CN114262720B - Signal peptide of baculovirus expression system and application thereof - Google Patents
Signal peptide of baculovirus expression system and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to a signal peptide of a baculovirus expression system and application thereof. The invention promotes the expression of exogenous proteins in insect cells by utilizing the signal peptide of HA, and can smoothly guide the expressed proteins to the outside of cells.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a signal peptide of a baculovirus expression system and application thereof.
Background
Baculoviruses (also known as polyhedra viruses or granuloviruses) have two types of virions, budding Virions (BV) and polyhedra-derived virions (polyhedron derived virion, PDV). In the viral replication process, BV is produced first, BV nucleocapsid is released from cells in a budding mode after production, other cells are infected again, PDV is produced in a replication later period, PDV nucleocapsid is produced, envelope is obtained in cell nucleus and is coated in protein inclusion body, and the BV nucleocapsid is released to the surrounding environment after cell lysis, and other cells are infected again.
Baculovirus is a vector system widely used in recent years for efficiently expressing foreign proteins, and in vitro gene expression systems include prokaryotic cell systems and eukaryotic cell systems. The prokaryotic cell system is mainly an escherichia coli cell system, is simple and convenient to operate, short in period, high in benefit and stable in expression product, but the molecular weight of the expressed gene is limited, the expressed gene is not excessively large, and some post-translational processing effect on the expression product cannot be performed. Eukaryotic cell systems include expression systems such as COS cells, CHO cells, yeast cells, and insect cells. The unique biological properties of insect cell expression systems (i.e., baculovirus expression systems) are becoming increasingly important.
The insect cell culture and operation are simple and convenient, and the cost is low, so the method is widely used for producing genetic engineering products. However, the Gp64 baculovirus can be generally used in the system at present, the production efficiency of Gp64 membrane protein is not very high, and in some cases, the signal peptide can not better guide target protein to realize efficient secretion, so that a novel efficient secretion guide signal peptide is necessary to be explored for large-scale production.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a signal peptide of a baculovirus expression system and application thereof.
The invention provides application of polypeptide with an amino acid sequence shown in SEQ ID NO. 1 in preparing a signal peptide of a baculovirus expression system foreign protein.
The invention verifies a plurality of signal peptides, including a signal peptide from Melittin, a signal peptide from GP64, a signal peptide from HIV-ENV, and a signal peptide from HA.
Specifically, the amino acid sequence of the Melittin-derived signal peptide is MKFLVNVALVFMVVYISYIYA; the amino acid sequence of the GP 64-derived signal peptide was MVSAIVLYVLLAAAAHSAFA; the amino acid sequence of the HIV-ENV derived signal peptide is MNIKFLVNVALVFMWYISYIYADPINMTGS; the amino acid sequence of the HA-derived signal peptide was MKTIIALSYIFCLVF.
The result shows that the HA-derived signal peptide can promote the expression of exogenous proteins more efficiently.
The present invention also provides a fusion protein comprising: the polypeptide comprises polypeptide with an amino acid sequence shown in SEQ ID NO. 1, a linker and foreign protein with signal peptide removed.
In the invention, the amino acid sequence of the linker is ASGR.
In some embodiments, the signal peptide of the exogenous protein is removed, and in some embodiments, the exogenous protein is Cap protein of a circovirus. According to the invention, the N-terminal of the Cap proteins with different lengths is removed, and experiments show that the expression quantity of the fusion protein is highest after the Cap proteins with 1-41aa amino acids at the N-terminal are removed.
In order to facilitate the purification of the protein, a tag is also attached to the C-terminus of the fusion protein. In the present invention, the tag is a 10×His tag.
In some embodiments, the amino acid sequence of the fusion protein provided by the invention is shown as SEQ ID NO. 2.
The invention also provides nucleic acids encoding the fusion proteins.
In the embodiment of the invention, the coding nucleic acid sequence of the signal peptide or the foreign protein is subjected to codon optimization. The present invention optimizes codons of the encoding nucleic acid according to insect cell SF 9.
Wherein the nucleic acid sequence of the polypeptide shown in SEQ ID NO. 1 is shown in SEQ ID NO. 3. Specifically, the nucleic acid sequence is atgaagaccatcattgctttgagctacattttctgtctggtgttc.
The nucleic acid sequence of the fusion protein shown in SEQ ID NO. 2 is shown in SEQ ID NO. 4.
The invention also provides a plasmid vector, the skeleton vector of which is pFASTbac, comprising the nucleic acid of the invention. In the invention, the pFASTbac vector is a baculovirus shuttle vector. The insertion site of the nucleic acid is between Not I and Xho I.
Hosts transformed or transfected with the plasmid vectors are also provided in the present invention.
The host cell in the invention is sf9 cell or E.coli cell.
The plasmid vector is proliferated and preserved in escherichia coli, and is used for sf9 insect cells.
The preparation method of the fusion protein comprises the step of culturing a host to obtain a culture containing the fusion protein.
The invention promotes the expression of exogenous proteins in insect cells by utilizing the signal peptide of HA, and can smoothly guide the expressed proteins to the outside of cells. Experiments show that the HA signal peptide is fused with Cap protein, and the density of the cultured host cells is 1.5X10 6 The expression quantity is 15-20 mg/ml.
Drawings
FIG. 1 illustrates a build mode;
FIG. 2 shows a plasmid map;
FIG. 3 shows comparison of different signal peptide-directed Cap expression abilities;
FIG. 4 shows secretory expression of HA-guided circular Cap;
FIG. 5 shows wb results for HA-guided circular Cap proteins;
fig. 6 shows a TEM electron microscope image of the circular Cap protein.
Detailed Description
The invention provides a signal peptide of a baculovirus expression system and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The invention firstly uses specific primers to amplify the exocrine signal peptide for guiding HA protein from influenza virus, and simultaneously introduces enzyme cutting sites. Then, specific primers are used for amplifying the circular virus Cap genes from the circular virus genome, and enzyme cutting sites are introduced for subsequent construction. The pFASTbac plasmid stored in a laboratory is used for recovery, expanded, extracted and prepared, and the concentration is detected by an ultraviolet spectrophotometer to meet the requirement of subsequent enzyme digestion.
The mentioned fragments and vectors were digested with NotI and XhoI and the gels were identified by recovery. The resulting fragments were enzymatically ligated using T4 ligase to construct pFASTbac-HA-Cap and pFASTbac-Cap. Transferring the obtained recombinant plasmid into TOP10 clone strain, picking positive clone, sequencing and identifying. DH10Bac E.coli competent cells were removed from the cultures at-80℃and thawed on ice.
Slowly adding 200ng of pFastBac1-gene transfer vector into competent cells, gently mixing, standing on ice for 20min, and then performing heat shock at 42 ℃ for 90s; 8. after rapid ice-rest for 5min, 1ml of nonreactive medium is added to the EP tube and shaken at 37℃for 2h at 200 rpm. 10 mu l of bacterial liquid is smeared on an LB plate containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline, 100mg/ml x-gal and 40mg/ml IPTG, and after the tinfoil is wrapped completely, the bacterial liquid is inversely cultured for 2 to 3 days at 37 ℃.
And (5) observing the density degree of the bacterial colony 12-24 hours after plating, properly adjusting the bacterial liquid amount of the plating according to the result, and re-plating the plate for standby. The white plate can be aligned to the white plate for picking verification 48-60 hours after the plate is coated, and the pure white bacterial colony is a theoretical recombinant bacterial strain. At the moment, 3-5 white monoclonal colonies can be picked according to the color of the colonies, inoculated into 5ml of bacteria liquid which contains 50mg/ml kanamycin, 7mg/ml gentamicin and 10mg/ml tetracycline and shake bacteria at 37 ℃ for 10-12 hours to be completely turbid. And (3) primarily screening positive clone bacteria by bacterial liquid PCR, and primarily screening recombinant baculovirus bacterial liquid with single band by using an M13 primer. And (3) adding the identified correct bacterial liquid into 3-5 mL of a three-antibody culture medium (sodium carbazate, gentamicin and tetracycline), and shaking at 37 ℃ for 12-15 hours. Collecting bacterial liquid, centrifuging at 4000rpm for 5-10 min, and completely discarding clean supernatant.
The cells were completely resuspended with 300. Mu.l of bufferP1 in the plasmid extraction kit. The bufferP2 was added to 300. Mu.l of the plasmid extraction kit and gently mixed and thoroughly lysed. The bufferP3 was added to 300. Mu.l of the plasmid extraction kit and gently mixed and completely neutralized. Centrifuge at 12000rpm for 10min, transfer the supernatant to a 1.5ml sterile centrifuge tube containing 600. Mu.l of pre-chilled isopropyl alcohol, mix well and then cool in a-20deg.C refrigerator for 30min. Centrifuge at 12000rpm at 4℃for 10min, discard supernatant, at which point small white spots are visible attached to the bottom of the tube. 1ml of pre-chilled 75% ethanol was added, rinsed, centrifuged at 12000rpm for 10min, and the supernatant carefully discarded. 1ml of precooled 100% ethanol is added, washed, centrifuged at 12000rpm for 10min, the supernatant carefully discarded, and left to dry in ultra-clean for 20-30 min until the small white spots completely disappear.
30 μl of sterile water is added, the bottom of the tube is gently sprung, the dissolved DNA is allowed to stand for 20-30 min, and then M13 primer is used for identification, in theory, if the target gene is successfully transposed into Bacmid, the size of the amplified product should be (2300 bp+target gene length). The extracted DNA can be used for transfection or stored at-20 ℃ for later use.
Inoculation of six well plates with 0.6X10 6 ～0.8×10 6 Culturing individual cells/2 ml/hole at 27 ℃ for 30-60 min to ensure that the cells are completely adhered, and the overall adhesion is about 70-80%; during this period, bacmid and cellfectnreactive complexes were prepared:
A. 1. Mu.g of recombinant Bacmid (about 10. Mu.l) was gently diluted with 100. Mu.l of incomplete Grace' smooth (no double antibody, FBS); B. before use, the Cellfectingreagent is gently inverted for 5-10 times, fully and uniformly mixed, 6 mu l CellfectinReagent is taken and gently diluted with 100 mu l of incomplete Grace' smooth (without double antibody and FBS);
C. mixing the two dilutions (the total volume is about 210 ul), gently mixing, and incubating for 30-45 min at room temperature;
during preparation of Bacmid and cellfectin reagent complexes, medium in six well plates was aspirated, washed once with 2ml of incomplete Grace' medium (without double antibody, FBS), and the original medium was removed; gently mixing 210 μl of the compound, gently adding into each hole, and gently mixing; six-hole plates are coated by preservative films and then incubated for 100-120 h in a constant temperature incubator at 27 ℃ until the cells show typical signs of virus infection, which indicates that the viruses have proliferated, the transfection is successful, and the P0 generation has been coated. When the cells show signs of infection, transferring the cell supernatant into a 15ml centrifuge tube, centrifuging for 10min at 1000g to remove the cells and large fragments, and filtering with a 0.2 μm filter membrane with low protein binding rate, wherein the titer loss is less than 10%; the virus-containing supernatant was transferred to another sterile capped EP tube and the resulting virus liquid was placed in a refrigerator at 4 ℃ in the dark (short term). If the product is stored for a long time, 1ml of the product is packaged and stored at-80 ℃ in a dark place.
Primary virus titer (P0) was low at 1×10 5 About, the titer after amplification can reach 1X 10 7 ～1×10 8 .15cmDish (1.6X10 total) 7 And b) adding a proper amount of P0 generation virus to amplify the virus. sf9 cells at 2×10 6 Inoculating the bottle into a 500ml cell culture bottle; after the cells grow to the logarithmic phase, inoculating P2 generation virus to infect sf9 cells; cells and supernatant were collected over 120h for detection of recombinant protein expression. The proteins from the above procedure were collected for subsequent SDS-PAGE detection and secreted protein expression.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
1. Plasmid construction
The exogenous gene segment is codon optimized to include the sequence encoding signal peptide, linker and Cap gene segment (the signal peptide is removed).
Wherein, the liquid crystal display device comprises a liquid crystal display device,
the Linker sequence was gctagcggccgc and,
cap gene fragment (1-41 aa amino acids are deleted in the expression sequence) is:
ASGRNGIFNTRLSRTFGYTIKKTTVRTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPLEGSDEVDAGS。
the amino acids of single Cap are:
NGIFNTRLSRTFGYTIKKTTVRTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNP
adding linker+Cap
ASGRNGIFNTRLSRTFGYTIKKTTVRTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNP
HA+LINKER+CAP+ vector sequence
MKTIIALSYIFCLVFASGRNGIFNTRLSRTFGYTIKKTTVRTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPLEGSDEVDAGSHHHHHHHHHH*
The sequence encoding the signal peptide includes:
HA signal peptide:
atgaagaccatcattgctttgagctacattttctgtctggtgttc
gp64 signal peptide:
atggtgagcgcgattgtgctgtatgtgctgctggcggcggcggcgcatagcgcgtttgcg
HIV signal peptide:
atgaacattaaatttctggtgaacgtggcgctggtgtttatgtggtatattagctatatttatgcggatccgattaacatgaccggcagc
MINI signal peptide:
atgaaatttctggtgaacgtggcgctggtgtttatggtggtgtatattagctatatttatgcg
as shown in FIG. 1, the exogenous gene fragment was ligated between NotI and XhoI of pFast-bac1 vector using a double cleavage ligation protocol to obtain pFastBac1-gene, designated pFastBac1-HA-Cap, pFastBac1-Gp64-Cap, pFastBac1-HIV-Cap, pFastBac1-MINI-Cap, respectively.
The vector map is shown in FIG. 2; the obtained recombinant plasmid is transferred into TOP10 clone strain, and positive cloned seeds are picked for sequencing.
2. Baculous strain transformation
2.1 seed at-80℃DH10Bac E.coli competent cells were removed and thawed on ice.
2.2, slowly adding 200ng of the pFastBac1-gene transfer vector into competent cells, gently mixing, standing on ice for 20min, and then performing heat shock at 42 ℃ for 90s;
2.3, after rapid ice-rest for 5min, 1ml of the nonreactive medium is added into the EP tube, and shaking is carried out for 2h at 200rpm at 37 ℃.
2.4, 10 mu l of bacterial liquid is coated on an LB plate containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline, 100mg/ml x-gal and 40mg/ml IPTG, and after the tinfoil is completely wrapped, the bacterial liquid is inversely cultured for 2 to 3 days at 37 ℃.
3. Screening and identification of recombinant Bacmid plasmid
And 3.1, observing the density of the bacterial colony 12-24 hours after plating, properly adjusting the bacterial liquid amount of the plating according to the result, and re-plating the plate for standby.
And 3.2, carrying out spot picking verification on the white plate in 48-60 hours after plating, wherein the pure white bacterial colony is a theoretical recombinant bacterial strain. At the moment, 3-5 white monoclonal colonies can be picked according to the color of the colonies, inoculated into 5ml of bacteria liquid which contains 50mg/ml kanamycin, 7mg/ml gentamicin and 10mg/ml tetracycline and shake bacteria at 37 ℃ for 10-12 hours to be completely turbid.
3.3, primarily screening positive clone bacteria by bacterial liquid PCR, and primarily screening recombinant baculovirus bacterial liquid with single band by using M13 primer.
4. Culture of sf9 cells
SF9 cell culture was cultured using SF900II medium, typically every 3 days in flasks for passaging. Sf9 cells in log phase and with cell viability greater than 95% were used in transfection experiments with a cell doubling cycle of 24h.
5. Sf9 cell cryopreservation method:
the cells were cultured to log phase with viability exceeding 95% and counted to give a storage concentration of 1X 10 7 Ml to 2X 10 7 /ml; preparing a required amount of storage culture solution, adding DMSO to 10% and FBS to 30%, and preserving precooled culture solution at 4 ℃; centrifuging the suspension cells or the monolayer cells for 100 Xg for 5min, and suspending the suspension cells or the monolayer cells to the required density by using a precooled freezing solution; mixing, and packaging into frozen storage tubes; placing the frozen storage tube into a foam box filled with absorbent cotton, and standing at-80 ℃ for 1 day; transferring into a liquid nitrogen tank for preservation.
6. Collection of P1 virus liquid
When the cells show signs of infection, transferring the cell supernatant into a 15ml centrifuge tube, centrifuging for 10min at 1000g to remove the cells and large fragments, and filtering with a 0.2 μm filter membrane with low protein binding rate, wherein the titer loss is less than 10%; the virus-containing supernatant was transferred to another sterile capped EP tube and the resulting virus liquid was placed in a refrigerator at 4 ℃ in the dark (short term). If the product is stored for a long time, 1ml of the product is packaged and stored at-80 ℃ in a dark place.
7. P2 virus amplification and harvesting
Primary virus titer (P0) was low at 1×10 5 About, the titer after amplification can reach 1X 10 7 ～1×10 8 .15cmDish (1.6X10 total) 7 And b) adding an appropriate amount of the P0 generation virus. The amplification of viruses can be calculated by the following formula, the MOI is between 0.01 and 0.1, the inoculum size (nearly 100 times the amplification of viruses collected in 72h infected cells, the quality of viruses collected in more than 120h is lower, the collection time of each virus is different, for example, the collection time is collected in 72h, but the proliferation of viruses is influenced with the lysis of cells.
8. Virus titer detection
120 ten thousand cells/well in six-well plate, static culture for 1h, virus gradient dilution for 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 Removing supernatant from six-hole plate, collecting 10 6 、10 7 、10 8 Three titer viruses were added to six well plates, control group 2 in parallel, incubated for 1h, plaque medium was prepared, and 2mL of plaque medium was added per well; adding neutral red staining solution in the fourth day; plaque numbers were counted 7-10 days to calculate virus titers. The titre of the P2 generation is 10 6 The titre of the generation P3 is 10 7 -10 8 Between them.
9. Recombinant protein expression and purification
sf9 cells at 2×10 6 Inoculating the bottle into a 500ml cell culture bottle; after the cells grow to the logarithmic phase, inoculating P2 generation virus to infect sf9 cells; cells and supernatant were collected over 120h for detection of recombinant protein expression.
9.1, amplifying culture according to the condition method. Centrifuging at 10000g for 20min at 4deg.C, collecting supernatant;
9.2. loading the supernatant onto Ni-IDA-Sepharose CL-6B affinity chromatography column pre-equilibrated with Ni-IDABinding-Buffer at a flow rate of 0.5ml/min by using a low pressure chromatography system;
9.3. flushing with Ni-IDABinding-Buffer at a flow rate of 0.5ml/min until the OD280 value of the effluent reaches a baseline;
9.4. washing with Ni-IDAWAbearing-Buffer (20 mM Tris-HCl,20mM imidazole, 0.15M NaCl, pH 8.0) at a flow rate of 1ml/min until the effluent OD280 reached baseline;
9.5. eluting the target protein with Ni-IDAEluation-Buffer (20 mM Tris-HCl,250mM imidazole, 0.15M NaCl, pH 8.0) at a flow rate of 1ml/min, and collecting the effluent;
9.6. adding the collected protein solution into a dialysis bag, and dialyzing overnight by using PBS (pH 7.4);
9.7. 10% SDS-PAGE analysis showed that expression obtained by fusion of HA signal peptide with Cap protein was optimal for Gp64 signal peptide, HIV signal peptide or MINI signal peptide, and therefore HA signal peptide was used to promote expression of Cap protein (FIG. 3). SDS-PAGE was performed on the eluate after secretory expression of HA-guided circular Cap and BSA standards at different concentrations, and the results are shown in FIG. 4.
10. WesternBlot detection
10.1. Samples of HA signal peptide after secretory expression of the circular Cap were sampled at 0.01. Mu.g.
10.2. After the sample is loaded, the polyacrylamide gel runs out of the laminating adhesive at 90V, and then the voltage is increased to 200V until the electrophoresis is finished.
10.3. After electrophoresis, the gel was removed and transferred to a membrane at a constant pressure of 100V for about 1.5 hours.
10.4. After the end of the electrotransfer, the membranes were removed and washed with PBS 4 times for 5 minutes. Then put in 5% skim milk powder sealing liquid for sealing for 1 hour at 37 ℃.
10.5. The primary antibody was diluted with blocking solution and the membrane was reacted in the primary antibody diluent at 37℃for 1 hour.
10.6. Washing the membrane for 4 times, each time for 5 minutes; the secondary antibody was diluted with a blocking solution containing 5% milk. The membrane was reacted in a secondary antibody at 37℃for 1 hour.
10.7. Film-washing ECL development (figure5). The result shows that the HA signal peptide can well guide the secretion expression of the circular Cap. After cultivation, cell density was 1.5X10 6 The expression quantity is 15-20 mg/ml. The expression parameters are further optimized, and the protein expression quantity is further improved.
In addition, the product was subjected to electron microscopy, and the result showed that the obtained protein was virus-like particles (FIG. 6).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Henan Xinghua Biotechnology Co., ltd
<120> Signal peptide of baculovirus expression system and application thereof
<130> MP21033706
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe
1 5 10 15
<210> 2
<211> 232
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Val Phe Ala
1 5 10 15
Ser Gly Arg Asn Gly Ile Phe Asn Thr Arg Leu Ser Arg Thr Phe Gly
20 25 30
Tyr Thr Ile Lys Lys Thr Thr Val Arg Thr Pro Ser Trp Ala Val Asp
35 40 45
Met Met Arg Phe Asn Ile Asn Asp Phe Leu Pro Pro Gly Gly Gly Ser
50 55 60
Asn Pro Arg Ser Val Pro Phe Glu Tyr Tyr Arg Ile Arg Lys Val Lys
65 70 75 80
Val Glu Phe Trp Pro Cys Ser Pro Ile Thr Gln Gly Asp Arg Gly Val
85 90 95
Gly Ser Ser Ala Val Ile Leu Asp Asp Asn Phe Val Thr Lys Ala Thr
100 105 110
Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr Ser Ser Arg His Thr Ile
115 120 125
Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr Phe Thr Pro Lys Pro Val
130 135 140
Leu Asp Ser Thr Ile Asp Tyr Phe Gln Pro Asn Asn Lys Arg Asn Gln
145 150 155 160
Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn Val Asp His Val Gly Leu
165 170 175
Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp Gln Glu Tyr Asn Ile Arg
180 185 190
Val Thr Met Tyr Val Gln Phe Arg Glu Phe Asn Leu Lys Asp Pro Pro
195 200 205
Leu Asn Pro Leu Glu Gly Ser Asp Glu Val Asp Ala Gly Ser His His
210 215 220
His His His His His His His His
225 230
<210> 3
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atgaagacca tcattgcttt gagctacatt ttctgtctgg tgttc 45
<210> 4
<211> 699
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atgaagacca taatcgctct ctcgtacatt ttttgcctgg tgttcgccag cggccgcaac 60
ggaatcttta atacaagatt gtctcgcacg ttcggataca ccatcaagaa gactaccgtg 120
cgaacaccaa gttgggcggt agacatgatg cgtttcaaca tcaatgattt cctaccccct 180
gggggcgggt cgaaccctcg tagtgtgccc tttgaatact atcgcattcg gaaagtgaaa 240
gttgagtttt ggccgtgttc gcccattacc cagggggacc gtggagtcgg aagtagcgca 300
gtaattttgg acgacaattt cgtgactaaa gcgacggcac ttacatatga cccgtatgtc 360
aactattcca gcaggcacac tataacgcaa cctttctcct accattcacg atatttcaca 420
ccgaagccag tactggattc gaccatcgat tattttcagc cgaacaataa gaggaatcaa 480
ttatggcttc gcctgcagac gacaggtaac gttgaccacg ttggcctagg tactgccttt 540
gagaattcta tatacgatca agagtacaac ataagagtca ccatgtatgt ccagtttcgg 600
gaattcaatt taaaagatcc tcctctcaac ccactcgaag gctctgacga ggttgatgct 660
ggttcacacc atcaccatca tcatcatcac caccactga 699
Claims (2)
1. The use of a signal peptide to promote expression of a foreign protein in an insect cell, wherein the signal peptide is linked to the foreign protein to obtain a fusion protein, and the foreign protein is expressed in the insect cell by a baculovirus vector;
the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1, and the nucleic acid sequence is shown as SEQ ID NO. 3;
the amino acid sequence of the exogenous protein is shown as SEQ ID NO. 2, and the nucleic acid sequence is shown as SEQ ID NO. 4;
the fusion protein sequentially comprises polypeptide with an amino acid sequence shown as SEQ ID NO. 1, linker and foreign protein with signal peptide removed, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 2;
the insect cells are sf9 cells.
2. The use according to claim 1, wherein the baculovirus vector is pFASTbac.
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