CN101348800A - Method for efficiently obtaining animal membrane protein by means of togavirus budding characteristic - Google Patents
Method for efficiently obtaining animal membrane protein by means of togavirus budding characteristic Download PDFInfo
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Abstract
The invention discloses a method for highly efficiently obtaining animal membrane proteins by utilization of the budding characteristic of togavirus. The method comprises the following steps: an animal host cell is infected with recombinant baculovirus, namely BacmidCMV-G-HA which is then proliferated; compositions of a host cell membrane are brought away when the virus is budded and then a self cyst membrane is formed; virion is separated and then cyst membrane compositions are obtained; and the membrane proteins are obtained after repeated frost thawing of the cyst membrane compositions. The invention has the advantages that the invention provides a novel technological method for separating and purifying the membrane proteins; the animal host cell is infected with the togavirus, and HA fusion genes carried by the virus are expressed and then positioned on the host cell membrane; and the HA is taken as a mark on the membrane proteins, and the compositions of the cell membrane exist if the HA activity exists, thereby contributing to finding acting targets of drug proteins.
Description
Technical field
The present invention relates to the protein expression in a kind of protein engineering and the field of separation and purification, particularly a kind of togavirus characteristic of sprouting of utilizing efficiently obtains the method for membranin.
Background technology
Microbial film has important biological function, as the cell recognition site is provided, being connected or the like between mediated cell and cell, cell and the matrix.The research of membranin is also just becoming the focus of proteome research.Membranin is the target site point of many medicines, and the research membranin not only helps the research of low-abundance protein, and more the diagnosis of medicament research and development and disease provides target body and labelled protein.Yet the separation of membranin is membranin research " bottleneck ", studies the research that proteic high efficiency method bidirectional electrophoresis technique also is not suitable for membranin.
Can take away host cell part film component when baculovirus sprouts, but that baculovirus has is restricted, can only infected insect cell.Yet nineteen ninety-five, after Hofmann etc. discovered and insert CMV (human cytomegalovirus immediate-early region) in baculovirus DNA, baculovirus can be expressed in human liver cell.1997, Barsoum etc. studied report, insert VSV-G (vesicular stomatitis virus G protein) and can enlarge host range and increase transfection efficiency in baculovirus DNA.
Summary of the invention
At the deficiencies in the prior art part, the invention provides the new technological method of a kind of membranin isolation and purification.
A kind of togavirus characteristic of sprouting of utilizing of the present invention efficiently obtains the proteic method of animal membrane, be by breeding behind the recombinant baculovirus BacmidCMV-G-HA infection animal host cell, when virus is sprouted, take away the host cell film component and form self cyst membrane, virus particle is isolated, the cyst membrane composition of reentrying, multigelation obtains membranin.
Utilize the togavirus characteristic of sprouting efficiently to obtain the method for membranin, specifically may further comprise the steps:
(1) recombinant baculovirus BacmidCMV-G-HA is with 5MOI infection animal host cell, 37 ℃, 5%CO
2Cultivated under the condition 3～4 days, and washed cell with PBS, harvested cell;
(2) 1000rpm is centrifugal, remove the fragment of above-mentioned steps (1) gained cell, supernatant liquor carries out ultracentrifugation, 35000rpm, 30～60min collects virus particle, the resuspended dissolving of PBS damping fluid of sedimentary virus particle, be used for sucrose density gradient ultracentrifugation, 35000rpm presses the peak and collects sample after centrifugal 3 hours;
(3) the activated virus particle sample that the density gradient of above-mentioned steps (2) is collected multigelation 3～5 times between liquid nitrogen and 37 ℃ breaks cyst membrane, 12000rpm, and centrifugal 20～60min precipitates large stretch of cyst membrane, obtains membranin.
The construction process of described recombinant baculovirus BacmidCMV-G-Ha: at pFastBac
TMAfter inserting CMV-VSV-G sequence and HA fusion gene sequence in the carrier, utilize Bac-to-Bac
System obtains BacmidCMV-G-HA.
The construction process of described recombinant baculovirus BacmidCMV-G-Ha may further comprise the steps:
(1) fusion gene HA and plasmid pFastBac
TMCarry out EcoR I and Xho I double digestion simultaneously, the HA fusion gene is inserted in the pFastBacTM plasmid, is transformed among the competent cell E.coli DH5 α picking list bacterium colony, extract plasmid, enzyme is cut the plasmid pFastBac-HA that order-checking identifies correct connection;
(2) the sequences Design one couple of PCR primers by pHCMV-VSV-G, amplify the sequence of CMV-VSV-G, the upstream primer that contains BamH I site: atggatccgagcttggcccattgcatac, the downstream primer that contains EcoR I site: gcgaattcgacggatccttatcactttc, after the PCR reaction finishes, electrophoresis, purifying reclaims reaction product;
(3) PCR product behind step (2) purifying and step (1) gained plasmid pFastBac-HA carry out BamH I and EcoR I double digestion respectively, under the effect of T4 ligase enzyme, make the PCR product cloning to plasmid pFastBac-HA, be converted among the competent cell DH5 α, picking list bacterium colony, extract plasmid, enzyme is cut and is checked order and identify the correct recombinant plasmid pFastBacCMV-G-HA that inserts;
(4) according to Bac-to-Bac
System gets 5ul step (3) gained recombinant plasmid pFastBacCMV-G-HA and is transformed into DH10Bac
TMIn the competent cell, dull and stereotyped in 37 ℃ cultivate 24-48h after picking white clone, streak inoculation to flat board, incubated overnight, confirmation is a positive colony; Second day, picking list colony inoculation was cultivated in liquid nutrient medium, obtained recombinant plasmid BacmidCMV-G-HA;
(5) get the about 5ul of the DNA of recombinant plasmid BacmidCMV-G-HA and add 100ul Sf-900IISFM, get cellfectin
Reagent joins the Sf-900II SFM of 100ul, and again with both mixings, room temperature is placed 15～30min, Sf9 cultivates in the SFM of penicillin that contains 50 units and Streptomycin sulphate, cell transfer to 27 ℃ is placed 1h at least, with after not containing antibiotic SFM substratum washed twice, add above-mentioned DNA mixture again, cultivate 5h for 27 ℃, remove the DNA mixture, add and contain antibiotic SFM, cultivate 72h in 27 ℃, harvested cell obtains recombinant baculovirus BacmidCMV-G-HA.
The structure of described fusion gene HA may further comprise the steps:
(1) with gp64DNA be template, pcr amplification gp64 signal peptide gene, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Psp2：5’tgtgtaacagtaacgttcttttccgcaaaggcagaatgcgccgc?3’；
(2) with gp64DNA be template, pcr amplification gp64 membrane-spanning domain gene, two kinds of used primers of PCR are:
Ptm1：5’cgttacaatgcagaatttgcattggtgatactgggctatccaaaa?3’
Ptm2：5’gtgagctcttactttccaagtcggttcatc?3’；
(3) be template with avian influenza virus H 5 N 1 HA gene order, amplification HA gene, PCR used two
Planting primer is:
Pha1：5’aaagaacgttactgttacac?3’
Pha2：5’atgcaaattctgcattgtaa?3’；
(4) be template with gp64 signal peptide gene and HA gene, utilize PCR method to amplify sp-HA fusion gene sequence, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Pha2：5’atgcaaattctgcattgtaa?3’
(5) be template with fusion gene sp-HA and gp64 membrane-spanning domain gene, construct the HA fusion gene, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Ptm2：5’gtgagctcttactttccaagtcggttcatc?3’。
DH10BacTM competent cell described in the step (4) contains the Bacmid plasmid.
Flat board described in the step (4) contains Bluo-gal, kantlex, gentamicin, tsiklomitsin, IPTG.
Liquid nutrient medium described in the step (4) contains kantlex, gentamicin, tsiklomitsin.
Contain antibiotic SFM described in the step (5), described microbiotic is penicillin and Streptomycin sulphate.
Usefulness of the present invention is: the technological method that the invention provides a kind of new membranin separation and purification, by togavirus infection animal host cell, the HA fusion gene that virus is carried will be positioned on host's the cytolemma after expressing, be a sign on the membranin with HA, having the HA activity then to show has cell membrane component to exist.The research of membrane protein is the bottleneck of proteomics research always, and the present invention is primarily aimed at membranin, can promote the development of membranin group research, helps to find the target spot of pharmaceutical protein effect, promotes the development of pharmacology, medical science.
Embodiment
The present invention utilizes baculovirus to sprout and takes away the characteristic of host cell part film component, isolates the virus of sprouting (togavirus) earlier, the cyst membrane of reentrying (membranin).The technological method that the purpose of this invention is to provide a kind of new membranin separation and purification, by togavirus infection animal host cell, the HA fusion gene that virus is carried will be positioned on host's the cytolemma after expressing, be a sign on the membranin with HA, having the HA activity then to show has cell membrane component to exist.When virus take away the cytolemma of host cell in the mode of sprouting and form virus cyst membrane the time, isolate togavirus, multigelation breaks cyst membrane, centrifugal results envelope protein, again envelope protein is carried out high performance liquid chromatography (HPLC) and mass spectroscopy, thereby identify and obtain membranin.
In order to make the cell of baculovirus energy infection animal, the present invention has made up the BacmidCMV-G-HA recombinant baculovirus, by from plasmid pHCMV-VSV-G, amplifying the sequence of CMV-VSV-G, construct HA fusion gene (be connected signal peptide respectively and stride the film district with the C end) simultaneously at the N of HA gene end, both are inserted in the pFastBacTM carrier respectively again, form the pFastBacCMV-G-HA plasmid, according to Bac-to-Bac
Baculovirus expression system and obtain Bacmi dCMV-G-HA recombinant baculovirus.
Below in conjunction with embodiment technical scheme of the present invention is described:
Embodiment 1
The effect of HA gene is equivalent to a molecule marker, and HA can replace with other gene or marker in this invention, and as green fluorescent protein GFP, the present invention is an example with the HA gene only, but these embodiment and be not used in qualification the present invention.
1, the structure of fusion gene HA
Is connected coding baculovirus envelope protein gp64 signal peptide gene sequence and gp64 respectively at 5 ' and 3 ' end of HA gene (Genbank DQ520855) and strides film district gene order, and hold at 5 ' and 3 ' of fusion gene and to introduce EcoR I and Xho I restriction enzyme site respectively.Respectively with baculovirus (AcNPV) membrane glycoprotein (gp64) signal peptide gene sequence (silkworm baculovirus genome, Genbank NP 074525), avian influenza virus H 5 N 1 HA gene order (avian influenza virus H 5 N 1 Hangzhou epidemic strain, GenbankDQ520855) and AcNPV gp64 stride film district gene order (silkworm baculovirus genome, Genbank NP 074525) is 3 pairs of primers of stencil design, the purpose that at first increases fragment gp64 signal peptide (hereinafter to be referred as: sp), HA and gp64 membrane-spanning domain (hereinafter to be referred as: tm), utilize each purpose fragment primer and the synthetic fusion gene HA of template each other then.The design primer is as follows:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Psp2：5’tgtgtaacagtaacgttcttttccgcaaaggcagaatgcgccgc?3’
Pha1：5’aaagaacgttactgttacac?3’
Pha2：5’atgcaaattctgcattgtaa?3’
Ptm1：5’cgttacaatgcagaatttgcattggtgatactgggctatccaaaa?3’
Ptm2：5’gtgagctcttactttccaagtcggttcatc?3’
(1) amplification of gp64 signal peptide (sp)
With gp64DNA (silkworm baculovirus genome, Genbank NP 074525) is template, carries out the PCR reaction, and 94 ℃ of pre-sex change 3min carry out 30 circulations then earlier, and its cycling condition is: 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 30s; After the loop ends, 68 ℃ are extended 5min again.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR?Buffer 10μl
25mmol?MgCl2 8μl
2.5mmol?dNTPs 8μl
Psp1 1μl
Psp2 1μl
Template 1 μ l
KOD-PlusDNA polysaccharase (TOYOBO company, Japan) 1 μ l
Add aseptic double-distilled water to 100l
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut glue simultaneously and reclaimed the purpose segment.
(2) amplification of gp64 membrane-spanning domain (tm)
With gp64DNA is template, carries out the PCR reaction, and 94 ℃ of pre-sex change 3min carry out 30 circulations then earlier, and its cycling condition is: 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 30s; After the loop ends, 68 ℃ are extended 5min again.
The reaction system of 100ul is the same, and the primer is Ptm1 and Ptm2, behind each component mixing, puts into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut glue simultaneously and reclaimed the purpose segment.
(3) amplification of HA gene
With avian influenza virus H 5 N 1 HA gene order (extract from ill chicken serum, sequence is seen GenbankDQ520855) is template, carries out the PCR reaction, 94 ℃ of pre-sex change 3min of elder generation, carry out 30 circulations then, its cycling condition is: 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min; After the loop ends, 68 ℃ are extended 5min again.In an aseptic 0.5ml centrifuge tube, mix following ingredients: 10 * PCR Buffer, 10 μ l, 25mmol MgCl2 8 μ l, 2.5mmol dNTPs 8 μ l, primer Pha1 1 μ l, primer Pha21 μ l, template 1 μ l, KOD-Plus archaeal dna polymerase 1 μ l adds aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut glue simultaneously and reclaimed the purpose segment.
(4) gp64 signal peptide sp gene and HA gene Fusion
Carry out the PCR reaction, 94 ℃ of pre-sex change 5min carry out 30 circulations then earlier, and its cycling condition is: 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min; After the loop ends, 68 ℃ are extended 7min again.In an aseptic 0.5ml centrifuge tube, mix following ingredients: 10 * PCR Buffer, 10 μ l, 25,mmo,lMg,Cl2 8 μ l, 2.5mmol dNTPs 8 μ l, primer Psp1 1 μ l, primer Pha2 1 μ l, template sp 1 μ l, template HA 1 μ l, KOD-Plus archaeal dna polymerase (TOYOBO company, Japan) 1 μ l adds aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut glue simultaneously and reclaimed the purpose segment.
Because added the partial sequence of HA gene 5 ' end during the downstream primer Psp2 of sp design, so it is overlapping that sp segment 3 ' terminal sequence that 0CR amplifies and HA gene 5 ' terminal sequence exist, utilize above-mentioned 0CR method just can amplify sp-HA fusion gene sequence once more.
(5) fusion gene sp-HA once more with the fusion of tm, construct the HA fusion gene
The PCR reaction parameter is 94 ℃ of pre-sex change 5min, carries out 30 circulations then, and its cycling condition is: 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1.5min; After the loop ends, 68 ℃ are extended 10min again.In an aseptic 0.5ml centrifuge tube, mix following ingredients: 10 * PCR Buffer, 10 μ l, 25mmol MgCl28 μ l, 2.5mmol dNTPs 8 μ l, primer Psp1 1 μ l, primer Ptm2 1 μ l, template sp-HA 1 μ l, template tm 1 μ l, KOD-Plus archaeal dna polymerase 1 μ l adds aseptic double-distilled water to 100 μ l.
Behind each component mixing, put into the PCR instrument, by 30 circulations of above-mentioned reaction parameter design.After question response finished, electrophoresis was identified the amplification segment, cut glue simultaneously and reclaimed the purpose segment.
The same, because the upstream primer Ptm1 of tm design the time has added the partial sequence of HA gene 3 ' end,, utilize PCR method just can amplify fusion gene HA once more so that tm segment 5 ' terminal sequence that pcr amplification goes out and HA gene 3 ' terminal sequence exist is overlapping.
2. make up the pFastBac-HA plasmid
Above-mentioned fusion gene HA that obtains and plasmid pFastBac
TM(the Bac-to-Bac of invitrogen company
Baculovirus expression system[kits]) carry out EcoR I and Xho I double digestion simultaneously, under the effect of T4 ligase enzyme (TAKARA company test kit, the by specification operation is carried out), make the HA fusion gene be inserted into pFastBac
TMIn the plasmid.According to specification sheets operation among the kits, be transformed among the competent cell E.coli DH5 α, picking list bacterium colony extracts plasmid, and enzyme is cut the plasmid pFastBac-HA that order-checking identifies correct connection.
3. make up the pFastBacCMV-G-HA plasmid
By the sequences Design one couple of PCR primers of pHCMV-VSV-G (Genebank NO.AJ318514), amplify the sequence of CMV-VSV-G.Upstream primer (containing BamH I site): atggatccgagcttggcccattgcatac, downstream primer (containing EcoR I site): gcgaattcgacggatccttatcactttc.The PCR programdesign is as follows: 94 ℃ of pre-sex change 7min, and 94 ℃ of sex change 1min, 68 ℃ of renaturation are extended 3min, 30 circulations, 68 ℃ are extended 10min.In an aseptic 0.5ml centrifuge tube, mix following ingredients: 10 * PCR Buffer, 10 μ l, 25mmol MgCl2 8 μ l, 2.5mmol dNTPs 8 μ l, each 3 μ l of primer, plasmid template 1 μ l, KOD-Plus archaeal dna polymerase 1 μ l adds aseptic double-distilled water to 100 μ l.
After the PCR reaction finishes, electrophoresis, purifying reclaims reaction product.
The PCR product of purifying and plasmid pFastBac-HA carry out BamH I and EcoR I double digestion respectively, make the PCR product cloning under the effect of T4 ligase enzyme to plasmid pFastBac-HA.Be converted among the competent cell E.coli DH5 α, picking list bacterium colony extracts plasmid, and enzyme is cut and checked order and identify the correct recombinant plasmid pFastBacCMV-G-HA that inserts.
4. baculovirus expression plasmid Bacmi dCMV-G-HA
According to Bac-t o-Bac
Baculovirus expression system process specifications carries out, and the DNA that gets 5ul (about 1ng) recombinant plasmid pFastBacCMV-G-HA is transformed into the DH10Bac that contains the Bacmid plasmid
TMCompetent cell (the invi trogen Bac-to-Bac of company
Baculovirusexpression system[kits]) in, dull and stereotyped (contain Bluo-gal, kantlex, gentamicin, tsiklomitsin, IPTG) picking white is cloned behind 37 ℃ of cultivation 24-48h.Streak inoculation to flat board, incubated overnight, confirmation is a positive colony.Second day, picking list colony inoculation was cultivated in liquid nutrient medium (containing kantlex, gentamicin, tsiklomitsin), was used for separating reorganization Bacmid plasmid, obtained recombinant plasmid BacmidCMV-G-HA according to the operation of specification sheets.PCR identifies that the recombinant plasmid sequence is correct.
5. contain the acquisition of the recombinant baculovirus BacmidCMV-G-HA of recombinant plasmid
According to bac-to-bac
The operation of baculovirus express ion systems specification sheets.The about 5ul of DNA that gets recombinant plasmid BacmidCMV-G-HA adds 100ul Sf-900I I SFM and (does not contain microbiotic, invitrogen, Bac-to-Bac
Baculovirus expression system[kits]), get cellfectin
Reagent (invitrogen, Bac-to-Bac
Baculovirus expressionsystem[kits]) join the Sf-900II SFM (not containing microbiotic) of 100ul, again with both mixings, room temperature is placed 15-30min.Sf9 (9 * 105cells/35mm well) cultivates in the SFM of penicillin that contains 50 units and Streptomycin sulphate, cell transfer to 27 ℃ is placed 1h at least, with after not containing antibiotic SFM substratum washed twice, add above-mentioned DNA mixture again, cultivate 5h for 27 ℃.Remove the DNA mixture with pipettor, add SFM (contain microbiotic: penicillin and Streptomycin sulphate), cultivate 72h in 27 ℃.Harvested cell obtains recombinant baculovirus BacmidCMV-G-HA.
6. recombinate shape virus infection mouse liver cell
This recombinant baculovirus can infect the mammal cell, and the present invention is example with the mouse liver cell.
Recombinant baculovirus is with 5MOI infecting mouse liver cell cell (purchasing in Shanghai wheat Sha biology), cultivates 3-4 days under 37 ℃, 5%CO2 condition, washes cell with PBS, harvested cell and smudge cells.
7. recombinant baculovirus separates
1000rpm is centrifugal, removes cell debris.Supernatant liquor carries out ultracentrifugation, 35000rpm, and 30-60min collects virus particle.Sedimentary virus particle is used for sucrose density gradient ultracentrifugation with the resuspended dissolving of PBS damping fluid.The last sample of density gradient is PBS, the resuspended sample of virus particle, 20% sucrose, 30% sucrose, 52% sucrose in proper order, and 35000rpm presses the peak and collects sample after centrifugal 3 hours.
8.HA active the detection
Measuring method for activity adopts hemagglutination test (HA test).Get 24 porocyte culture plates, after each row's 1 to 6 hole (A1-A6) adds 250 μ l physiological saline (0.85%), add 250 μ l samples in first hole, do doubling dilution, negative control hole is set simultaneously, adds the liquid that does not contain sample and do doubling dilution, add 1% chicken erythrocyte suspension, 250 μ l again to each hole, the light shaking culture plate, in 37 ℃ hatch 4 hours after observations.The result shows it is positive, illustrates that virus particle has cyst membrane.
9. the separation of cyst membrane (membranin)
The activated virus particle sample that density gradient is collected liquid nitrogen and+37 ℃ between multigelation 3-5 time, cyst membrane is broken.12000rpm, centrifugal 20-60min precipitates large stretch of cyst membrane, and the HPLC that is used for the back analyzes.
10.HPLC analyze cyst membrane with Q-TraP LC-MS/MS
After sedimentary cyst membrane dissolves repeatedly with 20% acetonitrile (acetonitrile), get supernatant and be used for upward sample of HPLC, the 0.5ml/ pipe is collected sample.
(trypsin sigma) carries out enzymolysis to the sample of collecting, and spends the night in 37 ℃ of reactions with mass spectrum level pancreatin.Sample behind the enzymolysis carries out HPLC again to be analyzed, and presses the peak or collects sample by pipe, is used for mass spectroscopy.
The sample of analyzing through HPLC carries out Q-Trap LC-MS/MS (ABI company) analysis, and obtaining may aminoacid sequence.
11. data retrieval
The measured possibility aminoacid sequence of mass spectrum is retrieved in the murine protein database on NCBI, simultaneously by NPS@ provide stride the diaphragm area analysis software and TMHMM server (http://www.cbs.dtu.dk/services/TMHMM) is striden film analysis to the albumen that retrieves, analytical results is found, albumen more than 90% is membranin, has the film of striding district.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQID?NO?1：
28atggatccga?gcttggccca?ttgcatac
SEQ?ID?NO?2：
28gcgaattcga?cggatcctta?tcactttc
SEQ?ID?NO?3：
26gcgaattcat?gctactagta?aatcag
SEQID?NO?4：
44tgtgtaacag?taacgttctt?ttccgcaaag?gcagaatgcg?ccgc
SEQ?ID?NO?5：
45cgttacaatg?cagaatttgc?attggtgata?ctgggctatc?caaaa
SEQ?ID?NO?6：
30gtgagctctt?actttccaag?tcggttcatc
SEQ?ID?NO?7：
20aaagaacgtt?actgttacac
SEQ?ID?NO?8：
20atgcaaattc?tgcattgtaa
Claims (9)
1, a kind of togavirus characteristic of sprouting of utilizing efficiently obtains the proteic method of animal membrane, it is characterized in that: breed behind the recombinant baculovirus BacmidCMV-G-HA infection animal host cell, when virus is sprouted, take away the host cell film component and form self cyst membrane, virus particle is isolated, the cyst membrane composition of reentrying, multigelation obtains membranin.
2, the togavirus characteristic of sprouting of utilizing according to claim 1 efficiently obtains the method for membranin, and its feature may further comprise the steps successively:
(1) recombinant baculovirus BacmidCMV-G-HA is with 5MOI infection animal host cell, 37 ℃, 5%CO
2Cultivated under the condition 3～4 days, and washed cell with PBS, harvested cell;
(2) 1000rpm is centrifugal, remove the fragment of above-mentioned steps (1) gained cell, supernatant liquor carries out ultracentrifugation, 35000rpm, 30～60min collects virus particle, the resuspended dissolving of PBS damping fluid of sedimentary virus particle, be used for sucrose density gradient ultracentrifugation, 35000rpm presses the peak and collects sample after centrifugal 3 hours;
(3) the activated virus particle sample that the density gradient of above-mentioned steps (2) is collected multigelation 3～5 times between liquid nitrogen and 37 ℃ breaks cyst membrane, 12000rpm, and centrifugal 20～60min precipitates large stretch of cyst membrane, obtains membranin.
3, the togavirus characteristic of sprouting of utilizing according to claim 1 and 2 efficiently obtains the method for membranin, it is characterized in that the construction process of described recombinant baculovirus BacmidCMV-G-Ha: at pFastBac
TMAfter inserting CMV-VSV-G sequence and HA fusion gene sequence in the carrier, utilize
System obtains Bacmi dCMV-G-HA.
4, the togavirus characteristic of sprouting of utilizing according to claim 3 efficiently obtains the method for membranin, it is characterized in that the construction process of described recombinant baculovirus BacmidCMV-G-Ha may further comprise the steps:
(1) fusion gene HA and plasmid pFastBac
TMCarry out EcoR I and Xho I double digestion simultaneously, under the effect of T4 ligase enzyme, make the HA fusion gene be inserted into pFastBac
TMIn the plasmid, be transformed among the competent cell E.coli DH5 α, picking list bacterium colony extracts plasmid, and enzyme is cut the plasmid pFastBac-HA that order-checking identifies correct connection;
(2) the sequences Design one couple of PCR primers by pHCMV-VSV-G, amplify the sequence of CMV-VSV-G, the upstream primer that contains BamH I site: atggatccgagcttggcccattgcatac, the downstream primer that contains the EcoRI site: gcgaattcgacggatccttatcactttc, after the PCR reaction finishes, electrophoresis, purifying reclaims reaction product;
(3) PCR product behind step (2) purifying and step (1) gained plasmid pFastBac-HA carry out BamH I and EcoR I double digestion respectively, under the effect of T4 ligase enzyme, make the PCR product cloning to plasmid pFastBac-HA, be converted among the competent cell DH5 α, picking list bacterium colony, extract plasmid, enzyme is cut and is checked order and identify the correct recombinant plasmid pFastBacCMV-G-HA that inserts;
(4) basis
System gets 5ul step (3) gained recombinant plasmid pFastBacCMV-G-HA and is transformed into DH10Bac
TMIn the competent cell, dull and stereotyped in 37 ℃ cultivate 24-48h after picking white clone, streak inoculation to flat board, incubated overnight, confirmation is a positive colony; Second day, picking list colony inoculation was cultivated in liquid nutrient medium, obtained recombinant plasmid BacmidCMV-G-HA;
(5) get the about 5ul of the DNA of recombinant plasmid BacmidCMV-G-HA and add 100ul Sf-900IISFM, get
Reagent joins the Sf-900 II SFM of 100ul, and again with both mixings, room temperature is placed 15～30min, Sf9 cultivates in the SFM of penicillin that contains 50 units and Streptomycin sulphate, cell transfer to 27 ℃ is placed 1h at least, with after not containing antibiotic SFM substratum washed twice, add above-mentioned DNA mixture again, cultivate 5h for 27 ℃, remove the DNA mixture, add and contain antibiotic SFM, cultivate 72h in 27 ℃, harvested cell obtains recombinant baculovirus BacmidCMV-G-HA.
5, the togavirus characteristic of sprouting of utilizing according to claim 4 efficiently obtains the method for membranin, it is characterized in that the structure of described fusion gene HA may further comprise the steps:
(1) with gp64DNA be template, pcr amplification gp64 signal peptide gene, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Psp2：5’tgtgtaacagtaacgttcttttccgcaaaggcagaatgcgccgc?3’；
(2) with gp64DNA be template, pcr amplification gp64 membrane-spanning domain gene, two kinds of used primers of PCR are:
Ptm1：5’cgttacaatgcagaatttgcattggtgatactgggctatccaaaa?3’
Ptm2：5’gtgagctcttactttccaagtcggttcatc?3’；
(3) be template with avian influenza virus H 5 N 1 HA gene order, amplification HA gene, two kinds of used primers of PCR are:
Pha1：5’aaagaacgttactgttacac?3’
Pha2：5’atgcaaattctgcattgtaa?3’；
(4) be template with gp64 signal peptide gene and HA gene, utilize PCR method to amplify sp-HA fusion gene sequence, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Pha2：5’atgcaaattctgcattgtaa?3’；
(5) be template with fusion gene sp-HA and gp64 membrane-spanning domain gene, construct the HA fusion gene, two kinds of used primers of PCR are:
Psp1：5’gcgaattcatgctactagtaaatcag?3’
Ptm2：5’gtgagctcttactttccaagtcggttcatc?3’。
6, the togavirus characteristic of sprouting of utilizing according to claim 4 efficiently obtains the method for membranin, it is characterized in that: the DH10Bac described in the step (4)
TMCompetent cell contains the Bacmid plasmid.
7, the togavirus characteristic of sprouting of utilizing according to claim 4 efficiently obtains the method for membranin, and it is characterized in that: the flat board described in the step (4) contains Bluo-gal, kantlex, gentamicin, tsiklomitsin, IPTG.
8, the togavirus characteristic of sprouting of utilizing according to claim 4 efficiently obtains the method for membranin, and it is characterized in that: the liquid nutrient medium described in the step (4) contains kantlex, gentamicin, tsiklomitsin.
9, the togavirus characteristic of sprouting of utilizing according to claim 4 efficiently obtains the method for membranin, it is characterized in that: contain antibiotic SFM described in the step (5), described microbiotic is penicillin and Streptomycin sulphate.
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