CN101260411B - Recombination bacillary viral vector skeleton plasmid and application thereof - Google Patents

Abstract

The invention belongs to the animal genetic engineering technical field, in particular relating to a construction for a framework plasmid of a recombinant baculovirus vector and application thereof. The invention acquires a framework plasmid pFB-G-SFV of a recombinant baculovirus transfer vector by cloning, and the plasmid is collected in China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: M207070. The invention also discloses the application of the plasmid pFB-G-SFV on the package of the recombinant baculovirus.

Description

A kind of recombination bacillary viral vector skeleton plasmid and application thereof

Technical field

The invention belongs to animal gene engineering technology field, be specifically related to a kind of recombination bacillary viral vector skeleton plasmid and application thereof.

Background technology

Baculovirus vector is to use one of virus vector very widely at present, is widely used in fields such as gene therapy, gene functional research, vaccine development.Baculovirus is maximum in an insect viruses section, and its host range mainly concentrates on the Insecta lepidopteran.At present, the insect baculovirus that is used for exogenous gene expression only limits to nucleopolyhedrosis virus and belongs to, wherein (Autographa californica multiple nucleopolyhedrosis virus is the rhabdovirus expression vector systematic study model that generally adopts in the world with the greedy frugiperda cell system in meadow AcMNPV) to many embedding body nuclear of clover crazing moth multiangular virus.Reported first such as nineteen eighty-three Smith are utilized AcMNPV to covet frugiperda cell on the meadow and have successfully been expressed people β-2 Interferon, rabbit, (the Luckow et al of bac-to-bac system that the baculovirus decorum is transformed into of successes such as Luckow in 1993,1993), be mainly used in mass expressing external gene in insect cell.Baculovirus expression system is as a kind of eukaryotic expression system, aspect exogenous gene expression, have glycosylation, phosphorylation and albumen cutting processing modification, the biological characteristics of expression product is similar to natural product, and expression efficiency height, it is reported, the Recombinant Protein Expression level can reach 50% (Luckowand Summers, 1989) of total protein of cell in cells infected.

For a long time, because its host specificity, the carrier of this high expression level is received insect cell inner expression only for being used for mediate foreign gene at it.Discovering in recent years, baculovirus can enter some mammalian cell, but it does not duplicate and genetic transcription after entering cell.Therefore in baculovirus vector, introduce promotor such as RSV, the CMV etc. that can in mammalian cell, act on, can foreign gene be changed in the mammalian cell by baculovirus and express.Nineteen ninety-five, reported first such as Hofmann will recombinate to the AcMNPV genome by the luciferase genes that cytomegalovirus utmost point early promoter (CMV-IE) drives, and with this reorganization AcMNPV infect several different mammalian cells, the result show this reorganization AcMNPV can infector for human liver cell and expressed reporter gene efficiently, the expression efficiency of the reporter gene of its mediation is higher than with calcium phosphate precipitation method and liposome transfection method.At present with baculovirus as carrier, utilize different promoters to realize different plant species, the transgenosis of a lot of clones of different tissue sources and expression.Baculovirus becomes outer another of traditional methods such as the liposome that continues, recombinant retrovirus, adenovirus and Mammals is carried out the instrument of transgenosis.

Make up baculovirus expression system several different methods is arranged, mainly comprise NPV DNA linearization technique, the bac-to-bac expression system, recombination method in the yeast, baculovirus-S2 system etc., wherein the bac-to-bac expression system by the exploitation of GIBCO company is transposon-mediated baculovirus recombination system, and it is the baculovirus recombination system that is most widely used at present.This system mainly is made up of donor plasmid pFastBac and prokaryotic cell prokaryocyte (DH10Bac).Contain adorned AcMNPV and be referred to as Bacmid in DH10Bac, Bacmid both can infect lepidopteran insect cell, can duplicate in intestinal bacteria again.The construction strategy of Bacmid is for cloning into kalamycin resistance gene in the corresponding site of AcMNPV polyhedron gene, Tn7 bacterial transposon target site attTn7 derives from the LacZ peptide section encoding gene of pUC plasmid and can guarantee the genome self-replicating and be present in mini-F replicon in the bacterium with stable low copy number.PFastBac is a donor plasmid, is the polyhedrin promotor between its Tn7 left and right sides sequence.Foreign gene in the donor plasmid is inserted among the Bacmid under the transposase effect of helper plasmid coding, thereby disturb the expression of LacZ, so just can directly extract reorganization Bacmid plasmid DNA in a small amount, can obtain recombinant virus with liposome transfection again by the blue white screening of bacterial colony.This system's recombination fraction is almost 100%, and reorganization rapidly.

Alphavirus is the RNA viruses of sub-thread, normal chain, and its member comprises sindbis alphavirus, Semliki forest virus, Venezuelan equine encephalitis virus etc., existing all be developed to replicon and become the carrier for expression of eukaryon that is subjected to extensive concern.The amount that replicon carrier utilizes the distinctive rna replicon enzyme of replicon to improve translation template mRNA realizes foreign gene high level expression (Kohnoet al, 1998).

The SFV replicon carrier is based on the carrier for expression of eukaryon of Semliki forest virus, mainly comprises based on the SFV replicon system of RNA with based on the SFV replicon system of DNA, and the latter is because its higher security and stability and adopted widely.SFV replicon carrier system constructing strategy based on DNA is the early promoter/enhanser that the cDNA of SFV is placed carrier for expression of eukaryon promotor downstream (as human cytomegalic inclusion disease virus (CMV))), with foreign gene substitute structure protein gene, in case it is synthetic by the replicative enzyme mixture that the Alphavirus Nonstructural Protein of strong promoter driving is encoded, the massive duplication of recombinant RNA in just but mediated cell is starched, thereby high level expression (the Hsu et al that causes the mRNAs of foreign gene coding, 2004), simultaneously, the Alphavirus replicon not only has the self-replicating function, and have the characteristic of inducing Apoptosis of Host Cells identical with complete Alphavirus, make transfected cell that apoptosis take place at short notice.Because these significant advantages, the SFV replicon has been widely used in expression alien gene and has made up fields such as replicon vaccine and the preparation of gene therapy oriented carrier, especially aspect vaccine research, existing virus and tumour replicon candidate vaccine enter preclinical phase and clinical trial, have shown its good prospects for application.

The mammalian genes of baculovirus mediation shifts and expresses and is subjected to many-sided condition effect, and for obtaining efficiently expressing of foreign gene, the investigator has attempted adopting multiple promotor to transform baculovirus vector.As the RSV promotor, the CMV-IE promotor, chicken β-actin promotor etc. is widely used in expression alien gene in mammalian cell, the present invention utilizes the inferior promotor of SFV to make up baculovirus, be intended to make up one and can realize efficiently expressing of foreign gene, carrier that can be apoptosis-induced again is for gene therapy provides safety, an expression vector efficiently.

Summary of the invention

The object of the present invention is to provide a kind of novel bacillary viral vector skeleton plasmid pFB-G-SFV, utilize this skeleton plasmid, can with the common a kind of novel baculovirus packaging system of forming of the intestinal bacteria that contain Bacmid (DH10Bac) and insect cell (sf-9 cell).This plasmid pFB-G-SFV provides two restriction enzyme site BamH I and Smal I to insert for foreign gene.After exogenous gene cloning gone into this carrier, conversion contains the intestinal bacteria (DH10Bac) of Bacmid, screens the reorganization Bacmid that can obtain to change over to foreign gene by blue hickie, extracts this Bacmid, transfection insect cell (sf-9 cell) can obtain to contain the recombinant baculovirus of foreign gene.The major advantage of carrier of the present invention is: the packaging system that (1) recombinant baculovirus skeleton plasmid of the present invention is constituted possesses the Bac-to-bac system and goes out poison soon, good stability, the characteristics that productive rate is high.The recombinant baculovirus of packing out can be in mammalian cell self-replicating, thereby a large amount that realizes foreign gene is expressed.(2) in the process of recombinant baculovirus self-replicating in mammalian cell of packing out of the packaging system that constituted of recombinant baculovirus skeleton plasmid of the present invention, can produce dsRNA, the generation of dsRNA can promote the generation of immunomodulatory and cell active factor.(3) recombinant baculovirus packed out of the packaging system that constituted of recombinant baculovirus skeleton plasmid of the present invention can be induced by the apoptosis of transducer cell, thereby promotes immune response, reduces DNA and is incorporated into the host chromosome risk.

Skeleton plasmid of the present invention is with pFastBac-Dual (available from Invitrogen company), pSFV1 (available from Invitrogen company) and pCI-neo (available from Invitrogen company) are material, obtain by following method: cut pSFV1 with SphI and SpeI enzyme, recovery comprises the fragment of the 8.7Kb of SFV Nonstructural Protein, the Pp of displacement pFastBac-Dual ₁₀And Pp _HPromotor, interstitial granules pFB-G-SFV1 in obtaining.Be increase respectively HCMV IEEnhancer/Promoter element and SV40 poly in late period (A) termination signal sequence of template with pCI-neo, insert in the SFV fragment the single restriction enzyme site SpeI in A69 downstream among the single restriction enzyme site SphI in Sp6 promotor upstream and SFV fragment respectively.The final plasmid pFB-G-SFV that obtains, (intestinal bacteria (Escherichia coli) DH5 α/pFB-G-SFV that carries this plasmid is preserved in Chinese typical culture collection center C CTCC in the Wuhan University of Wuhan City, Hubei Province on May 16th, 2007, and deposit number is CCTCCNO:M207070).Therefore on the pFB-G-SFV plasmid, between Tn7, contain expression cassette and the celebrating smalt mycin resistant gene formed by CMV-SFV.Remain with of the insertion of two restriction enzyme sites of BamHI and SmalI on the SFV for foreign gene.The packaging system that recombinant baculovirus skeleton plasmid of the present invention is constituted has kept the Bac-to-Bac system and has gone out poison soon, good stability, the characteristics that productive rate is high, the recombinant baculovirus of packing out has been integrated the foreign gene high expression level efficient of SFV carrier, produce the dsRNA intermediate, the apoptosis-induced good characteristic that waits.For vaccine development provides a new tool.

Description of drawings

Fig. 1: be that skeleton plasmid disclosed by the invention makes up flow process

Fig. 2: be that skeleton plasmid disclosed by the invention is compared with the pFastBac-Dual skeleton plasmid, the synoptic diagram of sequence between two restriction enzyme sites of SphI and SpeI

What 2A showed among the figure is the synoptic diagram of sequence between skeleton plasmid SphI disclosed by the invention and two restriction enzyme sites of SpeI, Nonstructural Protein nsP1, the nsP2, nsP3 and the nsP4 that between two restriction enzyme sites, have comprised HCMV IE Enhancer/Promoter element, SFV, and SV40 poly in late period (A) termination signal sequence.Fig. 2 B shows is not through the synoptic diagram of sequence between the pFastBac-Dual of the Bac-to-Bac system skeleton plasmid SphI that transforms and two restriction enzyme sites of SpeI, between two restriction enzyme sites, comprise two promotor Pp10 and Pph, and a plurality of restriction endonuclease sites.

Fig. 3: the comparison synoptic diagram of recombinant plasmid pFB-G-SFV-EGFP and recombinant plasmid pFB-G-CMV-EGFP.

Wherein: Fig. 3 A is recombinant plasmid pFB-G-SFV-EGFP, Nonstructural Protein nsP1, the nsP2, nsP3 and the nsP4 that mainly contain HCMV IE Enhancer/Promoter element, SFV, and SV40 poly in late period (A) termination signal sequence, and foreign gene EGFP.Fig. 3 B is recombinant plasmid pFB-G-CMV-EGFP, only contains HCMV IE Enhancer/Promoter element and foreign gene EGFP.

Fig. 4 is skeleton plasmid disclosed by the invention and DH10Bac intestinal bacteria, and the synoptic diagram of insect cell SF-9 packing recombinant baculovirus.

These intestinal bacteria contain shuttle plasmid Bacmid, the target sequence attTn7 that contains the Tn7 transposon on this shuttle plasmid, with skeleton plasmid transformed into escherichia coli disclosed by the invention, obtain recombinant shuttle plasmid by swivel base, with this recombinant shuttle plasmid transfection insect cell, can pack out recombinant baculovirus then.

Fig. 5 is the analytical results that utilizes fluorescence microscope and flow cytometer behind the recombinant baculovirus inoculation HEK-293 cell of the expressing green fluorescent protein (EGFP) that the skeleton plasmid among the present invention make up to obtain and the BHK-21 cell.

Wherein: Fig. 5 A is the observations of the HEK-293 cell of inoculation recombinant baculovirus under optical condition on the fluorescent microscope, Fig. 5 B is the Fluirescence observation result of the BHK-21 cell of the recombinant baculovirus that EGFP is expressed in inoculation under the fluorescent microscope, Fig. 5 C is a BHK-21 positive cell ratio of utilizing flow cytometry analysis expression alien gene EGFP, and Fig. 5 D is the average fluorescent strength of flow cytometry analysis expression alien gene EGFP.

Fig. 6 is detected by the apoptosis behind the recombinant baculovirus Bac-G-SFV-EGFP inoculation BHK-21 cell of the structure of the skeleton plasmid among the present invention acquisition.

Wherein: Fig. 6 A is the gel electrophoresis picture of DNA-Ladder, and Fig. 6 B is that the activity of caspase-3 detects.The result shows that significant apoptosis phenomenon takes place the BHK-21 cell of inoculating recombinant baculovirus.

Enzyme linked immunological absorption antibody (ELISA) level behind recombinant baculovirus Bac-G-SFV-EGFP that Fig. 7 the present invention is obtained and the contrast recombinant baculovirus Bac-G-CMV-EGFP difference immune mouse relatively.

Embodiment

Below in conjunction with accompanying drawing the present invention is further described.

Embodiment 1: the structure of the pFB-G-SFV skeleton plasmid among the present invention

With pFastBac-Dual shown in Figure 1 (available from Invitrogen company) and pSFV1 (available from Invitrogen company) is material, by flow process shown in Figure 1, makes up the said plasmid of the present invention, and concrete steps are as follows:

1, with pFastBac-Dual and pSFV1 plasmid respectively with SphI and SpeI double digestion, reclaim big fragment and the linearizing pFastBac-Dual that contains the baculovirus transfer vector skeleton and comprise the Nonstructural Protein nsPs1-4 of SFV respectively, interstitial granules I was pFB-G-SFV1 during these two fragments connection structures were obtained;

2, the HCMV IE Enhancer/Promoter element that has the SphI restriction enzyme site with pCI-neo for the template amplification two ends, intermediate segment I as shown in Figure 1;

3, with the SphI site of interstitial granules I in the HCMV IE Enhancer/Promoter element insertion of step 2 gained, interstitial granules II is pFB-G-SFV2 in the acquisition;

4, have SV40 poly in late period (A) termination signal sequence of SpeI restriction enzyme site with pCI-neo for the template amplification two ends, obtain intermediate segment II as shown in Figure 1;

5, with the SpeI site of the middle interstitial granules II shown in step 4 gained SV40 poly in late period (A) the termination signal sequence inserting step 3, obtain plasmid pFB-G-SFV of the present invention.This plasmid is preserved in Chinese typical culture collection center C CTCC in the Wuhan University of Wuhan City, Hubei Province on May 16th, 2007, and deposit number is CCTCC NO:M207070.

Embodiment 2: utilize skeleton plasmid of the present invention to pack out the recombinant baculovirus that has foreign gene green fluorescent protein (EGFP)

1, pFB-G-SFV-EGFP construction of recombinant plasmid

In the present embodiment, (the Genebank sequence number: the transformation carrier that 1377915) will be used to directly, objective evaluation the present invention obtains is expression alien gene correctly, relatively transforms plasmid and the difference of baculovirus skeleton plasmid on transfection efficiency and expression level commonly used at present simultaneously for fluorescence report gene EGFP.With plasmid pEGFP-C1 (available from ClonTech company) is EGFP sequence (the Genebank sequence number: 1377915) that the template amplification two ends have BamHI and SmalI restriction enzyme site, insert BamHI and the SmalI restriction enzyme site of the plasmid pFB-G-SFV that obtains among the embodiment 1, obtain recombinant plasmid pFB-G-SFV-EGFP.The result is shown in accompanying drawing 3A.

2, the swivel base that comprises the pFB-G-SFV-EGFP recombinant plasmid

2.1 take out DH10Bac ^TMCompetent cell (available from Invitrogen company) melts on ice, draws 100 μ lDH10Bac with liquid-transfering gun ^TMCompetent cell is in the 1.5ml of precooling EP pipe;

2.2 the recombinant plasmid pFB-G-SFV-EGFP that adds about 1ng gently beats tube wall gently and makes it mixing in competent cell;

2.3 place 30min on ice.Static 45 seconds of 42 ℃ of circulator baths are placed 2min on ice fast;

2.4 in above-mentioned EP pipe, add 900 μ l S.O.C. substratum (available from big day source biotech inc of Wuhan force), place 37 ℃ of shaking tables (225r/min) jolting to cultivate 4h the EP pipe;

2.5 dilute above-mentioned cell to 10 with the S.O.C. substratum ^-1, 10 ^-2, 10 ^-3

2.6 at every LB substratum (kantlex 50 μ g/ml, gentamicin 7 μ g/ml, tsiklomitsin 10 μ g/ml, Bluo-gal100 μ g/ml, isopropyl-(IPTG) 40 μ g/ml) add the described diluent 100 μ l of step 2.5 in, coating evenly;

2.7 cultivate 24-48h for 37 ℃.The picking hickie is purifying on the LB flat board, is purified to the third generation.

3, the separation and Extraction of reorganization Bacmid DNA

3.1 picking white is cloned in 2ml LB liquid nutrient medium (containing 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tsiklomitsins), 37 ℃ of shaking table 250-300r/min cultivate more than the 24h;

3.2 get the described culture of 1.5ml step 3.1 in 1.5ml EP pipe, the centrifugal 1min of 14000 * g.Remove supernatant liquor, stand upside down on thieving paper.The solution I (Tris-HCl (15Mm), EDTA (10Mm), RNA enzyme (100 μ g/ml)) that adds 0.3ml is is then blown and beaten re-suspended cell gently with the rifle head;

3.3 add 0.3ml solution II (NaOH 0.2N, sodium laurylsulfonate 1%), mixing gently, room temperature is placed 5min (solution becomes is clear);

3.4 slowly add 0.3ml solution III (KAc (3M)), mixing forms albumen and DNA precipitation gently, places 5-10min on ice;

3.514000 * g, 25 ℃ of centrifugal 10min, during get a 2ml centrifuge tube in addition, add 800 μ l Virahols;

3.6 gently supernatant liquor is changed over to the above-mentioned centrifuge tube that contains Virahol, avoids white precipitate is brought into.The centrifuge tube that reverses gently for several times.Place 5min (this process can be placed on-20 ℃ and spend the night) on ice.25 ℃ of centrifugal 15min;

3.7 remove supernatant, be inverted centrifuge tube on thieving paper, add 70% washing with alcohol precipitation then for several times, 14000 * g, 25 ℃ of centrifugal 5min.Abandon supernatant;

Step 3.7 is described to be deposited in seasoning 5-10min in the air 3.8 make.Add 40 μ l TE solution, rap the pipe end, make solution be positioned at the pipe bottom.

4 reorganization Bacmid transfection sf9 cells

4.1 recovery is numbered GDC008 insect cell line SF-9 and (purchases the commercial cell material at Chinese typical culture collection center in Chinese Wuhan University, see Http:// www.bio-equip.com/showlequip.asp? equipid=14757﹠amp; Division=2604).

4.1.1 get one in frozen sf-9 cell in liquid nitrogen, in 35-42 ℃ of warm water, melt rapidly, with cell at 25 ℃, centrifugal 5min under the 1000r/min;

4.1.2 abandon supernatant, add Grace ' the s substratum (available from Sigama company) that 1ml contains 10% foetal calf serum, blow and beat re-suspended cell gently;

4.1.3 the 25cm that 5ml contains Grace ' the s substratum of 10% foetal calf serum is equipped with in resuspended cell adding ²In the culturing bottle, 27 ℃ of overnight incubation.

4.2 transfection

4.2.1 in six orifice plates with 9 * 10 ⁵The density inoculation sf-9 cell in individual cell/2ml/ hole is cultivated 1h, is made cell attachment for 27 ℃;

4.2.2 with the reorganization bacmid transfection sf-9 cell that obtains (Liang Changyong, different baculoviruss are induced the comparative analysis of mammalian cell antiviral effect, Chinese science C collects life science 2006,36 (3), 261-266);

4.2.3 the cell in six orifice plates is hatched 5h, 27 ℃, changes fresh Grace ' s substratum.In 27 ℃ of incubators, hatch 72h or slight cytopathy sign occurs up to cell.Promptly contain the recombinant baculovirus (Bac-G-SFV-EGFP) that carries foreign gene EGFP in the nutrient solution of culturing cell.

Embodiment 3 (Application Example 1)

With the Bac-G-SFV-EGFP recombinate shape virus infection breast hamster nephrocyte (BHK-21) that utilizes embodiment 2 described methods to pack out.

1, in six orifice plates with 9 * 10 ⁵The density inoculation in individual cell/2ml/ hole is numbered GDC010 Syria hamster kidney cell line BHK-21 and (available from the commercial cell material at Chinese typical culture collection center in Chinese Wuhan University, sees Http:// www.bio-equip.com/showlequip.asp? equipid=14757﹠amp; Division=2604) cell, cultivate 1h, make cell attachment for 37 ℃.

2, with containing Ca ²⁺, Mg ²⁺Phosphate buffer soln (PBS, available from Sigma company) wash cell three times, dosage (MOI=50) with 50 infection multiplicities inserts baculovirus Bac-G-SFV-EGFP in the six orifice plate cells, 27 ℃ were infected after 4 hours, discard supernatant, add the fresh DMEM that contains 10% new-born calf serum (available from Gibico company).

3, six orifice plates are placed 37 ℃ CO ₂In the incubator, cultivated 48 hours, detect the expression of foreign gene EGFP with fluorescent microscope and flow cytometer.

Embodiment 4 (Application Example 2)

The Bac-G-SFV-EGFP recombinate shape virus infection HEK-293 cell of packing out with embodiment 2 described methods.

1, in six orifice plates with 9 * 10 ⁵It is that HEK-293 (available from the commercial cell material at Chinese typical culture collection center in Chinese Wuhan University, sees that the density inoculation in individual cell/2ml/ hole is numbered GDC067 people source embryonic kidney cells Http:// www.bio-equip.com/showlequip.asp? equipid=14757﹠amp; Division=2604), cultivate 1h, make cell attachment for 37 ℃.

2, with containing Ca ²⁺, Mg ²⁺Phosphate buffer soln (PBS, available from Sigama company) wash cell three times, with the dosage (MOI=50) of 50 infection multiplicities baculovirus Bac-G-SFV-EGFP is inserted in the six orifice plate cells, 27 ℃ infect 4h after, discard supernatant, add the fresh DMEM that contains 10% foetal calf serum.

3, six orifice plates are placed 37 ℃ of 5%CO ₂In the incubator, cultivate 48h, detect the expression of EGFP with fluorescent microscope and flow cytometer.

Embodiment 5 (Application Example 3)

Utilizing the Bac-G-SFV-EGFP recombinant baculovirus inoculation BHK-21 cell that obtains among the embodiment 3 to carry out apoptosis detects.

1, dna ladder degree fragment detects (DNA-ladder, method is with reference to reference 8)

1.1 BHK-21 cell phosphate buffer soln (PBS with inoculation baculovirus Bac-G-SFV-EGFP among the embodiment 3, available from Sigma company)) after the washing three times, add the cell pyrolysis liquid that contains 10mM Tris-HCl (pH8.0), 10mM EDTA and 0.5%TritonX-100 and make lysis.

Be placed in 37 ℃ of incubators 1.2 add the 0.1mg/mlRNA enzyme in the cell lysate that step 1.1 is collected, take out behind the 1h.

1.312000 the centrifugal 25min of * g makes the chromosomal DNA precipitation.

1.4 transfer supernatant, the sodium laurylsulfonate of adding 1% uses Proteinase K (available from Takara company) behind 50 ℃ of digestion 1h, with the DNA in the imitative extracting supernatant of phenol, the dehydrated alcohol precipitation, heavy molten with the damping fluid that contains 10mM Tris (pH 8.0) and 1mM EDTA then.

1.5 do the morphological analysis of dna fragmentationization again with 1.5% sepharose.Shown in accompanying drawing 5A, tangible fragmentation appears in the bhk cell chromosomal DNA of inoculation recombinant baculovirus Bac-G-SFV-EGFP, and the bhk cell chromosomal DNA of inoculation baculovirus wild-type still is kept perfectly.This shows that the baculovirus Bac-G-SFV-EGFP of reorganization can cause the apoptosis of bhk cell.

2, colorimetric method is measured the activity (detection kit is available from Nanjing KaiJi Biology Science Development Co., Ltd, and concrete operations are undertaken by product description) of caspase-3

2.1 utilize the BHK-21 cell harvesting of cell spatula with inoculation baculovirus among the embodiment 3, the centrifugal 5min of 1500g discards and asks, and keeps cell;

2.2 add the cell lysis buffer solution (available from Nanjing KaiJi Biology Science Development Co., Ltd, test kit is subsidiary) of 50 μ L precoolings in the sedimentation cell of collecting, piping and druming evenly;

2.3 put cracking 30min on ice, vortex vibration therebetween 3～4 times, each 10s; Or freeze thawing 2～3 times, use 4 ℃ of centrifugal 1min of 10000r/min afterwards;

Be transferred in the new pipe 2.4 carefully draw supernatant (containing cracked protein), and place stand-by on ice;

The supernatant 2.5 take a morsel (1～2 μ L), (two kinds of measuring methods of regrouping human interleukin-11 protein concentration compare ordinary method for Braford method, Zhang Tieyu etc., research and development of natural products, 2005,17:150-151) mensuration protein concentration wherein;

Contain the proteic lysis supernatant of 100～200 μ g 2.6 draw 50 μ L; As volume less than 50 μ L, complement to cumulative volume 50 μ L with lysis buffer (available from Nanjing KaiJi Biology Science Development Co., Ltd, test kit is subsidiary);

2.7 add 2 * reaction buffer (available from Nanjing KaiJi Biology Science Development Co., Ltd, test kit is subsidiary) of 50 μ L; 2.8 add 5 μ L caspase-3 substrate (available from Nanjing KaiJi Biology Science Development Co., Ltd, test kit is subsidiary), and hatch 4h in 37 ℃ of lucifuges;

2.9 finishing the back, substrate reactions measures its light absorption value at λ=405nm or 400nm with microplate reader or spectrophotometer (cuvettes of 100 μ L).By calculating OD _Inductor/ OD _{Negative right}According to multiple determine inducer of apoptosis group caspase-3 activation degree.Shown in Fig. 5 B, the intracellular caspase-3 activity of inoculation recombinant baculovirus Bac-G-SFV-EGFP has significantly than other untreated cells and increases, because Caspase family plays important effect in the process of mediating apoptosis, wherein caspase-3 is crucial execution molecule.Form relevant with dna break, karyomit(e) cohesion and apoptotic body.Therefore the raising of the hydrolase of proteolysis of caspase-3 can be used as the apoptosis signal.This result shows that significant apoptotic effect has taken place the cell of inoculation recombinant baculovirus Bac-G-SFV-EGFP.

Embodiment 6 (Application Example 4)

Utilize the comparison of the Bac-G-SFV-EGFP recombinant baculovirus immune effect that obtains among recombinant baculovirus that conventional commercial skeleton plasmid pFastBac-Dual (available from Invitrogen company) packs out and the embodiment 3.

1, the acquisition of common recombinant baculovirus Bac-G-CMV-EGFP

1.1 with reference to (Tong Cheng, et al, A rapid and efficient method to express target genes inmammalian cells by baculovirus, World J Gastroenterol 2004 such as Cheng Tong; 10 (11): the technological line that 1612-1618) provides, with plasmid pFastBac-Dual (purchasing company) in Invitrogen, plasmid pCI-neo (purchasing company) in Invitrogen, plasmid pEGFP-C1 (purchasing the company in ClonTech) constructs the reorganization skeleton plasmid pFB-G-CMV-EGFP that inserts foreign gene EGFP, shown in accompanying drawing 3B.

1.2 with reference to the step of embodiment 2, the reorganization skeleton plasmid pFB-G-CMV-EGFP that utilizes step 1.1 to obtain, pack out can expression alien gene the coventional type baculovirus Bac-G-CMV-EGFP of EGFP.

2, the enzyme linked immunological of immune mouse absorption antibody (ELISA) level relatively

With recombinant baculovirus Bac-G-SFV-EGFP that the present invention obtained and in contrast baculovirus Bac-G-CMV-EGFP respectively with 10 ¹⁰The Balb/c mouse (available from the disease prevention and control center, Wuhan) in the dosage of pfu/mL immunity 4-6 age in week, 6 every group, 100 μ l/ are only, in the back leg intramuscular injection, immunity is 2 times altogether, each 3 weeks at interval, and establish phosphoric acid buffer (PBS) immune group as negative control group.Exempt from 3 weeks of back in two and gather mouse blood, detect the specific enzyme linked immunological absorption of EGFP antibody horizontal in the serum.The result as shown in Figure 7, the 6th week after first immunisation, two kinds of baculovirus immune group antibody horizontals raise rapidly, wherein, the recombinant baculovirus Bac-G-SFV-EGFP immune group average antibody titre level that the present invention obtained reaches 1: 8250, and contrast Bac-G-CMV-EGFP immune group antibody titers level only is 1: 5000, and phosphoric acid buffer (PBS) immune group does not detect the specific enzyme linked immunological absorption of EGFP antibody.This result shows the baculovirus that the baculovirus skeleton plasmid packaging that obtained by the present invention goes out, and at expression alien gene, promotes many-side such as immune response will significantly be better than common baculovirus skeleton plasmid.

Reference:

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Claims (2)

1. a recombinant baculovirus transfer vector skeleton plasmid pFB-G-SFV is deposited in Chinese typical culture collection center, and preserving number is CCTCC NO:M207070.

2. the application of the described plasmid of claim 1 on the recombinant baculovirus packing.

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