CN1268573A - Carrier for expressing Chiense chicken pox virus vaccine strain - Google Patents
Carrier for expressing Chiense chicken pox virus vaccine strain Download PDFInfo
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Abstract
The present invention provides 14 henpox virus 282 E4 strain reconstittion expression carrier, of which 8 are constituted by inserting two henpox virus promotors respectively into 282 E4 strain TK gene or 1.5 kbB fragment nonessential region. Take bacillus coli LacZ gene or green fluorescent protein gene as report gene and insert into 6 carrier of above mentioned expression carrier to construct another six Chinese henpox virus 282 E4 reconstitution expression carrier containing report gene. By using these expression carrier can obtain reconstituted virus for expressing different exogenous gene, these reconstituted virus can be used as vaccine for preventing and treating human, poultry and animal diseases.
Description
The invention provides 14 Chinese bird pox virus 282E4 vaccine strain recombinant expression vectors, belong to biological technical field.
The technology of clone, recombinant DNA has been expanded the method that people make vaccine greatly in bacterial plasmid.Because poxvirus exists the reorganization phenomenon between homologous sequence in reproduction process, and in the genome of poxvirus, exist the nonessential zone of many virus replications, so, people can at first be cloned into the nonessential region of virus replication in the bacterial plasmid, again start exogenous gene promoter in the cytoplasm of host cells infected (because poxvirus utilizes the RNA polymerase of oneself to duplicate, therefore essential promotor) insert nonessential region, be built into recombinant expression vector with poxvirus.Then wanting expressed exogenous gene to be inserted into the promotor downstream in the virus replication nonessential region, obtain recombinant expression plasmid, utilize this plasmid and viral parent plant that homologous recombination takes place when time multiplexed cell system, can obtain the recombinant virus of expression alien gene.During with this recombinant virus immune animal, virus then can be expressed the foreign gene that carries in the reproduction process in animal body, stimulates the specific immune response of animal body generation to foreign protein.Therefore can be used as the diseases prevention and treatment that the live recombinant vectors vaccine is used for the animal and human.
On the whole, poxvirus is a virus maximum in the animal virus, can see under opticmicroscope.Under electron microscope, poxvirus is the avette outward appearance of bird.The topmost composition of virus is protein, thymus nucleic acid and lipoid.Polyacrylamide gel electrophoresis is analyzed virion protein and is shown the viral associated protein that kind more than 100 comprises the enzyme of structural protein, translation mRNA and relevant enzymes such as RNA is synthetic, dna replication dna.The poxvirus genome group is made up of the unit molecule double-stranded DNA, and G+C content is about 32%～64%, the about 140kb～280kb of genome length.So far, and vaccinia virus (Vaccinia Virus, genome sequence VV) is clear and definite, and therefore as the carrier of carrying foreign gene, it is that to use the earliest also be to use maximum virus vector.
So far, the protein of existing polytype biologically active has obtained expression in reorganization VV.And the reorganization VV of the various cause of diseases of thus obtained expression becomes prevention and the live recombinant vectors vaccine for the treatment of people and multiple communicable disease of animal and even tumour.But because VV infects the danger that produces viremia behind the individual inoculation of side reaction, the especially immune deficiency VV that produces behind the VV of host range and individual one inoculation widely and diffusion virus is arranged.So, be that the security of the recombiant vaccine of carrier has caused that people more and more worry with VV.
(Fowlpox Virus FPV) belongs to the Poxviridae Avipoxvirus to bird pox virus.An infected poultry under its natural condition, but can produce abortive infection to mammalian cell, do not produced infective virus particle, but can express, offer the FPV expressed proteins, stimulate specific cellular immunity and the humoral immunization of body generation to the FPV expressing protein, therefore FPV not only can be used as bird genetically engineered living vaccine carrier, and can be used as the Mammals non-replicating living vaccine carrier more safer than VV.
Structure is the recombinant Borrel virus of carrier with FPV, at first to make up the bird pox virus expression vector, foreign gene could be inserted the vector construction recombinant expression plasmid then, again the recombinant virus of the homologous recombination construction expression alien gene by recombinant expression plasmid and parent FPV.
From the end of the eighties, FPV as bird and Mammals living vaccine carrier since, existing abroad various exogenous genes has obtained expression in reorganization FPV.At present, though the domestic report that has foreign gene to express in reorganization FPV, used carrier or carrier element are from external introduction.
The weak malicious 282E4 vaccine strain of China's bird pox virus quailization successfully uses for many years in China, and existing effect has been controlled chicken pox in the generation of China and popular.Exploitation has good prospects for application as carrier for it.The present patent application people clones the TK gene (bird pox virus dispensable gene) of having identified the 282E4 strain first, and set up the segmental part of 282E4 pnca gene group part BamH I library, and therefrom Screening and Identification 1.5kb B fragment duplicate nonessential region for the 282E4 strain, can be used for making up the FPV expression vector.In addition, the artificial synthetic of the present patent application people two vaccination viral promotors are that single promotor and combined promoter have been proved to be very strong activity is arranged in VV.The similarity on genetic transcription, regulatory mechanism based on VV and FPV, two kinds of promotors are used in expression alien gene among the FPV.
The objective of the invention is to, Chinese bird pox virus 282E4 strain expression vector is provided, is the recombiant vaccine of various viruses, bacterium and the parasitosis of live vector with exploitation with Chinese bird pox virus.
The solution of the present invention is achieved in that
14 are used in the expression vector of expressing foreign protein in the recombinant Borrel virus.Foreign gene is inserted in single promotor in expression vector or combined promoter downstream, by obtaining to express the recombinant Borrel virus of foreign protein with bird pox virus parent plant homologous recombination.
Can be that Chinese bird pox virus 282E4 strain contains the 2.2kb HindIII-ClaI fragments sequence of TK gene and is positioned at genome proximal end 1.5kb B fragment sequence with the flanking sequence of bird pox virus parent plant generation homologous recombination in these carriers.
Being used to regulate and control exogenous gene promoter in these carriers is the single promotor or the combined promoter of synthetic.
Being used in these carriers expressed exogenous gene can be virus, bacterium and parasitic antigen gene, Disease-causing gene etc.; Tumor antigen gene, the gene involved in immunity of immunomodulatory gene such as people, animal, fowl and other animal.
Advantage of the present invention and positively effect are:
1, be that the expression vector of flanking sequence not only can homologous recombination take place with 282E4 strain parent FPV with 282E4 strain TK gene, and can with other FPV strain generation homologous recombination, can make up reorganization FPV strain as required with different biological characteristic.
2, with 1.5kb B fragment be the expression vector or the recombinant plasmid of flank, only can with nucleotide sequence in genome and 282E4 strain 1.5kb B fragment sequence homologous FPV strain generation homologous recombination, making up with these strains is the recombinant virus of parent plant.
3, when containing the expression vector establishment recombinant virus of LacZ reporter gene, can be by in the presence of X-Gal, the blue recombinant virus plaque screening acquisition recombinant virus of LacZ is expressed in screening.
4, when not containing the expression vector establishment recombinant virus of LacZ reporter gene, can obtain recombinant viruses by technology screenings such as plaque hybridization in situ technique or immune plaques.When utilization with TK is the expression vector establishment recombinant virus of flank, also can be by utilizing drug screening recombinant viruses such as Budr.
Utilize these expression vectors can be according to different purposes, the recombinant Borrel virus of the different foreign proteins of construction expression, these recombinant Borrel virus promptly can be used as the live recombinant vectors vaccine, be used for the control of various disease, also can be used as the research tools such as expression, processing, structure and function relationship of the different biological activity proteins of research.
Description of drawings is as follows:
Shown in Figure 1 is clone's process of single promotor.Concrete grammar is to prepare single promotor with restriction enzyme BamHI digestion vaccinia virus expression vector pSFJ2-16, is inserted into the BamHI site of plasmid pSK (-) then, identifies single promotor direction by nucleotide sequencing.
Shown in Figure 2 is the nucleotide sequence of each repeating unit of single promotor.
Shown in Figure 3 is clone's process of combined promoter.Concrete grammar is to digest vaccinia virus expression vector pSFJ1-20 simultaneously with restriction enzyme EcoRI and HindIII to prepare combined promoter, its orientation is inserted between the EcoRI and HindIII site of plasmid pSK (-) then.
Shown in Figure 4 is clone's process of TK gene and two nonessential regions of B fragment.Concrete grammar is that 2.2kb is contained between the genomic fragment of TK gene and two the pVUII sites that 1.5kb B fragment is inserted plasmid pUC19 respectively.
Shown in Figure 5 is that single promotor is inserted TK gene and the segmental process of B.Concrete grammar is the NcoI site and the segmental EcoRI of the B site of single promotor being inserted the TK gene respectively, obtains 4 expression vectors that do not contain reporter gene, pUT16-3, pUT16-4, pUB16-3, pUB16-4.
Shown in Figure 6 is that combined promoter is inserted TK gene and the segmental process of B.Concrete grammar is the NcoI site and the segmental EcoRI of the B site of combined promoter being inserted the TK gene respectively, obtains 4 expression vectors that do not contain reporter gene, pUTA-2, pUTA-5, pUBA-7, pUBA-9.
Shown in Figure 7 is the replacement process of the promotor of GFP and LacZ.
Fig. 8 is single promotor FPV carrier, combined promoter FPV carrier, contains GFP combined promoter FPV carrier and contain LacZ combined promoter FPV carrier.
Shown in Figure 9 is to make up the expression vector process that contains single promotor and reporter gene.Concrete grammar is with the combined promoter upstream among the LacZ gene under the single promoter regulation or GFP gene insertion pUTA-2 or the pUBA-7, makes its transcriptional orientation opposite with combined promoter.Obtaining two is the carrier pUTA-2-F716 and the pUBA-7-F716 of reporter gene with GFP.Two is the expression vector pUBA-7-16LacZ and the pUTA-2-16LacZ of reporter gene with LacZ.
Shown in Figure 10 is the process that makes up the expression vector that contains combined promoter and reporter gene.Concrete grammar is that LacZ reporter gene and single promotor are inserted TK gene NcoI site or the segmental EcoRI of B site together, obtains two expression vector pUB1616LacZ and pUT1616LacZ.
Shown in Figure 11 is to contain the segmental nucleotide sequence of the genomic 2.2kb HindIII-ClaI of 282E4 strain TK.
Shown in Figure 12 is the segmental nucleotide sequence of 282E4 pnca gene group 1.5kb B.
Shown in Figure 13 is the nucleotide sequence of the formation and the vaccinia virus A type inclusion body promotor of combined promoter.
TK gene among the present invention is meant thymidine nucleoside kinase gene, and 282E4 strain TK gene is arranged in its genome 2.2kb HindIII-ClaI fragment, and its nucleotide sequence as shown in Figure 7.
B fragment among the present invention is meant the fragment that is positioned at the subterminal long 1.5kb of 282E4 pnca gene group, and its nucleotide sequence as shown in figure 12.
Single promotor among the present invention is meant the promotor of being made up of 16 the placed in-line vaccinia mutant type of head and the tail P7.5 early promoters, and the sequence of its each repeating unit as shown in Figure 2.
Combined promoter among the present invention is meant by vaccinia virus A type inclusion body promotor (ATI) and 20 promotors that the placed in-line mutant vaccinia virus of head and the tail p7.5 early promoter is composed in series from beginning to end, wherein the nucleotide sequence of ATI promotor as shown in figure 13, the sequence of each repeating unit is as shown in Figure 2 in 20 placed in-line p7.5 early promoters of head and the tail.
LacZ gene among the present invention refers to intestinal bacteria β-D galactosidase gene.
GFP gene among the present invention is meant green fluorescence protein gene.
Structure and application method narration embodiments of the invention below by expression vector:
(1) DNA reorganization elementary operation method
1. the preparation of competent escherichia coli cell and conversion
The calcium chloride preparation method is adopted in the competent escherichia coli cell preparation, and concrete grammar is translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 55th～56 page.Just adopting by 0.75mmol/L Tris-HCl replacement water is solvent preparation 0.1mol/L CaCl
2The method for transformation of competent cell carries out with reference to above-mentioned reference.
2. (except that specified otherwise was arranged, reagent was prepared all with reference to " molecular cloning experiment guide " second edition, Science Press (1992) in a large amount of preparations of plasmid
Translate " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), adopts the alkaline lysis preparation, with PEG precipitator method purifying by the 26th～28 page.Concrete operation method is as follows, and the thalline of centrifugal results after plasmid transforms, cultivates suspends, washs thalline once with STE, uses special TE (10mmol/L Tris, 50mmol/LEDTA pH8.0) then, and precipitation suspends.Add 2 times to the solution II cracking of TE volume, add the solution III neutralization of 1.5 times of volumes, get supernatant after centrifugal, add the isopropanol precipitating of 0.7 times of volume.The centrifugal supernatant of abandoning.With TE (pH8.0) dissolution precipitation, add and wait doubly to the 5mol/L of TE volume LiCl centrifugal removal macromole RNA.Shift out in the new centrifuge tube of supernatant to, add 2 times to the dehydrated alcohol of supernatant volume, deposit D NA.Add an amount of 70% washing with alcohol precipitation once, airing.Use the TE dissolution precipitation, the RNA enzyme final concentration that wherein contains no DNA enzyme is 20Mg/ml, to remove RNA.Add to wait doubly to the PEG of TE volume solution (20%PEG6000,2.5mol/L NaCl) mixing, it is centrifugal to put 4 ℃ of backs of spending the night, and abandons supernatant.Use the TE dissolution precipitation, with waiting doubly to full phenol, the phenol of closing of TE volume: chloroform, each extracting is once used the dehydrated alcohol deposit D NA of 2 times of volumes then in order, and with 70% washing with alcohol precipitation once, airing is dissolved among the TE (pH8.0) standby at last.
3. the recovery of the digestion with restriction enzyme of plasmid and dna fragmentation
The digestion with restriction enzyme of plasmid all carries out with reference to following reaction conditions, every microgram plasmid DNA digests with 5 unit limit restriction endonucleases, the restriction enzyme reaction damping fluid that adds 1/10 volume, adding sterilization two, to heat up in a steamer water adjustment reaction cumulative volume be 10 times of used restriction enzyme volume, the temperature of reaction reaction of recommending by the supplier of various restriction enzymes 2～3 hours.Plasmid with digestion with restriction enzyme after, DEAE-cellulose membrane electrophoresis absorption method is adopted in the recovery of dna fragmentation, concrete grammar is translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 319th～321 page.This research adopts DE-81 ion exchange paper (Whatman company product) to replace the DEAE-cellulose membrane.
4. the terminal dephosphorylation of linear plasmid and being connected of dna fragmentation
Enzyme is cut the terminal dephosphorylation of back linear plasmid, adopts calf intestinal alkaline phosphatase (CIP), and concrete operations are translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 40th～41 page.After the dephosphorylation reaction, the deactivation of CIP adopts 5mmol/L EDTA (PH8.0) to exist down, 75 ℃ of heating 10min.Use phenol then: CIP is removed in the chloroform extracting, and the linear plasmid behind the ethanol sedimentation dephosphorylation is dissolved in two the heating up in a steamer in the water of sterilizing, and is used for ligation.
The T that the ligation of linear plasmid and dna fragmentation provides by GIBCO-BRL company
4The condition that dna ligase is recommended is carried out.Plasmid vector is pressed in the connection of sticky end: the mol ratio of target DNA fragment=1: 2,25 ℃ connect 1h.Plasmid vector is adopted in flat terminal connection: the ratio of target DNA fragment mol ratio=1: 5, and 14～16 ℃, connect 17～24h, will connect product transformed into escherichia coli competent cell then.
5. the screening of recombinant plasmid and evaluation
With ligation thing transformed competence colibacillus bacterium, be applied to and contain on the antibiotic flat board, select single bacterium colony that incubated overnight grows, be inoculated in the 2ml LB liquid nutrient medium, cultivate 16～20h.Translate " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc. then, Science Press (1992), the alkaline lysis of the 19th～22 page of introduction prepares plasmid in a small amount, compare the agarose gel electrophoresis that carries out plasmid with the empty carrier plasmid, select recombinant plasmid, make digestion with restriction enzyme, agarose gel electrophoresis is identified goal gene and the direction of insertion thereof inserted.
(2) FPV expression vector establishment method
1. the clone of single promotor and combined promoter
Digest pSFJ2-16 with BamHI, with HindIII and EcoRI digestion pSFJ1-20, agarose gel electrophoresis reclaims single promotor or compound promotor wherein respectively, then it is inserted respectively between the BamHI site or HindHI site and EcoRI site of plasmid pSK (-), be built into plasmid pSK16 and pKAT.Its construction strategy is such as accompanying drawing 1,3 shown in.
2. the sequencing of single promotor
Translate " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., Science Press (1992), the 632nd～637 page of preparation denaturing polyacrylamide gel.According to the T7 Sequence Kit operation instruction that pharmacia company provides, be template with the recombinant plasmid that contains single promotor, adopt the T7 primer on the pSK (-) to make the reaction primer, adopt the terminal cessation method of two deoxidations to carry out sequencing reaction.React with the about 2ug of template plasmid at every turn, get sample electrophoresis on the 3 μ l reaction mixtures.Electrophoresis finishes, and takes off behind the glue gel fixing 10min in 10% acetate methanol solution, presses the X-ray sheet, radioautograph 24h, and promoter sequence is read in punching, identifies the promotor direction.Through sequencing and analysis, the sequence of single promotor and direction are as shown in Figure 2 among the pSK16.
3.TK the clone of gene and two nonessential regions of B fragment.
Plasmid pSL with HindIII and ClaI digestion, is reclaimed the 2.2kb fragment that contains the TK gene.PKFB0 digests with Xbal with plasmid, reclaims 1.5kb B fragment, mends two fragments flat terminal with Klenow respectively then.Plasmid pUC19 with Pvu II digestion removal multiple clone site wherein, is carried out terminal dephosphorylation with CIP and handles.Insert respectively among the pUC19 that removes multiple clone site mending flat terminal 2.2kb fragment that contains the TK gene and 1.5kb B fragment, obtain plasmid pUT and pUB, construction strategy as shown in Figure 4.
4. different promoters is inserted two nonessential regions respectively
Digest plasmid pSK16 and the pKAT that two kinds of promotor subclones are obtained later on respectively with NotI and Xhol I, reclaim wherein single promotor and combined promoter, Klenow carries out end-filling.Digest pUT with NcoI respectively then, digest pUB, carry out end-filling and dephosphorylation subsequently with EcoRI.With the NcoI site that the single promotor that reclaims and combined promoter insert the TK gene respectively, screening is not contained the LacZ reporter gene, is the expression vector pUTA-2 that contains combined promoter of flank with TK, contains the expression vector pUT16-4 of single promotor.The single promotor and the combined promoter that reclaim being inserted the segmental EcoRI of B site, filter out and do not contain the LacZ reporter gene, is the expression vector pUBA-7 that contains combined promoter of flank with the B fragment, contains the expression vector pUB16-1 of single promotor.Construction strategy such as Fig. 5 are shown in 6.
5, the replacement of reporter gene promotor
Plasmid pSK16 with EcoRV and SmaI digestion, is removed EcoRV, EcoRI, sites such as SmaI, PstI between two sites, and self connects then.With Xba I digestion, Klenow carries out end-filling again, and then with HindIII digestion, reclaims single promotor wherein.
Plasmid pCMVGFP is digested with SpeI and BamHI, reclaim the GFP gene of CMV promoter regulation.Be inserted into the KS (-) that is digested and carry out end-filling by HindIII, the directed CMV of replacement of the single promotor promotor that will from pSK16, reclaim then.Obtain being contained in the plasmid pKSF716 of the GFP gene under the single promoter regulation.Construction strategy as shown in Figure 7.
Psv-β-Galacosidase is digested with SalI and HindIII, reclaim LacZ gene wherein, its orientation is substituted into GFP gene location among the pKSf716, obtains being contained in the plasmid pKS16LacZ of the LacZ gene under the single promoter regulation, construction strategy as shown in Figure 8.
6. the structure that contains the expression vector of combined promoter and LacZ reporter gene
GFP gene orientation is inserted into the HindIII site of combined promoter upstream among the pUTA-2, and direction is opposite with the combined promoter direction.Being built into what contain combined promoter is that flank is the plasmid pUTA-2-f716 of reporter gene with GFP with TK.With quadrat method be built into the B fragment be flank contain combined promoter, be the plasmid pUBA-7-f716 of reporter gene with GFP.Construction strategy is seen Fig. 9.
PUTA-2-f716 and pUBA-7-f716 are digested with Sal I and Hind III respectively, remove GFP gene wherein.With Sal I and Hind III digestion pSV-β-Galactosidase, reclaim LacZ gene wherein, LacZ gene orientation is substituted on the position of GFP gene, obtain is that flank is regulated and control goal gene with combined promoter with TK and B fragment respectively, with LacZ is the expression vector pUTA-2-16LacZ of reporter gene, pUBA-716 LacZ.Construction strategy is seen Fig. 9.
7. the structure that contains the expression vector of single promotor and LacZ reporter gene
With the LacZ gene under the single promoter regulation, be inserted into single promotor upstream among the pSK16, direction is opposite with single promotor direction, be built into the expression vector element that contains LacZ reporter gene and regulation and control foreign gene promotor, then with NotI and this expression vector element of XhoI digestion preparation, be inserted into the Nco I site of pUT plasmid, obtain with TK be flank be reporter gene with LacZ, start the expression vector pUT1616LacZ of goal gene with single promotor.With the EcoR I site that the expression vector element that makes up inserts the pUB plasmid, obtaining with the B fragment is flank, is reporter gene with LacZ, starts the expression vector pUB1616LacZ of goal gene with single promotor.Construction strategy is seen Figure 10.
(3) characteristics of these expression vectors
1, be that the expression vector of flanking sequence not only can homologous recombination take place with 282E4 strain parent FPV with 282E4 strain TK gene, and can with other FPV strain generation homologous recombination, can make up reorganization FPV strain as required with different biological characteristic.
2, with 1.5kb B fragment be the expression vector or the recombinant plasmid of flank, only can with nucleotide sequence in genome and 282E4 strain 1.5kb B fragment sequence homologous FPV strain generation homologous recombination, making up with these strains is the recombinant virus of parent plant.
3, when containing the expression vector establishment recombinant virus of LacZ reporter gene, can be by in the presence of X-Gal, the blue recombinant virus plaque screening acquisition recombinant virus of LacZ is expressed in screening.
4, when not containing the expression vector establishment recombinant virus of LacZ reporter gene, can obtain recombinant viruses by technology screenings such as plaque hybridization in situ technique or immune plaques.When utilization with TK is the expression vector establishment recombinant virus of flank, also can be by utilizing drug screening recombinant viruses such as Budr.
Utilize these expression vectors can be according to different purposes, the recombinant Borrel virus of the different foreign proteins of construction expression, these recombinant Borrel virus promptly can be used as the live recombinant vectors vaccine, be used for the control of various disease, also can be used as the research tools such as expression, processing, structure and function relationship of the different biological activity proteins of research.
(4) structure of recombinant virus and screening method
Utilize the method for recombinant virus of these expression vector establishment expression alien genes as follows.
At first in the downstream of these expression vectors, insert the structure gene that desire is expressed by correct method, make up recombinant expression plasmid, then with these recombinant expression plasmid transfections by the suitable cell (as chick embryo fibroblast CEF) that FPV is duplicated therein of FPV parent virus strain infection, homologous recombination takes place at the homologous sequence place by recombinant expression plasmid and FPV parent plant, generation is carried, the recombinant virus of expression alien gene, by various suitable methods recombinant virus is screened then.But the expressed exogenous gene sequence must be an encoding sequence in these carriers, can not contain non-coding sequences such as intron, and will have complete atg start codon ATG, and first ATG of promotor downstream must be an atg start codon of wanting expressed exogenous gene.And the foreign gene inside in early promoter downstream can not contain the TTTTTNT sequence, and wherein N is any in four kinds of deoxyribonucleotides.
(5) constructed embodiment of recombinant virus
1, the cultivation of cell and virus (except that the chick embryo fibroblast that the present invention introduces, the cell that also available other bird pox virus can duplicate therein)
Utilize the SPF egg to incubate chicken embryo to 9～11 ages in days, with reference to Yin Zhen, Liu Jinghua chief editor " animal virology " second edition, Science Press (1997), the method for the 223rd～224 page of introduction prepares the CEF primary cell.Employing contains 37 ℃ of 5%CO of MEM nutrient solution of 2% calf serum
2Cultivate under the condition.
FPV HIPRA strain is adopted CEF monolayer cell or chick chorioallantoic membrane to cultivate and is gone down to posterity.Virus PFU measures
2, preparation CEF monolayer cell is with 10
6The cell/ml subculture is cultured to cell and grows up to individual layer in the every hole 3ml of 6 porocyte culture plates (aperture 3.5cm), inhales and removes nutrient solution, washes cell 2 times with PBS liquid, and FPV virus liquid is done 10 with PBS liquid
-1～10
-5Go forward one by one 10 times and dilute, every then hole meets viral dilution liquid 300 μ l, establishes a hole and does the cell contrast, 37 ℃ of absorption 1-1.5h.Add the MEM nutrient solution that contains 2% calf serum and cultivate 72～96h, inhale and remove nutrient solution, wash twice with PBS, every then hole covers 1ml formalin Viola crystallina dye liquor (compound method: add 10ml 5% Viola crystallina among every 50ml, 37.5ml 0.85%NaCl, 1ml formaldehyde, 1.5ml H
2O) room temperature dyeing 30min inhales and removes dye liquor, to heat up in a steamer water rinse twice, calculates the PFU of plaque number and virus stock solution used.
3, the structure of recombinant expression plasmid
With restriction endonuclease sma I digestion expression vector, will be digested to linear carrier then and carry out being connected Screening and Identification recombinant expression plasmid, extraction, plasmid DNA purification then after terminal dephosphorylation is handled with goal gene.
4, cell transfecting method (except that the transfection method that the present invention introduced, also available other enters the DNA transfection method of cell)
Preparation CEF cell monolayer is with 1 * 10
5The cell/ml subculture is in 6 porocyte culture plates, every hole 3ml, when treating that cell grows to 70～80% fusion individual layers, nutrient solution is removed in suction, PBS washes cell twice, press 0.2PFU/cell inoculation FPV HIPRA strain then, 37 ℃ of absorption 1～1.5h, 30～50 μ g plasmids are diluted among the 50 μ l serum-free MEM therebetween, 20 μ l DOTAP transfection reagents are diluted among the MEM of 100 μ l serum-frees, press DOTAP Liposomal operation instruction, will be used for the plasmid of homologous recombination and DoTAP transfection reagent mixing gently, room temperature is placed more than the 30min.After treating virus absorption, inhale the viral liquid that goes in the cell cultures hole, the mixed solution of plasmid and transfection reagent is mended to volume 1ml with serum-free MEM, be added in the cultivation plate hole on the cell monolayer that is infected by FPV 37 ℃ of 5% CO
2Cultivate 14～18h, the MEM nutrient solution that 2ml contains 2% calf serum is added in every then hole, continues to cultivate 32～56h results virus, measures viral PFU.
5, the screening method of recombinant virus (except that the method that the present invention introduces, also the screening method of available other recombinant virus)
(1) with LacZ is the screening of the recombinant virus of reporter gene
Preparation CEF individual layer in six porocyte culture plates, to be that the virus gathered in the crops behind the carrier of reporter gene and the FPV HIPRA cotransfection is with every hole 300～500PFU inoculation CEF cell with LacZ, 37 ℃ of absorption 1.5h, cultivate 48h with the MEM that contains 2% calf serum, the MEM nutrient solution is removed in suction, cover then and contain the nutrient agar medium sugar of forming by 2 * MEM (containing 2% calf serum) and 1% agarose (the two ratio 1: 1) that final concentration is 200 μ g/ml X-gal, continue lucifuge and cultivate 24～48h, the blue viral plaque of picking then, and use with quadrat method and carry out the locus coeruleus purifying three times.
(2) with TK be the screening method of the recombinant virus of flanking sequence
Preparation CEF individual layer in 6 well culture plates, 24h cultivates with the MEM nutrient solution that contains 40 μ g/ml Budr before connecing poison, inoculates the virus of gathering in the crops after the recombinant expression plasmid transfection with the 1PFU/cell amount then, behind 37 ℃ of absorption 1.5h, continue to cultivate with the nutrient solution that contains 40 μ g/ml Budr again, observe CPE.Meet poison back 120h, harvested cell after the freeze thawing 3 times, inoculates the CEF monolayer cell of handling with Budr, continues to cultivate 120h in the substratum that contains 40 μ g/ml harvested cell then, freeze thawing 3 times.With the virus inoculation CEF cell of results, in no Budr nutrient agar medium sugar (seeing experiment two), cultivate again, select the single virus plaque, and carry out plaque purification twice, amplification.
6, the proteic evaluation of expression of recombinant virus (also available other suitable method)
With the recombinant virus of screening, purifying, press kind of a CEF cell with 5PFU/cell, infect back 36h harvested cell, with cell cell pyrolysis liquid (10mmol/L Tris-HCl, pH7.4～8.0, the 1mmol/L MgCl of results
2, 0.5%NP-40,20 μ g/ml Dnase I) after the cracking, add equivalent 2 * SDS loading Buffer and mix, 3min is boiled in 100 ℃ of water-baths, does to transfer on the nitrocellulose filter behind the SDS-PAGE electrophoresis.(prepare 3%BSA with TBS, 0.05%Tween-20) room temperature sealing 1～2h uses TBS (10mmol/LTris.Cl (pH7.5) then with confining liquid, 150mmol/L NaCl) the liquid rinsing is twice, make 2h with the antibody room temperature sense of anti-target protein again, with TBS liquid rinsing three times, each 10 minutes.Make 2h with second antibody (1: 500) the room temperature sense of alkali phosphatase enzyme mark then, use TBS liquid rinsing three times again, NBT and the BCIP colour developing liquid with the preparation of alkaline phosphatase damping fluid develops the color color development stopping reaction in good time at last.
Claims (4)
1,14 are used in the expression vector of expressing foreign protein in the recombinant Borrel virus.It is characterized in that foreign gene is inserted in single promotor in expression vector or combined promoter downstream, by obtaining to express the recombinant Borrel virus of foreign protein with bird pox virus parent plant homologous recombination.
2, expression vector according to claim 1 is characterized in that: can be that Chinese bird pox virus 282E4 strain contains the 2.2kb HindIII-ClaI fragments sequence of TK gene and is positioned at genome proximal end 1.5kb B fragment sequence with the flanking sequence of bird pox virus parent plant generation homologous recombination in these carriers.
3, expression vector according to claim 1 is characterized in that being used to regulate and control single promotor or the combined promoter that exogenous gene promoter is a synthetic in these carriers.
4, expression vector according to claim 1, it is characterized in that: being used in these carriers expressed exogenous gene can be virus, bacterium and parasitic antigen gene, Disease-causing gene, the tumor associated antigen gene, the immunogene of people, animal, fowl immunomodulatory gene and other animal.
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| CN 00103298 CN1268573A (en) | 2000-03-23 | 2000-03-23 | Carrier for expressing Chiense chicken pox virus vaccine strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00103298 CN1268573A (en) | 2000-03-23 | 2000-03-23 | Carrier for expressing Chiense chicken pox virus vaccine strain |
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| CN1268573A true CN1268573A (en) | 2000-10-04 |
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| CN 00103298 Pending CN1268573A (en) | 2000-03-23 | 2000-03-23 | Carrier for expressing Chiense chicken pox virus vaccine strain |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330376C (en) * | 2001-09-30 | 2007-08-08 | 扬州大学 | Recombinant Borrel virus combined vaccine |
| CN100465280C (en) * | 2003-02-28 | 2009-03-04 | 巴斯德研究院 | Chicken RNA polymerisate I promoter and the use thereof |
| CN101220375B (en) * | 2007-01-11 | 2011-07-27 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PT7.5N) |
| CN102321637A (en) * | 2011-09-09 | 2012-01-18 | 中国人民解放军军事医学科学院军事兽医研究所 | Chinese fowlpox virus 282E4 low virulent strain complete genome sequence and application thereof |
| CN101220374B (en) * | 2007-01-11 | 2012-02-01 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PG7.5N) |
-
2000
- 2000-03-23 CN CN 00103298 patent/CN1268573A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330376C (en) * | 2001-09-30 | 2007-08-08 | 扬州大学 | Recombinant Borrel virus combined vaccine |
| CN100465280C (en) * | 2003-02-28 | 2009-03-04 | 巴斯德研究院 | Chicken RNA polymerisate I promoter and the use thereof |
| CN101220375B (en) * | 2007-01-11 | 2011-07-27 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PT7.5N) |
| CN101220374B (en) * | 2007-01-11 | 2012-02-01 | 华南农业大学 | Fowl pox virus double-gene expression carrier (PG7.5N) |
| CN102321637A (en) * | 2011-09-09 | 2012-01-18 | 中国人民解放军军事医学科学院军事兽医研究所 | Chinese fowlpox virus 282E4 low virulent strain complete genome sequence and application thereof |
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