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CN110964749A - Method for efficiently expressing foreign protein in baculovirus expression system and application thereof - Google Patents

Method for efficiently expressing foreign protein in baculovirus expression system and application thereof Download PDF

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CN110964749A
CN110964749A CN201811151129.7A CN201811151129A CN110964749A CN 110964749 A CN110964749 A CN 110964749A CN 201811151129 A CN201811151129 A CN 201811151129A CN 110964749 A CN110964749 A CN 110964749A
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田克恭
王同燕
孙进忠
张许科
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Luoyang Huizhong Biotech Co ltd
Pulaike Biological Engineering Co Ltd
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Abstract

The invention provides a method for efficiently expressing exogenous protein in a baculovirus expression system, which clones exogenous protein genes respectively behind a p10 promoter and a polyhedron promoter in a pFastbac Dual plasmid, and inserts a homologous repetitive sequence between a p10 promoter and the polyhedron promoter, so that the amount of the expressed exogenous protein is greatly increased, and the expressed exogenous protein keeps the original immunogenicity. The method for expressing the foreign protein has high efficiency and low cost, and can be used for preparing a large amount of vaccines.

Description

Method for efficiently expressing foreign protein in baculovirus expression system and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and relates to a method for efficiently expressing exogenous protein by baculovirus.
Background
Baculoviruses are a class of double-stranded DNA viruses that infect lepidopteran, hymenopteran, and dipteran insects in nature. The baculovirus genome can be inserted with a large segment of exogenous gene and express exogenous protein, the expressed exogenous protein has better modification processing and good biological activity, thousands of proteins expressed by baculovirus so far exist, and a baculovirus expression system is known as an excellent expression system.
However, how to increase the expression level of foreign proteins expressed by a baculovirus expression system is always the direction of research of scholars at home and abroad, and the improvement of the expression level of foreign proteins is mostly realized by modifying baculovirus. Although the modified baculovirus improves the expression level of the foreign protein to a certain extent, the modification of the baculovirus is specific modification aiming at specific foreign genes, is not suitable for high-efficiency expression of other foreign proteins, has no universality, and needs to modify other foreign genes again when other foreign genes need to be expressed. Therefore, it is desirable to provide a method for highly efficient baculovirus expression for different proteins with a wider application range.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for efficiently expressing a foreign protein in a baculovirus expression system, wherein the method comprises: step (1) inserting the homologous repetitive sequence in AcMNPV genome into P10 promoter (P) of AcMNPVp10) And polyhedrin promoter (P)PH) Obtaining P with homologous repeat sequence inserted betweenp10And PPHA sequence; step (2) inserting one copy of each foreign protein gene into pFastbac Dual plasmid Pp10And PPHThereafter, obtaining a composition comprising said Pp10And foreign protein gene, said PPHAnd exogenous protein gene Dual expression box pFastbac Dual plasmid; step (3) inserting the P obtained in the step (1) with homologous repeat sequences inserted between the Pp10And PPHSequence replacement of adjacent P on the pFastbac Dual plasmid in step (2)p10And PPHObtaining a solution containing said Pp10And foreign protein gene, said PPHAnd a double expression cassette of a foreign protein gene and a plasmid with a homologous repetitive sequence inserted between the double expression cassettes; transforming the plasmid containing the double-copy exogenous gene and the homologous repetitive sequence obtained in the step (3) into a DH10Bac competent cell to obtain a recombinant Bacmid plasmid; step (5) of subjecting the above-mentioned step toTransfecting insect cells by the recombinant Bacmid plasmid obtained in the step (4) to obtain a recombinant baculovirus; and (6) culturing the recombinant baculovirus obtained in the step (5), and harvesting supernatant to obtain the expressed foreign protein.
The method of the invention leads the foreign protein to be expressed with high efficiency by transforming the baculovirus, can obviously improve the expression quantity of the foreign protein, and the expression quantity can be improved by one time or more. The method can be applied to the high-efficiency expression of various exogenous proteins and has wide application range.
In one embodiment of the present invention, in the method for efficiently expressing a foreign protein in a baculovirus expression system, the homologous repeat sequence in step (1) is Hr3, and Hr3 is SEQ id No. 7; or the homologous repetitive sequence is Hr1, and Hr1 is shown in SEQ ID No. 8.
As an embodiment of the present invention, in the method for efficiently expressing a foreign protein in a baculovirus expression system according to the present invention, the sequence of P10 and PH promoter between which the homologous repeat sequence is inserted in step (1) is shown in SEQ ID No.1 or SEQ ID No. 2.
As an embodiment of the present invention, in the method for expressing a foreign protein efficiently in a baculovirus expression system according to the present invention, the foreign protein gene in the step (2) comprises an avian adenovirus Penton protein, an avian adenovirus Fiber-2 protein, an avian egg-loss syndrome virus Penton protein, an avian egg-loss syndrome virus Fiber protein, a chicken infectious bursal disease virus VP2 protein, a porcine circovirus type 3 Cap protein, a porcine circovirus type 2 Cap protein, a porcine pseudorabies virus gB protein, a porcine pseudorabies virus gD protein, a porcine parvovirus VP2 protein, a classical swine fever virus E2 protein, a bovine infectious rhinotracheitis virus gB protein, a bovine infectious rhinotracheitis virus gD protein, a foot and mouth disease virus VP0 protein, a foot and mouth disease virus VP3 protein, a foot and mouth disease virus VP1 protein, a plague virus VP60 protein, a Japanese schistosome GALE protein and a Japanese schistosome Wnt5 protein.
In one embodiment of the present invention, in the method for efficiently expressing a foreign protein in a baculovirus expression system according to the present invention, the insect cell in the step (5) is sf21, sf9, or High five cell.
The invention also relates to the use of said method for producing foreign proteins.
The present invention relates to a method for preparing a vaccine, wherein the method comprises: : step (1) inserting the homologous repetitive sequence in AcMNPV genome into P of AcMNPVp10And PPHObtaining P with homologous repeat sequence inserted betweenp10And PPHA sequence; step (2) inserting one copy of each foreign protein gene into pFastbac Dual plasmid Pp10And PPHThereafter, obtaining a composition comprising said Pp10And foreign protein gene, said PPHAnd exogenous protein gene Dual expression box pFastbac Dual plasmid; step (3) inserting the P obtained in the step (1) with homologous repeat sequences inserted between the Pp10And PPHSequence replacement of adjacent P on the pFastbac Dual plasmid in step (2)p10And PPHObtaining a solution containing said Pp10And foreign protein gene, said PPHAnd a double expression cassette of a foreign protein gene and a plasmid with a homologous repetitive sequence inserted between the double expression cassettes; transforming the plasmid containing the double-copy exogenous gene and the homologous repetitive sequence obtained in the step (3) into a DH10Bac competent cell to obtain a recombinant Bacmid plasmid; transfecting insect cells with the recombinant Bacmid plasmid obtained in the step (4) to obtain a recombinant baculovirus; culturing the recombinant baculovirus obtained in the step (5), and harvesting a supernatant to obtain the expressed foreign protein; and (7) adding an adjuvant into the expressed foreign protein, and emulsifying.
The method for preparing the vaccine has high expressed antigen content, and the expressed antigen protein keeps the original immunogenicity, so the method is suitable for preparing the vaccine in large scale in industry.
In one embodiment of the present invention, in the method for preparing a vaccine according to the present invention, the homologous repeat sequence in step (1) is Hr3, Hr3 is SEQ id No. 7; or the homologous repetitive sequence is Hr1, and Hr1 is shown as SEQ ID No. 8.
As an embodiment of the present invention, in the method for preparing a vaccine according to the present invention, the P between which the homologous repeat sequence is inserted in the step (1) isp10And PPHThe sequence is shown in SEQ ID No.1 or SEQ ID No. 2.
As an embodiment of the present invention, in the method for preparing a vaccine according to the present invention, the foreign protein gene in step (2) includes an avian adenovirus Penton protein, an avian adenovirus Fiber-2 protein, an avian egg-loss syndrome virus Penton protein, an avian egg-loss syndrome virus Fiber protein, an infectious bursal disease virus VP2 protein, a porcine circovirus type 3 Cap protein, a porcine circovirus type 2 Cap protein, a porcine pseudorabies virus gB protein, a porcine pseudorabies virus gD protein, a porcine parvovirus VP2 protein, a porcine pestivirus E2 protein, an infectious rhinotracheitis virus gB protein, an infectious bovine rhinotracheitis virus gD protein, a foot and mouth disease virus VP0 protein, a foot and mouth disease virus VP3 protein, a foot and mouth disease virus VP1 protein, a rabbit plague virus VP60 protein, a japanese blood fluke GALE gal protein, a japanese schistosoma japonicum Wnt5 protein.
As an embodiment of the present invention, in the method for preparing a vaccine according to the present invention, the adjuvant in the step (7) comprises: (1) white oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
the adjuvant content is 5% -70% V/V.
The adjuvant may be present in an amount selected from the group consisting of 5% V/V, 6% V/V, 7% V/V, 8% V/V, 9% V/V, 10% V/V, 15% V/V, 20% V/V, 25% V/V, 30% V/V, 35% V/V, 40% V/V, 45% V/V, 50% V/V, 55% V/V, 60% V/V, 65% V/V, 66% V/V, 67% V/V, and 70% V/V.
The invention also relates to a vaccine composition prepared from the prepared foreign protein, and a subunit vaccine composition is prepared by adding a pharmaceutically acceptable carrier into the foreign protein prepared by the method.
The vaccine compositions of the present invention may be formulated using available techniques, preferably together with a pharmaceutically acceptable carrier. For example, the oil may help stabilize the formulation and additionally serve as a vaccine adjuvant. The oil adjuvant can be natural source or obtained by artificial synthesis. The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponin (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions, or (4) MontanideTMGel。
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The instant and Practical applications of Adjuvants" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on factors such as the ingredients used and the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
For example, the compositions of the present invention may also contain agents such as drugs, immunostimulants (e.g., α -interferon, β -interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.
The subunit vaccine compositions according to the invention may be formulated as oral or non-oral dosage forms.
Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
P10 promoter (P)p10) Is a promoter carried by a baculovirus transfer vector pFastbac Dual plasmid, and the base position is 4338-4459.
Polyhedrin promoter (P)PH) Is a promoter carried by a baculovirus transfer vector pFastbac Dual plasmid, and has the base position of 4478-4606.
In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 baculovirus engineering and preparation of subunit vaccine of Leporis Virus VP60 protein
1. P10 promoter inserted with homologous repeat sequence (P)p10) And polyhedrin promoter (P)PH) Gene synthesis
According to a homologous repeat sequence (Hr 3 for short) in the AcMNPV gene with the accession number NC _001623 on Genbank, the sequence of the homologous repeat sequence is shown as SEQ ID No.7, and the Hr3 gene is inserted into P in a synthetic modep10And PPHThe synthetic sequence p10-Hr3-PH is shown in SEQ ID No. 2.
2. Obtaining the Gene of Leporis Bimaculata Virus VP60
VP60 containing enzyme cutting sites is amplified by using a synthesized VP60 gene I template shown in SEQ ID No.3, the primer sequences are shown in Table 1, and the PCR reaction conditions are shown in Table 2.
TABLE 1 primer sequences
Primer name Primer sequence (5 '-3')
VP60-F(Sma I) TCCCCCGGGATGGAGGGAAAAGCGAGGACAGCACC
VP60-R(Kpn I) GGGGTACCCTACACATACGAGAAACCGTTGGTGG
VP60-F(BamH I) CGGGATCCATGGAGGGAAAAGCGAGGACAGCACC
VP60-R(EcoR I) CGGAATTCCTACACATACGAGAAACCGTTGGTGG
TABLE 2PCR reaction conditions
Figure BDA0001817986810000071
3. Construction of Donor plasmids 1, 2 and 3
The VP60 gene was inserted into P by digestion with BamHI and EcoRI using a commercial pFastbac Dual plasmid as the original vectorPHNext, the plasmid was designated as pFastBac Dual-VP 60-PH; the VP60 gene is inserted into P by cutting SmaI and KpnI endonucleasep10Next, the plasmid was named pFastBac Dual-VP60-p 10; the VP60 gene was inserted into P by digestion with BamHI and EcoRIPHNext, another copy of VP60 gene was inserted into P by SmaI and KpnI endonucleasesp10Next, the plasmid was designated as pFastBac Dual-VP 60X 2.
4. Construction of Donor plasmids 4, 5 and 6
The P10-Hr3-PH sequence synthesized in example 1.1 was digested with SmaI and BamHI, ligated and substituted for the promoter in pFastBacDual-VP60-PH, and the plasmid was named pFastBac Dual-Hr3-VP 60-PH; the promoter in the pFastBac Dual-VP60-p10 is replaced by SmaI and BamHI enzyme digestion and connection, and the plasmid is named as pFastBac Dual-Hr3-VP60-p 10; the promoter in pFastBac Dual-VP 60X 2 was replaced by SmaI and BamHI cleavage and ligation, and the plasmid was named pFastBac Dual-Hr3-VP 60X 2.
5. Acquisition of recombinant Bacmid
Respectively adding plasmids of 2ul of pFastBac Dual-VP60-PH, pFastBac Dual-VP60-p10, pFastBac Dual-VP60 × 2, pFastBac Dual-Hr3-VP60-PH, pFastBac Dual-Hr3-VP60-p10 and pFastBac Dual-Hr3-VP60 × 2 into DH10Bac competent cells, flicking and mixing, incubating on ice for 30min, performing heat shock at 42 ℃ for 45s, adding 400 ul of SOC culture medium at 37 ℃ for 200rpm 4h after incubating on ice for 5min, taking 100 ul of bacterial liquid to coat the liquid on a plate containing IPTG/X-gal/tetracycle/Qingda three-antibody, culturing at 37 ℃ for at least 48h, and taking white single colony to 5ml of liquid culture medium containing kanamycin/tetracycle/Luzhongqingda three-antibody when the cyanobacteria fall, and shaking overnight. Taking 1 mul as a template for PCR identification of bacteria liquid the next day. The PCR identification is correct, the reagent in the Tiangen plasmid miniprep kit is used for extracting the recombinant Bacmid, and the positive Bacmid are respectively named as Bacmid-VP60-PH, Bacmid-VP60-p10, Bacmid-VP 60X 2, Bacmid-Hr3-VP60-PH, Bacmid-Hr3-VP60-p10 and Bacmid-Hr3-VP 60X 2.
6. Acquisition and passage of recombinant baculovirus
Recombinant Bacmid-VP60-PH, Bacmid-VP60-p10, Bacmid-VP 60X 2, Bacmid-Hr3-VP60-PH, Bacmid-Hr3-VP60-p10 and Bacmid-Hr3-VP 60X 2 were transfected into insect cells sf 9. Reference to
Figure BDA0001817986810000081
II Regent instructions for transfection, 72h after transfection, and after cytopathic effect, the harvested cell supernatants were labeled rBac-VP60-PH, rBac-VP60-p10, rBac-VP 60X 2, rBac-Hr3-VP60-PH, rBac-Hr3-VP60-p10 and rBac-Hr3-VP 60X 2. Namely generation P1.
Sf9 cells in logarithmic growth phase were grown at 0.9X 106And (3) inoculating the cell/dish with 10cm of cell culture dish, adding the recombinant baculovirus of P1 generation into the cell culture dish paved with sf9 according to the volume ratio of 1:20-1:40 after the cells are completely attached to the wall, continuously culturing at 27 ℃, harvesting the supernatant marked as the recombinant baculovirus of P2 generation when the cytopathic effect is obvious about 72 hours, wrapping the recombinant baculovirus with tinfoil paper, and keeping the recombinant baculovirus in a refrigerator at 4 ℃ in a dark place for later use. The steps are repeated to inoculate according to the ratio of 1:100-200 to obtain P3 and P4 generation recombinant baculovirus.
7. Expression of proteins
Recombinant virus delivered to P4 was inoculated with Sf9 cells at a volume ratio of 1:100, harvested for about 96h, and centrifuged to obtain supernatant for HA assay. The results are shown in Table 3.
TABLE 3 Effect of different modification patterns of baculovirus on protein expression
HA titre (log2)
rBac-VP60-PH 9
rBac-VP60-p10 9
rBac-VP60×2 10
rBac-Hr3-VP60-PH 11
rBac-Hr3-VP60-p10 11
rBac-Hr3-VP60×2 16
The above results show that the expression level of the foreign protein is improved to some extent regardless of the double expression cassette modification or insertion of the homologous repeat sequence modification, but is far less affected than the combined modification of the double expression cassette and the homologous repeat sequence modification.
This example demonstrates that baculovirus engineered with a combination of dual expression cassettes and homologous repeats can significantly increase the expression level of foreign proteins.
Example 2 Effect of homologous repeat sequences (Hr1) on baculovirus expression
According to a homologous repeat sequence (Hr1 for short) in the AcMNPV gene with the accession number NC _001623 on Genbank, the sequence of the AcMNPV gene is shown as SEQ ID No.8, and the Hr1 gene is inserted into P in a synthetic modep10And PPHThe synthetic sequence P10-Hr1-PH is shown in SEQ ID No. 1. Referring to the method of example 1, Hr3 in the recombinant baculovirus rBac-Hr3-VP 60X 2 was replaced with Hr1 to obtain recombinant baculovirus rBac-Hr1-VP 60X 2.
The recombinant baculovirus rBac-Hr1-VP 60X 2 is inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after about 96h of inoculation, and the supernatant obtained by centrifugation is subjected to HA determination. The HA titer reached 16log 2.
This example demonstrates that the same effect of Hr3 in increasing the expression level of foreign proteins in baculovirus can be achieved by the homologous repeat Hr 1.
Example 3 preparation of subunit vaccine of Leporis Virus VP60 protein
VP60 protein expressed by the recombinant baculovirus rBac-Hr3-VP 60X 2 in example 1 and the recombinant baculovirus rBac-Hr1-VP 60X 2 in example 2 were mixed with an aluminum hydroxide gel adjuvant in proportion, and 1% thimerosal solution was added to the mixture until the stirring was stopped, so that the final concentration was 0.01%. Wherein the vaccine 1 is prepared from protein expressed by recombinant baculovirus rBac-Hr3-VP 60X 2, and the vaccine 2 is prepared from protein expressed by recombinant baculovirus rBac-Hr1-VP 60X 2, which is shown in Table 4.
TABLE 4 Rabbit plague virus VP60 protein subunit vaccine ratio
Components Vaccine 1 Vaccine 2
VP60 protein (HA titer) 9log2 9log2
Aluminum hydroxide gel adjuvant (V/V%) 10％ 10％
Example 4 Leporphobia virus VP60 protein subunit vaccine immunogenicity test
15 healthy susceptible rabbits (RHDV HI antibody titer is lower than 1:4) with the age of 60-90 days (1.5-3.0 kg) are randomly divided into 3 groups, and each group comprises 5 rabbits. The 1 st group and the 2 nd group are respectively a vaccine 1 and a vaccine 2 immunization group prepared in the embodiment 3 of the invention, and the 3 rd group is a challenge control group. The immunization group was injected subcutaneously with 1ml of vaccine, and the challenge control group was injected with an equal amount of PBS. 14 days after inoculation, 1ml of Lepidoptera virus (containing 1000 LD) was injected subcutaneously into each rabbit50) The disease, death and protection were recorded on day 7. The results are shown in Table 5.
TABLE 5 Leporis Virus VP60 protein subunit vaccine immunogenicity test results
Figure BDA0001817986810000101
The results show that the group 3 virus challenge control group is completely dead, while the group 1 and the group 2 immunization group generate better immune protection to the immunized rabbits, and the immune effect is good. The rabbit fever virus VP60 protein subunit vaccine prepared by the method can provide effective immune protection for rabbits.
EXAMPLE 5 preparation of avian adenovirus Fiber-2 protein subunit vaccine
Referring to the method of example 1, a recombinant baculovirus rBac-Hr 3-Fiber-2X 2 containing a homologous repeat sequence Hr3 and an avian adenovirus Fiber-2 gene double expression cassette, a recombinant baculovirus rBac-Fiber-2X 2 containing only an avian adenovirus Fiber-2 gene double expression cassette without a homologous repeat sequence, and an avian adenovirus Fiber-2 gene sequence shown in SEQ. ID NO 4 were constructed.
Recombinant baculovirus rBac-Hr 3-Fiber-2X 2 and rBac-Fiber-2X 2 are inoculated with Sf9 cells according to the volume ratio of 1:100 respectively, the cells are harvested after being inoculated for about 96 hours, and the target protein avian adenovirus Fiber-2 is confirmed to be expressed by Western Blot in the supernatant obtained by centrifugation. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-Fiber-2X 2 protein is 6.0 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr 3-Fiber-2X 2 protein is 10.0 mu g/ml. The recombinant baculovirus constructed by the invention is shown to obviously increase the expression level of the exogenous protein avian adenovirus Fiber-2 protein.
Respectively mixing the recombinant baculovirus rBac-Hr 3-Fiber-2X 2 and the avian adenovirus Fiber-2 protein expressed by the rBac-Fiber-2X 2 with a white oil adjuvant in proportion, and adding a 1% thimerosal solution before stopping stirring to ensure that the final concentration is 0.01%. Wherein the vaccine 3 is prepared by the protein expressed by the recombinant baculovirus rBac-Hr 3-Fiber-2X 2, and the vaccine 4 is prepared by the protein expressed by the recombinant baculovirus rBac-Fiber-2X 2, which is shown in Table 6.
TABLE 6 avian adenovirus Fiber-2 protein subunit vaccine ratios
Group of Vaccine 3 Vaccine 4
Fiber-2 protein (AGP potency) 1:4 1:4
White oil adjuvant (V/V%) 66％ 66％
EXAMPLE 6 avian adenovirus Fiber-2 protein subunit vaccine immunogenicity assay
30 SPF chickens of 21 days old are divided into 3 groups, each group comprises 10 SPF chickens, the 4 th group to the 5 th group are respectively injected with the vaccine 3 and the vaccine 4 prepared in the immunization example 5 through the neck part subcutaneously, the immunization dose is 0.3ml, and the 6 th group is injected with 0.3ml of physiological saline subcutaneously to serve as a challenge control. All test chickens were kept separately, and 21 days after immunization, virus liquid was attacked by intramuscular injection with FAV-HN strain (fowladenovirus, FAV-HN strain (fowlavirus, strain FAV-HN), having a preservation number of CCTCC No. v201609, having a preservation unit of china type culture collection, having a preservation address of university of wuhan, china, a preservation time of 2016, 2 months and 29 days, disclosed in chinese patent application CN107523556A), and observed for 14 days, and the number of diseases, deaths, and protections was recorded. The results are shown in Table 7.
TABLE 7 immunogenicity test results for two avian adenovirus Fiber-2 protein subunit vaccines
Figure BDA0001817986810000121
The results show that the 6 th group of the challenge control group is completely killed, and the 4 th to 5 th groups of the immunization groups generate better immune protection to the immunized chickens, so that the immunization effect is good. The poultry adenovirus Fiber-2 protein subunit vaccine prepared by the method can provide effective immune protection for chicken flocks.
Example 7 preparation of a Fiber protein subunit vaccine of avian egg drop syndrome Virus
By referring to the method of example 1, the recombinant baculovirus rBac-Hr3-Fiber x 2 containing the homologous repeat sequence Hr3 and the avian egg drop syndrome virus Fiber gene double expression cassette, and the recombinant baculovirus rBac-Fiber x 2 containing only the avian egg drop syndrome virus Fiber gene which is shown in SEQ ID NO 5 and does not contain the homologous repeat sequence, are constructed.
Recombinant baculovirus rBac-Hr3-Fiber X2 and rBac-Fiber X2 are respectively inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96 hours, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein avian egg-loss syndrome virus Fiber is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-Fiber X2 protein is 5.0 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr3-Fiber X2 protein is 8.0 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can obviously increase the expression level of foreign protein avian egg-loss syndrome virus Fiber protein.
Respectively and uniformly mixing the poultry egg drop syndrome virus Fiber proteins expressed by the recombinant baculovirus rBac-Hr3-Fiber X2 and the rBac-Fiber X2 with a white oil adjuvant in proportion, and adding a 1% thimerosal solution before stopping stirring to ensure that the final concentration is 0.01%. Wherein the vaccine 5 is prepared by the protein expressed by the recombinant baculovirus rBac-Hr3-Fiber X2, and the vaccine 6 is prepared by the protein expressed by the recombinant baculovirus rBac-Fiber X2, which is shown in Table 8.
TABLE 8 avian egg drop syndrome Virus Fiber protein subunit vaccine ratios
Group of Vaccine 5 Vaccine 6
Fiber protein (HA potency) 1:32 1:32
White oil adjuvant (V/V%) 66％ 66％
Example 8 immunogenicity test of avian egg drop syndrome Virus Fiber protein subunit vaccine
30 SPF chickens of 21 days old were divided into 3 groups of 10 chickens, and the vaccine 5 and the vaccine 6 prepared in immunization example 7 were injected subcutaneously into the neck of 7 th to 8 th groups, respectively, and the immunization dose was 0.5ml, and the 9 th group was injected subcutaneously with 0.5ml of physiological saline as a blank control. All test chickens were kept separately, and 21 days after immunization, each chicken was bled separately, serum was separated, and serum HI antibody titer of avian egg drop syndrome was determined. The results are shown in Table 9.
TABLE 9 immunogenicity test results for two avian egg drop syndrome Virus Fiber protein subunit vaccines
Figure BDA0001817986810000131
The results show that the HI antibody titer of the control group 9 at 21 days after immunization is 0, and the HI antibody titers of the immunization groups 7 to 8 are high and the immunization effect is good. The Fiber protein subunit vaccine of the avian egg drop syndrome virus prepared by the method can provide effective immune protection for chicken flocks.
EXAMPLE 9 preparation of infectious bursal disease Virus VP2 protein
Referring to the method of example 1, the construction of the double expression cassette recombinant baculovirus rBac-Hr3-VP 2X 2 containing the homologous repeat sequence Hr3 and the infectious bursal disease virus VP2 gene and the double expression cassette recombinant baculovirus rBac-VP 2X 2 containing only the infectious bursal disease virus VP2 gene without the homologous repeat sequence and the infectious bursal disease virus VP2 gene sequence is disclosed in the Chinese patent application CN 103849631A.
Recombinant baculovirus rBac-Hr3-VP2 x 2 and rBac-VP2 x 2 are inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96 hours, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein chicken infectious bursal disease virus VP2 is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-VP 2X 2 protein is 5.5 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr3-VP 2X 2 protein is 9.0 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can significantly increase the expression level of foreign protein chicken infectious bursal disease virus VP2 protein.
Example 10 preparation of porcine circovirus type 3 Cap protein subunit vaccine
With reference to the method of example 1, the recombinant baculovirus rBac-Hr3-PCV3-Cap x 2 containing the homologous repetitive sequence Hr3 and the double expression cassette of the porcine circovirus type 3 Cap gene, and the recombinant baculovirus rBac-PCV3-Cap x 2 containing only the porcine circovirus type 3 Cap gene without the homologous repetitive sequence were constructed, and the porcine circovirus type 3 Cap gene sequence was shown in SEQ.ID NO 6.
Recombinant baculovirus rBac-Hr3-PCV3-Cap x 2 and rBac-PCV3-Cap x 2 are respectively inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96 hours, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein porcine circovirus type 3 Cap is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of the rBac-PCV3-Cap X2 protein is 4.2 mu g/ml, and the expression quantity of the rBac-Hr3-PCV3-Cap X2 protein is 7.2 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can significantly increase the expression level of exogenous protein porcine circovirus type 3 Cap protein.
Porcine circovirus type 3 Cap proteins expressed by recombinant baculovirus rBac-Hr3-PCV3-Cap x 2 and rBac-PCV3-Cap x 2 are respectively mixed with a water-soluble adjuvant Gel adjuvant (French Saybox) in proportion. The vaccine 7 is prepared from proteins expressed by recombinant baculovirus rBac-Hr3-PCV3-Cap X2, and the vaccine 8 is prepared from proteins expressed by recombinant baculovirus rBac-PCV3-Cap X2, which are shown in Table 10.
TABLE 10 porcine circovirus type 3 Cap protein subunit vaccine ratios
Components Vaccine 7 Vaccine 8
Cap protein (μ g/ml) 30 30
Gel adjuvant (V/V%) 10％ 10％
Example 11 porcine circovirus type 3 Cap protein subunit vaccine immunogenicity assay
15 healthy piglets which are detected by ELISA for PCV2 and PCV3 antigens and negative antibodies at the age of 28-30 days are randomly divided into 3 groups and 5 groups, and the porcine circovirus type 3 Cap protein subunit vaccine prepared in the immune example 10 is immunized. The 10 th to 11 th groups are immune vaccines 7 to 8 respectively, and the 12 th group is immune and serves as a challenge control group. Each immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of physiological saline. Performing virus attack 28 days after immunization, wherein the virus attack dose is SG strain porcine circovirus (porcine circovirus type 3 SG strain, strain SG), the porcine circovirus type 3 SG is preserved in China center for type culture Collection with the preservation number of CCTCCNO.V201712, the preservation date of 2017, 3 months and 23 days, and the preservation address of Wuhan university in China) 105.0TCID50And/or continuously observing each piglet after the challenge, and judging according to the clinical symptoms, pathological changes and virus detection results of each piglet, wherein the specific results are shown in a table 11.
TABLE 11 immunogenicity test results for two porcine circovirus type 3 Cap protein subunit vaccines
Figure BDA0001817986810000151
The results show that the porcine circovirus type 3 Cap protein subunit vaccine can provide 100 percent (5/5) protection for piglets after being immunized by the piglets, and the control piglets are all attacked after being attacked by the virus. The porcine circovirus type 3 Cap protein subunit vaccine prepared by the method can provide effective immune protection for swinery.
Example 12 preparation of porcine circovirus type 2 Cap protein
Referring to the method of example 1, a recombinant baculovirus rBac-Hr3-PCV2-Cap x 2 containing a homologous repeat sequence Hr3 and a double expression cassette of a porcine circovirus type 2 Cap gene, and a recombinant baculovirus rBac-PCV2-Cap x 2 containing only a porcine circovirus type 2 Cap gene but not containing a homologous repeat sequence were constructed, and the porcine circovirus type 2 Cap gene sequence was disclosed in Chinese patent application CN 101920012A.
Recombinant baculovirus rBac-Hr3-PCV2-Cap x 2 and rBac-PCV2-Cap x 2 are respectively inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96 hours, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein porcine circovirus type 2 Cap is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of the rBac-PCV2-Cap X2 protein of the recombinant baculovirus is 4.5 mu g/ml, and the expression quantity of the rBac-Hr3-PCV2-Cap X2 protein of the recombinant baculovirus is 7.6 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can significantly increase the expression level of exogenous protein porcine circovirus type 2 Cap protein.
Example 13 preparation of porcine pseudorabies Virus gD protein
Referring to the method of example 1, the method for constructing the recombinant baculovirus rBac-Hr 3-gDx 2 containing the homologous repeat sequence Hr3 and the double expression cassette of the porcine pseudorabies virus gD gene and the recombinant baculovirus rBac-gDx 2 containing only the porcine pseudorabies virus gD gene but not the homologous repeat sequence is disclosed in the Chinese patent application CN 104004774A.
Recombinant baculovirus rBac-Hr 3-gDx 2 and rBac-gDx 2 are respectively inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96h, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein porcine pseudorabies virus gD is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-gDx2 protein is 6.0 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr 3-gDx2 protein is 10.0 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can significantly increase the expression level of the exogenous protein porcine pseudorabies virus gD protein.
Example 14 preparation of porcine parvovirus VP2 protein
Referring to the method of example 1, the construction of the double expression cassette recombinant baculovirus rBac-Hr3-PPV-VP2 x 2 containing the homologous repeat sequence Hr3 and the porcine parvovirus VP2 gene and the double expression cassette recombinant baculovirus rBac-PPV-VP2 x 2 containing only the porcine parvovirus VP2 gene without the homologous repeat sequence, and the porcine parvovirus VP2 gene sequence are disclosed in the Chinese patent application CN 103908664A.
Recombinant baculovirus rBac-Hr3-PPV-VP2 x 2 and rBac-PPV-VP2 x 2 are inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after inoculation for about 96 hours, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein porcine parvovirus VP2 is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-PPV-VP 2X 2 protein is 5.5 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr3-PPV-VP 2X 2 protein is 9.2 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can significantly increase the expression level of the foreign protein porcine parvovirus VP2 protein.
Example 15 preparation of classical swine fever Virus E2 protein
Referring to the method of example 1, the double expression cassette recombinant baculovirus rBac-Hr 3-E2X 2 containing the homologous repeat sequence Hr3 and the classical swine fever virus E2 gene and the double expression cassette recombinant baculovirus rBac-E2X 2 containing only the classical swine fever virus E2 gene and the classical swine fever virus E2 gene sequence are constructed and disclosed in Chinese patent application CN 105527442A.
Recombinant baculovirus rBac-Hr 3-E2X 2 and rBac-E2X 2 were inoculated with Sf9 cells at a volume ratio of 1:100, cells were harvested after about 96 hours of inoculation, and the supernatant obtained by centrifugation was subjected to Western Blot to confirm that the target protein classical swine fever virus E2 was expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-E2X 2 protein is 3.8 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr 3-E2X 2 protein is 6.0 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can obviously increase the expression level of the exogenous protein classical swine fever virus E2 protein.
Example 16 preparation of the schistosoma japonicum GALE protein
Referring to the method of example 1, a recombinant baculovirus rBac-Hr3-GALE X2 containing a homologous repeat sequence Hr3 and a schistosoma japonica GALE gene double expression cassette and a recombinant baculovirus rBac-GALE X2 containing no homologous repeat sequence but only a schistosoma japonica GALE gene double expression cassette were constructed, and the schistosoma japonica GALE gene sequence was disclosed in the Chinese patent application CN 102079783A.
Recombinant baculovirus rBac-Hr3-GALE X2 and rBac-GALE X2 are inoculated with Sf9 cells according to the volume ratio of 1:100, the cells are harvested after being inoculated for about 96h, and the supernatant obtained by centrifugation is subjected to Western Blot to confirm that the target protein schistosoma japonicum GALE is expressed. Protein quantification was performed by His affinity chromatography and molecular sieve purification, according to the BCA protein concentration assay kit of Biyunshi. The expression quantity of recombinant baculovirus rBac-GALE X2 protein is 3.6 mu g/ml, and the expression quantity of recombinant baculovirus rBac-Hr3-GALE X2 protein is 6.0 mu g/ml. This shows that the recombinant baculovirus constructed by the invention can obviously increase the expression level of the foreign protein schistosoma japonicum GALE protein.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Puleco bioengineering GmbH
Luoyang Huzhong Biotechnology Co., Ltd
<120> method for efficiently expressing foreign protein in baculovirus expression system and application thereof
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>875
<212>DNA
<213> Autographa california multiple polyhedrosis virus (Autographa california multiple polyhedrosis virus)
<400>1
cccggtaccg catgctatgc atcagctgct agcaccatgg ctcgagatcc cgggtgatca 60
agtcttcgtc gagtgattgt aaataaaatg taatttacag tatagtattt taattaatat 120
acaaatgatt tgataataat tcttatttaa ctataatata ttgtgttggg ttgaattaaa 180
ggtccgtata ctccggaata gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa 240
tgacatcatt tgtataatga catcatcccc tgattgtgtt ttacaagtag aattctatcc 300
gtaaagcgag ttcagttttg aaaacaaatg agtcatacct aaacacgtta ataatcttct 360
gatatcagct tatgactcaa gttatgagcc gtgtgcaaaa catgagataa gtttatgaca 420
tcatccactg atcgtgcgtt acaagtagaa ttctactcgt aaagccagtt cggttatgag 480
ccgtgtgcaa aacatgacat cagcttatga ctcatacttg attgtgtttt acgcgtagaa 540
ttctactcgt aaagcgagtt cggttatgag ccgtgtgcaa aacatgacat cagcttatga 600
gtcataatta atcgtgcgtt acaagtagaa ttctactcgt aaagcttaat agatcatgga 660
gataattaaa atgataacca tctcgcaaat aaataagtat tttactgttt tcgtaacagt 720
tttgtaataa aaaaacctat aaatattccg gattattcat accgtcccac catcgggcgc 780
ggatcccggt ccgaagcgcg cggaattcaa aggcctacgt cgacgagctc actagtcgcg 840
gccgctttcg aatctagagc ctgcagtctc gacaa 875
<210>2
<211>1096
<212>DNA
<213> Autographa california multiple polyhedrosis virus (Autographa california multiple polyhedrosis virus)
<400>2
cccggtaccg catgctatgc atcagctgct agcaccatgg ctcgagatcc cgggtgatca 60
agtcttcgtc gagtgattgt aaataaaatg taatttacag tatagtattt taattaatat 120
acaaatgatt tgataataat tcttatttaa ctataatata ttgtgttggg ttgaattaaa 180
ggtccgtata ctccggaata gatttacgcg tagaattcta cttgtaaagc aagttaaaat 240
aagccgtgtg caaaaatgac atcagacaaa tgacatcatc tacctatcat gatcatgtta 300
ataatcatgt tttaaaatga catcagctta tgactaataa ttgatcgtgc gttacaagta 360
gaattctact cgtaaagcga gtttagtttt gaaaaacaaa tgagtcatca ttaaacatgt 420
taataatcgt gtataaagga tgacatcatc cactaatcgt gcgttacaag tagaattcta 480
ctcgtaaagc gagttcggtt ttgaaaaaca aatgacatca tttcttgatt gtgttttaca 540
cgtagaattc tactcgtaaa gtatgttcag tttaaaaaac aaatgacatc attttacaga 600
tgacatcatt tcttgattat gttttacaag tagaattcta ctcgtaaagc aagtttagtt 660
ttaaaaaaca aatgacatca tctcttgatt atgttttaca agtagaattc tactcgtaaa 720
gcgagtttag ttttgaaaaa caaatgacat catctcttga ttatgtttta caagtagaat 780
tctactcgta aagcgagttt agttttcaaa aacaaatgac atcatccctt gatcatgcgt 840
tacaagtaga attctactcg taaagcttaa tagatcatgg agataattaa aatgataacc 900
atctcgcaaa taaataagta ttttactgtt ttcgtaacag ttttgtaata aaaaaaccta 960
taaatattcc ggattattca taccgtccca ccatcgggcg cggatcccgg tccgaagcgc 1020
gcggaattca aaggcctacg tcgacgagct cactagtcgc ggccgctttc gaatctagag 1080
cctgcagtct cgacaa 1096
<210>3
<211>1740
<212>DNA
<213> Lepidemic Virus (Rabbit viral hemorrhagic disease)
<400>3
atggagggaa aagcgaggac agcaccacag ggcgaagccg caggcaccgc aaccacagcg 60
agcgtaccag ggaccacaac cgatgggatg gaccccggtg ttgtggctgc cacatccgtc 120
gtaacggccg agaactcttc cgcatcagtc gctaccgccg gaatcggtgg acctccccag 180
caagtagatc agcaagaaac ctggcgcact aacttctact acaacgacgt tttcacttgg 240
agtgtggcag atgcgcctgg ttctatcctt tacactgttc agcactcacc tcaaaacaat 300
cccttcacag ctgtgttgtc gcagatgtac gctggatggg ccggcgggat gcaattcaga 360
tttatcgtcg ctggttcagg agtattcggt ggaaggctgg ttgcagctgt tatcccaccg 420
ggtatcgaga ttggccccgg tttggaagtc cgccagttcc cacacgttgt gatcgacgct 480
agatctcttg agcctgtaac aattacgatg ccagacttaa gaccgaacat gtaccatcct 540
acaggagatc ccggccttgt tccaaccttg gtcctttccg tatataacaa tctcatcaac 600
cccttcggcg gttctaccaa tgctattcag gtcacagtag agacgaggcc ttctgacgat 660
ttcgagttcg ttatgatccg agctccctca tcgaagactg tggactcaat ttcgccagcc 720
ggtctcctga ccactcctgt cctcacagga gtaggcaacg ataatcgttg gaacggccag 780
atcgttggtc tgcaacctgt gcccggtgga ttctcaacct gcaatcgtca ctggaacttg 840
aatggatcta cttacggctg gagcagtccc cgctttgcag acatcgatca tcgtcgcggt 900
tctgccagtt actctggcaa caattccact aacgtgctcc agttctggta tgctaacgcc 960
ggttccgcta tagataatcc catctcacaa gttgccccgg acggattccc tgatatgtcg 1020
ttcgtgccat ttaacagccc taatatcccc acagctggtt gggttggatt cggcgggatt 1080
tggaactcaa acaatggtgc tcctgctgcc acaacggtgc aggcctacga actaggtttc 1140
gcaaccggag cgcccaacaa tttgcagcca accactaaca cctcgggtgc tcaaactgtc 1200
gccaagagca tctacgctgt cgtaaccggt actaaccaga atccaacagg actgttcgtc 1260
atggcctccg gcgtaatctc aacccctaac gcatctgcgg tcacatacac gccacaaccg 1320
gaccgtattg taacaacgcc aggtacccca gctgctgctc cagtcggaaa aaacactccc 1380
atcatgttcg cttccgttgt gagaaggact ggtgacgtta acgctgctgc tggctcgacc 1440
aatggtactc agtacggtac tggatctcaa cctctccccg tgacaatcgg cttatcccta 1500
aacaattact cttctgctct tatgccagga cagttcttcg tgtggcaatt aacattcgcc 1560
agtggattta tggagattgg cctgagcgtc gacggttact tctatgcagg cacaggtgct 1620
tccaccactc tcattgacct gaccgaattg atagatgttc gacctgtggg tccacggccg 1680
tcaaagtcca cgctcgtctt caatctgggc ggcaccacca acggtttctc gtatgtgtag 1740
<210>4
<211>1440
<212>DNA
<213> avian adenovirus (avidenovirus)
<400>4
atgctccgag cccctaaaag aagacattcc gaaaacgggc agcccgagac tgaagcggga 60
ccttccccgg ctccaatcaa gcgcgcgaaa cgcatggtga gagcatccca gcttgacctg 120
gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttgggc 180
ggctccggac ccctagtgga ccagggcggt cagcttacgc tcaacgtcac cgatcccatc 240
atcatcaaga acagatcggt ggacttggcc cacgatccca gtctcgatgt caacgcccaa 300
ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360
gacgtcaagg tcgacggagt aaccgtgatg gtcaacgatg actgggaact ggccgtaaaa 420
gtcgacccgt ccggcggatt ggattccact gcgggcggac tgggggtcag cgtggacgac 480
accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540
gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600
accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccggcagt 660
tcgggagtgg gcgtttccgt ggatgaaagc ctagagattg tcaacaacac gctggaagtg 720
aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780
agcaataccc tggcggtgac cgcgggcgct ttgaccgtag taggaggggg aagcgtctcc 840
acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgatc 900
gtcaattcca gctcgcaccc cttctcttgt gcctactacc ttcaacagtg gaacgtacaa 960
gggctccttt ttacctccct ctacgtgaaa ctggacagca ccaccatggg gactcgccct 1020
ggggacaaca gctccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080
caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc cgccctcgcg 1140
gactttgaac ccatggccaa taggagcgtg tccagcccat ggacgtactc ggccaatgca 1200
tactatcaac catccagcgg agaattccaa gtgttcaccc cggtggtaac gggtgcctgg 1260
aacccgggaa acatagggat ccgcgtcctc ccagtgccgg ttacggcctc tggagaccgc 1320
tacacccttc tatgctacag tttgcagtgc acgaactcga gcatttttaa tccagccaac 1380
agcggaacta tgatcgtggg acccgtgctc tacagctgtc cagcagcctc cgtcccgtaa 1440
<210>5
<211>1932
<212>DNA
<213> avian Egg Drop syndrome Virus (Egg Drop syndrome Virus)
<400>5
atgaagcgac tacggttgga ccctgatcct gtttatccct tcgggacgag cgagacgatc 60
ccaatgcctc cgttcatcga agctgggtca ggtctagcag taaatggact gcagctttat 120
ataacagctc aagctccggt gggcttcacc aacaaagctg taacattaaa atatggagat 180
ggattggaag taaatgaaaa tggagaactc atagctacgg cttcttcggc agtaaagcca 240
ccactccatt ttgatagggg ttatatagtg ttaaatcttc aggatccatt gggtgttatt 300
gatgggaagc ttggggtcaa gttaggccct ggggttcaca tcaatggtga aggggctgtg 360
gcggtagaat cccctgtgga ccccattaca cttgatacgg ctggtagaat tactttaaat 420
tatggcacag gtttaaatgt gagtgatgga aaattacgac tagtaagtcc tgaaagtccg 480
ctcacacttc ttggaaatgg caaggttgct cttaattttg gtaattcaat ggagcttgtg 540
caagggacct tgcaactgaa agctccgcta aatcctttgt tcatgacccc cgcgggtgcg 600
atcggcttaa gggtggatga catgtttaac atttctgaag gtttactctc cttcaagatg 660
ccatccgatc caatttcgtt taatgctgat ggtatgttgt ctttgaacac aaatgacaca 720
ttgcaaacaa ctggtgggct gttagggttg accgaacctg ccaagccgtt aaaattggcc 780
gatggcaagt taggtgtaaa tgtgggcctt gggttagcgg tttctaatgg gtcattgact 840
gtaaatgcag ggcaggggtt gactattcga aataatgcgg tggcagttaa tgggggcaac 900
acgcttgctt ttaataatta tggagaggtg gaacttaaaa accctagaaa ccccataggc 960
ctgacccaag atggtgaatt ggctttgata atcggttatg gcctaacaac ccttgatgga 1020
cggctcactc tacttaccgc ttcgacctct ccgatagctg tagggccaac cggtgttaca 1080
tttaatgtta caccgagtga tttttacttt ttatctagta aattagctct caatgttgag 1140
acccgtggcg gcttagaaaa aagtgacact ggtttaaaaa ttaaacgtgc ggcccctctc 1200
agtatcacat ctgatggtga gttgactttg gcttatgatt ccacggattt tcaggtgaca 1260
gaaaacggcc tagccctaaa ggtatctccg acgcagaccc ctctcaccag aataatttct 1320
atgggaaata acttgtttga ttctggttat gagatttttg cttcatgtcc gcagaacaaa 1380
gcagcaaagg ttgcagggta tgtgtattta acatcggttg gtgggcttgt acatgggacc 1440
attcagatta aagctactgc ggggtattgg tttacggggg gaaacagcgt gcaggaaagt 1500
atcaggtttg gattggtgtt gtgtcctttt agtgctcgcg accccactgc taacctgtca 1560
ggctggccag cgccagtagt gtggagtggt gatagcaata ctcccctata ttttgcggcc 1620
aatgccatta gttataccaa taaccgtgta aatcttgcag ttaccggtaa cttttacaag 1680
gaggaaaccg aattgccggg ttacactcgt cattctttct gccctaccgg gaccaccgga 1740
atgaatttta cagggggtaa tttgtatgtg tgtccgtgca ctgtaaatac aggggcaacc 1800
acactgaatg ccatttatat ggtgtttgtg attactcaat cagctttggg aactaatttc 1860
tttgcttcta acacccctcc caacacattc tttttaactc cccccattcc ctttacatat 1920
gttggagcac ag 1932
<210>6
<211>645
<212>DNA
<213> Porcine circovirus type 3 (Porcine circovirus 3)
<400>6
atgagacaca gagctatatt cagaagaaga ccccgcccaa ggagacgacg acgccacaga 60
aggcgctatg ccagaagaaa actattcatt aggaggccca cagctggcac atactacaca 120
aagaaatact ccaccatgaa cgtcatatcc gttggaaccc ctcagaataa caagccctgg 180
cacgccaacc acttcattac ccgcctaaac gaatgggaaa ctgcaatttc ttttgaatat 240
tataagatac taaagatgaa agttacactc agccctgtaa tttctccggc tcagcaaaca 300
aaaactatgt tcgggcacac agccatagat ctagacggcg cctggaccac aaacacttgg 360
ctccaagacg acccttatgc ggaaagttcc actcgtaaag ttatgacttc taaaaaaaaa 420
cacagccgtt acttcacccc caaaccactt ctggcgggaa ctaccagcgc tcacccagga 480
caaagcctct cttttttctc cagacccacc ccatggctca acacatatga ccccaccgtt 540
caatggggag cactgctttg gagcatttat gtcccggaaa aaactggaat gacagacttc 600
tacggcacca aagaagtttg gattcgttac aagtccgttc tctaa 645
<210>7
<211>666
<212>DNA
<213> Autographa california multiple polyhedrosis virus (Autographa california multiple polyhedrosis virus)
<400>7
gatttacgcg tagaattcta cttgtaaagc aagttaaaat aagccgtgtg caaaaatgac 60
atcagacaaa tgacatcatc tacctatcat gatcatgtta ataatcatgt tttaaaatga 120
catcagctta tgactaataa ttgatcgtgc gttacaagta gaattctact cgtaaagcga 180
gtttagtttt gaaaaacaaa tgagtcatca ttaaacatgt taataatcgt gtataaagga 240
tgacatcatc cactaatcgt gcgttacaag tagaattcta ctcgtaaagc gagttcggtt 300
ttgaaaaaca aatgacatca tttcttgatt gtgttttaca cgtagaattc tactcgtaaa 360
gtatgttcag tttaaaaaac aaatgacatc attttacaga tgacatcatt tcttgattat 420
gttttacaag tagaattcta ctcgtaaagc aagtttagtt ttaaaaaaca aatgacatca 480
tctcttgatt atgttttaca agtagaattc tactcgtaaa gcgagtttag ttttgaaaaa 540
caaatgacat catctcttga ttatgtttta caagtagaat tctactcgta aagcgagttt 600
agttttcaaa aacaaatgac atcatccctt gatcatgcgt tacaagtaga attctactcg 660
taaagc 666
<210>8
<211>445
<212>DNA
<213> Autographa california multiple polyhedrosis virus (Autographa california multiple polyhedrosis virus)
<400>8
gaattctacc cgtaaagcga gtttagtttt gaaaaacaaa tgacatcatt tgtataatga 60
catcatcccc tgattgtgtt ttacaagtag aattctatcc gtaaagcgag ttcagttttg 120
aaaacaaatg agtcatacct aaacacgtta ataatcttct gatatcagct tatgactcaa 180
gttatgagcc gtgtgcaaaa catgagataa gtttatgaca tcatccactg atcgtgcgtt 240
acaagtagaa ttctactcgt aaagccagtt cggttatgag ccgtgtgcaa aacatgacat 300
cagcttatga ctcatacttg attgtgtttt acgcgtagaa ttctactcgt aaagcgagtt 360
cggttatgag ccgtgtgcaa aacatgacat cagcttatga gtcataatta atcgtgcgtt 420
acaagtagaa ttctactcgt aaagc 445

Claims (10)

1. A method for efficiently expressing a foreign protein in a baculovirus expression system, wherein the method comprises: step (1) inserting the homologous repetitive sequence in AcMNPV genome into P of AcMNPVp10And PPHObtaining P with homologous repeat sequence inserted betweenp10And PPHA sequence; step (2) inserting one copy of each foreign protein gene into pFastbac Dual plasmid Pp10And PPHThereafter, obtaining a composition comprising said Pp10And foreign protein gene, said PPHAnd exogenous protein gene Dual expression box pFastbac Dual plasmid; step (3) inserting the P obtained in the step (1) with homologous repeat sequences inserted between the Pp10And PPHSequence replacement of the adjacent P on the pFastbacDual plasmid in step (2)p10And PPHObtaining a solution containing said Pp10And foreign protein gene, said PPHAnd a double expression cassette of a foreign protein gene and a plasmid with a homologous repetitive sequence inserted between the double expression cassettes; transforming the plasmid containing the double-copy exogenous gene and the homologous repetitive sequence obtained in the step (3) into a DH10Bac competent cell to obtain a recombinant Bacmid plasmid; transfecting insect cells with the recombinant Bacmid plasmid obtained in the step (4) to obtain a recombinant baculovirus; and (6) culturing the recombinant baculovirus obtained in the step (5), and harvesting supernatant to obtain the expressed foreign protein.
2. The method according to claim 1, wherein the homologous repeat sequence in step (1) is Hr3, Hr3 is shown in SEQ ID No. 7; or the homologous repetitive sequence is Hr1, and Hr1 is shown in SEQ ID No. 8.
3. The method according to claim 1, wherein said P of step (1) has a homologous repeat sequence inserted therebetweenp10And PPHThe sequence is shown in SEQ ID No.1 or SEQ ID No. 2.
4. The method according to claim 1, wherein the foreign protein gene in the step (2) comprises an avian adenovirus Penton protein, an avian adenovirus Fiber-2 protein, an avian egg-loss syndrome virus Penton protein, an avian egg-loss syndrome virus Fiber protein, an avian infectious bursal disease virus VP2 protein, a porcine circovirus type 3 Cap protein, a porcine circovirus type 2 Cap protein, a porcine pseudorabies virus gB protein, a porcine pseudorabies virus gD protein, a porcine parvovirus VP2 protein, a classical swine fever virus E2 protein, a bovine infectious rhinotracheitis virus gB protein, a bovine infectious rhinotracheitis virus gD protein, a foot and mouth disease virus VP0 protein, a foot and mouth disease virus VP3 protein, a foot and mouth disease virus VP1 protein, a rabbit plague virus VP60 protein, a Japanese schistosome GALE protein, a Japanese schistosome Wnt5 protein.
5. The method according to claim 1, wherein the insect cell in step (5) is sf21, sf9, or Highfive cell.
6. A method of making a vaccine, wherein the method comprises: : step (1) inserting the homologous repetitive sequence in AcMNPV genome into P of AcMNPVp10And PPHObtaining P with homologous repeat sequence inserted betweenp10And PPHA sequence; step (2) inserting one copy of each foreign protein gene into pFastbac Dual plasmid Pp10And PPHThereafter, obtaining a composition comprising said Pp10And foreign protein gene, said PPHAnd exogenous protein gene Dual expression box pFastbac Dual plasmid; step (3) inserting the P obtained in the step (1) with homologous repeat sequences inserted between the Pp10And PPHSequence replacement of adjacent P on the pFastbac Dual plasmid in step (2)p10And PPHObtaining a composition comprisingPp10And foreign protein gene, said PPHAnd a double expression cassette of a foreign protein gene and a plasmid with a homologous repetitive sequence inserted between the double expression cassettes; transforming the plasmid containing the double-copy exogenous gene and the homologous repetitive sequence obtained in the step (3) into a DH10Bac competent cell to obtain a recombinant Bacmid plasmid; transfecting insect cells with the recombinant Bacmid plasmid obtained in the step (4) to obtain a recombinant baculovirus; culturing the recombinant baculovirus obtained in the step (5), and harvesting a supernatant to obtain the expressed foreign protein; and (7) adding an adjuvant into the expressed foreign protein, and emulsifying.
7. The method according to claim 6, wherein the homologous repeat sequence in step (1) is Hr3, Hr3 is shown in SEQ ID No. 7; or the homologous repetitive sequence is Hr1, and Hr1 is shown in SEQ ID No. 8.
8. The method according to claim 6, wherein said P of step (1) has inserted between them a homologous repeat sequencep10And PPHThe sequence is shown in SEQ ID No.1 or SEQ ID No. 2.
9. The method according to claim 6, wherein the foreign protein gene in the step (2) comprises an avian adenovirus Penton protein, an avian adenovirus Fiber-2 protein, an avian egg-loss syndrome virus Penton protein, an avian egg-loss syndrome virus Fiber protein, an avian infectious bursal disease virus VP2 protein, a porcine circovirus type 3 Cap protein, a porcine circovirus type 2 Cap protein, a porcine pseudorabies virus gB protein, a porcine pseudorabies virus gD protein, a porcine parvovirus VP2 protein, a classical swine fever virus E2 protein, a bovine infectious rhinotracheitis virus gB protein, a bovine infectious rhinotracheitis virus gD protein, a foot and mouth disease virus VP0 protein, a foot and mouth disease virus VP3 protein, a foot and mouth disease virus VP1 protein, a rabbit plague virus VP60 protein, a Japanese schistosome GALE protein, a Japanese schistosome Wnt5 protein.
10. The method of claim 6, wherein the adjuvant in step (7) comprises: (1) white oil, alumina gel adjuvant, saponin, alfvudine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) a copolymer of a polymer of acrylic acid or methacrylic acid, maleic anhydride and an alkenyl derivative; and one or more of RIBI adjuvant system, Blockco-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide, Montanide ISA 206, and Gel adjuvant; preferably, the saponin is Quil A, QS-21, GPI-0100;
the adjuvant content is 5% -70% V/V.
CN201811151129.7A 2018-09-29 2018-09-29 Method for efficiently expressing foreign protein in baculovirus expression system and application thereof Pending CN110964749A (en)

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