CN114262720A

CN114262720A - Signal peptide of baculovirus expression system and application thereof

Info

Publication number: CN114262720A
Application number: CN202111614860.0A
Authority: CN
Inventors: 牛旻; 杨瑞华; 贾宾; 乔洋洋; 单玲玲
Original assignee: Henan Xinghua Biotechnology Co ltd
Current assignee: Henan Xinghua Biotechnology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-04-01
Anticipated expiration: 2041-12-27
Also published as: CN114262720B

Abstract

The invention relates to the technical field of biology, in particular to a signal peptide of a baculovirus expression system and application thereof. The invention utilizes the signal peptide of HA to promote the expression of foreign protein in insect cells and can smoothly guide the expressed protein to the outside of the cells.

Description

Signal peptide of baculovirus expression system and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a signal peptide of a baculovirus expression system and application thereof.

Background

Baculovirus (also known as polyhedrosis virus or granulosis virus) has two types of virions, Budding Virions (BV) and Polyhedrosis Derived Virions (PDV). In the virus replication process, BV is generated firstly, and is released from cells in a budding mode after the BV nucleocapsid is generated, then other cells are infected, PDV is generated in the replication later stage, an envelope is obtained in the cell nucleus after the PDV nucleocapsid is generated, and then the envelope is encapsulated in a protein inclusion body, and is released to the surrounding environment until the cells are cracked, and then other cells are infected.

Baculovirus is a vector system widely used for high-efficiency expression of foreign proteins in recent years, and in vitro gene expression systems comprise a prokaryotic cell system and a eukaryotic cell system. Prokaryotic cell system is mainly colibacillus cell system, it is easy and simple to handle, the cycle is short, the income is big, the expression product is stable, but the molecular weight of the expression gene is limited, should not be too big, and can't carry on some post-translational processing effects to the expression product. 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 gaining increasing attention.

Insect cell culture and operation are simple and convenient, and cost is low, so that the method is widely used for producing gene engineering products. However, the Gp64 baculovirus is commonly used in the system, the production efficiency of the Gp64 membrane protein is not very high, and in some cases, the signal peptide cannot well guide the target protein to realize high-efficiency secretion, so that a novel guide signal peptide for high-efficiency secretion needs to be researched 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 baculovirus expression system and its application.

The invention provides application of polypeptide of an amino acid sequence shown in SEQ ID NO. 1 in preparing a signal peptide of a foreign protein of a baculovirus expression system.

The invention verifies a plurality of signal peptides, including a signal peptide from Melitin, a signal peptide from GP64, a signal peptide from HIV-ENV and a signal peptide from HA.

Specifically, the amino acid sequence of the signal peptide derived from Melitin is MKFLVNVALVFMVVYISYIYA; the amino acid sequence of the GP64 derived signal peptide is MVSAIVLYVLLAAAAHSAFA; the amino acid sequence of the HIV-ENV derived signal peptide is MNIKFLVNVALVFMWYISYIYADPINMTGS; the amino acid sequence of the HA-derived signal peptide is MKTIIALSYIFCLVF.

The result shows that the HA-derived signal peptide can more efficiently promote the expression of the foreign protein.

The present invention also provides a fusion protein comprising: sequentially comprises polypeptide with an amino acid sequence shown in SEQ ID NO. 1, linker and foreign protein with a signal peptide removed.

In the invention, the amino acid sequence of the linker is ASGR.

In the present invention, the signal peptide of the foreign protein is deleted, and in some embodiments, the foreign protein is a Cap protein of a circovirus. According to the invention, the N ends of the Cap proteins with different lengths are removed, and experiments show that the expression quantity of the fusion protein is the highest after the Cap proteins with 1-41aa amino acids at the N ends are removed.

To facilitate purification of the protein, a tag is attached to the C-terminus of the fusion protein. In the invention, the label is a 10 × His label.

In some embodiments, the amino acid sequence of the fusion protein provided by the invention is shown in SEQ ID NO 2.

The invention also provides nucleic acids encoding the fusion proteins.

In embodiments of the invention, the nucleic acid sequence encoding the signal peptide or the foreign protein is codon optimized. The invention optimizes codons of the encoding nucleic acid according to insect cells SF 9.

Wherein the nucleic acid sequence for coding 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 the 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 and comprises the nucleic acid. In the invention, the pFASTbac vector is a baculovirus shuttle vector. The insertion site of the nucleic acid of the invention is between Not I and Xho I.

Also provided in the present invention is a host transformed or transfected with the plasmid vector.

The host cell is sf9 cell or Escherichia coli cell.

The plasmid vector was propagated and stored in E.coli and then introduced into sf9 insect cells.

The preparation method of the fusion protein, and the culture host thereof are cultured to obtain a culture containing the fusion protein.

The invention utilizes the signal peptide of HA to promote the expression of foreign protein in insect cells and can smoothly guide the expressed protein to the outside of the cells. Experiments show that the HA signal peptide is fused with Cap protein, and the density of the cultured host cells is 1.5 multiplied by 10⁶The expression amount is 15-20 mg/L.

Drawings

FIG. 1 illustrates a build mode;

FIG. 2 shows a plasmid map;

FIG. 3 shows a comparison of the ability of different signal peptides to direct Cap expression;

FIG. 4 shows HA-directed secretory expression of circular Cap;

FIG. 5 shows wb results for HA-directed circular Cap protein;

FIG. 6 shows TEM electron micrographs of circular Cap proteins.

Detailed Description

The invention provides a signal peptide of a baculovirus expression system and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention firstly uses specific primers to amplify and guide the secreted signal peptide of HA protein from influenza virus, and simultaneously introduces enzyme cutting sites. Then, specific primers are used for amplifying the Cap gene of the circovirus from the genome of the circovirus, and enzyme cutting sites are introduced for subsequent construction. The pFASTbac plasmid preserved in a laboratory is used for recovering, expanding and then extracting and preparing, and an ultraviolet spectrophotometer is used for detecting the concentration and then meeting the requirement of subsequent enzyme digestion.

The mentioned fragments and vector were cleaved with NotI and XhoI and identified by gel recovery. The resulting fragments were enzymatically ligated using T4 ligase to construct pFASTbac-HA-Cap and pFASTbac-Cap. The obtained recombinant plasmid is transferred into TOP10 clone bacterial strain, positive clone is selected for sequencing and identification. Coli competent cells were thawed on ice at-80 ℃ from DH10Bac e.

Slowly adding 200ng of pFastBac1-gene transfer vector into the competent cells, gently mixing uniformly, standing on ice for 20min, and then thermally shocking for 90s at 42 ℃; 8. standing on ice for 5min, adding 1ml of non-antibiotic culture medium into an EP tube at 37 deg.C, shaking at 200rpm for 2 h. And (3) coating 10 mu l of bacterial liquid on an LB flat plate containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline, 100mg/ml x-gal and 40mg/ml IPTG, and performing inverted culture at 37 ℃ for 2-3 days after the bacterial liquid is completely wrapped by tinfoil.

Observing the density degree of the bacterial colonies 12-24 hours after coating, properly adjusting the amount of the bacteria for coating according to the result, and coating the plates again for later use. And (5) carrying out point picking verification on a white board 48-60 hours after the board is coated, wherein a pure white bacterial colony is a theoretically recombined bacterial strain. At the moment, 3-5 white monoclonal colonies can be selected according to the colony colors and inoculated to 5ml of bacterial liquid containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline and shaking at 37 ℃ for 10-12 h until the bacterial liquid is completely turbid. The PCR of the bacterial liquid is used for primarily screening positive clone bacteria, and M13 primer is used for primarily screening recombinant baculovirus bacterial liquid with single band. And adding the correctly identified bacterial liquid into 3-5 mL of a three-antibody culture medium (sodium bicarbonate, gentamicin and tetracycline), and shaking at 37 ℃ for 12-15 hours. Collecting bacterial liquid, centrifuging at 4000rpm for 5-10 min, and completely discarding the supernatant.

The cells were completely resuspended in 300. mu.l of buffer P1 in the plasmid extraction kit. Add 300. mu.l of buffer P2 from plasmid extraction kit, mix gently and lyse well. Add 300. mu.l of buffer P3 from plasmid extraction kit and mix gently to neutralize completely. Centrifuging at 12000rpm for 10min, transferring the supernatant into a 1.5ml sterile centrifuge tube containing 600. mu.l of pre-cooled isopropanol, mixing, and freezing in a refrigerator at-20 deg.C for 30 min. Centrifuge at 12000rpm for 10min at 4 ℃ and discard the supernatant, at which time small white spots were visible adhering to the bottom of the tube. 1ml of pre-cooled 75% ethanol was added, rinsed, centrifuged at 12000rpm for 10min and the supernatant carefully discarded. Adding 1ml of precooled 100% ethanol, washing, centrifuging at 12000rpm for 10min, carefully discarding the supernatant, and placing in ultra-clean to dry for 20-30 min until small white spots completely disappear.

Adding 30 mu l of sterile water, gently bouncing up the tube bottom, dissolving DNA, standing for 20-30 min, and identifying by using an M13 primer, wherein theoretically, if the target gene is successfully transposed into Bacmid, the size of an amplification product should be (2300bp + the length of the target gene). The extracted DNA may be used for transfection or stored at-20 ℃ for future use.

Inoculation in six well plates 0.6X 10⁶～0.8×10⁶Culturing each cell/2 ml/hole at 27 ℃ for 30-60 min to ensure that the cells are completely attached to the wall, and the whole attachment is about 70-80%; during this period Bacmid and cellfectin reagent complexes were prepared:

A. 1 μ g of recombinant Bacmid (approximately 10 μ l) was gently diluted with 100 μ l of incomplete Grace' smedium (no double antibody, FBS); B. the Cellffectin reagent is turned upside down for 5-10 times before use, so that the Cellffectin reagent is fully and uniformly mixed, 6 mu l of the Cellffectin reagent is taken and diluted by 100 mu l of incomplete Grace' plasmid (without double antibody and FBS);

C. combining the two dilutions (the total volume is about 210ul), gently mixing the dilutions, and incubating the dilutions at room temperature for 30-45 min;

during the preparation of Bacmid and cellfectin reagent complexes, the medium in the six-well plate was aspirated off, washed once with 2ml of incomplete Grace' smedium (without double antibody, FBS) and the original medium was removed; gently mixing the compound containing 210 μ l, gently adding into each well, and gently mixing; and (3) coating the six-hole plate with a preservative film, and then incubating the six-hole plate in a constant temperature incubator at 27 ℃ for 100-120 h until the cells show typical virus infection signs, which indicate that the virus is propagated and the transfection is successful, and the P0 generation is finished. When the cells show infection signs, transferring cell supernatant into a 15ml centrifuge tube, centrifuging for 10min at 1000g to remove cells and large debris, and filtering with a filter membrane with low protein binding rate of 0.2 μm to obtain a titer loss of less than 10%; the virus-containing supernatant was transferred to another sterile capped EP tube and the resulting virus liquid was placed in a 4 ℃ freezer in the dark (short term). If the product is stored for a long time, the product is subpackaged by 1ml and stored at minus 80 ℃ in a dark place.

Primary virus titers (P0) were low at 1X 10⁵About, the titer after amplification can reach 1 × 10⁷～1×10⁸.15 cmDish (total requirement is 1.6X 10)⁷Individual) was added with an appropriate amount of P0 generation virus to amplify the virus. sf9 cells at 2X 10⁶Inoculating in 500ml cell culture bottle; after the cells grow to the logarithmic phase, inoculating the P2 generation virus to infect sf9 cells; cells and supernatant were collected over 120h for detection of recombinant protein expression. And collecting the protein in the process for subsequent SDS-PAGE detection to detect the expression condition of the secreted protein.

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

Exogenous gene fragments were codon optimized, including sequences encoding signal peptides, linker, Cap gene fragments (removal of the signal peptide itself).

Wherein the content of the first and second substances,

the Linker sequence is gctagcggccgc which is,

the Cap gene segment (1-41 aa amino acid removed in the expression sequence) is:

ASGRNGIFNTRLSRTFGYTIKKTTVRTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTTGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPLEGSDEVDAGS。

the amino acids of a 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, a double-enzyme digestion connection scheme is adopted to connect the exogenous gene fragment to the position between Not I and Xho I of a pFast-bac1 vector to obtain pFastBac1-gene which is respectively marked as pFastBac1-HA-Cap, pFastBac1-Gp64-Cap, pFastBac1-HIV-Cap and pFastBac 1-MINI-Cap.

The vector map is shown in FIG. 2; the obtained recombinant plasmid is transferred into TOP10 clone strain, and positive clone is picked for sequencing.

2. Transformation of bacmid strains

And (4) taking out DH10Bac E.coli competent cells at the temperature of between 2.1 and 80 ℃ and unfreezing the cells on ice.

2.2, slowly adding 200ng of pFastBac1-gene transfer vector into the competent cells, gently mixing uniformly, standing on ice for 20min, and then thermally shocking at 42 ℃ for 90 s;

2.3, standing on ice for 5min, adding 1ml of non-antibiotic culture medium into an EP tube at 37 ℃, shaking the strain at 200rpm for 2 h.

2.4, spreading 10 mul of bacterial liquid on an LB plate containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline, 100mg/ml x-gal and 40mg/ml IPTG, and carrying out inverted culture at 37 ℃ for 2-3 days after the complete wrapping of the tin foil.

3. Screening and identification of recombinant Bacmid plasmid

3.1 observing the density degree of the bacterial colonies 12-24 hours after coating, properly adjusting the amount of the bacteria for coating according to the result, and coating the plates again for later use.

3.2, after the plate is coated, the plate can be aligned to a white plate for point picking verification 48-60 hours, and the pure white bacterial colony is a theoretically recombined bacterial strain. At the moment, 3-5 white monoclonal colonies can be selected according to the colony colors and inoculated to 5ml of bacterial liquid containing 50mg/ml kanamycin, 7mg/ml gentamicin, 10mg/ml tetracycline and shaking at 37 ℃ for 10-12 h until the bacterial liquid is completely turbid.

3.3, primarily screening positive clone bacteria by bacteria liquid PCR, and primarily screening recombinant baculovirus bacteria liquid with single bands by using M13 primers.

4. Culture of sf9 cells

SF9 cell cultures were grown in SF900II medium and were typically passaged in flasks every 3 days. Sf9 cells in logarithmic growth phase with cell viability greater than 95% were used for transfection experiments, this cell doubling cycle was 24 h.

5. Sf9 cell cryopreservation method:

the cells were cultured to logarithmic growth phase, the viability exceeded 95%, counted so that the storage concentration was 1X 10⁷From ml to 2X 10⁷Per ml; preparing a required amount of storage culture solution, adding DMSO to 10% and FBS to 30%, and preserving the precooled culture solution at 4 ℃; centrifuging suspended cells or monolayer cells at 100 Xg for 5min, and suspending with pre-cooled frozen stock solution to desired density; mixing, and packaging into freezing tube; placing the freezing tube into a foam box filled with absorbent cotton, and standing at-80 ℃ for 1 day; transferring into liquid nitrogen tank for preservation.

6. Collection of P1 virus liquid

When the cells show infection signs, transferring cell supernatant into a 15ml centrifuge tube, centrifuging for 10min at 1000g to remove cells and large debris, and filtering with a filter membrane with low protein binding rate of 0.2 μm to obtain a titer loss of less than 10%; the virus-containing supernatant was transferred to another sterile capped EP tube and the resulting virus liquid was placed in a 4 ℃ freezer in the dark (short term). If the product is stored for a long time, the product is subpackaged by 1ml and stored at minus 80 ℃ in a dark place.

7. Amplification and harvesting of P2 Virus

Primary virus titers (P0) were low at 1X 10⁵About, the titer after amplification can reach 1 × 10⁷～1×10⁸.15 cmDish (total requirement is 1.6X 10)⁷One) was added with an appropriate amount of P0 passage virus. The virus amplification can be calculated by the following formula, wherein the MOI is between 0.01 and 0.1, the inoculation amount (nearly 100 times of the virus collected in 72h of infected cells is amplified, the virus collected in more than 120h has lower quality, the collection time of each virus is different, for example, the virus is collected in 72h, but the virus proliferation is influenced along with the cell lysis.

8. Virus titer detection

120 million cells per well in a six-well plate, standing and culturing for 1h, and performing gradient dilution on the virus by 10¹、10²、10³、10⁴、10⁵、10⁶、10⁷、10⁸Removing supernatant from six-hole plate, and taking 10⁶、10⁷、10⁸Adding the three titer viruses into a six-well plate, parallelly controlling the six titer viruses in a group 2, incubating for 1h, preparing a plaque culture medium, and adding 2mL of the plaque culture medium into each well; adding neutral red staining solution on the fourth day; the number of plaques was counted for 7-10 days and the virus titer was calculated. Titer at P2 passage 10⁶P3 passage titer at 10⁷-10⁸In the meantime.

9. Recombinant protein expression and purification

sf9 cells at 2X 10⁶Inoculating in 500ml cell culture bottle; after the cells grow to the logarithmic phase, inoculating the P2 generation virus to infect sf9 cells; cells and supernatant were collected over 120h for detection of recombinant protein expression.

9.1, carrying out amplification culture according to the condition method. Centrifuging at 4 deg.C 10000g for 20min, and collecting supernatant;

9.2. loading the supernatant to Ni-IDA-Sepharose CL-6B affinity chromatography column pre-balanced by Ni-IDABinding-Buffer at flow rate of 0.5ml/min by using low pressure chromatography system;

9.3. flushing with Ni-IDABinding-Buffer at flow rate of 0.5ml/min until effluent OD280 value reaches baseline;

9.4. flushing with Ni-IDAWashing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min until the effluent OD280 reached baseline;

9.5. eluting the target protein with Ni-IDAElution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min, and collecting the eluate;

9.6. adding the collected protein solution into a dialysis bag, and dialyzing overnight with PBS (pH 7.4);

9.7. 10% SDS-PAGE analysis showed that the best expression would be obtained for Gp64 signal peptide, HIV signal peptide or MINI signal peptide when HA signal peptide was fused to Cap protein, thus using HA signal peptide to promote Cap protein expression (FIG. 3). SDS-PAGE detection is carried out on the eluate after the secretory expression of the HA-guided circular Cap and BSA standards with different concentrations, and the result is shown in FIG. 4.

10. WesternBlot assay

10.1. Samples after HA signal peptide guided secretion expression of the circular Cap were loaded at 0.01. mu.g.

10.2. After the sample loading is finished, the polyacrylamide gel runs out the laminated gel at 90V, and then the voltage is increased to 200V until the electrophoresis is finished.

10.3. After the electrophoresis was finished, the gel was removed and the membrane was transferred at a constant pressure of 100V for about 1.5 hours.

10.4. After the end of the electrotransfer, the membrane was removed and washed first with PBS 4 times for 5 minutes each. Then placing the mixture in 5% skimmed milk powder sealing solution, and sealing the mixture for 1 hour at 37 ℃.

10.5. The primary antibody was diluted with blocking solution and the membrane was reacted in the primary antibody dilution at 37 ℃ for 1 hour.

10.6. Washing the membrane for 4 times, 5 minutes each time; the secondary antibody was diluted with blocking solution containing 5% milk. The membrane was reacted in a secondary antibody at 37 ℃ for 1 hour.

10.7. Washed film ECL developed (fig. 5). The result shows that the HA signal peptide can well guide the secretory expression of the circular Cap. After cultivation, the cell density was 1.5X 10⁶The expression amount is 15-20 mg/L. Further optimizing the expression parameters and further improving the protein expression.

In addition, electron microscope detection is performed on the expression product, and the result shows that the obtained protein is virus-like particles (figure 6).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Henan Xinghua Biotechnology Ltd

<120> Signal peptide of baculovirus expression System and use 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

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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

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Leu Asn Pro Leu Glu Gly Ser Asp Glu Val Asp Ala Gly Ser His His

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His His His His His His His His

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atgaagacca tcattgcttt gagctacatt ttctgtctgg tgttc 45

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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

1, the application of the polypeptide of the amino acid sequence shown in SEQ ID NO. 1 in preparing the signal peptide of the foreign protein of the baculovirus expression system.

2. A fusion protein, comprising: sequentially comprises polypeptide with an amino acid sequence shown in SEQ ID NO. 1, linker and foreign protein with a signal peptide removed.

3. The fusion protein of claim 2, wherein the foreign protein is a Cap protein of a circovirus.

4. The fusion protein of claim 3, wherein the amino acid sequence is set forth in SEQ ID NO 2.

5. Nucleic acid, which is characterized in that the nucleic acid codes the fusion protein of any one of claims 2 to 4, wherein the nucleic acid sequence of the polypeptide shown in SEQ ID NO. 1 is shown in SEQ ID NO. 3.

6. The nucleic acid of claim 5, wherein the nucleic acid sequence encoding the fusion protein of SEQ ID NO. 2 is set forth in SEQ ID NO. 4.

7. A plasmid vector whose backbone vector is pFASTbac comprising the nucleic acid of claim 5 or 6.

8. A host transformed or transfected with the plasmid vector of claim 7.

9. The host of claim 8, wherein the host cell is a sf9 cell.

10. A method for producing the fusion protein according to any one of claims 2 to 3, characterized in that the host according to claim 8 is cultured to obtain a culture containing the fusion protein according to any one of claims 2 to 3.