CN109280675A

CN109280675A - For enhancing expression cassette, carrier, virus and the vaccine of protein expression

Info

Publication number: CN109280675A
Application number: CN201710598695.1A
Authority: CN
Inventors: 杨昊翰; 罗永村
Original assignee: Bai Sanitary Biotechnology Co Ltd
Current assignee: Bai Sanitary Biotechnology Co Ltd
Priority date: 2017-07-21
Filing date: 2017-07-21
Publication date: 2019-01-29

Abstract

The present invention provide it is a kind of for enhancing the bidirectional promoter expression cassette of protein expression, the bidirectional promoter expression cassette include the first expression cassette and operationally reverse link in the second expression cassette of the first expression cassette.Wherein the first expression cassette sequentially includes: the first bacilliform virus promoter, at least a pulse train, baculoviral GP64 gene order and stop；Wherein the second expression cassette sequentially includes: the second bacilliform virus promoter, transcription factor and terminator.Using can be by different foreign protein great expressions on the mantle of baculoviral, to be applied to the preparation of different subunit vaccines comprising the bidirectional promoter expression cassette expression vector.

Description

For enhancing expression cassette, carrier, virus and the vaccine of protein expression

Technical field

The present invention relates to a kind of expression cassettes, can espespecially reinforce the expression cassette of recombinant protein expression；The present invention also relates to A kind of expression vector espespecially includes aforementioned expression cassette and the expression vector that can reinforce recombinant protein expression；The present invention also relates to A kind of gene recombination baculovirus espespecially includes the gene recombination baculovirus of foregoing expression vectors.The present invention also relates to one The purposes for reinforcing recombinant protein expression is planted, reinforces recombinant protein expression espespecially by foregoing expression vectors to reach Purposes.The present invention also relates to a kind of vaccine comprising gene recombination baculovirus as the aforementioned.

Background technique

It can produce the virus of two kinds of patterns after insect baculovirus infected insect cell, one is budding pattern virus (budded Virion, BV), as extracellular virus (extracellular virus, ECV) or non-Viruses With Inclusion Body (nonoccluded Virus, NOV), another kind buries type viral (occluded body-derived virus, OV) or polyhedrosis virus for envelope (polyhedra-derived virus, PDVs).It is polygonal in nineteen eighty-three Smith and Summer et al. discovery autographa california moth core Polyhedrosis gene in precursor virus (Autographa californica nucleopolyhedrosisvirus, AcMNPV) is deleted It will not influence other viral protein expressions and virus infection ability after removing, and utilize later period of infection promoter p10 and polyhedral body Promoter (polyhedron) is capable of the characteristic of great expression albumen, is produced with insect cell as bioreactor exogenous Albumen.Gene recombination baculovirus expression system is to belong to eukaryotic system, in addition to modifying after mass production extrinsic protein, translation (post-translational modification) and the protein of production have the characteristics that outside bioactivity, most important It is that albumen after expression has good folding configuration, there is high similarity with the albumen of other eukaryocytes, possess proteinogen The characteristic of beginning.

In gene recombination baculovirus expression system, common selected cell is autumn armyworm (Spodoptera Frugiperda pupa time ovary tissue (pupal ovarian tissue) cell) includes two kinds of cells of Sf-9 and Sf-21, Mainly both cells are higher for the sensitivity of AcMNPV；Additionally comprising BTI-TN-5B1-4 cell, (Invitrogen is public The commodity of department are High Five^TM), cell origin is cabbage looper (Trichoplusia ni).As for rhabdovirus system institute Virus, having developed the Strain used at present is AcMNPV and bombyx nuclear polyhedrosis baculovirus (Bombyx mori Nuclear polyhedrosis virus, BmNPV).And difference, AcMNPV advantage are a large amount of using cell between the two Produce extrinsic protein；BmNPV is then suitable for mass production extrinsic protein in larva body.Past research has proven to expression albumen With good immunity, there is the potential for preparing subunit vaccine.Such as: enteric virus71 type (Enterovirus 71) vaccine Viral (avian reovirus) subunit vaccine difficult to understand in (Chung et al., 2010), poultry (Lin et al., 2008) and Avian influenza vaccine (H5N1) vaccine (Wu et al., 2011).Although rhabdovirus system has plurality of advantages, eukaryotic expression Culture medium or additive required for system can all promote unit cost production, and expressing quantity is not so good as Escherichia coli (E.coli), therefore preparation cellularity subunit vaccine or other immune formulations or the factor, do not meet probably business sell it is low Cost requirement.So improving the expression quantity of eukaryotic expression system becomes development priority.

Summary of the invention

In view of commercially available carrier eukaryotic system low output, it is unfavorable be applied to commercially produce the disadvantages of, mesh of the invention Be provide it is a kind of for increasing bidirectional promoter expression cassette (the Dual expression of recombinant protein expression Cassette), the bidirectional promoter expression cassette include the first expression cassette and operationally reverse link in the first expression cassette Second expression cassette.Wherein the first expression cassette sequentially includes: the first bacilliform virus promoter, at least a pulse train (burst Sequence, BS), baculoviral GP64 gene order and stop, and stop includes, but are not limited to simian virus 40 40 polyadenylic acids (40 poly A, SV40 pA of Simian vacuolating virus).Wherein the second expression cassette sequentially wraps It includes: the second bacilliform virus promoter, transcription factor (transcription factor) and terminator；The transcription factor is pole The late transcription factor (very late factor-1, VLF-1)；The terminator includes, but are not limited to herpes simplex virus thymidine Kinases gathers polyadenylation A (herpes simplex virus thymidine kinase polyadenylation, HSV tk pA)。

Preferably, first bacilliform virus promoter includes polyhedrin protein promoter (polyhedron Promoter, Pph).

Preferably, the baculoviral GP64 gene order sequentially include signal peptide (signal peptide, SP), across Diaphragm area (transmembrane domain, TM) and cytosolic domain (cytoplasmic domain, CTD).

Preferably, further including a variety of limitations between the signal peptide and trans-membrane region of the baculoviral GP64 gene order Digestion position (multiple cloning site, MCS).

Preferably, further including the cyclic annular disease of pig between the signal peptide and trans-membrane region of the baculoviral GP64 gene order Poison two type structural housing albumen deletion 41 amino acid of N-terminal [porcine circovirus 2-capsid protein d41, PCV2-Cap(d41)]。

Preferably, second bacilliform virus promoter includes P10 promoter.

Preferably, the quantity of an at least pulse train is one, the pulse train and baculoviral GP64 gene It is further included between sequence the first mutant nucleotide sequence (mutant signal 1, ms1), the pulse train and first mutant nucleotide sequence (BSms1) as shown in SEQ ID No:8.

Preferably, the quantity of an at least pulse train is one, the pulse train and baculoviral GP64 gene It is further included between sequence the second mutant nucleotide sequence (mutant signal 2, ms2), the pulse train and second mutant nucleotide sequence (BSms2) as shown in SEQ ID No:11.

According to the present invention, " expression cassette " is able in referred to herein as by recombinant technique or the nucleotide sequence being synthetically produced Gene expression is carried out in host cell.In the embeddable plastid of expression cassette or chromosome.In general, the expression cassette of expression vector Part is comprising promoter, the nucleotide sequence to be transcribed and gathers polyadenylation.According to the present invention, " bidirectional promoter expression cassette " is in this Place refers to a kind of expression cassette, contained by nucleotide sequence coded two or two or more gene products being transcribed.It is specific and Speech, the second baculovirus promoter of this case second expression cassette of reverse link after the first bacilliform virus promoter of the first expression cassette Son.

The present invention more provides a kind of expression vector comprising bidirectional promoter expression cassette above-mentioned, by expression vector All genes containing GP64 facilitate and target antigen segment are brought to cell membrane.

In addition, expression vector above-mentioned is placed in two pulse trains (i.e. BS2), a pulse in polyhedron promoter downstream Sequence and the first mutant nucleotide sequence BSms1 or a pulse train and the second mutant nucleotide sequence BSms2 etc., reversely construct p10 promoter Expression infection very late transcription factor Ⅴ lf-1, transcription factor positive regulation promoter PphBS2, PphBSms1, PphBSms2, Strengthen albumen translational efficiency.

The present invention provides a kind of gene recombination baculovirus comprising expression vector above-mentioned again, is by table as the aforementioned It is formed up to carrier transfection (transfection) to insect cell.

Preferably, the type of the insect cell includes, but are not limited to Sf-9 (Spodoptera fugiperda) carefully Born of the same parents or Hi-5 (BTI-TN-5B1-4) cell.

The present invention more provides the purposes that a kind of expression vector as the aforementioned is used to enhance protein expression, is that will express load Body is transfected to insect cell.

The present invention provides a kind of vaccine comprising gene recombination baculovirus as the aforementioned again, to produce among receptor Raw immune response.Preferably, the receptor is pig.

The present invention usesBaculovirus Expression System(Invitrogen, Carlsbad, CA, USA) carry out preparation and reorganization baculoviral, while cloning GP64 (comprising SP, TM, CTD domain) gene and making Carrier has protein surface and characteristic is presented.Allogenic gene down is entered between the SP of GP64 and TM again, utilizes baculoviral advanced stage Promoter polyhedron or p10 start downstream gene expression, and the fusion protein after expression is presented on cell membrane with tripolymer And on baculoviral mantle.Then, the present invention is using Sf-9 cell strain as host, will Bacmid DNA that building is completed transfect to The cell preparation and reorganization baculoviral.Finally, the present invention selects Hi-5 cell strain as host, by gene recombination baculovirus sense Host is contaminated in intracellular great expression target protein PCV2-Cap (d41), and GP64PCV2Cap is presented in metainfective cell surface (d41) albumen, then to be rapidly frozen thawing mode lytic cell, a large amount of cell fragments are generated, containing in fragment can be effective The recombinant protein of immune response is induced, and the cell pyrolysis liquid is PCV2 subunit vaccine.

Detailed description of the invention

Fig. 1 is BSms1 and BSms2 genetic fragment of the present invention by Overlapping PCR amplification；Wherein M indicates DNA Marker, BSms1 and BSms2 genetic fragment clip size about 115bp respectively.

Fig. 2 is pFastBac used in the present invention^TMThe schematic diagram of Dual carrier.

Fig. 3 A is the schematic diagram of pBV1 expression vector of the present invention.

Fig. 3 B is the important regulating and controlling segment of pBV1 expression vector of the present invention, is constructed in transcription initial point (+1) upstream Position BS2 about -10bp to -94bp；ATG in section's letter gram (Kozak) sequence is transcription initial point (+1), and archaeal dna polymerase is then past Downstream direction expresses the schematic diagram of albumen such as GP64SP, PCV2-Cap (d41).

Fig. 4 A is the schematic diagram of pBV2 expression vector of the present invention.

Fig. 4 B is the important regulating and controlling segment of pBV2 expression vector of the present invention, is constructed in transcription initial point (+1) upstream Position BS2 about -10bp to -94bp；ATG in Kozak sequence is transcription initial point (+1), and archaeal dna polymerase is then toward downstream direction Express the schematic diagram of albumen such as GP64SP, PCV2-Cap (d41).

Fig. 5 A is the schematic diagram of pBV10 expression vector of the present invention.

Fig. 5 B is that the important regulating and controlling segment of pBV10 expression vector of the present invention is constructed in transcription initial point (+1) upstream Position BS2 about -10bp to -94bp；BSms1 about -10bp to -107bp；ATG in Kozak sequence is transcription initial point (+1), Archaeal dna polymerase is then toward the schematic diagram of the albumen such as downstream direction expression GP64SP, PCV2-Cap (d41).

Fig. 6 A is the schematic diagram of pBV11 expression vector of the present invention.

Fig. 6 B is the important regulating and controlling segment of pBV11 expression vector of the present invention, is constructed in transcription initial point (+1) Swim position BS2 about -10bp to -94bp；BSms2 about -10bp to -107bp；ATG in Kozak sequence be transcription initial point (+ 1), archaeal dna polymerase is then toward the schematic diagram of the albumen such as downstream direction expression GP64SP, PCV2-Cap (d41).

Fig. 7 A is pBV1 expression vector of the present invention, pBV2 the expression vector electrophoretogram after cutting respectively, wherein GP64Cap (d41) genetic fragment meets expected size 885bp, PphEGFPSV40 pA genetic fragment and meets expected size 1176bp；For pBV2 expression vector after cutting, Vlf-1 promoter gene segment meets expected size about 1140bp.

Fig. 7 B is that pBV10 expression vector of the present invention, pBV11 expression vector penetrate the electricity that PCR mode is detected respectively Swimming figure, it is 115bp that wherein the size of pBV10 expression vector, which is the size of 115bp, pBV11 expression vector,.

Fig. 7 C be the present invention by the website NCBI (nucleotide blast) by the BSms1 sequence of pBV10 expression vector (Sbjct) with the comparison chart of sequencing result (Query) sequence.

Fig. 7 D be the present invention by the website NCBI (nucleotide blast) by the BSms2 sequence of pBV11 expression vector (Sbjct) with the comparison chart of sequencing result (Query) sequence.

Fig. 8 is baculovirus vector Bacmid-BD (BD pFastBac of the present invention^TMDual's writes a Chinese character in simplified form), Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11 transfect the visible light to after Sf-9 insect cell 3 days With fluorogram, figure layer scale bar is 200 μm.

Fig. 9 A is baculoviral BD, BV1, BV2, BV10 and BV11 of the present invention with MOI0.1 infection Hi-5 cell (10⁶Total cell) expressed by Cap (d41) protein expression efficiency comparison in difference electrophoretogram；Mock indicates that negative control group does not have Virus.

Fig. 9 B is the digitization histogram that Fig. 9 A is quantitative with software.

Figure 10 A is that virus BV11 of the present invention test-manufactures the line chart for analyzing its survival rate in fermentation tank, wherein Hi-5 Cell survival rate after infection 24 hours declines to a great extent, and continues to 72 hours about 50%.

Figure 10 B is that virus BV11 of the present invention test-manufactures the line chart for analyzing its total number of cells in fermentation tank, wherein always Cell number is then presented to be risen by a small margin, and about slightly increased 40% after 72 hours.

Figure 11 is that virus BV11 of the present invention test-manufactures the histogram for analyzing its expressing quantity in fermentation tank, wherein 1L Fermentation tank can produce 138mg PCV2-Cap (d41) antigen protein.

Specific embodiment

The present invention is used as by following embodiments and illustrates, and the scope of the invention made and technical characteristic is more clear Chu, but it is not construed as the limitation of limitation the scope of the present invention.

Preparation example 1 recombinates plastid

The source 1.DNA

PCV2-Cap used in this case (d41) segment (as shown in SEQ ID NO.1) (Accession number: AY885225,576bp), Vlf-1 segment (as shown in SEQ ID NO.2) (office of Accession number:NC_001623.1 Portion's segment, 1140bp), pulse train (burst sequence) segment (as shown in SEQ ID NO.3), Kozak Sequence-GP64SP-His6 segment (as shown in SEQ ID NO.4), GP64 genetic fragment (GP64TMCTD, such as SEQ ID Shown in NO.5) (local segment of Accession number:NC_001623.1,222bp) all commission Invitrogen progress are entirely Gene chemical synthesis, while by codon modification as the sequence for being suitble to insect cell expression.BSms1 and BSms2 are then with primer counterweight Multiple polymerase chain reaction (primer overlapping PCR) completes segment amplification；Wherein PCV2-Cap (d41) segment is Refer to that the Capsid albumen of Porcine circovirus type2 (porcine circovirus type 2, PCV2) deletes 41 amino acid, more Specifically, this case is the expression protein molecular using PCV2-Cap (d41) segment as the i.e. expression vector of expression cassette.

2.BSms1 polymerase chain reaction

By 1 microlitre (μ l) [concentration be 10 micro- mole concentration (μM)] P10BSms1F1 primer (as shown in SEQ ID No:6), 1 μ l (10 μM) P10BSms1R1 primer (as shown in SEQ ID No:7), 2.5 μ l 10X pfx buffer [50 milli mole concentration (mM)Mg⁺], 2.5 μ l 10X enhancer, 2 μ l dNTP (2.5mM), primer pair, 0.5 μ l pfx DNA polymerase, Deionized water is eventually adding to 25 μ l of total volume, PCR machine is put into and is reacted.It imposes a condition as follows: 95 DEG C of denaturations (denaturation) it carries out 3 minutes, subsequent to be carried out 30 seconds with 95 DEG C, 55 DEG C of adhesive effects (annealing) carry out 40 seconds, and 72 DEG C amplification effect (extension) carries out 15 seconds, carries out 35 circulations altogether；It is finally reacted 5 minutes with 72 DEG C, to obtain BSms1 (as shown in SEQ ID No:8).

3.BSms2 polymerase chain reaction

By 1 μ l (10 μM) P10BSms2F1 primer (as shown in SEQ ID No:9), 1 μ l (10 μM) P10BSms2R1 primer (as shown in SEQ ID No:10), 2.5 μ l (50mM Mg⁺)10X pfx buffer、2.5μl(2.5mM)10X enhancer、2 μ l dNTP, primer pair, 0.5 μ l pfx DNA polymerase, are eventually adding deionized water to 25 μ l of total volume, are put into PCR Machine is reacted.Impose a condition as follows: 95 DEG C of denaturations carry out 3 minutes, and subsequent to be carried out 30 seconds with 95 DEG C, 55 DEG C of bondings are made With carrying out 40 seconds, 72 DEG C amplification effect 15 seconds, carry out 35 circulations altogether；It is finally reacted 5 minutes with 72 DEG C, to obtain BSms2 (such as Shown in SEQ ID No:11).

4.PCR DNA identification and sequencing

The DNA expanded after the completion of PCR is with 1% Ago-Gel (agarose gel) containing SYBR in 1X TAE DNA electrophoresis is carried out in buffer, and is analyzed with UV light as a result, to carry out sequencing after confirmed clip size is errorless again true Recognize.As shown in Figure 1, M indicates DNA marker (Bio-100bp Ladder)；BSms1 expression is amplified through Overlapping PCR BSms1 genetic fragment；BSms2 indicates the BSms2 genetic fragment amplified through Overlapping PCR, and product accords with as the result is shown Close expected clip size about 115bp.

5.DNA purifying and engagement reaction

Plastid and PCR DNA are cut 1 hour with restriction enzyme in 37 DEG C of water baths respectively, then by the DNA of end of reaction in 1.5%agarose gel race glue, which is analyzed, determines its Insert Fragment (insert) and carrier segments (vector) size, then with Clean/Gel Extraction Kit purification and recovery DNA.The adhesive tape cut is added the DNA that target sizes are cut from colloid Binding buffer [100 milligrams (mg): 100 μ l] is placed in 55 DEG C of water baths and carries out colloidal sol, by its liquid after colloid dissolution It is added to spin column, is centrifuged 13000rpm, 30 seconds, remove lower clear liquid, add 700 μ l Wash buffer, be centrifuged 13000rpm 30 seconds, remove lower clear liquid.Later by spin column with 13000rpm idle running 1 minute, it is therefore an objective to steam alcohol It is dry.40 μ l deionized waters (D3W) are added and stand 2 minutes to spin column, 13000rpm is centrifuged 2 minutes, and lower clear liquid body is The DNA of purifying.Molar ratio (insert:vector ratio is 1:3) is calculated, the T4ligase of equivalent is added in 22 DEG C of water Bath reacts 15 minutes.

6. transition acts on (transformation)

This case is acted on using DH5 α bacterial strain as transition.Firstly, DH5 α is taken out from -80 DEG C, it is placed in and slowly thaws on ice, to The mixing of ligation product is added after dissolution half, and is placed 30 minutes on ice, and DNA is enable to be attached at the cell wall and dimension of bacterium Thallus is held under 4 DEG C of states, later place 42 DEG C of two minutes progresss heat shock, will DNA feeding thallus in, finally put again to Hole on Membranes is set within 10 minutes to heal on ice.100 μ l bacterium solutions are taken to be coated in containing antibiotic [containing 50 μ g/ml Ampicillins (ampicillin)] LB agar disk, 37 DEG C are cultivated 16 hours.

7. extraction recombination plastid DNA

White colony after transition effect is shaken with 10ml LB broth (containing 50 μ g/ml ampicillin) in 37 DEG C 250rpm liquid culture 14 hours to 16 hours.It takes bacterium solution to be centrifuged later 3000rpm 10 minutes, finishes removal supernatant wait be centrifuged Liquid carries out plastid extraction using Plasmid Miniprep Kit, is firstly added 200 μ l of R3buffer and fungus block is suspended, then plus Enter 200 μ l of L7buffer gently mixing up and down, egg flower soup shape is presented, and stand 5 minutes to 10 minutes broken bacterium, is eventually adding 200 μ l of N4buffer gently mixing up and down, with 13000rpm centrifugation 10 minutes.Take supernatant to spin column centrifugation 13000rpm 1 minute (Labnet), lower clear liquid is removed, 500 μ l W10buffer are added and are centrifuged 13000rpm 30 seconds, it is clear under abandoning Liquid is added 600 μ l W9buffer and is centrifuged 13000rpm 30 seconds, then dallies 13000rpm 10 minutes and be evaporated alcohol, finally Spin column is put into 50 μ l deionized waters (D3W) of 1.5ml centrifuge tube addition to be stored at room temperature 2 minutes, and 13000rpm is centrifuged 2 minutes, the liquid being collected into was to recombinate plastid.

Preparation example 2 constructs gene recombination baculovirus carrier

1.pFastBac^TMDual carrier

This case uses the pFastBac of Invitrogen exploitation^TMDual carrier, as shown in Fig. 2, including two expression cassettes (expression cassette), one of expression cassette are sequentially p10 promoter, more limitation digestion position (multiple Cloning site, MCS), end termination codon segment be HSV tk pA；Another expression cassette is then sequentially polyhedral body starting Sub (Pph) (sequence is as shown in SEQ ID No:12), MCS, end termination codon segment are SV40 pA.The following table 1 lists each section Characteristic and application thereof.

Table 1, pFastBac^TMThe characteristic and application thereof of each section of Dual carrier

2. constructing pBV1 expression vector

(1) by pFastBac^TMDual carrier with BamHI and HindIII restriction enzyme after 37 DEG C are cut 1 hour, product with Gel/PCR DNA fragments extraction kit purifies DNA as Vector from colloid；GP64TMCTD conduct insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pFastBac^TMDual- GP64TMCTD carrier.

(2) by above-mentioned pFastBac^TMDual-GP64TMCTD carrier cuts 1 in 37 DEG C with Bstz17I and NotI restriction enzyme After hour, product purifies DNA as Vector using Gel/PCR DNA fragments extraction kit from colloid, incites somebody to action PH-BS2GP64SP is constructed to carrier.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pFastBac^TMDual-GP64 carrier.

(3) by pFastBac^TMDual-GP64 carrier with NotI and ApaI restriction enzyme after 37 DEG C are cut 1 hour, product with Gel/PCR DNA fragments extraction kit purifies DNA as Vector from colloid；PCV2-Cap (d41) makees For insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pFastBac^TMDual- GP64Cap (d41) carrier.

(4) by pFastBac^TMDual-GP64Cap (d41) carrier with HindIII restriction enzyme in 37 DEG C cut 1 hour after, Product purifies DNA as Vector using Gel/PCR DNA fragments extraction kit from colloid；PH-EGFP- SV40 pA is as insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete such as Fig. 3 A Shown in pBV1 expression vector, wherein the important regulating and controlling segment of pBV1 as shown in Figure 3B construct in transcription initial point (+1) upstream position Set BS2 about -10bp to -94bp；ATG in Kozak sequence is transcription initial point (+1), and archaeal dna polymerase is then toward downstream direction table Up to albumen such as GP64SP, PCV2-Cap (d41).The following table 2 lists the sequence starting point and terminal and fragment length in each region.

Table 2, pBV1 carrier information

Title	Sequence starting point	Sequence terminal	Fragment length
				Ampicillin	689	1549	861
His6	4829	4846	18
				Cap(d41)	4871	5446	576
EGFP	5931	6647	717
				GP64TMCTD	5465	5554	90
Tn7R	2611	2835	225
				Gentamicin	2902	3435	534
BS2	4619	4702	84
				Kozak	4709	4717	9
SV40 pA	6648	6888	241
				Tn7L	6917	7082	166
SV40 pA	5561	5801	241
				Pph	4478	4606	129
Pph	5802	5930	129
				f1 origin	102	557	456
pUC ori	1694	2367	674
				GP64SP	4718	4828	111
source	1	7164	7164

3. constructing pBV2 expression vector

By aforementioned resulting pBV1 expression vector with SmaI and KpnI restriction enzyme after 37 DEG C are cut 1 hour, product with Gel/PCR DNA fragments extraction kit purifies DNA as Vector from colloid；Vlf-1 conduct insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pBV2 table as shown in Figure 4 A Up to carrier, wherein the important regulating and controlling segment of pBV2 as shown in Figure 4 B is constructed in transcription initial point (+1) upstream position BS2 about -10bp To -94bp；ATG in Kozak sequence is transcription initial point (+1), archaeal dna polymerase then toward downstream direction expression GP64SP, The albumen such as PCV2-Cap (d41).The following table 3 lists the sequence starting point and terminal and fragment length in each region.

Table 3, pBV2 carrier information

Title	Sequence starting point	Sequence terminal	Fragment length
				f1 origin	102	557	456
Ampicillin	689	1549	861
				pUC ori	1694	2367	674
Tn7R	2611	2835	225
				Gentamicin	2902	3435	534
HSV tk pA	3992	4274	283
				Vlf-1	4281	5417	1137
p10	5433	5554	122
				Pph	5555	5683	129
BS2	5696	5779	84
				Kozak	5786	5794	9
GP64SP	5795	5905	111
				His6	5906	5923	18
Cap(d41)	5948	6523	576
				GP64TMCTD	6542	6631	90
SV40 pA	6638	6878	241
				Pph	6879	7007	129
EGFP	7008	7724	717
				SV40 pA	7725	7965	241
Tn7L	7994	8159	166
				source	1	8241	8241

4. constructing pBV10 expression vector

Aforementioned resulting pBV2 expression vector is produced after 37 DEG C are cut 1 hour with restriction enzyme NdeI and SpeI restriction enzyme Object purifies DNA as Vector using Gel/PCR DNA fragments extraction kit from colloid；BSms1 conduct insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pBV10 as shown in Figure 5A Expression vector, wherein the important regulating and controlling segment of pBV10 as shown in Figure 5 B construct in transcription initial point (+1) upstream position BS2 about- 10bp to -94bp；BSms1 about -10bp to -107bp；ATG in Kozak sequence is transcription initial point (+1), and archaeal dna polymerase is then Toward albumen such as downstream direction expression GP64SP, PCV2-Cap (d41).The following table 4 list each region sequence starting point and terminal and Fragment length.

Table 4, pBV10 carrier information

Title	Sequence starting point	Sequence terminal	Fragment length
				f1 origin	102	557	456
Ampicillin	689	1549	861
				pUC ori	1694	2367	674
Tn7R	2611	2835	225
				Gentamicin	2902	3435	534
HSV tk pA	3992	4274	283
				Vlf-1	4281	5417	1137
p10	5433	5554	122
				Pph	5555	5683	129
BS	5696	5737	42
				ms1	5738	5792	55
Kozak	5799	5807	9
				GP64SP	5808	5918	111
His6	5919	5936	18
				Cap(d41)	5961	6536	576
GP64TMCTD	6555	6644	90
				SV40 pA	6651	6891	241
Pph	6892	7020	129
				EGFP	7021	7737	717
SV40 pA	7738	7978	241
				Tn7L	8007	8172	166
source	1	8254	8254

5. constructing pBV11 expression vector

Aforementioned resulting pBV2 expression vector is produced after 37 DEG C are cut 1 hour with restriction enzyme NdeI and SpeI restriction enzyme Object purifies DNA as Vector using Gel/PCR DNA fragments extraction kit from colloid；BSms2 conduct insert.It is subsequent to be carried out engagement reaction, transition effect and recombination plastid screening purifying, to complete pBV11 as shown in Figure 6A Expression vector, wherein the important regulating and controlling segment of pBV11 as shown in Figure 6B construct in transcription initial point (+1) upstream position BS2 about- 10bp to -94bp；BSms2 about -10bp to -107bp；ATG in Kozak sequence is transcription initial point (+1), and archaeal dna polymerase is then Toward albumen such as downstream direction expression GP64SP, PCV2-Cap (d41).The following table 5 list each region sequence starting point and terminal and Fragment length.

Table 5, pBV11 carrier information

Title	Sequence starting point	Sequence terminal	Fragment length
				f1 origin	102	557	456
Ampicillin	689	1549	861
				pUC ori	1694	2367	674
Tn7R	2611	2835	225
				Gentamicin	2902	3435	534
HSV tk pA	3992	4274	283
				Vlf-1	4281	5417	1137
p10	5433	5554	122
				Pph	5555	5683	129
BS	5696	5737	42
				ms2	5738	5792	55
Kozak	5799	5807	9
				GP64SP	5808	5918	111
His6	5919	5936	18
				Cap(d41)	5961	6536	576
GP64TMCTD	6555	6644	90
				SV40 pA	6651	6891	241
Pph	6892	7020	129
				EGFP	7021	7737	717
SV40 pA	7738	7978	241
				Tn7L	8007	8172	166
source	1	8254	8254

Preparation example 3Sf-9 and BTI-TN-5B1-4 (Hi-5) cell culture

This case is Sf-9 (Spodoptera fugiperda) and Hi-5 (BTI-TN-5B1-4) thin using insect cell line Born of the same parents.Training method can use sticking type and floated, use Insect-XPRESS^TMProtein-free Insect Cell Medium is cultivated in 28 DEG C of incubators.About 3 days subcultures of Sf-9 cell are primary；The growth of Hi-5 cell is very fast, can 2 days to 3 days after In generation, is primary.Two kinds of training methods: sticking type is then incubated at flask, so that cell is suspended when cell subculture, then in a manner of beating, 3x10 is taken again⁶Cell number is to new T-75flask.It is floated, it is incubated at 1000mL Erlenmeyer flask and places the concussion of 200mL culture medium 100rpm is cultivated, cell concentration is 1 × 10⁶cells/mL。

Preparation example 4 prepares gene recombination baculovirus

1. constructing gene recombination baculovirus genosome (bacmid)

This preparation example is prepared using the bac-to-bac baculovirus expression system that Invitrogen company is developed Recombinant baculovirus can be screened more rapidly and construct recombinant baculovirus.Bac-to-bac baculovirus expression system is main It is to utilize competent cell (competent cell) (DH10Bac^TM) carry out recombinant baculovirus building, the gene of this bacterial strain Body portion includes baculovirus vector (shuttle vector) and helper plasmid, then has constructed completion for aforementioned PBV1 expression vector, pBV2 expression vector, pBV10 expression vector and pBV11 expression vector are acted on transition be sent into bacterium respectively Genetic recombination is carried out in vivo.Since the above carrier is with pFastBac^TMAs substrate, i.e., each carrier itself contains to be had Dual carrier The gene of Tn7R and Tn7L jumps credit point and Bacmid genosome contains mini-attTn7 sequence, when aforementioned each carrier is respectively fed to When in thallus, helper plasmid will translate out translocase (transposase), using this characteristic target gene with Bacmid is recombinated.After recombinating successfully, the lacZ gene of Bacmid genosome will be destroyed, and it is sweet can not to produce beta galactose Enzyme (β-galatosidase), then with 5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside (X- Gal) and isopropylthio- β-galactoside (IPTG) carries out blue and white screening, places 3 days to 4 days, that is, indigo plant can be observed White colony finally with liquid culture and is extracted recombination bacmid again by white bacterium colony.In this step, it can obtain respectively containing ill Poisonous carrier Bacmid-BD (negative control group), Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11.Though Baculovirus Gene body Bacmid of the present invention has Vlf-1 gene, but considers its rate of rotation efficiency, separately by genetic recombination bar Shape viral vectors pBV2, pBV10 and pBV11 construct expression Vlf-1, regulatory transcription initial point (+1) upstream BS2, BSms1 and BSms2 segment.

2. transition acts on

BV1 recombination Bacmid, BV2 recombination Bacmid, BV10 recombination Bacmid and BV11 recombination that building is completed Bacmid is transferred to DH10Bac in a manner of heat shock respectively^TM(being purchased from Invitrogen company), the step that makes the transition and above-mentioned phase Together, being competent at cell recovery incubation time is 3 hours to 5 hours, and 30 μ l of bacterium solution to 80 μ l is taken uniformly to be applied to containing three kinds of antibiotic LA plate on (50 μ g/ml Kanamycin, 7 μ g/ml Gentamycin, 10 μ g/ml Tetracycline), and be added IPTG (40 μ g/ml) and X-gal (100 μ g/ml) is cultivated 2 days to 3 days, then is selected its white colony and carried out liquid culture.

3. purified genes recombinate Bacmid

By the bacterium colony after Multiplying culture with Plasmid Purification Mini Kit extraction.First by 10mL bacterium solution with 13000rpm centrifugation, removes supernatant, and 300 μ l buffer P1 back dissolving fungus blocks are added, add 300 μ l buffer P2 or more Overturning mixing 10 times, stand room temperature act on 7 minutes, be eventually adding 300 μ l buffer P3 spin upside down after mixing with 13000rpm is centrifuged 10 minutes.It is centrifuged while having been set up QIAGEN-tip 20, is first added with 1mL Buffer QBT to balance pipe In column.It takes supernatant that equilibrated QIAGEN-tip20 is added after centrifugation, is then cleaned in balance tubing string with 2mL Buffer QC Membrane twice, then with 800 μ l Buffer QF the DNA in membrane is made to be extracted to 1.5mL centrifuge tube.500 μ are added L isopropanol or more is uniformly mixed, and with 13000rpm centrifugation 30 minutes, is cleaned with 1mL 70%wash buffer, with Supernatant is outwelled in 13000rpm centrifugation after ten minutes, in air-drying DNA in aseptic operating platform, finally with 20 μ l deionization sterile waters (D3W) back dissolving Bacmid DNA, and with PCR and carry out electrophoretic analysis, confirm extraction recombination Bacmid whether homologous recombination at Function.

4. transfecting (transfection)

By the Sf-9 cell subculture of culture to 6 porose discs, every hole culture 1 × 10⁶Cells/mL, prior to 28 DEG C stationary cultures 1 Hour.The aforementioned recombination bacmid of 2 μ g is taken to be diluted in 200 μ l Insect-XPRESS respectively^TMProtein-free Insect Cell Medium culture medium adds 4 μ l- Insect Transfection Reagent, stands room temperature after mixing 30 minutes.The mixing of 800 μ l culture mediums is added later, is added in 6 porose discs and is acted on 16 hours in 28 DEG C, remove transfection reagent more every other day Renew fresh Insect-XPRESS^TMProtein-free Insect Cell Medium.After persistently cultivating 3 days, Aspirate supernatant It can be obtained viral BV1, BV2, BV10 and BV11 (being all P1 virus).

5. amplified gene recombinant baculovirus

The purpose of virus of proliferation is in order to promote viral power valence, to facilitate the use and virus preservation in subsequent experimental.First By 3 × 10⁶Sf-9 cell kind infects elder brother to T25flask with the virus liquid of MOI 1 (multiplicity of infection) Worm cell is placed in 28 DEG C of incubator cultures 3 days, when cell generates fluorescence, collects supernatant and is centrifuged 10 minutes with 3000rpm, It is centrifuged purpose mainly to separate the Sf-9 cell of suspension, and by 3 generation of virus multiplication, virus is placed in -80 DEG C later and is saved or with 4 DEG C make of short duration preservation.

6. viral power valence measurement

Viral power valence mensuration mode is that quantity is fluoresced after infecting as judgement, reuses Endpoint Dilution Method and measures viral power Valence.15 μ l aforementioned viral liquid are taken to be added separately to 135 μ l Insect-XPRESS^TMProtein-free Insect Cell Medium culture solution, and do serial dilution 10 times, the virus liquid after dilution, 1215 μ l of every pipe extension rate addition (1 × 10⁵Cells/mL Sf-9 cell) takes 100 μ l that 96 porose discs are added after mixing, and 12 holes are added in different dilution ratios, finally 28 DEG C of incubators are placed in, cell can be observed within about 7 days to 10 days and generate fluorescence, count each dilution ratio luciferase expression degree, Viral power valence TCID is calculated with formula (Reed-Muench method)₅₀, multiplied by 0.69 coefficient conversion at pfu (plaque forming units)。

Embodiment 1 identifies pBV1 expression vector, pBV2 expression vector, pBV10 expression vector and pBV11 expression vector

PBV1 expression vector establishment process is detailed in preparation example 2.2, pBV2 expression vector establishment process is detailed in preparation example 2.3, PBV10 expression vector establishment process is detailed in preparation example 2.4, pBV11 expression vector establishment process is detailed in preparation example 2.5.PBV1 table Identification up to carrier is cut with NotI and HindIII；The identification of pBV2 expression vector is cut with SmaI and KpnI；PBV10 expression Carrier is based on pBV2 expression vector, and BS is replaced as BSms1, so identifying that the region segments are true with NdeI and SpeI cutting Recognize, but since segment is too small, is not easy object observing segment, therefore separately detect through PCR mode；PBV11 expression vector is Based on pBV2 expression vector, BS is replaced as BSms2, so identifying the region segments with NdeI and SpeI cutting confirmation i.e. It can.Since segment is too small, it is not easy object observing segment, therefore separately detect through PCR mode.As shown in Figure 7 A, pBV1 expression carries Body after cutting, GP64Cap (d41) genetic fragment meet expected size 885bp, PphEGFPSV40 pA genetic fragment meet it is pre- Phase size 1176bp；For pBV2 expression vector after cutting, Vlf-1 promoter gene segment meets expected size about 1140bp.Such as Shown in Fig. 7 B, pBV10 expression vector is detected through PCR mode, and display meets expected size 115bp, pBV11 expression vector and penetrates PCR mode is detected, and display meets expected size 115bp.As seen in figure 7 c, by the nucleotide blast ratio of the website NCBI To display, the BSms1 sequence (i.e. Sbjct shown in Fig. 7 C) and sequencing result (Query) sequence one of pBV10 expression vector It causes.As illustrated in fig. 7d, display, the BSms2 sequence of pBV11 expression vector are compared by the nucleotide blast of the website NCBI (i.e. Subjct shown in Fig. 7 D) is consistent with sequencing result (Query) sequence.

Embodiment 2 identifies viral BV1, BV2, BV10 and BV11

To recombinate successful baculovirus vector Bacmid-BD, Bacmid-BV1, Bacmid-BV2, Bacmid-BV10 with Bacmid-BV11 is transfected to Sf-9 insect cell (being detailed in preparation example 4), after transfection 3 days with fluorescence microscope, due to carrier On have building EGFP gene, so if having luciferase expression indicate successfully will carrier be sent into cell in start generate recombinant virus. By Fig. 8 the results show that Sf-9 insect cell all issues green fluorescence, therefore, it is determined that viral vectors Bacmid-BD, Bacmid-BV1, Recombinant baculovirus can be all generated after Bacmid-BV2, Bacmid-BV10 and Bacmid-BV11 transfection insect cell.Due to Baculoviral used in the present invention is AcMNPV, therefore the virus being completed can leave cell in such a way that gemmation is raw, because This supernatant contains gene recombination baculovirus.And this recombinant baculovirus is respectively designated as viral BD, virus BV1, virus BV2, virus BV10 and virus BV11.

The protein expression efficiency of 3 gene recombination baculovirus of embodiment

By Hi-5 cell culture in 250mL Erlenmeyer flask (being detailed in preparation example 3), cell density 10⁶Cells/mL, volume 25mL, 28 DEG C, 100rpm shake culture.Hi-5 cell 3 is infected with viral BV1, BV2, BV10, BV11, with MOI 0.1 respectively again It.The subsequent cell by infection is with 1500rpm centrifugation 5 minutes, the PBS of 1/10 fermentation volume of back dissolving.5X sample is taken again Buffer dye is diluted to 1X lytic cell, and is analyzed with protein electrophoresis and Western blot.

When carrying out protein electrophorese, group includes altogether: Hi-5 and FastBacDual negative control group；BV1,BV2,BV10, BV11 experimental group and the albumen of purification standard of E.coli expression are as positive controls.After recombinant cell protein matter is quantified With 5X sample buffer dye mixed diluting at 1X, it is denaturalized protein thoroughly within water-bath 10 minutes with 100 DEG C.Then with 10%SDS-PAGE carries out electrophoresis, injects 1X running buffer, upper layer glue was with 60V race 30 minutes, and lower layer's glue is then with 100V It runs 90 minutes.After colloid is run through, Western blot analysis albumen can be carried out by cutting adhesive tape.

It is complete after SDS-PAGE colloid, the nitrocellulose membrane (Nitrocellulose crossed by methyl alcohol process Membrane, NC membrane) film is covered on SDS-PAGE colloid.(film must be first soaked in by NC film before Transfer buffer 10 minutes).When above-mentioned steps are completed, setting power supply 400mA turns stain 45 minutes.It will turn the good film of stain It puts to blocking buffer (containing 5% skim milk), the effect of rocking at room temperature 1 hour, purpose reduces non-specific antibody In conjunction with.Subsequent addition anti-β actin monoclonal antibody dilution ratio is diluted to 10000X, and anti-His6 monoclonal antibody dilutes Multiplying power is 5000X, the room temperature effect of rocking 1 hour to 2 hours.After antibody combination, wash buffer (0.3%Tween is added 20in PBS buffer) cleaning 3 times, 20 minutes every time, wash away non-specific bond.Antibody (the goat- of dilution 5000X is taken again Anti-mouse (H+L) conjugate HRP, after the effect of rocking at room temperature 50 minutes, with appropriate wash buffer cleaning 3 It is secondary, 20 minutes every time.(Anti-His6 monoclonal antibody has then demarcated HRP enzyme, does not need to additionally incorporate secondary antibody).

By the NC membrane containing albumen, enhanced chemiluminescence (ECL) solution is added (be purchased from Amersham Pharmacia Biotech, New Territories company) is divided into A and B two and manages, respectively plus equivalent A with B is mixed, then takes 200 μ l to be covered on NC membrane after reaction appropriate time, then be placed on NC film with egative film, by shadow As being presented on egative film, imaging is finally carried out with developing fixing agent.

Finally result is quantified with software Photo-CaptMW, each group result data and positive controls work is relatively fixed Amount, calculates its expressing quantity.Data are mainly analyzed with t-test function between different groups, the P being calculated Value is used as statistical foundation, if the expression of P < 0.05 has significant difference；The table of P > 0.05 is data and there was no significant difference, Determine its validity of different data.Through the resulting data of Western blot, all by divided by β-actin data as quantitative basis.

As a result as shown in Fig. 9 A and Fig. 9 B, 4 kinds of gene recombination baculovirus have stable protein expression ability.With His6 Monoclonal antibody can detect Cap (d41) albumen of expected size in 28kDa and 33kDa, wherein separately including Cap (d41)- Two kinds of patterns of gp64 (TM-CTD) and Cap (d41)-gp64 (SP-TM-CTD).From BV1 compared with BV2, additional expression facilitates Regulate and control PCV2-Cap (d41) protein expression, and as different fragments are constructed such as in the end of promoter 3 ': BS, BSms1 and BSms2, hence it is evident that There is variable expression difference.Especially (it is by AT series jump by the specific region modification in the 55bp of the end of p10 promoter 5 ' GC it) constructs again in Pph promoter downstream, wherein optimal expression can be obtained with BV11.It is made through software quantitative analysis standardized Chart shows that expression quantity of Cap (d41) albumen in BV11 dramatically increases 1.3 times compared in BV1 expression quantity.

Embodiment 4 is with fermentation tank pattern analysis protein output

By Hi-5 cell culture in 5L fermentation tank, cell concentration 10⁶Cells/mL, 80rpm, DO maintain 50%, pH value 6.1, defoaming agentF-127 100ppm, 0.01 MOI, real attenuation volume 1000mL were with viral BV11 infection 3 days (such as the following table 6).As a result show such as Figure 10 A, cell survival rate after infection 24 hours declines to a great extent, in infecting 72 hours only about 50%, as shown in Figure 10 B, total cell number then about increased 40% compared to 0 hour.

Table 6,5L fermentation tank transport pattern formula

It is subsequent that part cell concentration is taken to carry out Western blot, while purifying protein carries out one as control for one group of addition Serial dilution does linearisation export function from the band content on 1 μ g, 2 μ g, 4 μ g, 8 μ g, 16 μ g electrophoretograms, finally by target The data of albumen PCV2-Cap (d41) substitute into function and obtain data.Such as the following table 7 and Figure 11 the results show that the quantitative rear total amount of BV11 About 138mg can be obtained.

Table 7, quantitative analysis PCV2-Cap (d41) albumen

Test group	Hi-5	BD	BV11
				Cap protein (milligram)	N.A.	N.A.	138
Cell number	5x10⁷	5x10⁷	5x10⁷

N.A. it indicates without detecting.

Sequence table

<110>hundred hygienic articles Science and Technology Co., Ltd.

<120>for enhancing expression cassette, carrier, virus and the vaccine of protein expression

<130> GAI17TW2572

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 576

<212> DNA

<213>pig second type Orbivirus (Porcine circovirus 2)

<400> 1

aacggaatct tcaacaccag gctgtctagg accttcggat acaccatcaa gaggaccacc 60

gtcaggaccc cctcttgggc cgtcgacatg atgaggttca acatcaacga cttcctcccc 120

cctggtggtg gatctaaccc ccgctctgtc cccttcgagt actacaggat ccgcaaggtc 180

aaggtcgaat tctggccctg ctctcccatc acccagggcg acaggggagt cggatcttct 240

gccgtcatcc tggacgacaa cttcgtcacc aaggccaccg ccctgaccta cgacccctac 300

gtcaactact cttctaggca caccatcact cagcccttct cttaccactc tcgctacttc 360

acccccaagc ccgtcctgga ctctaccatc gactacttcc agcccaacaa caagaggaac 420

cagctgtggc tgaggctgca gaccgccgga aacgtcgacc acgtcggact gggaaccgcc 480

ttcgaaaact ctaagtacga ccaggactac aacatccgtg tcactatgta cgtccagttc 540

cgcgaattca acctgaagga ccccccactg aagccc 576

<210> 2

<211> 1146

<212> DNA

<213>autographa california moth nucleopolyhedrosis virus (Autographa californica nucleopolyhedrovirus)

<400> 2

accatggtca acggattcaa cgtccgcaac gagaacaact tcaactcctg gaagatcaag 60

atccagtctg cccctcgctt cgaatctgtg ttcgacctgg ccaccgacag gcagaggtgc 120

acccccgacg aagtcaagaa caactctctg tggtctaagt acatgttccc caagcccttc 180

gcccccacca ccctgaagtc ttacaagtct cgcttcatca agatcgtcta ctgctctgtc 240

gacgacgtcc acctcgagga catgtcttac tccctggaca aggaattcga ctccatcgaa 300

aaccagaccc tgctgatcga cccccaagaa ctgtgcaggc gcatgctgga actgcgctct 360

gtcaccaagg aaaccctgca gctgaccatc aacttctaca ccaacatgat gaacctgccc 420

gagtacaaga tccccaggat ggtcatgctg cccagggaca aggaactgaa gaacatccgc 480

gaaaaggaaa agaacctgat gctgaagaac gtcatcgaca ccatcctgaa cttcatcaac 540

gacaagatca agatgctgaa ctccgactac gtccacgaca ggggactgat ccgcggagcc 600

atcgtgttct gcatcatgct gggaaccgga atgaggatca acgaagccag gcagctgtcc 660

gtcgacgacc tgaacgtcct gatcaagagg ggaaagctgc actctgacac catcaacctg 720

aagaggaaga ggtctaggaa caacaccctg aacaacatca agatgaagcc cctggaactg 780

gccagggaaa tctactctag gaaccccacc atcctgcaga tctctaagaa cacctctacc 840

cccttcaagg acttccgcag gctgctggaa gaatctggtg tcgaaatgga aaggcccagg 900

tctaacatga tcaggcacta cctgtcctcc aacctgtaca actctggtgt ccccctgcaa 960

aaggtcgcca agctgatgaa ccacgaatct tccgcctcta ccaagcacta cctgaacaag 1020

tacaacatcg gactggacga aacctcctcc gaggaagaaa acaacaacga cgacgacgac 1080

gcccagcaca accgcaactc ttccggatct agcggagaat ctctgctgta ctaccgcaac 1140

gaataa 1146

<210> 3

<211> 84

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

5' non-coding (leading) sequence of<223>two pulse trains

<400> 3

ctgttttcgt aacagttttg taataaaaaa acctataaat atctgttttc gtaacagttt 60

tgtaataaaa aaacctataa atat 84

<210> 4

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>optimize GP64SPHis6 gene

<400> 4

accatggtcc tggtcaacca gtctcaccag ggattcaaca aggaacacac ctctaagatg 60

gtgtccgcca tcgtcctgta cgtcctgctg gccgctgccg cccactctgc cttcgctgct 120

catcaccatc accaccac 138

<210> 5

<211> 90

<212> DNA

<400> 5

tctatgttcc acgtcgtcaa cttcgtcatc atcctgatcg tcatcctgtt cctctactgc 60

atgatccgca accgcaacag gcagtactaa 90

<210> 6

<211> 68

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>forward primer (P10 BSms1 F1)

<400> 6

agtccgcata tgctgttttc gtaacagttt tgtaataaaa aaacctataa atatatacgg 60

acctttaa 68

<210> 7

<211> 61

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>reverse primer (P10 BSms1 R1)

<400> 7

actagtaata attcttattt aactatccgg atccgtgttg ggttgaatta aaggtccgta 60

t 61

<210> 8

<211> 97

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>BSms1 sequence, mutated site ggatccgg

<400> 8

ctgttttcgt aacagttttg taataaaaaa acctataaat atatacggac ctttaattca 60

acccaacacg gatccggata gttaaataag aattatt 97

<210> 9

<211> 68

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>forward primer (P10 BSms2 F1)

<400> 9

agtccgcata tgctgttttc gtaacagttt tgtaataaaa aaacctataa atatatacgg 60

acctttaa 68

<210> 10

<211> 61

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>reverse primer (P10 BSms2 R1)

<400> 10

actagtaata attcttattt aactatccgg atccgcgccg ggttgaatta aaggtccgta 60

t 61

<210> 11

<211> 97

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>BSms2 sequence, mutated site ggcgcggatccgg

<400> 11

ctgttttcgt aacagttttg taataaaaaa acctataaat atatacggac ctttaattca 60

acccggcgcg gatccggata gttaaataag aattatt 97

<210> 12

<211> 129

<212> DNA

<400> 12

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa atattccgga ttattcatac cgtcccacca 120

tcgggcgcg 129

Claims

1. a kind of bidirectional promoter expression cassette (expression cassette) for enhancing protein expression, this is two-way to be opened Promoter cassettes include:

First expression cassette sequentially includes:

First bacilliform virus promoter；

An at least pulse train (burst sequence, BS)；

Baculoviral GP64 gene order；And

Stop comprising simian vacuolating virus 40 polyadenylic acid (Simian vacuolating virus 40polyadenylation, SV40pA)；And

Second expression cassette is that operationally reverse link is in the first expression cassette, and second expression cassette sequentially includes:

Second bacilliform virus promoter；

Transcription factor (transcription factor), wherein the transcription factor is the pole late transcription factor (very late Factor-1, VLF-1)；And

Terminator comprising herpes simplex virus thymidine kinase gathers polyadenylation A (herpes simplex virus thymidine Kinase polyadenylation, HSV tk pA).

2. bidirectional promoter expression cassette according to claim 1, wherein the first bacilliform virus promoter includes polyhedral body egg White matter promoter (polyhedron promoter, Pph).

3. bidirectional promoter expression cassette according to claim 1, wherein baculoviral GP64 gene order sequentially includes letter Number peptide (signal peptide, SP), trans-membrane region (transmembrane domain, TM) and cytosolic domain (cytoplasmic domain, CTD).

4. bidirectional promoter expression cassette according to claim 1, the wherein signal peptide of baculoviral GP64 gene order (SP) a variety of limitation digestion positions (multiple cloning site, MCS) is further included between trans-membrane region (TM).

5. bidirectional promoter expression cassette according to claim 1, the wherein signal peptide of baculoviral GP64 gene order (SP) porcine circovirus two type structural housing albumen (d41) [porcine is further included between trans-membrane region (TM) Circovirus 2-capsid protein d41, PCV2-Cap (d41)].

6. bidirectional promoter expression cassette according to any one of claim 1 to 5, wherein the second bacilliform virus promoter packet Include P10 promoter.

7. bidirectional promoter expression cassette according to claim 6, wherein the quantity of an at least pulse train is one, should The first mutant nucleotide sequence (mutant signal 1, ms1) is further included between pulse train and baculoviral GP64 gene order, it should Pulse train and first mutant nucleotide sequence (BSms1) are as shown in SEQ ID No:8.

8. bidirectional promoter expression cassette according to claim 6, wherein the quantity of an at least pulse train is one, should The second mutant nucleotide sequence (mutant signal 2, ms2) is further included between pulse train and baculoviral GP64 gene order, it should Pulse train and second mutant nucleotide sequence (BSms2) are as shown in SEQ ID No:11.

9. a kind of expression vector comprising the described in any item bidirectional promoter expression cassettes of claim 1 to 8.

10. a kind of gene recombination baculovirus is to transfect (transfection) by expression vector as claimed in claim 9 It is formed to insect cell.

11. gene recombination baculovirus according to claim 10, wherein the type of insect cell includes Sf-9 (Spodoptera fugiperda) cell or Hi-5 (BTI-TN-5B1-4) cell.

It is to turn expression vector 12. a kind of expression vector as claimed in claim 9 is used to enhance the purposes of protein expression It contaminates to insect cell.

13. a kind of vaccine comprising gene recombination baculovirus as claimed in claim 10, to be generated among receptor Immune response.