CN114107392A - Preparation method of CVB5 virus-like particles - Google Patents
Preparation method of CVB5 virus-like particles Download PDFInfo
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Abstract
The invention discloses a preparation method of CVB5 virus-like particles, which specifically comprises the following steps: step 1, amplifying a CVB5 genome to obtain a P1 and 3CD gene fragment, wherein the 5 'end and the 3' end of the 3CD gene respectively have Xho I enzyme cutting sites and Sph I enzyme cutting sites, enzyme cutting the synthesized gene with the enzyme cutting sites, connecting the enzyme cut site to the downstream of a P10 promoter of a pFast-Bac Dual vector subjected to enzyme cutting in advance, and constructing to obtain pFast-BacDual-3 CD; step 2, respectively carrying Not I and Xba I enzyme cutting sites at the 5 'end and the 3' end of the P1 gene, carrying out enzyme cutting on the synthesized gene with the enzyme cutting sites, connecting the synthesized gene with the enzyme cutting sites to a pFast-Bac Dual-3CD plasmid subjected to enzyme cutting in advance, and preparing to obtain a plasmid pFast-Bac Dual-3 CD-P1; and 3, transforming the shuttle expression vector which constructs the 3CD fused with the CVB5 and the P1 protein sequence into an escherichia coli DH10Bac competent cell to obtain positive clone, and extracting recombinant baculovirus genome DNA. The 3CD protein fused with the exogenous peptide fragment of the CVB5 provided by the invention cuts the P1 protein to form VLPs, and further, the VLPs form CVB5 virus-like particles with uniform size.
Description
Technical Field
The invention relates to the technical field of immunity, in particular to a preparation method of CVB5 virus-like particles.
Background
Hand-Foot-and-Mouth Disease (HFMD) is an acute infectious Disease in children caused by infection with various enteroviruses. Most infant patients have sudden onset of diseases, mainly symptoms of fever, rash, herpes, ulcer of hands, feet, oral cavity and other parts, a few infant patients can be complicated with aseptic meningitis, encephalitis, acute flaccid arthralgia, neurogenic pulmonary edema and serious complications of myocarditis, and individual serious infant patients can rapidly progress to die. Outbreaks and epidemics have occurred in various parts of the world in recent years, and the critical rate and the death rate are all rising, which poses serious threats to the health of children and causes huge economic losses. The pathogens of HFMD are picornaviridae, mainly including enterovirus 71, coxsackievirus A and B, echovirus, etc. More and more research evidence in recent years shows that coxsackie virus type B5 (abbreviated as CVB5) is one of the important pathogens of hand-foot-and-mouth disease.
CVB5 is a single-stranded positive-stranded RNA virus without an envelope, and the virus particles are of an icosahedral three-dimensional symmetrical spherical structure with a diameter of 24-30 nm. The CVB5 genome is 7,400bp in length and consists of one open reading frame and two untranslated regions. The CVB5 genome open reading frame encodes a polyprotein precursor of 2,194 amino acids, which is further hydrolyzed by the virus's own encoded proteases into three precursor proteins (P1, P2, P3). The P1 precursor protein gene encodes four structural proteins, namely VP1, VP2, VP3 and VP4, wherein three polypeptides, namely VP1, VP2 and VP3, are presented on the surface of the virus capsid, and the neutralizing antibody mainly acts on VP 1; VP4 is tightly attached to the viral core and embedded inside the virus. The P2 and P3 gene regions encode seven non-structural proteins-2A, 2B, 2C, 3A, 3B, 3C and 3D, where the main role of the 3CD protease is to cleave P1 and assemble into viral capsids. VLPs are genetic material-free shell-like structures composed of viral coat proteins. Because the vaccine does not contain nucleic acid of virus, the vaccine can not be autonomously replicated, and compared with other antigen components, the vaccine is closer to a natural conformation, can more effectively cause an organism to generate immune response, and provides a new idea for vaccine research.
Baculovirus is a double-stranded circular DNA virus with envelope package, its genome size is 80-180kb, and it can be used as specific host by arthropod to make infection and transmission in nature. One feature that baculovirus distinguishes from other viruses is that it has two distinct virion morphologies: one type of virion, a Budded Virus (BV), primarily mediates cell-to-cell systemic infection, invading cells by receptor-mediated endocytosis; the other is embedded virus particles (ODV), and in the process of oral infection of the virus, the shell of the embedded virus particles is separated by the alkaline environment of the intestinal tract of the arthropod, and the embedded virus particles germinate into the virus with infection capacity and infect intestinal cells to achieve virus transmission. Coli-insect cell shuttle vector (Bacmid) is the most convenient recombinant baculovirus screening system. Bacmid is able to replicate in e. The recombinant Bacmid transfects insect cells, packages out recombinant baculovirus and expresses exogenous genes at the same time.
Disclosure of Invention
Objects of the invention
In view of the above, the present invention aims to provide a method for preparing a CVB5 virus-like particle, which can obtain a high-purity uniform-form virus-like particle by fusing the P1 protein of CVB5 exogenous peptide fragment and 3CD protease, and can be used for preparing a hand-foot-and-mouth vaccine.
(II) technical scheme
In order to achieve the technical purpose:
in one aspect, we provide a polycistronic eukaryotic expression vector for expressing CVB5 type virus-like particles, comprising a P1 protein expression sequence under the control of a polyhedrin promoter and a non-structural protein expression sequence under the control of a P10 promoter, respectively.
Preferably, the protein expression sequence is a 3CD protein expression sequence.
Preferably, the CVB5 virus-like particle is a shuttle expression vector of P1 protein fused with exogenous peptide fragments and 3CD protein.
On the other hand, the preparation method of the CVB5 virus-like particle specifically comprises the following steps:
and 2, respectively carrying Not I and Xba I enzyme cutting sites at the 5 'end and the 3' end of the P1 gene, carrying out enzyme cutting on the synthesized gene with the enzyme cutting sites, connecting the synthesized gene with the enzyme cutting sites to a pFast-Bac Dual-3CD plasmid subjected to enzyme cutting in advance, and preparing to obtain a plasmid pFast-Bac Dual-3 CD-1.
Preferably, the method further comprises the following steps:
and 4, transfecting the recombinant baculovirus genome DNA prepared in the step 3 into an insect cell sf9, harvesting cell culture supernatant, and storing a first-generation virus solution.
And 5, infecting the insect cells sf9 with the first generation of virus solution obtained in the step 4, and repeating the steps to obtain a sixth generation of virus solution with high titer.
Preferably, the transfected cells are insect Spodoptera frugiperda sf9 cells, sf9 cells are uniform in size, convenient to operate and sensitive to baculovirus, and are generally used as insect expression cells and easy to produce high-level protein expression.
Preferably, the 3CD protease fused with the exogenous fragment is used for cutting the P1 protein to form VLP, and the VLP lays a foundation for CVB5 virus-like particle vaccine research.
According to the technical scheme, the method has the following beneficial effects:
the 3CD protein fused with the exogenous peptide fragment of the CVB5 provided by the invention cuts the P1 protein to form VLPs, and further, the VLPs form CVB5 virus-like particles with uniform size.
The invention develops a virus-like particle similar to CVB5 in structure as a vaccine antigen, a granular repetitive structure is observed under an electron microscope, and the granular antigen generally has stronger immunogenicity.
The prepared vaccine antigen does not contain virus genetic material, does not have potential infection possibility, and is more suitable for vaccine candidate antigens.
The CVB5 viroid particles of the invention obtain high-purity and uniform-shape viroid particles, and the induced immune response degree of the vaccine based on the viroid particles is close, and the subsequent antigen content determination is facilitated.
The invention can obtain virus-like particles with high purity and uniform shape, and is used for preparing hand-foot-mouth vaccines.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1: pFastBac Dual plasmid map.
FIG. 2: pFastBac Dual-3CD restriction map.
FIG. 3: pFastBac Dual-P1 restriction map.
FIG. 4: PCR detection of recombinant Bacmid-3CD-P1 baculovirus genome.
FIG. 5: SDS-PAGA detection result chart before purification of recombinant CVB5 virus-like particles.
FIG. 6: SDS-PAGA detection result graph after recombinant CVB5 virus-like particle purification
FIG. 7: electron microscope observation of recombinant CVB5 virus-like particle.
In fig. 2: 1: DL5000Marker 2: 3CD-pFastBacDual map before enzyme digestion
3, CD-pFastBacDual double enzyme digestion map.
In fig. 3: 1: DL5000Marker 2: P1-pFastBacDual map before enzyme digestion;
3-5, P1-pFastBacDual 3 single colony double enzyme cutting maps.
In fig. 4: 1: DL5000Marker 2: before double enzyme digestion, P1-pFastBacDual;
3-6, respectively verifying Bacmid-3CD-P1 baculovirus genome 3 CD;
7-10: respectively, the Bacmid-3CD-P1 baculovirus genome P1.
Detailed Description
The following description is merely exemplary in nature and is not intended to limit the present disclosure, application, or uses. It should be understood that throughout the drawings, identical or similar reference numerals indicate identical or similar parts and features. The drawings are only schematic representations of the concepts and principles of the embodiments of the disclosure, and do not necessarily show specific dimensions or proportions of the various embodiments of the disclosure. Certain features that are part of a particular figure may be exaggerated in order to illustrate relevant details or structures of embodiments of the present disclosure.
Referring to FIGS. 1-7:
example one
A polycistronic eukaryotic expression vector for expressing CVB5 virus-like particles contains a P1 protein expression sequence and a non-structural protein expression sequence, wherein the P1 protein expression sequence and the non-structural protein expression sequence are respectively regulated and controlled by a polyhedrin promoter.
Wherein the non-structural protein expression sequence is a 3CD protein expression sequence.
In addition, the CVB5 virus-like particle is a shuttle expression vector fusing P1 protein of CVB5 exogenous peptide segment and 3CD protein.
Example two
A preparation method of CVB5 viroid-like particles specifically comprises the following steps:
and 2, respectively carrying Not I and Xba I enzyme cutting sites at the 5 'end and the 3' end of the P1 gene, carrying out enzyme cutting on the synthesized gene with the enzyme cutting sites, connecting the synthesized gene with the enzyme cutting sites to a pFast-Bac Dual-3CD plasmid subjected to enzyme cutting in advance, and preparing to obtain a plasmid pFast-Bac Dual-3 CD-1.
Specifically, CVB5 virus solution stored in a laboratory is used for extracting CVB5 RNA by a Trizol method, the RNA is inverted into cDNA, and PCR amplification is carried out to obtain P1 and 3CD gene fragments, wherein the P1 carries Xba I and Not I enzyme cutting sites, and the 3CD gene fragment carries Xho I and Sph I enzyme cutting sites.
P1 amplification primer
An upstream primer: 5'-ATAAGAATGCGGCCGCATGGGAGCTCAAGTATCAACAC-3'
A downstream primer: 5'-TGCTCTAGATTAGGTGGTCTGCATGGTTGTTAT-3'
3CD amplification primer
An upstream primer: 5'-CCGCTCGAGATGGGTCCCGCTTTTGAGTTTGCT-3'
A downstream primer: 5'-ACATGCATGCTTAAAAGGAGTCCAGCCATTTCCT-3' are provided.
Further comprising the steps of:
and 4, transfecting the recombinant baculovirus genome DNA prepared in the step 3 to cells, harvesting the cells, culturing a supernatant, and storing a virus solution.
Specifically, a plasmid carrying CVB5 and fused with 3CD and P1 protein sequences of a foreign peptide fragment is transformed into an escherichia coli DH10Bac competent cell, and a positive clone is screened. Extracting recombinant bacterium genome DNA, transfecting insect Spodoptera frugiperda sf9 cells, harvesting cell culture supernatant in 5-7 days, and storing first-generation virus liquid. The sf9 cells are uniform in size, convenient to operate, sensitive to baculovirus, usually used as insect expression cells and easy to produce high-level protein expression.
And 5, infecting the insect cells sf9 with the first generation of virus solution obtained in the step 4, and repeating the steps to obtain a sixth generation of virus solution with high titer.
It should be noted that, in the examples of the present invention, the CVB5 virus, the pFast Bac Dual vector, the DH10Bac competent cell and Spodoptera frugiperda cell were self-preserved, the serum-free medium was purchased from Hyclone, and the fetal bovine serum was AusGeneX Australian fetal bovine serum.
The 3CD protease fused with the exogenous fragment is used for cutting the P1 protein to form VLP, and the VLP lays a foundation for CVB5 virus-like particle vaccine research.
In order to obtain a large amount of protein, the harvested supernatant containing the recombinant baculovirus is continuously infected with Spodoptera frugiperda cells with high density, next generation virus solution is harvested and stored, meanwhile, the cells are harvested, and sixth generation virus solution is harvested and stored according to the method. The protein of interest was purified by cesium chloride density gradient centrifugation.
The method comprises the following specific steps: repeatedly freezing and thawing the collected cells at-80 deg.C for three times to lyse protein, centrifuging at 4 deg.C and 10000rpm for 5 min, removing cell debris, and collecting supernatant. Adding 1.85g of cesium chloride powder into every 4mL of supernatant, mixing uniformly, ultracentrifuging at 36000rpm at 4 ℃ for 16h, and taking eight parts of components by 0.5mL from top to bottom after centrifugation. The fractions were then dialyzed 3 times at 4 ℃ for 2 hours against 1 XPBS, 3kD dialysis bags, and finally concentrated at 4 ℃ using polyethylene glycol 6000(PEG6000), the concentrate was collected and the protein concentration was determined using NanoDrop 2000, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western-Blot to determine the integrity and purity of the purified protein.
The mice immunized with the purified CVB5 VLP can produce specific serum antibody and simultaneously have protective effect on CVB5 infected suckling mice. CVB5 VLP was mixed with aluminum adjuvant and 6-8 week old Balb/C mice were immunized by intraperitoneal injection at 0,1 and 2 weeks, respectively. Mouse serum samples were collected at week 4. Subsequently, mice at 1 day of birth were vaccinated with CVB5 VLP by active immunization and 1 day later were challenged with CVB 5. Compared with a control group, the survival rate of mice inoculated with CVB5 VLP can reach more than 60 percent after 15 days of toxin challenge.
Exemplary embodiments of the solution provided by the present disclosure have been described in detail above with reference to preferred embodiments, however, it will be understood by those skilled in the art that many variations and modifications may be made to the specific embodiments described above, and that many combinations of the various technical features and structures provided by the present disclosure may be made without departing from the concept of the present disclosure, without departing from the scope of the present disclosure, which is defined by the appended claims.
Claims (7)
1. A polycistronic eukaryotic expression vector for expressing CVB5 virus-like particles is characterized by comprising a P1 protein expression sequence and a non-structural protein expression sequence, wherein the P1 protein expression sequence and the non-structural protein expression sequence are respectively regulated and controlled by a polyhedrin promoter and a P10 promoter.
2. The polycistronic eukaryotic expression vector of claim 1, wherein the nonstructural protein expression sequence is a 3CD protein expression sequence.
3. The polycistronic eukaryotic expression vector for expressing the CVB5 virus-like particle of claim 1 or 2, wherein the CVB5 virus-like particle is a shuttle expression vector fusing the P1 protein and the 3CD protein of the CVB5 foreign peptide fragment.
4. A preparation method of CVB5 virus-like particles is characterized by comprising the following steps:
step 1, performing reverse transcription and PCR amplification on CVB5 gene RNA to obtain P1 and 3CD gene fragments, wherein the 5 'end and the 3' end of the 3CD gene are respectively provided with Xho I enzyme cutting sites and Sph I enzyme cutting sites, performing enzyme cutting on the synthesized gene with the enzyme cutting sites, connecting the enzyme-cut synthesized gene with the enzyme cutting sites to the downstream of a P10 promoter of a pFast-Bac Dual vector subjected to enzyme cutting in advance, and constructing to obtain pFast-Bac Dual-3 CD;
and 2, respectively carrying Not I and Xba I enzyme cutting sites at the 5 'end and the 3' end of the P1 gene, carrying out enzyme cutting on the synthesized gene with the enzyme cutting sites, connecting the synthesized gene with the enzyme cutting sites to a pFast-Bac Dual-3CD plasmid subjected to enzyme cutting in advance, and preparing to obtain a plasmid pFast-Bac Dual-3 CD-1.
5. The method of claim 4, further comprising the steps of:
step 3, transforming the constructed shuttle expression vector fused with the 3CD of the CVB5 and the P1 protein sequence into an escherichia coli DH10Bac competent cell to obtain positive clone, and extracting recombinant baculovirus genome DNA;
and 4, transfecting the recombinant baculovirus genome DNA prepared in the step 3 into an insect cell sf9, harvesting cell culture supernatant, and storing a first-generation virus solution.
And 5, infecting the insect cells sf9 with the first generation of virus solution obtained in the step 4, and repeating the steps to obtain a sixth generation of virus solution with high titer.
6. The method for preparing the CVB5 virus-like particle as claimed in claim 4, wherein the transfected cells are insect Spodoptera frugiperda sf9 cells, sf9 cells are uniform in size, convenient to operate, sensitive to baculovirus and generally used as insect expression cells and easy to produce high-level protein expression.
7. The method of claim 5, wherein the 3CD protease fused with the foreign fragment cleaves the P1 protein to form VLPs.
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