CN102453711A - Establishment of protein secreted expression vector and application of same - Google Patents
Establishment of protein secreted expression vector and application of same Download PDFInfo
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- CN102453711A CN102453711A CN2010105238056A CN201010523805A CN102453711A CN 102453711 A CN102453711 A CN 102453711A CN 2010105238056 A CN2010105238056 A CN 2010105238056A CN 201010523805 A CN201010523805 A CN 201010523805A CN 102453711 A CN102453711 A CN 102453711A
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Abstract
The invention provides a baculovirus expression vector capable of realizing secreted expression of protein and a method for establishing the same, as well as a method for producing protein by using the vector.
Description
Technical field
The present invention relates to the genetically engineered field.Particularly, the present invention relates to a kind of rhabdovirus expression vector and construction process thereof that can make albumen carry out secreting, expressing, and utilize said carrier to produce proteic method.
Background technology
The exogenous protein expression system comprises prokaryotic cell prokaryocyte system and eukaryotic cell system.The prokaryotic cell prokaryocyte system mainly is a Bacillus coli cells, and it is easy and simple to handle, expression product is stable, but can not carry out some translation post-treatment and modifications to expressed proteins.Eukaryotic cell system comprises mammalian cell, yeast cell and insect cell etc.It is that foreign gene carrier, insect cell are the receptor expression system with the insect baculovirus that insect cell expression system (being baculovirus expression system) is one.Compare with bacterium, yeast and mammalian cell expression system, it has following characteristics:
-safe: baculovirus does not have infectivity to vertebrates;
-can hold bigger foreign gene: the exogenous segment that can carry can arrive 38kb greatly;
-efficiently expressing exogenous gene: compare with other eukaryotic expression system, the most outstanding characteristics of baculovirus expression system can make recombinant protein obtain high-caliber expression exactly, and the highest amount of target protein that makes reaches 50% of total protein of cell;
-having complete translation post-treatment modification system: baculovirus expression system has the complete translation post-treatment ability of protein, comprises that glycosylation, phosphorylation, acylations, signal peptide excise and the cutting of peptide section and decomposition etc.;
-recombinant protein has complete biological function: foreign protein can carry out correctly collocation folding, disulfide linkage in cell, expression product on structure and function near native protein.
The most frequently used baculovirus expression system is the Bac-to-Bac system at present; It is a kind of AcMNPV carrier (autogra multiple nuclear polyhedrosis virus that Luckow etc. utilizes phage replication to make up; Autogra-phacalifornica multiple nuclear polyhedro-sisvirus); Be by transposon-mediated baculovirus recombination system; Promptly substitute polyhedron gene and construction of recombinant virus with foreign gene, used transcriptional regulatory signal is single or the promotor of a plurality of baculoviruss, and wherein the most frequently used is PPH promotor and P10 promotor.The swivel base process is carried out in intestinal bacteria, is made up of donor plasmid, Bacmid shuttle vectors and helper plasmid three parts.Said Bacmid shuttle vectors contains F-factor replicon (can in intestinal bacteria, duplicate), kalamycin resistance gene and Tn7 swivel base site attTn7.In donor plasmid, foreign gene places under the bacilliform virus promoter, two ends be respectively Tn7 about the end.Contain the coli strain DH10Bac of Bacmid with its conversion, by helper plasmid trans-acting generation swivel base is provided, and forwards foreign gene the attTn7 position of Bacmid to.Foreign gene in the donor plasmid disturbs the expression of LacZ under the transposase effect, on the culture plate that IPTG, X-gal are arranged, form white colony so contain the DH10Bac bacterium of recombinant plasmid (reorganization Bacmid).Extract the reorganization Bacmid DNA that contains foreign gene from the bacterium colony culture, can obtain recombinant virus to carry out expression of exogenous gene with the liposome mediated-method transfection insect cell.Insect cell line mainly comprises Sf9, Sf21 and High 5 cells, and three kinds of cells are the sphere of rule; Sf9 cell size is rule, needs adherent growth, and Sf21 cell size is irregular; Need adherent growth; And High 5 cell sizes are irregular, can carry out suspension culture, adhere to more open during adherent culture.Sf9 or Sf21 are commonly used to prepare recombinant virus because of having higher transfection efficiency, also can be used for the recombinant protein that increases; And High 5 cell transfecting efficient are low, but can utilize its suspension culture characteristic to utilize the rolling bottle recombinant protein that increases in a large number.
Utilizing the white expression system of baculovirus to express foreign protein at present, all is cell inner expression basically, and expression vector does not contain protein signal peptide secretion signal; Do not contain purification tag yet; Make the recombinant protein of expressing to secrete the extracellular,, bring difficulty to protein purification because cell oneself protein content is high, complicated component; Also increase cost, also be unfavorable for the purifying of recombinant protein.
Gp67 is the baculovirus surface glycoprotein; The signal peptide sequence that the major function district of protein molecular is held by N, the commentaries on classics film set sequence of C end, and the outer zone of action of intermediary is formed; Wherein the gp67 signal peptide sequence is to instruct protein to carry out the strong signal peptide sequence of excretory (referring to Whitford M; Stewart S, Kuzio J, Faulkner P.Identification andsequence analysis of a gene encoding gp67; An abundant envelopeglycoprotein of the baculovirus Autographa californica nuclearpolyhedrosis virus.J Virol.1989,63:1393-9).Utilize the recombination principle; This signal peptide is carried out being recombined into rhabdovirus expression vector behind the synthetic; Introduce purification tag simultaneously, be expected to obtain the secretor type donor plasmid, promote the recombinant baculovirus recombinant protein to carry out secretion type expression; In conjunction with affinity tag, utilize affinity purification single step purification method just can obtain the higher target protein of purity.
Summary of the invention
The objective of the invention is to address the above problem through following embodiment.
According to an aspect of the present invention, the invention provides a kind of gp67 signal peptide sequence, it is characterized in that having the sequence shown in the SEQ ID NO:1.
According to another aspect of the present invention; The invention provides a kind of secretor type baculovirus donor plasmids pFastBacgp67; It is characterized in that said donor plasmid is the basis with the pFastBacI rhabdovirus expression vector, between its MCS, be connected with gp67 signal peptide sequence mentioned above.Preferably, said MCS is BssHII and MfeI.
According to another aspect of the present invention; The invention provides a kind of method that makes up above-mentioned secretor type baculovirus donor plasmids pFastBacgp67, comprise above-mentioned gp67 signal peptide sequence is inserted in the MCS of pFastBacI rhabdovirus expression vector.Preferably, said MCS is BssHII and MfeI.
According to another aspect of the present invention, the invention provides a kind of exogenous protein expression system, comprise above-mentioned secretor type baculovirus donor plasmids pFastBacgp67, Bacmid shuttle vectors, helper plasmid and insect cell.Preferably, said insect cell comprises Sf9 insect cell, Sf21 insect cell or High 5 insect cells.
According to another aspect of the present invention, the invention provides the method that a kind of above-mentioned exogenous protein expression system produces foreign protein, may further comprise the steps:
(1) will the encode gene of said foreign protein is inserted among the secretor type baculovirus donor plasmids pFastBacgp67, to obtain transfer vector plasmid;
(2) the transfer vector plasmid swivel base DH10Bac competent cell and the extraction reorganization Bacmid DNA that prepare with step (1);
(3) with the reorganization Bacmid DNA transfection insect cell of step (2) preparation to obtain recombinant virus;
(4) with step (3) the recombinant virus infection insect cell that obtained and cultivate;
(5) supernatant of harvested cell culture and therefrom purifying protein.
Preferably, insect cell described in comprises Sf9 insect cell, Sf21 insect cell or High5 insect cell.More preferably, wherein liposome method is adopted in the transfection in the step (3).
In one embodiment, in order to achieve the above object, the technical scheme that the present invention adopts is following:
Synthetic gp67 signal peptide sequence at first; Add 6 * His and 3 * flag label (this synthetic gene is called for short gp67) simultaneously; The gene gp67 of synthetic and pFastBac1 vector plasmid (American I nvitrogen Company products) are connected, transform DH5 α competent cell with BssH II with Mfe I (U.S. NewEngland Biolabs product) double digestion, after reclaiming; Make up a kind of new donor plasmid, and carry out sequencing.Then this donor plasmid is gone in 16 artificial synthetic influenza virus gene type HA1 gene clone; Extract reorganization Bacmid DNA behind the swivel base DH10Bac competent cell; Be packaged into recombinant baculovirus behind the transfection insect cell; Identify the form of protein expression then, the result proves that target protein is a secretion type expression.
Description of drawings
Fig. 1 is donor plasmid pFastBac 1 figure.
Fig. 2 is secretor type donor plasmid pFastBacgp67 figure.
Fig. 3 is that the PCR of secretor type recombination rhabdovirus expression vector pFBgp67-H1HA1-pFBgp67-H16HA1 identifies figure, and wherein each swimming lane is respectively: 1.pFBgp67-H1HA1; 2.pFBgp67-H2HA1; 3.pFBgp67-H3HA1; 4.pFBgp67-H4HA1; 5.pFBgp67-H5HA1; 6.pFBgp67-H6HA1; 7.pFBgp67-H7HA1; 8.pFBgp67-H8HA1; 9.pFBgp67-H9HA1; 10.pFBgp67-H10HA1; 11.pFBgp67-H11HA1; 12.pFBgp67-H12HA1; 13.pFBgp67-H13HA1; 14.pFBgp67-H14HA1; 15.pFBgp67-H15HA1; 16.pFBgp67-H16HA1.
Fig. 4 is that each hypotype HA gene of influenza A virus is identified at the Western blot of High 5 expressed in insect cells forms.Wherein swimming lane 1,3, and 5,7,9,11,13,15,17,19,21,23,25,27,29,31: the H1-H16HA1 albumen of expressing in the supernatant; Swimming lane 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32: the H1-H16HA1 albumen of expressing in the cell precipitation; The contrast of 0 swimming lane: High, 5 insect cells.
The purifying of each hypotype HA1 gene of Fig. 5 influenza A virus expressing protein in High 5 insect cell culture supernatant.Wherein each swimming lane is: 1.H2HA1; 2.H4HA1; 3.H6HA1; 4.H8HA1; 5.H11HA1; 6.H12HA1; 7.H13HA1; 8.H14HA1; 9.H15HA1; 10.H16HA1; 11.H3HA1; 12.H10HA1; 13.H1HA1; 14.H5HA1; 15.H7HA1; 16.H9HA1.
Fig. 6 is recombinant baculovirus preparation and protein expression operational flowchart
Embodiment
Further set forth the present invention below in conjunction with preferred embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.Unreceipted concrete experimental technique in the following example; Usually according to normal condition and method; The method that method or reagent manufacturers provide described in molecular cloning laboratory manual (Sambrook, et al.New York:Cold Spring Harbor Laboratory Press, 1989).
The structure of embodiment 1. secretor type baculovirus donor plasmids
At first the synthetic gp67 signal peptide sequence of design adds 6 * His and 3 * flag label simultaneously and is convenient to proteic evaluation (entrusting Shanghai to give birth to worker bio-engineering corporation synthesizes).Between 6 * His and 3 * Flag label, add the PreScissionTM protease cutting site, utilize the nicking activity of PreScission proteolytic enzyme can be behind the expressed fusion protein His and Flag label and target protein incision.The synthetic sequence length is 595bp, is inserted in the pUC57 carrier, and inserting the site is BssHII and MfeI, and preparation contains the recombinant plasmid of gp67 signal peptide sequence, called after pUC57-gp67.
With BssHII and MfeI (New England Biolabs company) double digestion rhabdovirus expression vector pFastBac 1 (American I nvitrogen company), system is: pFastBac 1 is 5 μ l, each 1.5 μ l of BssHII and MfeI, 10 * damping fluid, 5 μ l, H
2O 37 μ l place and hatched under 37 ℃ 2 hours, with the sepharose recovery test kit recovery carrier segments of Qiagen company.
Simultaneously with the plasmid pUC57-gp67 that contains the gp67 signal peptide gene of BssHII and the above-mentioned preparation of MfeI double digestion.Reaction system is: plasmid 10 μ l, each 1.5 μ l of BssHII and MfeI, 10 * damping fluid, 5 μ l, H
2O 32 μ l place and hatched under 37 ℃ 2 hours, with the sepharose recovery test kit recovery target gene fragment gp67 of Qiagen company.
Above-mentioned linearizing carrier pFastBac 1 is carried out ligation with goal gene gp67, and reaction system is: carrier pFastBac10.5 μ l, and purpose fragment 3 μ l connect damping fluid 2 μ l, and T4DNA ligase enzyme (Japanese TAKARA company) 1 μ l mends H
2O to 20 μ l places 16 ℃ water-bath to connect 12-16 hour.Get 10 μ l and connect product transformed into escherichia coli DH5 α competent cell (sky, Beijing root biochemical technology ltd).Transform the mono-clonal bacterium colony of picking ammonia benzyl resistance on the flat board next day at LB; Use the LB nutrient solution that contains penbritin to cultivate after 12～16 hours; Prepare plasmid in a small amount with alkaline lysis (referring to the molecular cloning laboratory manual); The new secretor type baculovirus donor plasmids called after pFastBacgp67 (being designated hereinafter simply as pFBgp67) that makes up, and through the exactness of sequencing checking positive colony.
With the secretor type baculovirus donor plasmids pFBgp67 of Xho I and Hind III (New England Biolabs company) the above-mentioned preparation of double digestion, system is: pFBgp67 is 5 μ l, each 1.5 μ l of Xho I and Hind III, 10 * damping fluid, 5 μ l, H
2O 37 μ l place and hatched under 37 ℃ 2 hours, with the sepharose recovery test kit recovery carrier segments of Qiagen company.
(entrusting Shanghai to give birth to worker bio-engineering corporation according to the corresponding gene order of GenBank synthesizes to contain 16 influenza virus gene type HA1 gene hypotypes with Xho I and Hind III double digestion simultaneously; Influenza virus is divided into 16 hypotypes according to the HA gene, and the HA gene must cut into HA1 and HA2 just has function, and these 16 HA1 genes are exactly the HA1 gene of 16 subtype influenza viruses; Their sequence has certain similarity; Homology is probably between 28-77%) recombinant plasmid pCDNAII/H1-HA1 to pCDNAII/H16-HA1, reaction system is: plasmid 10 μ l, each 1.5 μ l of Xho I and Hind III; 10 * damping fluid, 5 μ l, H
2O 32 μ l place and hatched under 37 ℃ 2 hours, reclaim test kit with the sepharose of Qiagen company and reclaim purpose fragment H1-HA1 to H16-HA1 respectively.
Above-mentioned linearizing carrier pFBgp67 is carried out ligation with goal gene H1-HA1 to H16-HA1 respectively, and reaction system is: carrier pFBgp67 is 0.5 μ l, and purpose fragment 3 μ l connect damping fluid 2 μ l, and T4DNA ligase enzyme (TAKARA company) 1 μ l mends H
2O to 20 μ l places 16 ℃ water-bath to connect 12-16 hour.Get 10 μ l and connect product transformed into escherichia coli DH5 α competent cell respectively.Transform flat board on picking ammonia benzyl resistance mono-clonal bacterium colony at LB next day; After 12～16 hours, prepare plasmid in a small amount with the LB nutrient solution cultivation that contains penbritin, carry out double digestion respectively with Xho I and Hind III and identify with alkaline lysis; System is: pFBgp67 is 5 μ l; Each 0.5 μ l of Xho I and Hind III, 10 * damping fluid, 2 μ l, H
2O 12 μ l; Place and hatched under 37 ℃ 2 hours; Enzyme is cut capable 1% agarose gel electrophoresis of product; Downcut about 950bp fragment respectively, obtain secretor type recombination rhabdovirus expression vector pFBgp67-H1HA1, pFBgp67-H2HA1, pFBgp67-H3HA1, pFBgp67-H4HA1, pFBgp67-H5HA1, pFBgp67-H6HA1, pFBgp67-H7HA1, pFBgp67-H8HA1, pFBgp67-H9HA1, pFBgp67-H10HA1, pFBgp67-H11HA1, pFBgp67-H12HA1, pFBgp67-H13HA1, pFBgp67-H14HA1, pFBgp67-H15HA1, pFBgp67-H16HA1.
The swivel base reaction of embodiment 3. secretor type recombination rhabdovirus expression vectors
Take out DH10Bac competent cell (American I nvitrogen company) from-80 ℃ of refrigerators and place on ice and dissolve, add 1 μ l recombinant expression vector plasmid pFBgp67-H1HA1 to pFBgp67-H16HA1 respectively, flick eppendorf pipe mixing; Place ice bath after 30 minutes; In 42 ℃ of following heat-shockeds 90 seconds, place ice bath to stablize then 5 minutes, add 900 μ lSOC nutrient solutions respectively; Place under 37 ℃ with 200rpm jolting 4-6 hour; Get 40 μ l bacterium liquid coating Luria Agar (American I nvitrogen company) selectivity culture plate (kantlex 50 μ g/ml, qingfengmeisu qiong 7 μ g/ml, tsiklomitsin 10 μ g/ml) respectively; Place 37 ℃ of following lucifuges to cultivate at least 48 hours, observe blue hickie growing state.
The separation of embodiment 4. reorganization Bacmid DNA
Each 3～4 of white colonies on the above-mentioned flat board of picking; Difference is streak culture spending the night on Luria agar (American I nvitrogen company) flat board again; Picking is the bacterium colony of hickie still, and the LB nutrient solution that adds to 3ml (contains kantlex 50 μ g/ml, qingfengmeisu qiong 7 μ g/ml; Tsiklomitsin 10 μ g/ml) in, place 37 ℃ of following joltings to cultivate 12-16 hour.Get the 1.0ml culture, centrifugal collection thalline adds 0.3ml solution I [15mmol/L Tris-HCl (pH 8.0); 10mmol/LEDTA, 100 μ g/ml RNase A (U.S. Sigma Company products)] resuspended thalline, add 0.3ml solution II (0.2N NaOH; 1%SDS) mixing; In room temperature held 5 minutes, add potassium acetate (pH5.5) and the mixing of the 3mol/L of 0.3ml, placed ice bath 5-10 minute.In 13, under the 000rpm centrifugal 10 minutes, get supernatant; The Virahol and the mixing that add 0.8ml place behind the ice bath 5-10min in 13 then, under the 000rpm centrifugal 15 minutes; To precipitate (the reorganization Bacmid DNA that promptly contains gp67 and influenza virus HA1 gene) washing with alcohol, place drying under the room temperature, be dissolved in aseptic TE damping fluid (the 10mmol/L Tris-HCl of 50 μ l with 70%; 1mmol/L EDTA, pH8.0) in.
The acquisition of embodiment 5. recombinant baculovirus
The Grace substratum (American I nvitrogen company) that contains 10% foetal calf serum (U.S. Hyclone company) and P.S. (penicillium mould 100U/ml, Streptomycin sulphate 100 μ g/ml, North China Pharmaceutical Factory) is adopted in the cultivation of insect cell Sf9 (American I nvitrogen company).Preceding 24 hours of transfection reaches cell in the 6 porocyte culture plates (U.S. Costar company), treats that cell carries out transfection when growing to about 50～70% abundance.Prepare following solution during transfection:
Solution A: the 1-2 μ g reorganization Bacmid DNA of preparation among the embodiment 4 is mixed with the Grace substratum (American I nvitrogen company) of 100 μ l serum-free antibiotic-frees;
Solution B: the Grace substratum of 6 μ l
transfection reagents (American I nvitrogen company) with 100 μ l serum-free antibiotic-frees mixed;
Above-mentioned solution A is mixed with solution B, flick the tube wall mixing, place and hatched under the room temperature 30 minutes.In the time of waiting for, wash cell 1 time with the Grace substratum of 2ml serum-free antibiotic-free.0.8ml Grace substratum is added in the mixture (A+B mixture) of DNA+ liposome; Behind the soft mixing; Be added to and use on the washed Sf9 cell of Grace substratum; Place hatch 5 hours under 27 ℃ after, absorb transfection liquid with suction pipe, be changed to the Grace cell culture fluid that contains 10% foetal calf serum and continue at 27 ℃ and cultivate down.Be changed to the Grace substratum that contains 2% foetal calf serum next day, place 27 ℃ to cultivate 72 hours down.After the transfection 72 hours; Collection contains substratum to the aseptic centrifuge tube of 15ml (U.S. Costar company) of cell; Place centrifugal 5 minutes of 500g room temperature to remove cell and fragment, the results supernatant divides to be filled in the aseptic frozen pipe; This is the former generation seed culture of viruses that contains recombinant virus, places 4 ℃ or-80 ℃ to preserve down.This virus infection Sf9 cell increased go down to posterity.
The evaluation of embodiment 6. target protein expression-forms
(American I nvitrogen company) is diluted to 0.5 * 10 with High 5 insect cells
6/ ml, the recombinant baculovirus supernatant of getting the above-mentioned preparation of 1～2ml is inoculated into 50ml and contains above-mentioned 0.5 * 10
6In the substratum of/ml High 5 cells, place 28 ℃ of following joltings to cultivate 72 hours, rotating speed is 90 rev/mins, gets culture supernatant and cell respectively and carries out Western-blot with the proteic expression of testing goal.
The processing of culturing cell: with the cell of pathology with the PBS washing after, cell precipitation contains RIPA lysate (50mmol/LTris-HCl, the 150mmol/L NaCl of proteinase inhibitor (U.S. Roche company) with 100 μ l; 0.1%SDS, 1%NP40,0.5% Sodium desoxycholate) in cracking on ice 30 minutes; Under 4 ℃ with 12; Centrifugal 10 minutes of 000rpm is transferred to supernatant in another new eppendorf pipe, is used for Western-blot and detects.The Western-blot detection method is pressed method described in the molecular cloning laboratory manual (Sambrook, et al.NewYork:Cold Spring Harbor Laboratory Press, 1989) and is operated.Used one anti-is mouse anti-His antibody (U.S. Sigma company) in the experiment, and two anti-ly are the goat anti-mouse antibody of horseradish peroxidase-labeled (U.S. Sigma company).
The processing of culture supernatant: get the 1ml culture supernatant, add 0.35g (NH
4)
2SO
4(35%, M/V) place 4 ℃ of settle to spend the night, place next day under 4 ℃ with centrifugal 30 minutes collecting precipitations of 18000rpm, add an amount of (about 100 μ l) resuspended deposition of PBS, be used for Western-blot and detect.The Western-blot detection method is pressed method described in the molecular cloning laboratory manual (Sambrook, et al.New York:Cold Spring Harbor Laboratory Press, 1989) and is operated.Used one anti-is mouse anti-His antibody (U.S. Sigma company) in the experiment, and two anti-ly are the goat anti-mouse antibody of horseradish peroxidase-labeled (U.S. Sigma company).
The Western-blot results suggest; Clone the influenza A virus HA1 gene of 16 hypotypes in the pFastBacgp67 carrier; Not only in insect cell, express through swivel base and transfection experiment step; And in cells and supernatant, realized the secretion expression, be convenient to next step purifying.
The insect cell culture supernatant is in order to 35% (NH
4)
2SO
4 Place 4 ℃ of settle to spend the night, next day under 4 ℃ with 18, centrifugal 30 minutes collecting precipitations of 000rpm; Deposition is with an amount of binding buffer liquid (20mM PBS+500mM NaCl+5mM imidazoles; PH7.4) dissolve, deposit then 4 ℃ with centrifugal 30 minutes of 18000rpm to remove insoluble sediment, centrifugal back supernatant combines Ni Sepharose 6Fast Flow affinity chromatography filler (U.S. GE Healthcare company); Use lavation buffer solution (20mM PB+500mM NaCl+70mM imidazoles then; PH7.4) the washing filler is used elution buffer (20mM PBS+500mM NaCl+500mM imidazoles, pH7.4) wash-out target protein at last
Purified albumen is carried out protein electrophoresis, and the albumen behind the purifying adds 6 * SDS protein electrophoresis sample loading buffer (concrete prescription see for details molecular cloning experiment guide) respectively, place 100 ℃ to boil 5 minutes after; Row 12%SDS-PAGE gel electrophoresis; Add 1 * SDS-PAGE electrophoretic buffer in the electrophoresis chamber, energized is carried out electrophoresis; Concentrate glue with 80 volts of constant voltages, behind the entering separation gel with 100 volts of constant voltages.Electrophoresis is pried open sheet glass after finishing, and with being placed on after the gel slab marked in the big petridish, adds staining fluid, dyes about 1 hour.Gel slab after the dyeing decolours with destainer with the zero(ppm) water rinsing for several times again, and it is clear to be with up to the district, puts in the gel imaging system and takes a picture.The protein purification results suggest is secreted into the target protein in the culture supernatant, earlier through ammonium sulfate precipitation, promptly can obtain the target protein (concrete steps are referring to the molecular cloning experiment guide) of higher degree (＞90%) again through the proteic method of a step affinitive layer purification.
Claims (10)
1. a gp67 signal peptide sequence is characterized in that having the sequence shown in the SEQ ID NO:1.
2. a secretor type baculovirus donor plasmids pFastBacgp67 is characterized in that said donor plasmid is the basis with the pFastBacI rhabdovirus expression vector, between its MCS, is connected with the described gp67 signal peptide sequence of claim 1.
3. the described secretor type baculovirus donor plasmids of claim 2 pFastBacgp67 is characterized in that said MCS is BssHII and MfeI.
4. a method for preparing the described secretor type baculovirus donor plasmids of claim 2 pFastBacgp67 comprises the described gp67 signal peptide sequence of claim 1 is inserted in the MCS of pFastBacI rhabdovirus expression vector.
5. the described method of claim 4 is characterized in that said MCS is BssHII and MfeI.
6. an exogenous protein expression system comprises the described secretor type baculovirus donor plasmids of claim 2 pFastBacgp67, Bacmid shuttle vectors, helper plasmid and insect cell.
7. the described exogenous protein expression of claim 6 system, wherein said insect cell comprises Sf9 insect cell, Sf21 insect cell or High 5 insect cells.
8. method of utilizing the described exogenous protein expression of claim 6 system to produce foreign protein may further comprise the steps:
(1) will the encode gene of said foreign protein is inserted among the secretor type baculovirus donor plasmids pFastBacgp67, to obtain transfer vector plasmid;
(2) the transfer vector plasmid swivel base DH10Bac competent cell and the extraction reorganization Bacmid DNA that prepare with step (1);
(3) with the reorganization Bacmid DNA transfection insect cell of step (2) preparation to obtain recombinant virus;
(4) with step (3) the recombinant virus infection insect cell that obtained and cultivate;
(5) supernatant of harvested cell culture and therefrom purifying protein.
9. the described method of claim 8, wherein said insect cell comprises Sf9 insect cell, Sf21 insect cell or High 5 insect cells.
10. claim 8 or 9 described methods, wherein liposome method is adopted in the transfection in the step (3).
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