CN110846326A - Raccoon parvovirus VP2 gene, expression vector, recombinant strain, method for preparing VP2 protein and assembly method - Google Patents

Raccoon parvovirus VP2 gene, expression vector, recombinant strain, method for preparing VP2 protein and assembly method Download PDF

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CN110846326A
CN110846326A CN201911197992.0A CN201911197992A CN110846326A CN 110846326 A CN110846326 A CN 110846326A CN 201911197992 A CN201911197992 A CN 201911197992A CN 110846326 A CN110846326 A CN 110846326A
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protein
raccoon
gene
parvovirus
raccoon parvovirus
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殷玉和
雷欢
孙博
赫玉芳
罗国良
李希辰
吴丛梅
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Changchun University of Technology
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Changchun University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14023—Virus like particles [VLP]

Abstract

The invention provides a raccoon parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method, and belongs to the technical field of vaccine preparation, wherein the raccoon parvovirus VP2 gene has a nucleotide sequence shown in SEQ ID No. 1. The gene of the raccoon parvovirus VP2 and the molecular chaperone are transformed into the ER2566 cell together, and the molecular chaperone improves the correct folding rate of the VP2 protein. The gene of the raccoon parvovirus VP2 can form high-quality raccoon parvovirus-like particles. The raccoon parvovirus virus-like provided by the inventionThe particle vaccine can reach the hemagglutination titer of 2 after being purified16Has similar hemagglutination with natural virus.

Description

Raccoon parvovirus VP2 gene, expression vector, recombinant strain, method for preparing VP2 protein and assembly method
Technical Field
The invention belongs to the technical field of vaccine preparation, and particularly relates to a raccoon parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method.
Background
Raccoon Dog Parvovirus (RDPV) can cause Raccoon dog parvovirus enteritis, which is an acute and high-contact infectious disease with high morbidity and mortality and is one of important infectious diseases harming the breeding and development of Raccoon dogs. At present, raccoon dog parvovirus enteritis is widely existed in various countries of the world. Raccoon dogs of different ages and varieties are susceptible, especially the raccoon dogs and young raccoon dogs of 50-60 days old are most susceptible, the incidence rate of the young raccoon dogs is more than 70%, and the mortality rate is up to 90%; the incidence rate of adult raccoon dogs is about 30 percent, and the death rate is below 30 percent. The sick raccoon dog and the raccoon dog with the disease resistance are main infection sources of the disease, and viruses are discharged out of the body through feces, urine, saliva and the like and are infected to susceptible healthy raccoon dogs through digestive tracts and respiratory tracts. The disease can occur all year round and is often endemic. Once transferred into a raccoon dog house, if veterinary epidemic prevention and sanitation measures are not perfect, diseases exist for a long time and occur periodically, and huge economic losses are brought to farmers.
RDPV is a main member of parvoviridae and parvoviridae, is an envelope-free single-stranded DNA virus, and the nucleocapsid is composed of three structural proteins of VP1, VP2 and VP3, and has a structure similar to that of the nucleocapsid of other members of the parvoviridae. Wherein VP2 with blood coagulation activity is the main component of virus capsid protein.
RDPV is originally generated by infecting raccoon dogs with canine parvovirus across hosts, is a congeneric xenovirus of CPV, and is close to the relatives of carnivorous animal parvovirus such as feline panleukosis syndrome virus, mink enteritis virus and the like. The RDPV is firstly found in China in Heilongjiang province in 1984, and the RDPV is successfully separated from enteritis type raccoon bodies in different regions in 1988 by using Xia salted columna in 1988, and the existence of the RDPV in China is firstly verified. Then, reports about the appearance of the raccoon parvovirus enteritis are reported in various places of China; obviously, the raccoon parvovirus has a more and more intense tendency in China.
Because no special parvoviral enteritis vaccine for raccoon dogs exists in the domestic market, the inactivated parvoviral enteritis vaccine for minks is used for immunoprophylaxis all the time, but the substituted vaccine has low immunoprotection rate and still high parvovirus morbidity of raccoon dogs, and the substituted inactivated vaccine often has the phenomenon of incomplete inactivation or excessive use of an inactivating agent (most of formaldehyde) in the production process, thereby bringing great hidden danger to the next step of using the vaccine. Meanwhile, the formaldehyde has high toxicity, so that the raccoon dog has certain toxicity when being used as an inactivating agent, local inflammatory reaction, even suppuration and ulcer are often caused at the inoculation part, the whole quality of the raccoon dog skin is affected, and economic loss is caused. Meanwhile, the inactivated vaccine can only mobilize humoral immunity of an organism and cannot stimulate generation of cellular immune response of the organism. Therefore, the safe and effective vaccine for treating the raccoon parvovirus enteritis is an effective method for controlling the raccoon parvovirus enteritis.
Currently, novel virus-like particle subunit vaccines are extensively studied. The virus-like particles formed by the RDPVVP2 protein have a structure similar to that of RDPV virus particles, do not contain viral genetic materials, cannot be autonomously replicated, have no infectivity, can present antigens in a natural conformation and mode, effectively stimulate humoral and cellular immunity, and induce raccoon dogs to generate good immune response.
At present, the main insect cell expression system for expressing virus-like particles is reported in research, but the production cost is high. In contrast, the prokaryotic expression system has high protein expression amount, low production cost and simple production operation, but in actual operation, most proteins cannot be correctly folded to form inclusion bodies, thereby bringing obstacles to subsequent research work and application.
Disclosure of Invention
In view of the above, the invention aims to provide a raccoon parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method thereof, the raccoon parvovirus VP2 gene and a molecular chaperone are jointly transformed into ER2566 cells, and the molecular chaperone improves the correct folding rate of VP2 protein.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a raccoon parvovirus VP2 gene, wherein the raccoon parvovirus VP2 gene has a nucleotide sequence shown in SEQ ID No. 1.
The invention also provides an expression vector, and the raccoon parvovirus VP2 gene in the technical scheme is inserted into the vector to obtain the expression vector.
Preferably, the vector comprises the pET30a vector.
The invention also provides a recombinant bacterium, and the expression vector and the molecular chaperone plasmid in the technical scheme are jointly transformed into an ER2566 cell to obtain the recombinant bacterium.
Preferably, the mass ratio of the expression vector to the molecular chaperone plasmid is 0.5-1.5: 0.5-1.5.
Preferably, the chaperone plasmid comprises the pTf6 plasmid.
The invention also provides a method for preparing the raccoon parvovirus VP2 protein, which comprises the following steps:
1) inoculating the recombinant strain in the technical scheme into a liquid culture medium containing L-arabinose to culture to obtain a culture;
2) OD of the culture obtained when said step 1) is performed600When the value is 0.6, mixing the mixture with isopropyl thiogalactose, inducing for 14-18 h, and collecting thalli;
3) and (3) mixing the thalli obtained in the step 2) with the lysate liquid, carrying out ice-bath ultrasonic crushing, centrifuging the obtained ultrasonic substance, and obtaining a precipitate as the protein VP2 of the raccoon parvovirus.
Preferably, the concentration of the L-arabinose in the liquid culture medium is 1.5-2.5 mg/ml, and the concentration of the isopropylthiogalactoside in the culture is 0.05-0.15 mmol/ml.
Preferably, the components of the lysate liquid comprise 50mM Tris and 150-500 mM NaCl, and the pH value of the lysate liquid is 8.
The invention also provides a method for assembling virus-like particles in vitro by using the raccoon parvovirus VP2 protein, which is characterized in that the raccoon parvovirus VP2 protein prepared by the technical scheme is placed in an assembly liquid for in vitro assembly, the components of the assembly liquid comprise 50mM Tris and 150-500 mM NaCl, and the pH value of the assembly liquid is 8.
The invention provides a raccoon parvovirus VP2 gene, an expression vector, a recombinant bacterium, a method for preparing VP2 protein and an assembly method, and the invention transforms the raccoon parvovirus VP2 gene and a molecular chaperone into ER2566 cells together, so that the molecular chaperone improves the correct folding rate of the VP2 protein.
The invention has the beneficial effects that:
the invention firstly proposes that chaperonin and RDPVVP2 protein are co-expressed in escherichia coli, the solubility of the expressed VP2 protein is obviously improved, and the VP2 protein can be self-assembled into virus-like particles (VLPs) under 50mM Tris, 150-500 mM NaCl and pH8.0, and has good immunocompetence.
The DNA sequence of the gene VP2 of raccoon parvovirus provided by the invention, and the VP2 protein expressed by the sequence has good immunocompetence.
According to the invention, the gene of the raccoon parvovirus VP2 is inserted into the vector and co-expressed with the chaperonin, so that the expression level of the soluble VP2 protein is improved; the selected chaperones not only prevent the VP2 protein from forming aggregates or inactive structures during folding, increasing the correct folding rate, but may also affect the folding pathway.
The gene of the raccoon parvovirus VP2 can form high-quality raccoon parvovirus-like particles.
After purification, the raccoon dog parvovirus virus-like particle vaccine provided by the invention has the hemagglutination titer reaching 216Has similar hemagglutination with natural virus.
Drawings
FIG. 1 is a map of pET30a vector;
FIG. 2 shows the restriction enzyme identification of recombinant plasmid pET30a-VP2, wherein M.DNA molecular mass standard; 1, recombinant plasmid pET30a-VP 2; 2. HindIII + Nde I double enzyme digestion identification product of the recombinant plasmid;
figure 3 is an expression product solubility assay in which m. protein marker; 1. before expression was induced by the empty plasmid pET30 a; 2. after the induction of expression of the empty plasmid pET30 a; before IPTG induction, pET30a-VP2 bacterial liquid; pET30a-VP2 thalli after IPTG induction; ultrasonic supernatant of pET30a-VP 2; pET30a-VP2 inclusion bodies;
FIG. 4 is an SDS-PAGE identification of co-expression products of chaperones and genes of interest, wherein M. protein marker; IPTG inducing expression of pre-bacterial liquid; 2, inducing thalli by IPTG; 3. ultrasonic supernatant; 4. inclusion bodies;
FIG. 5 is a Western-blot identification of co-expression products of molecular chaperones and target genes, wherein M. protein marker; IPTG inducing expression of pre-bacterial liquid; 2, inducing thalli by IPTG; 3. ultrasonic supernatant; 4. inclusion bodies;
fig. 6 shows the purification of Cap protein by ammonium sulfate precipitation, wherein m. protein Marker; 1. supernatant after ultrasonic treatment; s, supernatant fluid; p, precipitation;
fig. 7 shows ammonium sulfate precipitation of primarily purified VP2 protein, wherein m. protein Marker; 1. supernatant after ultrasonic treatment; s, supernatant fluid; p, precipitation;
fig. 8 shows sucrose density centrifugation purified VP2 protein, wherein m. protein marker; 1.30% sulfuric acid precipitation; 2-3.30% sulfuric acid precipitation of the sample layer; 4-7.30% of a sucrose layer; 8-11.40% of a sucrose layer; 12-15.50% of a sucrose layer; 16-18.60% of a sucrose layer;
FIG. 9 is an in vitro assembly dynamic light scattering assay of RDPVVLPs at different pHs (pH 7.0, pH8.0), where pH 7.0 in a and pH8.0 in b;
FIG. 10 is a graph of RDPVVLPs assembled dynamic light scattering assays in vitro at various NaCl concentrations (150mM,250mM,500mM) where the NaCl concentration in a is 150mM, the NaCl concentration in b is 250mM, and the NaCl concentration in c is 500 mM;
FIG. 11 is an electron micrograph of the formation of VLPs from RDPVVP2 protein;
FIG. 12 is a graph of the hemagglutination properties (after sucrose density chromatography) of RDPVVLPs, wherein A-D.RDPV VLPs; E-F. positive control; G-H negative control.
Detailed Description
The invention provides a raccoon parvovirus VP2 gene, which is characterized in that the raccoon parvovirus VP2 gene has a nucleotide sequence shown in SEQ ID No.1, and the nucleotide sequence is as follows:
atgagcgatggtgcggtgcaaccggatggtggccaaccggcggtgcgtaacgaacgtgcgaccggcagcggta acggcaccggtggcggtggcggtggcggtagcggcggtgtgggtatcagcaccggcacctttaacaaccaaacc gagtttaagttcctggaaaacggttgggttgagattaccgcgaacagcagccgtctggtgcacctgaacatgccgga gagcgaaaactaccgtcgtgtggttgtgaacaacatggacaaaaccgcggtgaacggcaacatggcgctggacg atatccacgcgcagattgttaccccgtggagcctggtggacgcgaacgcgtggggtgtttggttcaacccgggcga ttggcagctgatcgttaacaccatgagcgaactgcacctggtgagctttgagcaagaaattttcaacgttgtgctgaaa accgttagcgagagcgcgacccagccgccgaccaaagtgtacaacaacgacctgaccgcgagcctgatggttgc gctggatagcaacaacaccatgccgtttaccccggcggcgatgcgtagcgaaaccctgggtttctatccgtggaag ccgaccatcccgaccccgtggcgttactatttccaatgggaccgtaccctgattccgagccacaccggcaccagcg gcaccccgaccaacatttaccacggcaccgacccggacgatgtgcagttttataccattgaaaacagcgttccggtg cacctgctgcgtaccggtgacgagttcgcgaccggcaccttctttttcgattgcaagccgtgccgtctgacccacacc tggcaaaccaaccgtgcgctgggtctgccgccgtttctgaacagcctgccgcaggcggagggtgcgaccaacttc ggtgacatcggcgttcagcaagataaacgtcgtggtgtgacccaaatgggcaacaccaactacatcaccgaagcg accattatgcgtccggcggaagtgggttatagcgcgccgtactatagctttgaagcgagcacccagggtccgttcaa gaccccgattgcggcgggccgtggcggtgcgcagaccgacgagaaccaagcggcggacggtgatccgcgttac gcgtttggtcgtcagcacggccaaaaaaccaccaccaccggcgaaaccccggaacgtttcacctacattgcgcac caggacaccggtcgttatccggaaggcgattggattcagaacattaactttaacctgccggttaccaacgacaacgt gctgctgccgaccgatccgatcggcggtaaaaccggtatcaactacaccaacatttttaacacctatggcccgctga ccgcgctgaacaacgttccgccggtgtatccgaacggtcagatttgggataaggagttcgacaccgatctgaaacc gcgtctgcacgttaacgcgccgtttgtgtgccagaacaactgcccgggccaactgttcgttaaagtggcgccgaacc tgaccaacgagtacgacccggatgcgagcgcgaacatgagccgtatcgtgacctatagcgacttttggtggaagg gtaaactggttttcaaggcgaaactgcgtgcgagccacacctggaacccgatccagcaaatgagcattaacgtgga taaccaattcaactacctgccgagcaacatcggcggtatgaagattgtttacgagaaaagccagctggcgccgcgta aactgtattaa。
according to the invention, the DNA sequence of the gene VP2 of raccoon parvovirus is artificially modified and synthesized according to the preference of the codon of escherichia coli, and the VP2 protein expressed by the sequence has good immunocompetence. In the invention, the amino acid sequence of the VP2 protein is shown as SEQ ID No.2, and specifically comprises the following steps: MSDGAVQPDGGQPAVRNERATGSGNGTGGGGGGGSGGVGISTGTFNNQT EFKFLENGWVEITANSSRLVHLNMPESENYRRVVVNNMDKTAVNGNMA LDDIHAQIVTPWSLVDANAWGVWFNPGDWQLIVNTMSELHLVSFEQEIF NVVLKTVSESATQPPTKVYNNDLTASLMVALDSNNTMPFTPAAMRSETL GFYPWKPTIPTPWRYYFQWDRTLIPSHTGTSGTPTNIYHGTDPDDVQFYTI ENSVPVHLLRTGDEFATGTFFFDCKPCRLTHTWQTNRALGLPPFLNSLPQA EGATNFGDIGVQQDKRRGVTQMGNTNYITEATIMRPAEVGYSAPYYSFEA STQGPFKTPIAAGRGGAQTDENQAADGDPRYAFGRQHGQKTTTTGETPE RFTYIAHQDTGRYPEGDWIQNINFNLPVTNDNVLLPTDPIGGKTGINYTNI FNTYGPLTALNNVPPVYPNGQIWDKEFDTDLKPRLHVNAPFVCQNNCPG QLFVKVAPNLTNEYDPDASANMSRIVTYSDFWWKGKLVFKAKLRASHT WNPIQQMSINVDNQFNYLPSNIGGMKIVYEKSQLAPRKLY are provided.
The invention also provides an expression vector, and the raccoon parvovirus VP2 gene in the technical scheme is inserted into the vector to obtain the expression vector.
In the present invention, the vector preferably includes the pET30a vector. In the present invention, the origin of the pET30a vector is not particularly limited, and any of the conventional commercially available vectors can be used. In the present invention, the method for constructing the expression vector preferably comprises: inserting the gene of the raccoon parvovirus VP2 into a pUC vector to obtain pUC-VP2, carrying out double enzyme digestion on the pUC-VP2 vector and the pET30a vector respectively by restriction enzymes Nde I and Hind III, and then carrying out T4 ligase ligation to obtain the expression vector.
In the present invention, the double digestion is preferably carried out at 37 ℃ for 2 hours, and the double digestion system preferably comprises 30. mu.l of mixed vector, 1. mu.l of Hind III, 1. mu.l of NdeI, 5. mu.l of 10 XKBuffer and ddH per 50. mu.l2O13. mu.l. In the present invention, the mixed vector comprises the plasmid pUC-VP2 and the vector pET30a, the concentration of the plasmid pUC-VP2 is preferably 123.5. mu.g/. mu.l, and the concentration of the vector pET30a is preferably 101.8. mu.g/. mu.l.
In the present invention, the ligation is performed overnight at 16 ℃ in a system comprising, per 20. mu.l: 5. mu.l of the VP2 double-digested fragment, 4. mu.l of the pET30a double-digested vector fragment, 1. mu.l of T4 ligase and 10. mu.l of 2 XBuffer.
The invention provides a recombinant bacterium, which is obtained by transforming an expression vector and a molecular chaperone plasmid in the technical scheme into an ER2566 cell together.
In the invention, the mass ratio of the expression vector to the molecular chaperone plasmid is preferably 0.5-1.5: 0.5-1.5, and more preferably 1: 1. In the present invention, the chaperone plasmid includes pTf6 plasmid, and the source of pTf6 plasmid is not particularly limited, and may be any of those conventionally available on the market. In the present invention, the chaperone plasmid can not only prevent VP2 protein from forming aggregates or inactive structures during folding, improving the correct folding rate, but also may affect the folding pathway.
In the present invention, the source of the ER2566 cells is not particularly limited, and any conventional commercially available cells may be used. The method for co-transforming the expression vector and the molecular chaperone plasmid into the ER2566 cell is not particularly limited, and the conventional method is adopted.
The invention also provides a method for preparing the raccoon parvovirus VP2 protein, which comprises the following steps:
1) inoculating the recombinant strain in the technical scheme into a liquid culture medium containing L-arabinose to culture to obtain a culture;
2) OD of the culture obtained when said step 1) is performed600When the value is 0.6, mixing the mixture with isopropyl thiogalactose, inducing for 14-18 h, and collecting thalli;
3) and (3) mixing the thalli obtained in the step 2) with the lysate liquid, carrying out ice-bath ultrasonic crushing, centrifuging the obtained ultrasonic substance, and obtaining a precipitate as the protein VP2 of the raccoon parvovirus.
The recombinant strain is inoculated in a liquid culture medium containing L-arabinose and cultured to obtain a culture. The type of the liquid medium is not particularly limited in the present invention, and a conventional liquid medium may be used, and an LB liquid medium is preferred in the present embodiment. In the invention, the concentration of the L-arabinose in the LB liquid culture medium is preferably 1.5-2.5 mg/ml, more preferably 2mg/ml, and the L-arabinose induces the expression of chaperonin. In the present invention, the LB liquid medium also preferably contains kanamycin, preferably at a concentration of 50. mu.g/ml, and chloramphenicol preferably at a concentration of 20. mu.g/ml. In the present invention, the volume ratio of the recombinant bacterium to the liquid medium is preferably 1: 200.
In the present invention, OD of the resulting culture600When the value is 0.6, mixing the bacterial strain with isopropyl thiogalactose, inducing for 14-18 h, and collecting the bacterial strain. In the invention, the concentration of the isopropylthiogalactose in the culture is preferably 0.05-0.15 mmol/ml, more preferably 0.1mmol/ml, and the isopropylthiogalactose induces the expression of the VP2 protein of the raccoon parvovirus. In the invention, the induction time is 14-18 h, and more preferably 16 h.
The obtained thalli and the lysate liquid are mixed and then subjected to ice bath ultrasonic crushing, the obtained ultrasonic material is centrifuged, and the obtained precipitate is the protein VP2 of the raccoon parvovirus.
In the invention, the components of the lysate liquid preferably comprise 50mM Tris and 150-500 mM NaCl, the pH value of the lysate liquid is 8, and the concentration of NaCl is more preferably 250 mM.
In the present invention, the conditions for the ultrasonication preferably include: the time of the ultrasonic crushing is preferably 30min, the interval is 5s, and the power of the ultrasonic crushing is preferably 300W. In the present invention, the time of the centrifugation is preferably 25min, and the speed of the centrifugation is preferably 16000 rpm.
The invention also provides a method for assembling virus-like particles in vitro by the raccoon parvovirus VP2 protein, which is characterized in that the raccoon parvovirus VP2 protein prepared by the preparation method of the technical scheme is placed in an assembly liquid for in vitro assembly, the components of the assembly liquid comprise 50mM Tris and 150-500 mM NaCl, and the pH value of the assembly liquid is 8. In the present invention, the concentration of NaCl in the composition liquid is preferably 250 mM. In the present invention, the diameter of the virus-like particle is preferably 24nm, the virus-like particle has a size, shape and hemagglutination similar to natural parvovirus, and the hemagglutination titer reaches 216。
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Example 1
A method for prokaryotic expression of a raccoon parvovirus VP2 protein is characterized in that VP2 protein and chaperonin protein are co-expressed, and a recombinant expression protein with good solubility and high activity can be obtained. The method comprises the following specific steps:
1.1 construction of recombinant vector pET30a-VP2
1.1.1 Synthesis of an artificially modified RDPV Primary immunogenic Gene VP2
And (3) optimizing the sequence according to the codon preference of escherichia coli by referring to the RDPV gene sequence, inserting the artificially synthesized complete raccoon parvovirus VP2 gene into a vector pUC, and constructing a plasmid pUC-VP 2.
1.1.2 construction of pET30a-VP2 vector
Carrying out double enzyme digestion on the plasmid pUC-VP2 and the vector pET30a by restriction enzymes Nde I and Hind III respectively under the reaction condition of 37 ℃ for 2h, wherein the enzyme digestion system is shown in Table 1; subsequently, the double-digested products of the pET30a and VP2 genes were recovered separately and ligated with T4 ligase at 16 ℃ overnight, as shown in Table 2. The ligation products were transformed into E.coli ER2566 competent cells, as described in the kit instructions (purchased from Tiangen Biochemical technology Co., Ltd.).
TABLE 1 double enzyme digestion System
Reagent Volume (50. mu.l system)
Plasmid pUC-VP 2/vector pET30a 30μl
HindⅢ 1μl
NdeⅠ 1μl
10×KBuffer 5μl
ddH2O 13μl
TABLE 2 recombinant plasmid pET30a-VP2 ligation System
Reagent Volume (20. mu.l system)
VP2 double-enzyme digestion target fragment 5μl
pET30a double enzyme digestion carrier fragment 4μl
T4Ligase 1μl
2×Buffer 10μl
Since the vector pET30a has the kanamycin resistance gene, the transformed cells were plated on LB agar plates containing 50. mu.g/mL kanamycin resistance and cultured overnight at 37 ℃. Single colonies on a plate are randomly picked and inoculated in LB culture medium with kanamycin resistance, the culture is carried out overnight at 37 ℃, positive clones (the result is shown in figure 2) are obtained through enzyme digestion identification, the positive plasmids are named as pET30a-VP2, the recombinant plasmids pET30a-VP2 are sent to companies for sequencing, and the result shows that the sequence of the inserted fragment is correct, no mutation exists and the insertion direction is correct.
1.2 expression of RDPVVP2 in E.coli
1.2.1 expression of the protein of interest RDPVVP2
The obtained recombinant expression plasmid pET30a-VP2 was transformed into ER2566 competent cells, plated on LB agar plates containing 50. mu.g/mL kanamycin resistance, and cultured overnight at 37 ℃. Positive colonies were picked and inoculated into 5mLLB liquid medium containing 50. mu.g/mL kanamycin, incubated at 37 ℃ overnight at 220r/min, inoculated into 1L LB liquid medium (50. mu.g/mL kanamycin) at a ratio of 1:200, and incubated at 37 ℃ at 220r/min to an absorbance OD600When the value reaches 0.6-0.8, 0.2mmol/L IPTG is added, and the target protein RDPVVP216h is induced and expressed at 25 ℃. Centrifuging at 8000r/min for 15min to collect thallus. Adding a lysate (50mmol/LTris, 150mmol/LNaCl, pH value of 8.0) into the thallus according to a ratio of 1:10, suspending, carrying out ice-bath ultrasonic crushing, wherein the effective time is 30min, the intermittent time is 5s, and centrifuging for 25min at 16000r/min after crushing. The supernatant and pellet were separated, the pellet (inclusion bodies) was suspended in an equal volume of PBS, and the expression product was analyzed by SDS-PAGE. As shown in FIG. 3, compared with the empty plasmid pET30a control, a clear band is observed at 64ku after the recombinant plasmid induces the expression, which is consistent with the expected size, and the result shows that the expression of the target protein RDPVVP2 is successfully induced, but the soluble expression amount is not high, and most of the protein exists in the form of inclusion bodies.
1.2.2 Co-expression of chaperones with proteins of interest
The recombinant expression plasmid pET30a-VP2 and the molecular chaperone plasmid pTf16 are jointly transformed into ER2566 competent cells according to the mass ratio of 1:1, the competent cells are coated on an LB agar plate containing 50 mu g/mL kanamycin and 20 mu g/mL chloramphenicol resistance, the LB agar plate is cultured overnight at 37 ℃, and positive clones are screened, namely the recombinant strains which are used for co-expressing VP2 protein and chaperone protein.
The obtained RDPVVP 2-expressing strain 1:200 was inoculated into liquid LB medium containing 50. mu.g/mL kanamycin and 20 ug/mL chloramphenicol, while maintaining the stabilityAdding 2 mg/ml-arabinose to induce the expression of chaperonin, culturing at 37 deg.C to OD600When the concentration is 0.6, IPTG (isopropyl thiogalactopyranoside) with the final concentration of 0.1mmol/mL is added to induce the expression of VP2 protein, and after 16 hours of induction at 30 ℃, the cells are collected by centrifugation. Adding a lysate (50mmol/L Tris, 150mmol/L NaCl, pH value of 8.0) into the thallus according to a ratio of 1:10, suspending, carrying out ice-bath ultrasonic crushing for 30min, and carrying out centrifugation for 25min at 16000r/min after crushing, wherein the intermittent time is 5 s. Separating supernatant and sediment, suspending the sediment (inclusion body) by PBS with the same volume, and identifying the expression and solubility of the protein by SDS-PAGE and Western-blot. The results are shown in FIGS. 4-5, compared with the IPTG induction, clear bands are shown at 56ku and 64ku after IPTG and L-arabinose induction, which are respectively molecular chaperone Tf16 and RDPVVP2 proteins, and are consistent with the expected size of the target protein RDPVVP 2. Through Western-blot identification, when a canine small monoclonal antibody is used as a primary antibody, a specific band appears at 64ku, which indicates that the target protein RDPVVP2 can be specifically recognized. Compared with a system without molecular chaperone, the target protein is expressed in a large amount, most of the target protein is expressed in a soluble form, and only a small amount of the target protein exists in an inclusion body form; it is shown that the molecular chaperone Tf16 successfully promotes the soluble expression of target protein in Escherichia coli.
Example 2
Preparation of raccoon parvovirus virus-like particles
2.1 purification of VP2 protein and determination of VLP
2.1.1 purification of recombinant VP2 protein
A saturated ammonium sulfate solution was added to the supernatant of the ultrasonic lysis obtained in example 1 so that the final concentrations of ammonium sulfate were 10%, 15%, 20%, 25%, and 30% saturation, respectively. The mixture was stirred at 4 ℃ for 1h, centrifuged at 10000rpm for 30min, the supernatant was taken and the pellet was resuspended in lysate (50mM Tris, 150mM NaCl, pH 8.0). After each supernatant and precipitate sample is processed, SDS-PAGE detection shows that VP2 protein can remove a large amount of impurity protein after 30% ammonium sulfate as shown in FIGS. 6-7.
Adding 60%, 50%, 40% and 30% sucrose solutions into a 38.5mL sucrose density gradient centrifuge tube in sequence, wherein each gradient is 8 mL; adding 6.5mL of sample precipitated by 30% ammonium sulfate to the top of a gradient column, balancing, placing in an ultracentrifuge, and centrifuging at 4 ℃ and 35000r/min for 34 h; after completion of centrifugation, 1 fraction of 2mL was collected by pipetting from low concentration to high concentration, and 17 parts in total were collected. And SDS-PAGE is used for detection, the results are shown as a and b in figure 8, when the density of the sucrose is 40-50%, the impurity removal rate is maximized, and the recovery rate of the target protein is also maximized. After sucrose density gradient centrifugation, the purity of the VP2 protein can reach more than 80%, and the recovery rate can reach 50%.
2.1.2 in vitro Assembly of recombinant VP2 protein
The purified protein is filled into a dialysis bag and dialyzed at 4 ℃, and the optimal condition of the VP2 protein in vitro assembly is explored by changing the salt ion concentration and the pH value of the dialyzate. Effect of pH on VLP formation at the same salt ion concentration. The concentration of the salt ions is 500 mM; the pH values were 7.0 and 8.0, respectively. Effect of salt ion concentration on VLP formation at ph 8.0. The salt ion concentration is respectively 150mM,250mM and 500 mM. The morphology of the formed VLPs was observed by transmission electron microscopy, the particle size distribution of the VLPs was examined by a nano-particle sizer, and the immunocompetence of the VLPs was examined by a hemagglutination assay.
The results show that: the effect of pH on VLP formation at the same NaCl concentration, as shown by a and b in fig. 9, the diameter of most VLPs under acidic conditions was 19.10nm, smaller than that of native canine parvovirus (21-25nm), while alkaline conditions, in contrast, were more favorable for VLP formation, under which the diameter of most VLPs was 23.85 nm. The dialysate was adjusted to pH8.0 and analyzed for packaging effect at NaCl concentrations (150mM,250mM,500mM), as shown in FIGS. 10 a-c, and the size of VLPs increased with increasing salt concentration, but did not vary much, between 21.70-23.85nm, as measured by DLS. The diameters of VLPs (23.50nm) were closest to those of natural canine parvovirus particles at a salt concentration of 250 mM. To further determine the formation of VLPs, the VLPs were observed under a transmission electron microscope to have the same shape as the virions, and the hemagglutination test showed that the VLPs had hemagglutination properties identical to those of the native virions. Thus, it can be seen from the results that the prokaryotically expressed VP2 protein can be assembled in vitro into VLPs that are similar in morphology, size and hemagglutination activity to native RDPV viruses. VLP formed with better quality and higher formation rate under the assembly condition of 50mM Tris, 250mM NaCl and pH 8.0.
The specific detection method is as follows:
dynamic Light Scattering (DLs) detection: the RDPVVLP obtained by purification is detected by a nanometer particle size analyzer, the results are shown in figures 9-10, and the DLS results show that the VLP obtained by the invention has high purity which can reach 80 percent, uniform size and complete structure.
Transmission Electron Microscope (TEM) detection: the RDPV VLP obtained by the purification is negatively stained by 2% phosphotungstic acid, the shape of the VLP is observed by a transmission electron microscope, the diameter of the VLP is about 23-24nm, the size of the VLP is uniform, the VLP is in a hollow shape, and the result is shown in figure 11, the target protein RDPVVP2 can be spontaneously assembled into particles with the diameter of about 24nm, the limit is clear, the particle uniformity is high, and the shape of the particles is similar to the shape of natural virus-like particles of RDPV.
Hemagglutination Activity (HA) assay: a96-well microplate is taken, 25 mu L of PBS is plated, 25 mu L/well RDPV VLP is added into a first row, two multiple wells are made, the two multiple wells are diluted backwards twice, 25 mu L is abandoned at the end, 25 mu L of PBS is added into each well, 1% pig red blood cells are added into 50 mu L/well, and after 1h at 4 ℃, the experimental result is observed immediately on the plate (the result is shown in figure 12).
As RDPV has the characteristic of agglutinating pig red blood cells, the canine parvovirus is used as a positive control, and PBS is used as a negative control to measure the hemagglutination titer of VLP. The results show that the blood coagulation titer of RDPVVLP after sucrose gradient centrifugation is 216The hemagglutination titer of the positive control was 23Negative controls were free of clotting. This demonstrates that this VLP has hemagglutination properties with the same properties as the native virion.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Changchun university of industry
<120> racoon dog parvovirus VP2 gene, expression vector, recombinant bacteria, method for preparing VP2 protein and assembly method
<160>2
<170>SIPOSequenceListing 1.0
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atgagcgatg gtgcggtgca accggatggt ggccaaccgg cggtgcgtaa cgaacgtgcg 60
accggcagcg gtaacggcac cggtggcggt ggcggtggcg gtagcggcgg tgtgggtatc 120
agcaccggca cctttaacaa ccaaaccgag tttaagttcc tggaaaacgg ttgggttgag 180
attaccgcga acagcagccg tctggtgcac ctgaacatgc cggagagcga aaactaccgt 240
cgtgtggttg tgaacaacat ggacaaaacc gcggtgaacg gcaacatggc gctggacgat 300
atccacgcgc agattgttac cccgtggagc ctggtggacg cgaacgcgtg gggtgtttgg 360
ttcaacccgg gcgattggca gctgatcgtt aacaccatga gcgaactgca cctggtgagc 420
tttgagcaag aaattttcaa cgttgtgctg aaaaccgtta gcgagagcgc gacccagccg 480
ccgaccaaag tgtacaacaa cgacctgacc gcgagcctga tggttgcgct ggatagcaac 540
aacaccatgc cgtttacccc ggcggcgatg cgtagcgaaa ccctgggttt ctatccgtgg 600
aagccgacca tcccgacccc gtggcgttac tatttccaat gggaccgtac cctgattccg 660
agccacaccg gcaccagcgg caccccgacc aacatttacc acggcaccga cccggacgat 720
gtgcagtttt ataccattga aaacagcgtt ccggtgcacc tgctgcgtac cggtgacgag 780
ttcgcgaccg gcaccttctt tttcgattgc aagccgtgcc gtctgaccca cacctggcaa 840
accaaccgtg cgctgggtct gccgccgttt ctgaacagcc tgccgcaggc ggagggtgcg 900
accaacttcg gtgacatcgg cgttcagcaa gataaacgtc gtggtgtgac ccaaatgggc 960
aacaccaact acatcaccga agcgaccatt atgcgtccgg cggaagtggg ttatagcgcg 1020
ccgtactata gctttgaagc gagcacccag ggtccgttca agaccccgat tgcggcgggc 1080
cgtggcggtg cgcagaccga cgagaaccaa gcggcggacg gtgatccgcg ttacgcgttt 1140
ggtcgtcagc acggccaaaa aaccaccacc accggcgaaa ccccggaacg tttcacctac 1200
attgcgcacc aggacaccgg tcgttatccg gaaggcgatt ggattcagaa cattaacttt 1260
aacctgccgg ttaccaacga caacgtgctg ctgccgaccg atccgatcgg cggtaaaacc 1320
ggtatcaact acaccaacat ttttaacacc tatggcccgc tgaccgcgct gaacaacgtt 1380
ccgccggtgt atccgaacgg tcagatttgg gataaggagt tcgacaccga tctgaaaccg 1440
cgtctgcacg ttaacgcgcc gtttgtgtgc cagaacaact gcccgggcca actgttcgtt 1500
aaagtggcgc cgaacctgac caacgagtac gacccggatg cgagcgcgaa catgagccgt 1560
atcgtgacct atagcgactt ttggtggaag ggtaaactgg ttttcaaggc gaaactgcgt 1620
gcgagccaca cctggaaccc gatccagcaa atgagcatta acgtggataa ccaattcaac 1680
tacctgccga gcaacatcgg cggtatgaag attgtttacg agaaaagcca gctggcgccg 1740
cgtaaactgt attaa 1755
<210>2
<211>584
<212>PRT
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Met Ser Asp Gly Ala Val Gln Pro Asp Gly Gly Gln Pro Ala Val Arg
1 5 10 15
Asn Glu Arg Ala Thr Gly Ser Gly Asn Gly Thr Gly Gly Gly Gly Gly
20 25 30
Gly Gly Ser Gly Gly Val Gly Ile Ser Thr Gly Thr Phe Asn Asn Gln
35 40 45
Thr Glu Phe Lys Phe Leu Glu Asn Gly Trp Val Glu Ile Thr Ala Asn
50 55 60
Ser Ser Arg Leu Val His Leu Asn Met Pro Glu Ser Glu Asn Tyr Arg
65 70 75 80
Arg Val Val Val Asn Asn Met Asp Lys Thr Ala Val Asn Gly Asn Met
85 90 95
Ala Leu Asp Asp Ile His Ala Gln Ile Val Thr Pro Trp Ser Leu Val
100 105 110
Asp Ala Asn Ala Trp Gly Val Trp Phe Asn Pro Gly Asp Trp Gln Leu
115 120 125
Ile Val Asn Thr Met Ser Glu Leu His Leu Val Ser Phe Glu Gln Glu
130 135 140
Ile Phe Asn Val Val Leu Lys Thr Val Ser Glu Ser Ala Thr Gln Pro
145 150 155 160
Pro Thr Lys Val Tyr Asn Asn Asp Leu Thr Ala Ser Leu Met Val Ala
165 170 175
Leu Asp Ser Asn Asn Thr Met Pro Phe Thr Pro Ala Ala Met Arg Ser
180 185 190
Glu Thr Leu Gly Phe Tyr Pro Trp Lys Pro Thr Ile Pro Thr Pro Trp
195 200 205
Arg Tyr Tyr Phe Gln Trp Asp Arg Thr Leu Ile Pro Ser His Thr Gly
210 215 220
Thr Ser Gly Thr Pro Thr Asn Ile Tyr His Gly Thr Asp Pro Asp Asp
225 230 235 240
Val Gln Phe Tyr Thr Ile Glu Asn Ser Val Pro Val His Leu Leu Arg
245 250 255
Thr Gly Asp Glu Phe Ala Thr Gly Thr Phe Phe Phe Asp Cys Lys Pro
260 265 270
Cys Arg Leu Thr His Thr Trp Gln Thr Asn Arg Ala Leu Gly Leu Pro
275 280 285
Pro Phe Leu Asn Ser Leu Pro Gln Ala Glu Gly Ala Thr Asn Phe Gly
290 295 300
Asp Ile Gly Val Gln Gln Asp Lys Arg Arg Gly Val Thr Gln Met Gly
305 310 315 320
Asn Thr Asn Tyr Ile Thr Glu Ala Thr Ile Met Arg Pro Ala Glu Val
325 330 335
Gly Tyr Ser Ala Pro Tyr Tyr Ser Phe Glu Ala Ser Thr Gln Gly Pro
340 345 350
Phe Lys Thr Pro Ile Ala Ala Gly Arg Gly Gly Ala Gln Thr Asp Glu
355 360 365
Asn Gln Ala Ala Asp Gly Asp Pro Arg Tyr Ala Phe Gly Arg Gln His
370 375 380
Gly Gln Lys Thr Thr Thr Thr Gly Glu Thr Pro Glu Arg Phe Thr Tyr
385 390 395 400
Ile Ala His Gln Asp Thr Gly Arg Tyr Pro Glu Gly Asp Trp Ile Gln
405 410 415
Asn Ile Asn Phe Asn Leu Pro Val Thr Asn Asp Asn Val Leu Leu Pro
420 425 430
Thr Asp Pro Ile Gly Gly Lys Thr Gly Ile Asn Tyr Thr Asn Ile Phe
435 440 445
Asn Thr Tyr Gly Pro Leu Thr Ala Leu Asn Asn Val Pro Pro Val Tyr
450 455 460
Pro Asn Gly Gln Ile Trp Asp Lys Glu Phe Asp Thr Asp Leu Lys Pro
465 470 475 480
Arg Leu His Val Asn Ala Pro Phe Val Cys Gln Asn Asn Cys Pro Gly
485 490 495
Gln Leu Phe Val Lys Val Ala Pro Asn Leu Thr Asn Glu Tyr Asp Pro
500 505 510
Asp Ala Ser Ala Asn Met Ser Arg Ile Val Thr Tyr Ser Asp Phe Trp
515 520 525
Trp Lys Gly Lys Leu Val Phe Lys Ala Lys Leu Arg Ala Ser His Thr
530 535 540
Trp Asn Pro Ile Gln Gln Met Ser Ile Asn Val Asp Asn Gln Phe Asn
545 550 555 560
Tyr Leu Pro Ser Asn Ile Gly Gly Met Lys Ile Val Tyr Glu Lys Ser
565 570 575
Gln Leu Ala Pro Arg Lys Leu Tyr
580

Claims (10)

1. A raccoon parvovirus VP2 gene is characterized in that the raccoon parvovirus VP2 gene has a nucleotide sequence shown in SEQ ID No. 1.
2. An expression vector, which is obtained by inserting the gene VP2 of raccoon parvovirus of claim 1 into a vector.
3. The expression vector of claim 2, wherein the vector comprises the pET30a vector.
4. A recombinant bacterium obtained by co-transforming the expression vector of claim 2 or 3 and a chaperone plasmid into ER2566 cells.
5. The recombinant strain as claimed in claim 4, wherein the mass ratio of the expression vector to the molecular chaperone plasmid is 0.5-1.5: 0.5-1.5.
6. The recombinant bacterium of claim 4 or 5, wherein the chaperone plasmid comprises an pTf6 plasmid.
7. A method for preparing a raccoon parvovirus VP2 protein is characterized by comprising the following steps:
1) inoculating the recombinant strain of any one of claims 4 to 6 into a liquid culture medium containing L-arabinose, and culturing to obtain a culture;
2) OD of the culture obtained when said step 1) is performed600When the value is 0.6, mixing the mixture with isopropyl thiogalactose, inducing for 14-18 h, and collecting thalli;
3) and (3) mixing the thalli obtained in the step 2) with the lysate liquid, carrying out ice-bath ultrasonic crushing, centrifuging the obtained ultrasonic substance, and obtaining a precipitate as the protein VP2 of the raccoon parvovirus.
8. The method according to claim 7, wherein the concentration of L-arabinose in the liquid medium is 1.5 to 2.5mg/ml, and the concentration of isopropylthiogalactose in the culture is 0.05 to 0.15 mmol/ml.
9. The method of claim 7, wherein the components of the lysate comprise 50mM Tris and 150-500 mM NaCl, and the pH of the lysate is 8.
10. A method for in-vitro assembly of virus-like particles by using the raccoon parvovirus VP2 protein, which is characterized in that the raccoon parvovirus VP2 protein prepared by the preparation method of any one of claims 7 to 9 is placed in an assembly solution for in-vitro assembly, wherein the components of the assembly solution comprise 50mM Tris and 150-500 mM NaCl, and the pH value of the assembly solution is 8.
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Application publication date: 20200228

RJ01 Rejection of invention patent application after publication