfection insect cell
With LipofectaminTM2000 is cotransfection reagent; With the recombinant shuttle vector Bac-p10-ORF2-pH-ORF2 transfection of above-mentioned acquisition in the Sf9 insect cell; The transfection step is following: choose the good Sf9 cell of growth conditions; Add a small amount of Grace ' s complete culture solution and blow down cell gently, after cell counting, it being diluted to concentration is 1 * 10
6The Sf9 cell suspension of individual/mL inserts 6 orifice plates by the 2mL/ hole, is placed on 27 ℃ of incubator absorption 1h.Draw 3 μ g purified recombinant Bacmid plasmids (referring to recombinant shuttle vector Bac-p10-ORF2-pH-ORF2) and add respectively in the simple nutrient solution of 100 μ l serum-free Grace ' s, mixing gently is as A liquid.Mixing cotransfection reagent is 5 ~ 10 times repeatedly, draws 20 μ l then and adds in the simple nutrient solution of 100 μ l serum-free Grace ' s and dilute, as B liquid.With A liquid and B liquid gently behind the mixing incubated at room 45 ~ 60min as mixed solution.
Serum Grace ' the s nutrient solution that contains in 6 orifice plates is removed, and used the simple nutrient solution washing of serum-free Grace ' s 2 times.In the mixed solution of hatching, add the simple nutrient solution of serum-free Grace ' s of 0.8mL, add 6 orifice plates gently behind the mixing, cultivate 4 ~ 6h for 27 ℃.Transfection mixed solution in 6 orifice plates is abandoned in suction, and every hole adding 2mL contains serum Grace ' s complete culture solution and continues to cultivate 72h at least, or can receive poison up to observing cytopathy.Every 12h observes once after the transfection, when cytopathy is obvious, and collecting cell and supernatant, full substratum, the metamorphosis of attention cell.Collecting cell and supernatant after the pathology appear in the above or cell of transfection 72h, and after the freeze thawing once, 500 * g is centrifugal, and 5min gets supernatant, and 4 ℃ keep in Dark Place, and are P1 for recombinant baculovirus Ac.Dual-Cap.
2, P2, P3 are for the amplification of recombinant baculovirus
In 6 porocyte culture plates, carry out, virus amplification same day viable cell quantity is suspended with the Grace's perfect medium greater than 97% Sf9 cell, adjustment concentration is 1 * 10
6Individual/mL, every hole inoculation 2mL, 27 ℃ attach more than the 2h, inoculate a certain amount of P1 for viral liquid, cultivate 48 ~ 72h for 27 ℃, collect culture supernatant, and the centrifugal 5min of 500 * g removes cell and big cell debris, keeps in Dark Place in-80 ℃, is P2 for the shaft-like disease of reorganization.
P3 is for identical for virus of the amplification of virus and P2.
3, indirect immunofluorescence detects the expression of Cap albumen in the Sf9 cell
In 48 porocyte culture plates, P3 for recombinate shape virus infection Sf9 insect cell 96h after, inhale and to abandon culture supernatant; Every hole is with PBS (pH7.4) washing 2 times, with acetone-ethanol (3:2 volume ratio) stationary liquid-20 ℃ fixing 10 ~ 15min of-20 ℃ of precoolings, PBS washing 3 times; Adding mouse positive serum (available from Korea S JBT company) by the anti-PCV2 of 1:30 dilution and be one resists; 37 ℃ of effect 30min, PBS washes each 5min 3 times, adds two anti-(the anti-mouse IgG of FITC labelled goat) then; 37 ℃ of effect 30min, PBS washes each 5min 3 times.Under inverted fluorescence microscope, observe the fluorescence and the photograph of transfectional cell.The Sf9 insect cell that does not infect compares simultaneously.The result shows and successfully obtains recombinant baculovirus Ac.Dual-Cap, and in the Sf9 insect cell, obtains to express, and the Cap albumen major part of expression is present in (Fig. 3) in the nucleus.
4, SDS-PAGE and Western Blot detect the expression of Cap albumen in the Sf9 cell
In floorage is 75cm
2Tissue Culture Flask in (about 6 * 10
6Sf9cells), P3 for recombinate shape virus infection Sf9 insect cell 96h after, inhale and to abandon culture supernatant; Collecting cell; Cell is used 500 μ l PBS re-suspended cells at last with PBS (pH7.4) washing 2 times, gets the resuspended liquid spotting of 30 μ l and runs SDS-PAGE detection protein expression situation.The result shows that recombinant C ap albumen accounts for 15% of total protein of cell, and the Cap expressing quantity is about 1.5mg/6 * 10
6Sf9cells, recombinant C ap albumen obtain in the Sf9 insect cell than high expression level (Fig. 4,5).
5, purification of Recombinant Cap albumen
The Cap albumen that utilizes Ni-NTA purifying resin purification to express.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (3)
1. one kind is utilized the proteic method of pFast Bac Dual baculovirus expression PCV2Cap, it is characterized in that may further comprise the steps:
(1) DNA of extracting PCV2 virus; According to the sequences Design primer of PCV2ORF2, amplification obtains having the proteic gene fragment of coding PCV2Cap of His label;
(2) gene fragment that step (1) amplification is obtained is connected on the pFast Bac Dual plasmid, obtains recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2;
(3) with recombinant transfer plasmid pFast Bac-p10-ORF2-pH-ORF2 transformed into escherichia coli DH10Bac, transposition takes place, after blue hickie screening, obtain containing the recombinant shuttle vector Bac-p10-ORF2-pH-ORF2 of ORF2 gene;
(4) with liposome-mediated method with recombinant shuttle vector Bac-p10-ORF2-pH-ORF2 transfection insect cell, obtain recombinant baculovirus Ac.Dual-Cap;
(5) recombinant baculovirus Ac.Dual-Cap is connect poison in insect cell, cultivate the back and collect insect cell, recombinant C ap albumen obtains expression in the nucleus of insect cell and tenuigenin; With obtaining recombinant C ap albumen behind the expression product purifying;
The sequence of the said primer of step (1) is:
P10F：5’–
CCATGGATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
P10R：5’-TGCA
GGTACCTTAAGGGTTAAGTGGGGGGTCT-3’；
PHF：5’-
GGATCCATGCACCACCATCATCACCACACGTATCCAAGGAGGCGTTT-3’；
PHR：5’-CGC
AAGCTTTTAAGGGTTAAGTGGGGGGTCTT-3’；
The described purifying of step (5) is to use the NI-NTA resin purification.
2. the proteic method of pFast Bac Dual baculovirus expression PCV2Cap of utilizing according to claim 1; It is characterized in that: the described gene fragment that step (1) amplification is obtained of step (2) is connected on the pFast Bac Dual plasmid, is that the gene fragment that step (1) amplification obtains is inserted BamHI, the HindIII cloning site in NcoI, KpnI cloning site and the pH promotor downstream in pFast Bac Dual plasmid p10 promotor downstream respectively.
3. the proteic method of pFast Bac Dual baculovirus expression PCV2Cap of utilizing according to claim 1, it is characterized in that: step (4) and (5) described insect cell are the Sf9 insect cell.
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