CN117866995A - Method for expressing avian adenovirus protein by using escherichia coli and forming virus-like particles by in-vitro assembly - Google Patents
Method for expressing avian adenovirus protein by using escherichia coli and forming virus-like particles by in-vitro assembly Download PDFInfo
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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
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Abstract
The invention discloses a method for expressing avian adenovirus protein by using escherichia coli and forming virus-like particles by in vitro assembly, belonging to the technical field of animal vaccines. The invention expresses the fiber protein and the penton base protein of the avian adenovirus by using an escherichia coli expression system for the first time, and realizes self-assembly in vitro to form virus-like particles. The invention provides a method for forming virus-like particles by in vitro assembly, which is based on the mode of expressing E.coli, uses E.coli to express the penton base and fiber proteins of adenovirus 8b serotype and adenovirus 11 serotype, and successfully realizes the in vitro assembly of the penton base and fiber proteins expressed by an E.coli expression system to form VLP particles.
Description
Technical Field
The invention belongs to the technical field of animal vaccines, and particularly relates to a method for expressing avian adenovirus proteins by using escherichia coli and assembling the avian adenovirus proteins in vitro to form virus-like particles.
Background
Virus-like particles (VLPs) are considered safe and effective viral disease candidate vaccines [1] . VLPs are virus-sized particles having a supramolecular structure of rods or icosahedrons [2] . They consist of one or more recombinantly expressed viral structural proteins that spontaneously assemble into particles without incorporation of the viral genome. They display antigens in an orderly and repetitive manner, thereby inducing a rapid, powerful humoral immune response and an efficient T cell response [3] . Adenovirus is a large economic loss to the poultry industry as an infectious disease transmitted among birds, and thus development of a safer and more effective vaccine to prevent adenovirus transmission is urgently required. It has been shown that the expression of recombinant baculoviruses of the penton base and fiber proteins of adenoviruses in insect cells allows self-assembly into VLP particles [4][5] . However, no proteins have been expressed in E.coli and assembled in vitro to form VLP particles.
Reference is made to:
[1]Jennings Gary T,Bachmann Martin F,The coming of age of virus-like particle vaccines.[J].Biol Chem,2008,389:521-36.
[2]Johnson J E,Chiu W,Structures of virus and virus-like particles.[J].Curr Opin Struct Biol,2000,10:229-35.
[3]Grgacic Elizabeth V L,Anderson David A,Virus-like particles:passport to immune recognition.[J].Methods,2006,40:60-5.
[4]Wang Xinglong,Tang Qiuxia,Qiu Li et al.Penton-dodecahedron of fowl adenovirus serotype 4 as a vaccine candidate for the control of related diseases.[J].Vaccine,2019,37:839-847.
[5]Szolajska Ewa,Burmeister Wim P,Zochowska Monika et al.The structural basis for the integrity of adenovirus Ad3 dodecahedron.[J].PLoS One,2012,7:e46075.
disclosure of Invention
The invention aims to provide a method for expressing avian adenovirus proteins by using escherichia coli and assembling the avian adenovirus proteins in vitro to form virus-like particles.
The invention provides a method for forming virus-like particles by in vitro assembly, which comprises the following steps: (1) The coding genes of the fiber protein and the coding genes of the penton base protein from the avian adenovirus are respectively inserted into a prokaryotic expression vector to form a prokaryotic expression recombinant vector;
(2) Respectively converting the obtained two prokaryotic expression recombinant vectors into an escherichia coli expression strain, expressing target proteins under the induction of IPTG, and collecting bacterial body crushing supernatant to obtain two target protein solutions;
(3) And respectively purifying and mixing the two target protein solutions, and dialyzing to obtain virus-like particles.
Preferably, the avian adenovirus of step (1) comprises an avian adenovirus 8b serotype and an avian adenovirus 11 serotype.
Preferably, the fiber protein and the pentonbase protein of step (1) are derived from the same avian adenovirus serotype.
Preferably, before the coding gene of the fiber protein and the coding gene of the penton base protein are respectively inserted into the prokaryotic expression vector, the method further comprises codon optimization, the nucleotide sequence of the coding gene of the optimized fiber protein is shown as SEQ ID No.1 or SEQ ID No.2, and the nucleotide sequence of the coding gene of the optimized penton base protein is shown as SEQ ID No.3 or SEQ ID No. 4.
Preferably, the prokaryotic expression vector of step (1) comprises pET-28a, pET-28b or pCold-I.
Preferably, after the transformation in the step (2), the method further comprises culturing the transformed bacterium to OD 600 When the value is 0.6-0.8, IPTG is induced to express.
Preferably, the purification of step (3) comprises loading the column with Ni-NTA His band resin by affinity chromatography, and finally eluting the target protein with 500mM imidazole.
Preferably, the mass ratio of the two target proteins in the step (3) is 1:1.
Preferably, the dialysis in step (3) comprises dialysis in a dialysate with a pH of 8.0, the temperature of the dialysis being 4 ℃ and the time being 24 hours, the dialysate being replaced every 6 hours;
the composition of the dialysate included 0.15M NaCl and 20mM Tris-HCl.
The invention also provides virus-like particles prepared by the method for forming virus-like particles by in vitro assembly.
The beneficial effects are that: the invention provides a method for forming virus-like particles by in vitro assembly, which is based on the mode of expressing escherichia coli, and an escherichia coli expression system has the advantages of being most widely applied, economical, rapid and high in yield. The invention expresses the fiber protein and the penton base protein of the avian adenovirus by using an escherichia coli expression system for the first time, and realizes self-assembly in vitro to form virus-like particles.
Drawings
FIG. 1 is a graph showing the purification result of FADV-8b-fiber protein;
FIG. 2 is a graph showing the purification result of FADV-8b-pentonbase protein;
FIG. 3 is a graph showing the purification result of FADV-11-fiber protein;
FIG. 4 is a graph showing the purification result of FADV-11-penton base protein;
FIG. 5 is a graph showing peaks of FADV-8b assembled products passing through a molecular sieve (10 kD-600 kD);
FIG. 6 is a diagram of FADV-8b virus-like particles;
FIG. 7 is a graph showing peaks of FADV-11 assembled products passing through a molecular sieve (10 kD-600 kD);
FIG. 8 is a diagram of FADV-11 virus-like particles;
FIG. 9 is a SDS-PAGE chart of peaks after FADV-8b passes through a molecular sieve, wherein red arrows indicate FADV-8b-penton base proteins, and yellow arrows indicate FADV-8b-fiber proteins;
FIG. 10 is a graph of a peak western blot after FADV-8b passes through a molecular sieve, wherein a red arrow indicates FADV-8b-penton base protein, and a yellow arrow indicates FADV-8b-fiber protein;
FIG. 11 is a peak non-denaturing PAGE map of FADV-8b after passing through a molecular sieve;
FIG. 12 is a SDS-PAGE chart of peaks after FADV-11 has passed through a molecular sieve, wherein red arrows indicate FADV-11-penton base protein and FADV-11-fiber protein;
FIG. 13 is a peak western blot plot of FADV-11 after molecular sieve passage;
FIG. 14 is a diagram showing the non-denaturing PAGE of FADV-11 after passing through a molecular sieve, wherein the uppermost band of the gel in lane 1 corresponds to the peak of the virus-like particle, the bands indicated by the red arrows in lanes 1 and 2 correspond to peak 2, and the bands indicated by the yellow arrows in lanes 3 and 4 correspond to peak 3 of the protein which is not assembled into the virus-like particle.
Detailed Description
The invention provides a method for forming virus-like particles by in vitro assembly, which comprises the following steps: (1) The coding genes of the fiber protein and the coding genes of the penton base protein from the avian adenovirus are respectively inserted into a prokaryotic expression vector to form a prokaryotic expression recombinant vector;
(2) Respectively converting the obtained two prokaryotic expression recombinant vectors into an escherichia coli expression strain, expressing target proteins under the induction of IPTG, and collecting bacterial body crushing supernatant to obtain two target protein solutions;
(3) And respectively purifying and mixing the two target protein solutions, and dialyzing to obtain virus-like particles.
The coding genes of the fiber protein and the coding genes of the penton base protein from the avian adenovirus are respectively inserted into a prokaryotic expression vector to form a prokaryotic expression recombinant vector. The avian adenoviruses according to the invention preferably comprise an avian adenovirus 8b serotype (FADV-8 b) and an avian adenovirus 11 serotype (FADV-11), and the fiber protein and the pendon base protein are derived from the same serotype, wherein the amino acid of the FAdV-8b fiber protein has an accession number AWT08538.1 on NCBI, the amino acid of the FAdV-8b pendon base protein has an accession number ANJ02525.1 on NCBI, the amino acid of the FAdV-11fiber protein has an accession number AIS19830.1 on NCBI, and the amino acid of the FAdV-11 pendon protein has an accession number AIS19822.1 on NCBI. The invention preferably further comprises codon optimization of the coding gene of the protein according to the preference of the expression cells before the coding gene of the protein is inserted into a prokaryotic expression vector, and the nucleotide sequence of the optimized coding gene of the fiber protein is preferably shown as SEQ ID No.1 or SEQ ID No.2, wherein the nucleotide sequence of the FADV-8b-fiber protein after codon optimization (SEQ ID No. 1): ATGGCTACTTCAACACCCCACGCATTTAGTTTCGGTCAGATCGGTTCTCGTAAACGTCCGGCAGGCGGTGATGGTGAACGTGACGCAAGCAAAGTTCCGAAAATGCAGACTCCGGCTCCGAGCGCGACCGCTAACGGCAACGATGAGCTGGACCTGGTTTATCCGTTTTGGCTGCAAAACGGCAGCACCGGAGGCGGTGGCGGCGGCAGCGGTGGCAACCCGAGCCTGAATCCACCGTTCCTAGACCCGAATGGTCCGCTCGCGGTGCAAAACAGCCTGCTGAAGGTCAACACCGCCGCCCCGATCACCGTTACCAATAAAGCATTGACGCTGGCTTACGAGCCGGAGTCGCTTGAGTTGACCAATCAGCAGCAACTGGCGGTCAAGATCGATCCGGAAGGTCCGCTCAAGGCCACCACTGAAGGTATCCAGCTGTCTGTGGACCCGACCACCTTGGAGGTTGACGACGTGGACTGGGAACTGACAGTGAAGCTGGACCCGGATGGCCCGCTGGACAGCTCTGCTGCGGGCATTACAGTGCGCGTTGATGAAACCTTGTTGATTGAAGATGACGTGTCCGGTCAGGGCAAAGAATTAGGTGTTAATCTGAACCCGGCTGGCCCCATCACCGCGGATGAGCAGGGTCTCGACTTAGAGATTGATAACCAGACCCTGAAAGTGAACAGCGTGACTGGCGGTGGCGTACTGGCGGTTCAACTTAAGTCCCAAGGTGGTTTGACCGTGCAGACCGACGGCATTCAGGTTAACACCCAGAACTCCATTACTGTCACCAATGGTGCGCTGGACGTCAAAGTGGCCGCGAATGGCCCACTGGAGTCGACCGATACCGGTCTGACGTTGAACTACGATCCAGGTGATTTTACCGTCAATGCGGGTACGCTTTCCATCATTCGCGACCCGGCGCTGGTGGCAAACGCGTACCTGACGTCTGGCGCGAGCACCCTGCAACAATTTACTGCGAAAAGCGAAAATAGCAGCCAGTTCTCCTTCCCGTGTGCATACTACTTGCAACAATGGCTGAGCGATGGGCTGATTGTTAGCTCTCTGTATCTGAAGCTGGATCGTGCACAGTTCACCAACATGCCGACGGGTGCCAACTATCAGAACGCCCGTTACTTCACCTTTTGGGTTGGTGCGGGCACGTCCTTTAACCTGTCCGCATTGACGGCTCCGACCATTACGCCGAACACCACGCAATGGAATGCGTTTGCCCCGGCGCTAGACTACAGCGGTGCACCGCCTTTCATCTACGACGCATCTTCGGTTGTTACGATCTACTTTGAACCGACAAGTGGCCGTCTGGAGAGCTATTTACCAGTTCTGACCGACAACTGGTCACAGACCTATAATCCGGGTACGGTGACCTTATGCGTGAAGACCGTACGCGTGCAGCTGCGTAGCCAAGGTACATTTAGCACCTTGGTTTGTTATAACTTCCGCTGCCAGAATACCGGCATCTTCAATAACAACGCGACCGCGGGGACGATGACCCTGGGTCCAATCTTCTTTAGCTGCCCGGCTCTCAGTACCGCGAACGCGCCGCATCATCACCACCATCACCACCATCACCACTAA; the nucleotide sequence (SEQ ID No. 2) of the FADV-11-fiber protein after codon optimization is: ATGGCTAAATCAACTCCCTTCACATTTAGTATGGGTCAGCATAGCAGCCGTAAACGTCCGGCGGATAGCGAAAACACGCAGAATGCTAGCAAAGTTGCTAAAACCCAAACCTCAGCAACTCGCGCAGGTGTTGACGGCAATGATGACCTGAACCTGGTTTATCCGTTTTGGCTGCAAAATAGCACGTCCGGCGGCGGTGGGGGTGGCAGCGGCGGCAACCCGAGCCTTAACCCGCCTTTCATTGATCCGAACGGCCCACTGTACGTTCAAAACAGCCTCCTGTACGTGAAGACCACCGCACCGATCGAGGTCGAGAATAAGAGCCTGGCGCTGGCGTACGACAGCTCCCTGGATGTTGACGCGCAAAACCAGCTGCAGGTGAAGGTGGATGCGGAAGGTCCGATTCGCATTTCCCCGGACGGCCTGGACATCGCCGTCGACCCGAGCACCCTGGAGGTGGACGACGAATGGGAACTGACCGTTAAGCTGGACCCAGCGGGTCCGATCTCATCAAGCTCTGCTGGCATCAACATTCGTGTTGATGACACTCTGCTGATTGAAGATGACGACACCGCTCAAGTTAAGGAGCTGGGTGTTCATCTGAACCCGAACGGCCCTATCACCGCCGACCAGGATGGTCTGGACCTGGAGGTCGACCCGCAGACATTAACTGTAACTACGAGCGGTGCGACGGGGGGTGTTCTGGGCGTTCTGCTCAAGCCGTCCGGTGGCCTCCAAACCTCGATCCAGGGCATCGGCGTGGCGGTGGCGGATACGCTGACGATCAGCTCTAACACCGGTACGGTCGAAGTTAAGACCGATCCGAATGGCTCCATCGGTTCGAGCTCTAACGGCATCGCCGTGGTGACGGACCCGGCGGGTCCGCTGACCACCTCCTCTAACGGTTTGTCTCTCAAGCTGACCCCAAACGGCAGCATCCAGAGCAGCTCGACCGGCTTGTCTGTGCAAACCGACCCTGCCGGTCCGATTACCAGTGGTGCTAATGGCTTGAGCCTAAGCTACGACACCAGCGATTTTACCGTTAGCCAGGGTATGCTGTCCATTATTCGCAATCCGAGCGCATACCCGGATGCTTATCTTGAGAGCGGCACCAATTTGTTGAACAACTACACCGCGTACGCAGAGAACAGCTCGAATTATAAATTTAACTGCGCATACTTCTTGCAAAGCTGGTATTCCAACGGCTTAGTGACTTCTTCACTGTATCTGAAGATCAATCGTGATAACTTGACCAGCCTGCCGAGCGGTCAGCTGTCCGAAAACGCTAAATACTTCACCTTCTGGGTGCCGACATACGAGAGCATGAATTTGTCTAACGTTGCCACGCCGACCATCACCCCGAGCAGCGTTCCGTGGGGTGCGTTTCTGCCGGCGCAGAACTGTACCTCGAACCCAGCGTTCAAATACTATTTGACGCAACCGCCGTCGATCTATTTCGAGCCGGAAAGCGGTAGCGTGCAGACTTTCCAGCCCGTGCTGACCGGTGATTGGGATACCAATACCTACAATCCGGGTACCGTGCAAGTGTGCATCCTGCCACAGACCGTCGTGGGCGGCCAAAGCACTTTCGTGAATATGACCTGCTACAATTTTCGTTGTCAGAATCCGGGTATTTTTAAAGTTGCGGCGTCCTCCGGTACGTTTACCATTGGTCCGATCTTCTATAGCTGCCATCACCACCACCACCATCACCACCACCATTAA. The nucleotide sequence (SEQ ID No. 3) of the FADV-8b-penton base protein after codon optimization is: ATGCTAGGACATCACGAATTTGGGTTCAGGGAGGCAGGCCTGCAACGCCCGAGCTCCTCGTCTGCGGCGGCCACTAGCGTGCCGCCAGCGCCACCGAGCTCCCCGGTCTCCGGCGTACCGCCGACGATGACGATGGGTCAGTTGCAACAACAGCGCTACCCGGCTACCGTTCATGGTTACCCGCCGGTAATCCCGGCGCCCGACAGCGCGGTGGACAACGGTACTGAACTGTTCGTTCCCGTGCAACGTGTTATGGCACCGACCGGCGGCCGTAATTCGATTAAATATCGTGATTACGCTCCGTGCCAAAACACCACCAAACTGTTCTATGTGGACAACAAACTGAGCGACATCGACACCTTTAACCCGGAGGCAAATCATTCTAACTACCGCACCACCGTGATTCATAATCAAGATCTGGACCCGGCGACGGCGGCGACCGAGACAATTCAGCTGGACAACCGTAGCTGCTGGGGTGGTGATTTGAAGACCGCGGTTAAGACCAACTGCCCGAATGTCAGCTCTTTTTTCCAGTCTAACACGGTTCGTGTCAAGCTGATGTCCTCACGTGACCCGATCCCTCCGGGCACGCCAGAGCCGACCACTCCGGCTGCGGCGTACGCCCCGGCTGGCGCGCAGTATAAGTGGTATGATTTGACGATTCCGGAGGGCAACTATGCACTGCCGGAGATCATCGATTTACTGAACGAAGGCATCGTGCAACTGTATCTGAGCGAAGGTCGTCAGAATAATGTTCTGCGTAGCGATATTGGCGTTAAGTTCGATACCCGTTACCTGAATTTATTGCGCGATCCGGTGACCGGTCTGGTTACGCCTGGTACATACGTCTTTAAAGGCTATCATCCGGACATCATTTTGTTACCAGGGTGCGCAGTTGACTTCACCCATAGCCGTCTGAGCCTGGTTCTAGGTATTGCGAAACGTGAACCGTACAGCAAGGGTTTCGTGATCACCTACGAAGATCTTGAGGGCGGTAACGTGCCAGCTTTGCTTGACGTAGATGCTGCTCAGATGAGCGGTGCGGACCAAGACGTCATCGAGCTCGCCGACGCTCAGCCGCTGCTGAAGGACAGCAAAGGTGTTAGCTATAACGTTATTTACGACGCGGACCAACGTCCGGTGACCGCATACCGCAGCTGGTTGATTGCCTACAACCAGTCCGGCAGCGCAGCCAACAGCACTACACTCCTGACCGTTCCGGACGTCGGTGGTGGCATTGGTGCCATGTATACCAGCATGCCGGACACCTTTGTGGCACCAACGGGTTTCAAAGAAGATAACACCACGAACTTTGCGCCGGTCGTGGGCATGAATCTGTTCCCGGCGTTGAATAAAGTTTACTATCAGGGTGCGTCCGCGTACGTTCAGCAACTGGAGAATTCATGTCAGAGTGCGACGGCTGCGTTTAACCGCTTTCCGGAAAATGAAATTCTGAAGCAGGCACCGCCTATCAACGTGTCTGCGGTGTGTGATAACCAGCCGGCTGTGGTGCAGCAAGGTGTTCTGCCGCTGAAGAACTCCTTGGCGGGTCTGCAACGCGTGCTTATCACCGATGATCAGAGACGTCCGATCCCGTACGTGTATAAAAGCCTGGCCACCGTTCAGCCGCGTGTGCTCTCGTCCTCCACCCTGCAACATCACCACCACCACCACCACCACCACCACtaa; the nucleotide sequence (SEQ ID No. 4) of the FADV-11-pentonbase protein after codon optimization is: ATGAGACGTAATGGACGAAGGAGGCGGAAAAACACCCGTGGTGTGACCCGTGAGCTGACTGCGATTAGCCGTGCCATGCTGGGTCATCACGATTTTGGCTTCCGCGAGGCGGCACTCAACCGTGGTTCGAGCAGCAGCTTTGCAACCTCCATGCCGGCTGCTCCGCCACCGAGCCCGGTGTCCGGTGTGCCGCCGGCTATGGCCCCACAGAGATACCCGGCGACCGTGCATGGTTATCCGCCGGTCATCCCGCCACTGGACAGCGCGGCAGACGACACCAGCGAACTGTTCGTGCCGGTTCAACGTGTTATGGCACCGACCGGAGGCCGTAACTCTATTAAATATCGCGATTACGTGCCGTGTCAAAACACCACGAAACTGTTCTATGTCGACAACAAGCTGTCCGATATCGATACCTTCAATCCGGAAGCAAATCATAGCAACTTTCGTACTACGGTGATTCATAATCAGGATCTGGACCCGGCGACGGCTGCCACGGAAACCATTCAGCTGGACAACCGTAGCTGCTGGGGTGGTGAACTGAAAACCGCCGTGAAGACCAATTGTCCGAATGTTTCTTCGTTCTTCCAGTCTAACACCGTGCGTGTTAAGCTGATGAGCTACCGCGATCCGGTGCCGCCGGGTACTGCGGCGCCAACCAGTCCGCAGCCGTACGCACCAGCTGGTGCGCAATATAAGTGGTATGATTTGACGATTCCGGAAGGCAACTATGCGTTACACGAAATCATCGATCTCCTTAACGAGGGCGTCGTACAAATTTACCTGAAGGAGGGCCGTCAGAACAACGTACAGAGAAGCGACATCGGCGTGAAGTTTGATACTCGCTATTTCAACCTGCTGCAGGACCCGGTTACCGGTTTGGTTACTCCGGGTACATACGTTTACAAGGGCTACCATCCGGACGTTGTTTTGCTGCCGGGGTGCGCAGTTGACTTCACCTACTCCCGTCTATCTCTGATGCTGGGCATCGCCAAACGTGAGCCTTACAGCAAAGGTTTTATCATAACCTATGAAGATCTTGAGGGCGGAAACGTTCCGGCGTTGCTGGACGTGGACGCGGCGCAGATGACCGGCGTAGACCAGGATGTTATCGAATTGGCCGATGCAAAACCGCTGCTGAAAGATAGCAAAGGCGTTTCGTATAATGTGATCTACGACAGCAATAATCGCCCTGTTACCGCGTACCGCAGCTGGCTGATTGCATACAATCAAAGCGGTTCCCCAGCTAATGAAACCACTCTGCTTACCGTCCCGGACGTGGGTGGTGGCATTGGTGCCATGTATACCAGCATGCCGGACACCTTTGTTGCACCGACGGGCTTCAAAGAGGACAACACCACCAACTTGGCGCCTGTTGTGGGCATGAATTTGTTCCCGGCGCTGAACAAGGTGTACTATCAGGGTGCTAGCACCTACGTGCAACAACTGGAGAACAGTTGCCAGAGCGCTACCGCTGCGTTTAACCGCTTTCCGGAGAATGAGATCCTGAAGCAGGCGCCGCCGATCAACGTTTCTGCCGTGTGCGACAACCAACCGGCGGTCGTGCAGCAGGGCGTTTTACCGCTGAAAAACAGCTTGGCGGGTTTGCAACGTGTGCTGATCACCGATGATCAACGCCGTCCGATTCCGTATGTTTACAAGTCCTTAGCGACGGTTCAACCACGTGTCCTGAGCTCTTCCACCCTGCAACATCACCACCACCACCATCACCACCACCACtaa.
The kind of the prokaryotic expression vector is not particularly limited in the present invention, and it preferably includes pET-28a, pET-28b or pCold-I. In the examples of the present invention, the nucleotide sequence is preferably inserted into the pET-28b vector through both NdeI and XhoI cleavage sites.
After obtaining two prokaryotic expression vectors, the invention can obtainRespectively converting the prokaryotic expression recombinant vectors into escherichia coli expression strains, expressing target proteins under the induction of IPTG, and collecting bacterial body crushing supernatant to obtain two target protein solutions. The method of the transformation is not particularly limited, but the transformation is preferably carried out by a heat shock transformation method in examples. The invention preferably converts two recombinant expression vectors into Rosetta escherichia coli expression strain, and shakes the strain to OD of bacterial liquid 600 0.6 to 0.8. The invention carries out shaking culture on the transformed strain, the shaking culture is preferably carried out in an LB culture medium, the parameter of the shaking culture is preferably 37 ℃, and the shaking culture is carried out at 180rpm, the OD of the two bacterial solutions is the OD of the invention 600 After reaching the standard, preferably, IPTG with the concentration of 0.5mM is added to induce the protein expression at 25 ℃, the bacterial liquid is centrifuged at 8000rpm for 10min after 12 hours of induced expression, and the supernatant is discarded to obtain the bacterial cells.
The invention preferably uses 20mM Tris-HCl,500mM NaCl pH 8.0 liquid heavy suspension, fully mixing, high pressure crushing by a high pressure crusher, after crushing the obtained solution centrifugal 30min by 12000rpm, getting the supernatant, the supernatant contains soluble target protein.
After the target protein liquid is obtained, the two target protein liquids are respectively purified and then mixed, and virus-like particles are obtained after dialysis. The purification according to the present invention preferably comprises purifying the two protein supernatants by AKTA protein purification apparatus, using affinity chromatography, packing the column with Ni-NTA His band resin, and finally eluting the hetero-protein and the target protein with 20mM Tris-HCl containing 40, 60, 80, 100, 125, 500mM imidazole, respectively, in buffer with pH of 8.0. The present invention preferably further comprises the step of observing the content of the purified target protein by SDS-PAGE after eluting the target protein.
The purified fiber protein and the penton base protein with the same quality are preferably taken to be mixed together, dialyzed in a dialysis solution with the pH of 8.0 of 0.15M NaCl,20mM Tris-HCl and the temperature of 4 ℃ for 24 hours, the solution is required to be changed every 6 hours in the middle, and the two proteins realize self-assembly under the condition to form VLP particles.
The invention also provides virus-like particles prepared by the method for forming virus-like particles by in vitro assembly.
In the embodiment of the invention, the assembled products of the fiber protein and the penton base protein, which are all from FADV-8b serotype strains, are placed under a transmission electron microscope for observation, and particles with the size of 50 nm-75 nm can be seen.
To further illustrate the present invention, a method of expressing avian adenovirus proteins with E.coli and assembling them in vitro to form virus-like particles is provided in the present invention with reference to the following examples, which are not to be construed as limiting the scope of the present invention.
Example 1
1. Sequencing the nucleotide sequences of the fiber protein and the penton base protein of the FADV-8b serotype strain, and performing codon optimization on the nucleotide sequences: FADV-8b-fiber (SEQ ID No. 1), FADV-11-fiber (SEQ ID No. 2), FADV-8b-penton base (SEQ ID No. 3) and FADV-11-penton base (SEQ ID No. 4) were inserted into PET-28b vector at NdeI and XhoI sites, respectively.
2. Four vectors are transformed into Rosetta escherichia coli expression strain, and shaking is carried out until OD of bacterial liquid is reached 600 0.6 to 0.8, adding IPTG with the concentration of 0.5mM at 25 ℃ to induce and express protein, centrifuging bacterial liquid at 8000rpm for 10min after 12 hours of induced and expressed, and discarding the supernatant to obtain bacterial cells.
3. The four cells were resuspended in 20mM Tris-HCl,500mM NaCl, pH 8.0, and the mixture was subjected to high-pressure crushing by a high-pressure crusher, and the crushed solution was centrifuged at 12000rpm for 30 minutes to obtain a supernatant.
4. Purifying the four protein supernatants by an AKTA protein purifier, and adopting an affinity chromatography method, wherein the method comprises the following specific steps:
(1) the column was packed with Ni-NTA His band resin, 1.6cm.times.20cm, and the column bed volume was 10mL, and nickel ions and affinity groups were bound to the column packing to form a nickel column having affinity energy.
(2) Balancing 2-5 column bed volumes by using 20mM Tris-HCl buffer solution with pH of 8.0, wherein the flow rate is 2mL/min;
(3) filtering the bacterial cell crushed supernatant through a 0.45 mu m filter membrane, and loading the sample at a flow rate of 1mL/min;
(4) re-washing the column bed with 20mM Tris-HCl buffer solution with pH of 8.0 for 2-5 column bed volumes at a flow rate of 2mL/min;
(5) performing stage elution with 20mM Tris-HCl containing 40, 60, 80, 100, 125 and 500mM imidazole respectively and buffer solution with pH of 8.0 at flow rate of 2mL/min, collecting elution peaks at each stage, and detecting molecular weight and purity of target protein by SDS-PAGE;
(6) washing 5 column bed volumes with pure water, washing 3 column bed volumes with 20% ethanol flow at a flow rate of 2mL/min, and preserving the column in a low temperature environment.
(7) The purification result of the FADV-8b-fiber protein is shown in figure 1, the purification result of the FADV-8b-penton base protein is shown in figure 2, the purification result of the FADV-11-fiber protein is shown in figure 3, and the purification result of the FADV-11-penton base protein is shown in figure 4. The concentrations of the 4 proteins after purification are shown in Table 1.
TABLE 1 concentration of purified protein
| Name of the name | FADV-8b-fiber | FADV-8b-penton | FADV-11-fiber | FADV-11-penton |
| Concentration of | 80ug/ml | 214ug/ml | 324ug/ml | 64ug/ml |
5. Mixing purified fiber protein and penton base protein with the same quality, dialyzing in a dialysis solution of 0.15M NaCl,20mM Tris-HCl and pH 8.0 at 4 ℃ for 24 hours, and changing the solution every 6 hours in the middle to realize self-assembly of the two proteins to form VLP particles under the condition;
6. the assembled product was further purified by molecular sieves and observed under electron microscopy to verify if VLP particles were formed.
After the FADV-8b assembled product passes through a molecular sieve (the separation range is 10kD-600 kD), as shown in FIG. 5, a virus-like particle peak is shown, and the assembled product is observed under a transmission electron microscope, so that the fiber protein and the penton base protein from the FADV-8b serotype strain form particles with the size ranging from 50nm to 75nm (FIG. 6).
After the FADV-11 assembled product passes through the molecular sieve, a part of proteins are not assembled into virus-like particles due to poor assembly effect, so that the peak of the virus-like particles is higher than the peak of the virus-like particles when the FADV-11 assembled product passes through the molecular sieve. As shown in FIG. 7, after FADV-11 assembled products are further purified by molecular sieves, FADV-11 virus-like particle peak sections are collected and placed under a transmission electron microscope for observation, and fiber proteins and penton base proteins derived from FADV-11 serotype strains constitute particles with a size in the range of 20nm to 75nm (FIG. 8).
And (3) performing SDS-PAGE, western blot and non-denaturing PAGE on each peak section collected after the FADV-8b assembled product and the FADV-11 assembled product pass through a molecular sieve, and further verifying that the fiber and the pendon base proteins are self-assembled into virus-like particles. After the FADV-8b assembled product passes through the molecular sieve, a distinct virus-like particle peak is formed, 3-tube solutions are sequentially collected, SDS-PAGE is performed to show that the peak mainly consists of FADV-8b-fiber protein and FADV-8b-penton base protein (figure 9), WB is further performed to verify that the peak mainly consists of FADV-8b-fiber protein and FADV-8b-penton base protein (figure 10), non-denaturing PAGE is performed, virus-like particles (17 nm in size) assembled by adding porcine circovirus type 2 CAP protein and virus-like particles (17 nm in size) assembled by porcine parvovirus VP2 protein are used as a comparison, and the virus-like particles of FADV-8b are always at the top of the gel as the virus-like particles assembled by porcine circovirus type 2 CAP protein and the virus-like particles assembled by porcine parvovirus VP2 protein, and cannot pass through the gel (figure 11).
After the FADV-11 assembled product passes through the molecular sieve, three obvious peaks are formed, and from peak 1 to peak 3, 8-tube solutions are collected in turn, so that it is clear that this peak section 1 mainly consists of FADV-8b-fiber protein and FADV-8b-penton base protein, and because of the close size of FADV-8b-fiber protein and FADV-8b-penton base protein, two proteins cannot be distinguished on SDS-PAGE (FIG. 12) and WB (FIG. 13), on non-denaturing PAGE (FIG. 14), the uppermost band of lane 1 gel corresponds to the peak of virus-like particles, the bands indicated by red arrows in lanes 1 and 2 correspond to peak 2, and the bands indicated by yellow arrows in lanes 3 and 4 correspond to peak 3 of proteins not assembled into virus-like particles.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (9)
1. A method of in vitro assembly to form virus-like particles comprising the steps of:
(1) The coding genes of the fiber protein and the pentonbase protein from the avian adenovirus are respectively inserted into a prokaryotic expression vector to form a prokaryotic expression recombinant vector;
(2) Respectively converting the obtained two prokaryotic expression recombinant vectors into an escherichia coli expression strain, expressing target proteins under the induction of IPTG, and collecting bacterial body crushing supernatant to obtain two target protein solutions;
(3) And respectively purifying and mixing the two target protein solutions, and dialyzing to obtain virus-like particles.
2. The method of in vitro assembly to form virus-like particles according to claim 1, wherein said avian adenovirus of step (1) comprises an avian adenovirus 8b serotype and an avian adenovirus 11 serotype.
3. The method of in vitro assembly to form virus-like particles according to claim 1 or 2, wherein the fiber protein and the penton base protein of step (1) are derived from the same avian adenovirus serotype.
4. The method for forming virus-like particles by in vitro assembly according to claim 3, wherein the nucleotide sequence of the optimized fiber protein encoding gene is shown as SEQ ID No.1 or SEQ ID No.2, and the nucleotide sequence of the optimized penton base protein encoding gene is shown as SEQ ID No.3 or SEQ ID No.4, before inserting the fiber protein encoding gene and the penton base protein encoding gene into the prokaryotic expression vectors, respectively.
5. The method of in vitro assembly to form virus-like particles according to claim 1 or 4, wherein said prokaryotic expression vector of step (1) comprises pET-28a, pET-28b or pCold-I.
6. The method of claim 1, further comprising culturing the transformant to OD after the transforming in step (2) 600 When the value is 0.6-0.8, IPTG is induced to express.
7. The method of claim 1, wherein the mass ratio of the two proteins of interest in step (3) is 1:1.
8. The method of in vitro assembly to form virus-like particles according to claim 1, wherein the dialysis of step (3) comprises dialysis in a dialysate having a pH of 8.0, the dialysis being at a temperature of 4 ℃ for 24 hours, the dialysate being replaced every 6 hours;
the composition of the dialysate included 0.15M NaCl and 20mM Tris-HCl.
9. A virus-like particle prepared by the method of in vitro assembly of any one of claims 1 to 8 to form a virus-like particle.
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