CN101168743B - Recombination baculoviral and its preparation method and application - Google Patents
Recombination baculoviral and its preparation method and application Download PDFInfo
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Abstract
The invention relates to recombinant baculovirus, and the preparation method and the application thereof, in particular to the recombinant baculovirus which carries fusion genes formed by gp64 signal peptide, HA genes and gp64 trans-membrane domain genes. The recombinant baculovirus is preserved in China General Microbiological Culture Collection Center, and the accession number is CGMCC No.2033.High-level expression is performed after inoculation of larva and nymphs of domestic silkworms by utilizing the recombinant baculovirus, HA protein is displayed on cyst membranes of the baculovirus after the expression, and through separation, purification, and freeze drying, avian influenza vaccine with higher bioactivity, namely, lyophilized powder is produced.
Description
Technical field
The invention belongs to the genetically engineered producer gene recombiant vaccine technical field in the biotechnological pharmaceutics engineering.
Background technology
Bird flu is the acute respiratory transmissible disease that is caused by some strains in some hypotype of influenza virus A avian.Human and bird fluenza, promptly people's avian influenza is the acute respiratory transmissible disease that is caused by some strains in some hypotype of influenza virus A avian.In recent years, constantly there is the report human and bird fluenza to cause dead example.In case human and bird fluenza large-scale outbreak, its hazard rating be not second to SARS, even surpass SARS.The discovery of vaccine is the incident that has milestone significance on the human development history, and vaccine is a kind of for the generation that prevents, control transmissible disease, popular, is used for the premunitive vaccine preventive biological products of human body.And the human and bird fluenza vaccine is prevention and a kind of efficient ways of treatment human and bird fluenza.
Summary of the invention
One object of the present invention is to provide a kind of recombinant baculovirus;
Another object of the present invention is to provide a kind of method for preparing recombinant baculovirus of the present invention;
Another object of the present invention is to provide the application of recombinant baculovirus of the present invention in the preparation avian influenza virus vaccine;
Another object of the present invention is to provide a kind of method for preparing avian influenza vaccine.
On the one hand, the invention provides a kind of recombinant baculovirus, this virus has the fusion rotein that gp64 signal peptide (silkworm baculovirus membrane glycoprotein gp64 signal peptide), HA gene (avian influenza virus hemagglutinin gene) and gp64 membrane-spanning domain gene (silkworm baculovirus membrane glycoprotein gp64 membrane-spanning domain gene) form.This recombinant virus helps the separation and purification of vaccine, as the pseudo-virus of avian influenza virus, antigen can be illustrated on the cyst membrane of virus simultaneously, and being beneficial to stimulates body to produce immunne response.
In an embodiment, the concrete sequence of described gp64 signal peptide sequence is seen SEQID NO.1), the HA gene coded sequence sees SEQ ID NO.2; The whole membrane-spanning domain encoding sequences of gp64 membrane-spanning domain gene are seen SEQ ID NO.3.
In an embodiment, the invention provides recombinant baculovirus, this virus is silkworm baculovirus.Utilize silkworm baculovirus inoculation silkworm of the present invention, can obtain silkworm biological reactor.China's sericulture industry has a long history, and belongs to national industry, and silkworm biological reactor has traditional advantage and resources advantage; And this reactor can carry out scale operation, has cost advantage.Certainly, recombinant baculovirus of the present invention not only can be inoculated silkworm, can also inoculate other insect cells.
In an embodiment, the invention provides a kind of recombinant baculovirus, this virus is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is 2714 mailbox in the BeiJing ZhongGuanCun, preservation date is on April 28th, 2007, deposit number is CGMCC No.2033, classification called after Bombyx mori nuclear polyhydrosis virus Bombyxmori nucleopolyhedrovirus).
On the other hand, the invention provides a kind of method for preparing recombinant baculovirus, this method comprises:
Prepare gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene order respectively;
Prepared comprise gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene Fusion gene order are imported in the baculovirus, form recombinant baculovirus.
In an embodiment, the invention provides the method for recombinant baculovirus, wherein gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene order are formed fusion gene gp64HA, again this gp64HA is cloned into and obtains recombinant transfer vector in the transfer vector, make this recombinant transfer vector and baculovirus generation homologous recombination then and obtain recombinant baculovirus.Described recombinant transfer vector can carry out homologous recombination (the homologous recombination position taking place on the multiple clone site upstream and downstream homologous sequence of carrier) with the linearizing silkworm baculovirus, foreign gene is inserted in the silkworm baculovirus genome, improve the screening efficiency of recombinant virus simultaneously greatly.
On the other hand, the invention provides the application of a kind of recombinant baculovirus in the preparation avian influenza virus vaccine.
In an embodiment, the invention provides the application of a kind of recombinant baculovirus in the preparation avian influenza virus vaccine, wherein said vaccine is the human vaccine.
On the other hand, the invention provides a kind of method for preparing avian influenza vaccine, this method comprises utilizes recombinant baculovirus inoculation silkworm of the present invention (larva and/or the pupa of preferred silkworm), silkworm baculovirus sprouts and makes HA be showed in its surface, virus particle is carried out separation and purification obtain avian influenza vaccine.HA is showed in the silkworm baculovirus surface and not only is beneficial to purifying, but also helps stimulating body to produce immunne response.
In an embodiment, the invention provides and utilize recombinant baculovirus inoculation silkworm of the present invention, silkworm baculovirus sprouts and makes HA be showed in its surface, broken cell homogenate, the centrifugal removal impurity of multistep, Ultracentrifugation virus particle, the resuspended post precipitation of aseptic lysate, carry out the band centrifugation purification of samples, ultrafiltration desugar and lyophilize.
In an embodiment, the present invention utilizes silkworm as bio-reactor, adopt the surface display technology to make target protein HA be expressed in the recombinant baculovirus surface, in silkworm larva and pupa, efficiently express human and bird fluenza vaccine with high clinical value.The vaccine that present method is produced is real through mouse, monkey body pilot scale checking, and this vaccine produces antibody, has provide protection.Present method is applicable to scale operation, has reduced cost, and the output height, and the human and bird fluenza vaccine using value of being produced is big.
Description of drawings
Fig. 1: the recombinant plasmid pGEM-gp64HA that contains the gp64HA fusion gene;
Fig. 2: contain gp64HA fusion gene baculovirus transferring plasmid pBacgp64HA;
Wherein, P:polyhedrin, polyhedrin gene promoter; A:Polyhedin PolyA
+Signal, the polyhedron gene polyadenylation signal; M13 ori:M13 phage replication starting point; Amp: ampicillin resistance gene; The ori:pUC replication origin;
Fig. 3: modify C-type virus C BmBacPAK6;
Fig. 4: the evaluation of target protein, wherein arrow is depicted as the HA fusion rotein.
Embodiment
In a specific embodiment of the present invention, the method of utilizing silkworm biological reactor surface display technology to produce the human and bird fluenza vaccine of the present invention, it is method acquisition gp64HA fusion gene by PCR, and introduce BamH I and Not I restriction enzyme site respectively at its 5 ' and 3 ' end, after silkworm baculovirus carrier pBacPAK8 is connected, connect product and wild-type silkworm baculovirus BmBacPAK6 homologous recombination takes place in bombyx mori cell, pass through plaque select, the silkworm with recombinant baculovirus that acquisition has gp64HA (is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is 2714 mailbox in the BeiJing ZhongGuanCun, preservation date is on April 28th, 2007, deposit number is CGMCC No.2033, classification called after Bombyx mori nuclear polyhydrosis virus Bombyx mori nucleopolyhedrovirus), the artificial inoculation silkworm larva, pupa, after 5-7 days, collect larva body fluid and pupal cell, homogenate, carry out centrifugal, separation and purification, make human and bird fluenza vaccine freeze-drying powder under the lyophilize, aseptic condition.
In another embodiment of the present invention, utilize genetic engineering technique to obtain to have the fusion gene gp64HA recombinant baculovirus of avian influenza virus major antigen gene HA and silkworm baculovirus gene gp64, produce this recombinant virus by silkworm biological reactor, utilize the surface display technology to make target protein be expressed in surface of cell membrane, recombinant virus forms virus envelope and target protein is showed on the virus envelope by sprouting.Having fusion gene gp64HA recombinant baculovirus efficiently expresses behind inoculation silkworm larva, pupa, expression amount reaches 0.5mg/ml, expresses back HA albumen and is showed on the baculovirus cyst membrane, through separation and purification, lyophilize is made and is had higher bioactive human and bird fluenza vaccine freeze-drying powder.This recombinant baculovirus has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to, the address is in the BeiJing ZhongGuanCun, preservation date is on April 28th, 2007, deposit number is CGMCCNo.2033, classification called after Bombyx mori nuclear polyhydrosis virus Bombyx morinucleopolyhedrovirus.This method raw material is easy to get, and production cost is low, and vaccine can produce protection antibody, toxicological harmless, and implementary value is great.
Embodiment
Elaborate particular content of the present invention below in conjunction with embodiment, these embodiment also are not used in qualification the present invention.
1. the structure of fusion gene gp64HA.
In order to make HA albumen can be expressed in the baculovirus surface, 5 ' and 3 ' end at the HA gene is connected coding baculovirus envelope protein gp64 signal peptide gene sequence and gp64 membrane-spanning domain gene order respectively, and holds at 5 ' and 3 ' of fusion gene and to introduce BamH I and Not I restriction enzyme site respectively.Respectively with baculovirus BmNPV 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, Genbank DQ520855.) and gp64 membrane-spanning domain gene order (silkworm baculovirus genome Genbank NP 074525) be 3 pairs of primers of stencil design, the purpose that at first increases fragment gp64 signal peptide (sp), HA and gp64 membrane-spanning domain (tm) utilize each purpose fragment primer and the synthetic fusion gene gp64HA of template each other then.The design primer is as follows:
Psp1：5’gcggatcc atgctactagtaaatcagtc 3’(SEQ ID NO.4)
Psp2：5’catgtgtaacagtaacgttcttttccgcaaaggcagaatgcgccgccgcc 3’(SEQ ID NO.5)
Pha1：5’gaaaagaacgttactgttacacatgc 3’(SEQ ID NO.6)
Pha2：5’aatgcaaattctgcattgtaacgac 3’(SEQ ID NO.7)
Ptm1：5’gtcgttacaatgcagaatttgcatt ttcatgtttggtcatgtagc 3’(SEQID NO.8)
Ptm2：5’gagcggccgc ttaatattgtctactattacggttt 3’(SEQ ID NO.9)
(1) amplification of gp64 signal peptide (sp)
With gp64 DNA is template, and the PCR reaction parameter is designed to, 94 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 68 ℃ of renaturation are extended 30s, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR Buffer 10μl
25mmol MgCl
2 8μl
2.5mmol dNTPs 8μl
Psp1 1μl
Psp2 1μl
Template 1 μ l
KOD-Plus archaeal dna polymerase 1 μ l (available from TOYOBO company)
Add 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 amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.This purpose fragment can be used as template sp and is used for subsequent experimental.
(2) amplification of gp64 membrane-spanning domain (tm)
(preserve in silkworm baculovirus seed culture of viruses by this laboratory and to extract with gp64 DNA, the preparation method is with reference to " insect baculovirus molecular biology " Lv Hongsheng chief editor, Chinese agriculture science and technology press publishes) be template, the PCR reaction parameter is designed to, 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, 68 ℃ of renaturation are extended 30s, 30 circulations, 68 ℃ are extended 5min.
The reaction system of 100 μ l 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 amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.This purpose fragment can be used as template tm and is used for subsequent experimental.
(3) amplification of HA gene
With avian influenza virus H 5 N 1 HA gene (extract from ill porcine blood serum, clone's process is seen the amplification of HA gene) is template, and the PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCRBuffer 10μl
25mmol MgCl
2 8μl
2.5mmol dNTPs 8μl
Pha1 1μl
Pha2 1μl
Template 1 μ l
KOD-Plus archaeal dna polymerase 1 μ l
Add 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 amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.This purpose fragment can be used as template HA and is used for subsequent experimental.
(4) gp64 signal peptide sp gene and HA gene Fusion
The PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCR Buffer 10μl
25mmol MgCl
2 8μl
2.5mmol dNTPs 8μl
Psp1 1μl
Pha2 1μl
Template sp (available from preceding step 1) 1 μ l
Template HA (available from preceding step 3) 1 μ l
KOD-Plus archaeal dna polymerase 1 μ l
Add 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 amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.This purpose fragment can be used as template sp-HA and is used for subsequent experimental.
Because the downstream primer Psp2 of sp design the time has added the partial sequence of HA gene 5 ' end,, utilize PCR method just can amplify sp-HA fusion gene sequence once more so that sp fragment 3 ' terminal sequence that pcr amplification goes out and HA gene 5 ' terminal sequence exist is overlapping.
(5) fusion gene sp-HA once more with the fusion of tm, construct the gp64HA fusion gene
The PCR reaction parameter is 94 ℃ of pre-sex change 3min, 94 ℃ of sex change 30s, and 68 ℃ of renaturation are extended 1.5min, 30 circulations, 68 ℃ are extended 5min.
In an aseptic 0.5ml centrifuge tube, mix following ingredients:
10×PCRBuffer 10μl
25mmol MgCl
2 8μl
2.5mmol dNTPs 8μl
Psp1 1μl
Ptm2 1μl
Template sp-HA (available from preceding step 4) 1 μ l
Template tm (available from preceding step 2) 1 μ l
KOD-Dash archaeal dna polymerase 1 μ l (TOYOBO company)
Add 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 amplified fragments, cut glue simultaneously and reclaimed the purpose fragment.
The same, because added the partial sequence of HA gene 3 ' end during the upstream primer Ptm1 of tm design, so it is overlapping that tm fragment 5 ' terminal sequence that pcr amplification goes out and HA gene 3 ' terminal sequence exist, utilizing PCR method just can amplify the sp-HA-tm fusion gene once more is the gp64HA sequence.
2. the structure of cloned plasmids pGEM-gp64HA
The fusion gene gp64HA that pcr amplification is gone out is connected on the pGEM-T vector
PGEM-T vector (available from promage company) 0.5 μ l
2×Rapid Ligation buffer 5μl
T4DNA ligase (available from promage company) 1 μ l
ddH
2O 1.5μl
↓ 10μl
Mixing, 25 ℃ of reaction 1h
Reaction mixture is converted among the competent cell E.coli DH5 α
1. get 40 μ l with the aseptic suction nozzle of refrigerative from the competent cell suspension and transfer in the aseptic Eppendorf tube, add the connection product in the previous step, mixing was placed 30 minutes in ice gently;
2. centrifuge tube is put in the circulator bath that is warmed to 42 ℃ in advance, is placed on the test-tube stand, exactly placed 90 seconds, do not shake test tube;
3. fast pipe is transferred in the ice bath, made cell cooling 1-2 minute;
4. every pipe adds 400 μ l LB liquid nutrient mediums.Pipe is transferred on 37 ℃ of shaking tables, and incubation made bacteria resuscitation in 45 minutes, and the ammonia benzyl antibiotics resistance marker gene of expression plasmid coding;
5. the competent cell that proper volume (each 90mm flat board can reach 200 μ l) has been transformed is transferred to and is contained on the antibiotic LB solid medium of ammonia benzyl;
6. place room temperature to be absorbed flat board until liquid;
7. be inverted plate,, bacterium colony can occur after 12-16 hour in 37 ℃ of cultivations.
Picking list bacterium colony extracts plasmid, carries out enzyme and cuts preliminary evaluation, and through order-checking, sequence is entirely true.
3. contain the acquisition of gp64HA fusion gene baculovirus transferring plasmid pBacgp64HA
Plasmid pGEM-gp64HA downcuts gp64HA fusion gene fragment cloning in the pBacPAK8 of BamH I and Not I double digestion (available from Clontech company) through BamH I and Not I double digestion, make the gp64HA fusion gene place polyhedrin (polyhedrin, ph) under the gene promoter control, be built into recombinant transfer plasmid pBacgp64HA, correct through restriction analysis identified gene sequence.
4. contain the acquisition of the silkworm with recombinant baculovirus Bmgp64HA of gp64HA fusion gene
Get 5 μ l recombinant transfer plasmid pBacgp64HA DNA and 6 μ l through the modification C-type virus C BmBacPAK6 of Bsu36I linearization for enzyme restriction DNA (available from the biochemical cell in Shanghai institute), the TC-100 substratum (GIBCOBRL company) that adds 100 μ l serum-frees is mixed.The TC-100 substratum of getting 6 μ lDosper (Bao Ling Man) adding, 100 μ l serum-frees is mixed.The TC-100 substratum washed twice of BmN cell (available from the biochemical cell in Shanghai institute) the usefulness serum-free in the 35mm plate will be cultivated in advance, and dropwise add pBacgp64HA transferring plasmid and Dosper mixture, cultivated 4-5 days, and collected supernatant and carry out first round plaque screening for 27 ℃.Get the Bm N cell in the 5 μ l supernatants infection 35mm plate, supernatant discarded adds the TC-100 substratum and the low melting-point agarose of balanced mix after 1 hour.Picking plaque after 4-5 days, infected the BmN cell 3-4 days, preserve supernatant, cell is used for Southern blot dot hybridization with the NaOH cracking, make template random primer probe mark test kit (Bao Ling Man) label probe with the gp64HA fusion gene, hybridizing method is according to " molecular cloning " (Science Press, 1995).The supernatant of getting positive colony carries out second and takes turns plaque screening.Get the supernatant infected silkworm cell amplification of positive colony.Can obtain a large amount of recombinant baculovirus Bmgp64HA that contains the gp64HA fusion gene.This virus energy infected silkworm cell is the morbidity shape at the microscopically cell.This recombinant baculovirus has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to, the address is in the BeiJing ZhongGuanCun, preservation date is on April 28th, 2007, deposit number is CGMCC No.2033, classification called after Bombyx mori nuclear polyhydrosis virus Bombyx mori nucleopolyhedrovirus.
5. the expression of recombinant silkworm baculovirus Bmgp64HA in silkworm 5 instar larvaes and pupa
Recombinant virus Bmgp64HA is infected the BmN cell with 10 MOI carry out virus amplification, by 1 * 10
6PFU/ bar stab inoculation inserts silkworm and plays silkworm five ages, after infecting 5-7 days, take silkworm body fluid and pupal cell, through high speed centrifugation (12000rpm, 30min), get the HA activity (blood clotting method) in the supernatant survey hemolymph, the HA activity in the 6th day hemolymph that infects reaches 5 * 10
4U/ml.Supernatant behind the high speed centrifugation adds isopyknic 2 * protein sample-loading buffer (100Mm Tris.HCl, 4%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), and 100 ℃ of heating 10min get 20 μ l and carry out the SDS-PAGE analysis.The result shows that recombinant virus has been expressed the HA fusion rotein.
6. the acquisition of the proteic baculovirus Bmgp64HA of surface display HA
(1) separation and purification goes out Bmgp64HA virus particle (human and bird fluenza vaccine) from silkworm chrysalis
A. get the silkworm chrysalis sample that infects through Bmgp64HA in right amount, after thawing under 4 ℃, mixed in right amount with 0.85% physiological saline, homogenate evenly gets final product to slurries are careful;
B. homogenate is added in the 500ml centrifuge tube, trim, the centrifugal 20-60min of 3000rpm gets supernatant, carefully discards grease;
C. the 3000rpm centrifuged supernatant is poured in the new 500ml centrifuge tube, trim, the centrifugal 20-60min of 6000rpm gets supernatant, abandons grease;
D.6000rpm the centrifugal supernatant liquor is poured the 50ml centrifuge tube into, trim, and the centrifugal 30-120min of 12000rpm gets supernatant, abandons grease;
E.12000rpm centrifuged supernatant changes new 50ml centrifuge tube over to, trim, and the centrifugal 30-120min of 18000rpm gets supernatant, abandons grease;
F.18000rpm the supernatant after centrifugal is being sub-packed in the super in pipe of sterilization on the super clean bench, balance, and the centrifugal 30-90min of 35000rpm precipitates virus particle;
G.35000rpm the supernatant after centrifugal is packed in the bottle of sterilization, places 4 ℃ earlier, precipitation with aseptic lysate (0.05M PB, 0.5M NaCl, 0.04%EDTA) resuspended, blow and beat gently repeatedly with the rifle head, precipitation can fully be dissolved; Precipitation is settled to certain volume (300-500ml) after all dissolving, and is used for band centrifugation (Zonal Centrifuge), is further purified.
(2) band centrifugation
The required different concns sucrose of band centrifugation is all prepared with working fluid, application of sample is followed successively by 150-200ml sterilizing works liquid, 300-500ml sample, 300-500ml 20% sucrose, 300-500ml 30% sucrose in proper order, adds 52% sucrose at last and is full of rotary head (cumulative volume is 1690ml).35000rpm receives sample after centrifugal 3 hours, is higher than 52% sucrose with concentration and makes top liquid, and the sample in the rotary head is ejected, and detects with the nucleic acid-protein detector, and aseptic centrifuge tube is collected sample.
(3) desugar
Sugar degree is higher in the sample, need remove, and adopts ultra-fine filter, uses hollow-fibre membrane to carry out desugar.At first be to select suitable hollow-fibre membrane specification (molecular weight cut-off is 750kD), clean, application of sample desugar again, with the sucrose in the aseptic ultrapure water displacement sample, sucrose concentration is lower than 5% in sample.
(4) Bmgp64HA virus particle (human and bird fluenza vaccine) biological activity is identified
Sample after the band centrifugation desugar is lived by the pipe survey, and 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.
With Bradford method (Xylene Brilliant Cyanine G method) test sample protein content.
It is as follows that human and bird fluenza virus is calculated (mg/mL) formula than work: human and bird fluenza virus is than living=2
n* 4/ sample concentration, the hole count of red cell agglutination for taking place in n.The human and bird fluenza virus that this method obtained can reach more than 20 than work.
(5) preserve and detect
Than the sample of live higher and desugar, aseptic subpackaged, human and bird fluenza vaccine freeze-drying powder is made in-50 ℃ of lyophilizes.Simultaneously, identify lyophilized powder again, reach 20 above persons and be qualified samples than living.12%SDS-PAGE leakage of electricity swimming, test sample has the expression (see figure 4) of target protein at the 45kD place.
(6) vaccine effect
Select cleaning level BALB/C mice for use, male, 56, body weight 20-22g is provided by Beijing Vital River Experimental Animals Technology Co., Ltd., animal credit number: SCXK-(army) 2002-001.
According to experiment purpose and experimental design requirement, dosage group (10-100ug/kg), vaccine low dose group (0.1-10ug/kg) in blank group, adjuvant control group (1: 9 (w/v) mixed solution of aluminium hydroxide and physiological saline), vaccine high dose group (100-300ug/kg), the vaccine are established in experiment, and the principle of grouping is that body weight does not have significant difference between each treated animal.Each organizes dosage as follows
| Group | Sex | Number of animals |
| TA (vaccine high dose group) TB (dosage group in the vaccine) TC (vaccine low dose group) CN1 (solvent control group) | ♂ ♂ ♂ ♂ | 14 14 14 14 |
Route of administration simulation clinical administration approach, the neck subcutaneous injection divides 2 points, every some 0.5ml.Administration frequency and time limit: per two all immunity once, immunity detects antibody titer after 3 times.It is collunarium that animal is attacked malicious method
Detect the pharmacodynamics of comprehensive evaluation vaccine in mouse experiment by overview, pathologic finding, virus separation, antibody titer.The result shows: the human and bird fluenza vaccine carries out testing in the animal body, produces antibody in the mouse body, and its protection antibody titer was greater than 1: 200, and the challenge test result shows that this vaccine has obvious protection effect.
Select regular grade rhesus monkey (Rhesus macaque) for use, female 10, male 10, the age is 3-4 year, and body weight 3.5-5.0 kilogram is provided by the laboratory animal Beijing prosperous Biological resources of Xie Er institute, and the animal credit number is SCXK (capital) 2005-0005.With testing purpose and experimental design requirement factually, dosage group (80-300ug/kg), vaccine low dose group (0.1-80ug/kg) in solvent control group, vaccine high dose group (300-600ug/kg), the vaccine are established in this experiment, and the principle of grouping is that body weight does not have significant difference between each treated animal.
| Group | Sex | Number of animals |
| TA (vaccine high dose group) TB (dosage group in the vaccine) TC (vaccine low dose group) CN1 (solvent control group) | ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ | 22222222 |
Route of administration is the neck subcutaneous injection, divides 2 points, every some 0.5ml, per two all immunity once, the immunity 3 times.It is collunarium that animal is attacked malicious method.Detect the pharmacodynamics of comprehensive evaluation vaccine in the monkey experiment by overview, pathologic finding, virus separation, antibody titer.The result shows: produce antibody in the monkey body, its protection antibody titer was greater than 1: 100, and the challenge test result shows that this vaccine has the significant protection effect.Require to carry out the safety evaluation of vaccine according to national biological product safety pharmacology, the result shows that this vaccine does not have obviously influence to cardiovascular, the respiratory system of mouse and monkey, neural system, body temperature etc.Long malicious result shows that this vaccine is to rat and monkey nontoxicity.Anxious malicious result shows that this vaccine carries out subcutaneous injection to rat and mouse, and the rat maximum tolerated dose is greater than 94mg/kg, and the mouse maximum tolerated dose is greater than 141mg/ml.Mutagenicity test result shows that this vaccine does not have mutagenicity.
As seen, this vaccine can produce antibody in animal body, has obvious provide protection, has no side effect, and peace comments the result to show that this vaccine safety is effective.
Sequence table
＜110〉applicant: Zhongqi Biological Pharmaceutical Co., Ltd., Zhejiang
＜120〉recombinant baculovirus and its production and application
＜130〉file number: 071436-I-CP-NZJ
<160>9
<170>PatentIn version 3.3
<210>1
<211>111
<212>DNA
＜213〉silkworm baculovirus
<400>1
atgctactag taaatcagtc ataccaaggc ttcgataaga aacacacaag cgagatggta 60
ggcgctattg ttttatacgt gcttttggcg gcggcgcatt ctgcctttgc g 111
<210>2
<211>1593
<212>DNA
＜213〉avian influenza virus
<400>2
gaaaagaacg ttactgttac acatgcccaa gacatactgg aaaagacaca caacgggaag 60
ctctgcgatc tagatggagt gaaacctctg attttaagag attgtagtgt agctggatgg 120
ctcctcggga acccaatgtg tgacgaattc atcaatgtgc cggaatggtc ttacatagtg 180
gagaaggcca acccagccaa tgacctctgt tacccaggga atttcaacga ctatgaagaa 240
ctgaaacacc tattgagcag aataaaccat tttgagaaaa ttcagatcat ccccaaaagt 300
tcttggtccg atcatgaagc ctcatcaggg gtgagctcag catgtccata ccagggaacg 360
ccctcctttt tcagaaatgt ggtatggctt atcaaaaaga acaatacata cccaacaata 420
aagagaagct acaataatac caaccaggaa gatcttttga tactgtgggg gattcatcat 480
tctaatgatg cggcagagca gacaaagctc tatcaaaacc caaccaccta tatttccgtt 540
gggacatcaa cactaaacca gagattggta ccaaaaatag ctactagatc caaagtaaac 600
gggcaaagtg gaaggatgga tttcttctgg acaattttaa aaccgaatga tgcaatcaac 660
ttcgagagta atggaaattt cattgctcca gaatatgcat acaaaattgt caagaaaggg 720
gactcagcaa ttatgaaaag tgaagtggaa tatggtaact gcaacaccaa gtgtcaaact 780
ccaatagggg cgataaactc tagtatgcca ttccacaaca tacaccctct caccatcggg 840
gaatgcccca aatatgtgaa atcaaacaaa ttagtccttg cgactgggct cagaaatagt 900
cctctaagag aaagaagaag aaaaagagga ctatttggag ctatagcagg gtttatagag 960
ggaggatggc agggaatggt agatggttgg tatgggtacc accatagcaa tgagcagggg 1020
agtgggtacg ctgcagacaa agaatccact caaaaggcaa tagatggagt caccaataag 1080
gtcaactcga tcattgacaa aatgaacact cagtttgagg ccgttggaag ggaatttaat 1140
aacttagaaa ggagaataga gaatttaaac aagaaaatgg aagacggatt cctagatgtc 1200
tggacttata atgctgaact tctggttctc atggaaaatg agagaactct agacttccat 1260
gactcaaatg tcaagaacct ttacgacaag gtccgactac agcttaggga taatgcaaag 1320
gagctgggta acggttgttt cgagttctat cacaaatgtg ataatgaatg tatggaaagt 1380
gtaagaaacg gaacgtatga ctacccgcag tattcagaag aagcaagatt aaaaagagag 1440
gaaataagtg gagtaaaatt ggaatcaata ggaacttacc aaatactgtc aatttattca 1500
acagttgcga gttctctagc actggcaatc atggtggctg gtctatcttt gtggatgtgc 1560
tccaatgggt cgttacaatg cagaatttgc att 1593
<210>3
<211>96
<212>DNA
＜213〉silkworm baculovirus
<400>3
ttcatgtttg gtcatgtagc cacttttgta attgtattta ttgtaatttt atttttgtac 60
tgtatggtta gaaaccgtaa tagtagacaa tattaa 96
<210>4
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>4
gcggatccat gctactagta aatcagtc 28
<210>5
<211>50
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>5
catgtgtaac agtaacgttc ttttccgcaa aggcagaatg cgccgccgcc 50
<210>6
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>6
gaaaagaacg ttactgttac acatgc 26
<210>7
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>7
aatgcaaatt ctgcattgtaacgac 25
<210>8
<211>45
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>8
gtcgttacaa tgcagaattt gcattttcat gtttggtcat gtagc 45
<210>9
<211>35
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>9
gagcggccgc ttaatattgt ctactattac ggttt 35
Claims (8)
1. recombinant baculovirus, this virus is silkworm baculovirus, has the fusion gene that gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene form;
Wherein, described gp64 signal peptide is the sequence shown in the SEQ ID NO.1, and the HA gene is the sequence shown in the SEQ ID NO.2, and gp64 membrane-spanning domain gene is the sequence shown in the SEQ ID NO.3.
2. recombinant baculovirus as claimed in claim 1, this virus are deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2033.
3. method for preparing the described recombinant baculovirus of claim 1, this method comprises:
(a) prepare gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene order respectively, wherein, described gp64 signal peptide is the sequence shown in the SEQ ID NO.1, and the HA gene is the sequence shown in the SEQ ID NO.2, and gp64 membrane-spanning domain gene is the sequence shown in the SEQ ID NO.3;
(b) after prepared gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene order form fusion gene, import in the baculovirus, form recombinant baculovirus.
4. method as claimed in claim 3, wherein earlier gp64 signal peptide, HA gene and gp64 membrane-spanning domain gene order are formed fusion gene gp64HA, again this gp64HA is cloned into and obtains recombinant transfer vector in the transfer vector, make this recombinant transfer vector and baculovirus generation homologous recombination then and obtain recombinant baculovirus.
5. the application of recombinant baculovirus as claimed in claim 1 or 2 in the preparation avian influenza virus vaccine.
6. application as claimed in claim 5, wherein said vaccine are the human vaccine.
7. method for preparing avian influenza vaccine, this method comprise utilizes claim 1 or 2 described recombinant baculovirus inoculation silkworms, and silkworm baculovirus makes HA be showed in its surface by the mode of sprouting, and virus particle is carried out separation and purification.
8. method as claimed in claim 7, this method comprises: utilize claim 1 or 2 described recombinant baculovirus inoculation silkworms, silkworm baculovirus sprouts and makes HA be showed in its surface, broken cell homogenate, the centrifugal removal impurity of multistep, Ultracentrifugation virus particle, the resuspended post precipitation of aseptic lysate, carry out the band centrifugation purification of samples, ultrafiltration desugar and lyophilize.
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| CN102492662B (en) * | 2011-11-14 | 2013-10-09 | 武汉大学 | Method for marking live viral particles in multicolor |
| CN103667348B (en) * | 2012-09-03 | 2015-07-29 | 天津耀宇生物技术有限公司 | A kind of double-tagging recombinant Bombyx mori baculovirus and its preparation method and application |
| CN105779500B (en) * | 2016-04-15 | 2019-08-09 | 华南农业大学 | A method of improving recombinant baculovirus surface display efficiency |
| CN112359053B (en) * | 2020-11-10 | 2022-05-13 | 江苏科技大学 | Fusion gene and application thereof |
| CN114262720B (en) * | 2021-12-27 | 2023-07-25 | 河南兴华生物技术有限公司 | Signal peptide of baculovirus expression system and application thereof |
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| Title |
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| Ding-Gang Yang,等.Avian Influenza Virus Hemagglutinin Display onBaculovirusEnvelope: Cytoplasmic Domain Affects VirusProperties andVaccine Potential. 2007), 15(5.Molecular Therapy15 5.2007,15(5),正文994页左栏第1段、右栏第2-3段,附图1-6. * |
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