CN116769831A - Construction method of foot-and-mouth disease virus-like particle induced expression vector - Google Patents
Construction method of foot-and-mouth disease virus-like particle induced expression vector Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention relates to the biomedical field, in particular to a transposition-induced expression system for foot-and-mouth disease virus-like particles, which comprises nucleic acid for encoding transposase, a reverse transactivating element, an inducer and the foot-and-mouth disease virus-like particles, wherein the transposase can be transposase for encoding PiggyBac (PB) and variants thereof, the reverse transactivating element can be the reverse transactivating element and variants thereof, the inducer is tetracycline and derivatives thereof, and the nucleic acid of the foot-and-mouth disease virus-like particles is an optimized sequence P12A3Copti, as shown in SEQ ID NO: 3. The PB transposition-tetracycline induction expression system is constructed, and the optimized P12A3Copti is expressed in the system, so that the success rate of stably integrating the P12A and 3C genes into a cell genome is improved, and meanwhile, the expression quantity of FMDV capsid protein is increased by 15-30 times.
Description
Technical Field
The present invention relates to the biomedical field. In particular to a construction method of a foot-and-mouth disease virus particle-like expression vector.
Background
Foot-and-mouth disease (FMD) is an acute, febrile, high-contact infectious disease of human and animal co-morbid origin caused by foot-and-mouth disease virus, FMDV. FMDV belongs to the Picornaviridae family (Picornaviridae), the genus foot and mouth disease virus (Aphthotvrus). FMDV is spherical, has a regular icosahedral symmetry diameter of 25-30nm, is free of envelope, and consists of a single-stranded positive strand RNA of about 8500bp and capsid proteins, and comprises 3 parts of a 5 'non-coding region (untranslation region, UTR), a 3' -UTR and an open reading frame (open reading frame, ORF) in genomic RNA. The ORF encodes a large polyprotein that can be cleaved by viral proteases into 4 structural proteins (VP 4, VP2, VP3, VP 1) and 8 nonstructural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, 3D), with the 4 structural proteins of 60 molecules each constituting the capsid of the viral particle, and the 8 nonstructural proteins regulating viral replication, protein processing, and protein modification in the host. Proteases have leader proteins L, 2A, 3C and unknown enzymes, of which 3C protease (3 Cpro) plays a very important role in cleavage of FMDV structural proteins. During viral translation and modification, FMDV capsid protein precursor P12A is cleaved by 3CPr, yielding 1AB (VP 0), 1C (VP 3), 1D (VP 1) and 2A, with VP0 being further cleaved by 3CP into VP2 and VP4 during gene assembly. During assembly of the virions, VP0, VP3 and VP1 form 5S protomers first, then form the pentamer of 14S, and finally the empty FMDV capsids of 75S are assembled from 12 pentamers. VP0 is a precursor protein, which eventually self-cleaves into VP2 and VP4, thereby forming mature virions. Studies have shown that the structural protein VP1 is capable of inducing the production of neutralizing antibodies by host cells, while VP2, VP3, VP4 is involved in the assembly of the viral particles and affects the immunogenicity of the viral particles. The FMDV capsid protein precursor P1-2A contains main antigenic determinant of FMDV, and can induce organism to generate humoral immune response, thereby protecting animals from being attacked by FMDV. At present, many reports about constructing FMDV capsid protein live vector vaccines by utilizing different live vectors at home and abroad exist, and the used viral vectors are chicken pox virus, pseudorabies virus, adenovirus and baculovirus. Wherein, the FMDV capsid protein live vector vaccine constructed by the defective adenovirus type 5 is most studied intensively, and a good immune effect is obtained.
Virus-like particles (VLPs) are self-assembled from one or more recombinantly expressed structural proteins and are capable of rapidly inducing humoral and cellular immunity. FMDV virus-like particles (Virus like particles, VLPs), which are hollow particles composed of the mature capsid proteins VP0, VP3, VP1 of the virus, are closest to the native virus in morphology and conformation compared to other subunit vaccines, and have a wide range of uses, such as vaccines and drug-targeted delivery vehicles (Nooraei et al 2021). The production of VLPs using recombinant expression systems is a complex process. Vector construction, particularly the construction of the open reading frame (open reading frame, ORF), is one of the core and difficulties in recombinant expression of VLPs.
Expression of P12A and 3Cpro within the recombinant expression system allows cleavage of P12A to occur, thereby forming the mature capsid proteins VP0, VP3 and VP1 (Roosien et al, 1990). The joining of 3C downstream of P12A to P12A3C by means of the "ribosome jump function" of 2A is one of the usual modes of construction (Li et al, 2008) which co-express P12A and 3Cpro in a single ORF and which is capable of completely cleaving 2A short peptides, with simple features. However, the expression levels of the 2A upstream and downstream proteins appear to be different in different cells adding uncertainty to this manner of construction (Li et al 2008;Mignaqui et al, 2013 b). The placement of P12A, 3C downstream of the two promoters, respectively, for transcription is another way of construction. The dual promoter construction in insects showed lower yields of VLPs than P12A3C (Ruiz et al, 2014), a phenomenon that could be caused by the early massive transcription of the 3C gene placed downstream of the P10 promoter to inhibit P12A translation. Ribosome internal entry site elements (IRES) are also common building blocks for bicistronic vectors, but IRES have low translational initiation activity. The translation of 3Cpro in insect cells would be promoted with IRES significantly enhancing the expression of capsid proteins (Vivek Srinivas et al., 2016), indicating that a reduction in translation of 3Cpro to some extent is beneficial for expression of VLPs. Similar results have also been reported in mammalian cells, with some of them regulating the expression level of 3Cpro by co-transfection ratio of plasmids (Polacek et al, 2013), and with IRES elements reducing the expression of 3Cpro (Gullberg et al, 2013 b) to enhance the expression of mature capsid proteins. In addition, there have been studies on the enhancement of the expression level of P12A by recombinant vaccinia virus-T7 expression system to obtain a large number of VLPs (Porta et al, 2013 a). There are also researchers that enhance the expression level of capsid proteins by introducing mutations that reduce the activity of 3 Cpro. Efficient VLPs in insect cells were obtained by ligating HIV-1 frameshift and 3C (C142T) mutants downstream of 2A (pora et al, 2013a;Porta et al, 2013 b). The introduction of the 3Copti mutation also showed efficient expression of VLPs in mammalian cells under the construction of P12A3C (Puckette et al 2022). From this, it can be seen that rational control of the expression levels of P12A and 3Cpro for a particular 3Cpro (wild type, mutant) is highly efficient in producing FMD VLPs as precursors. In addition, removal of 3Cpro also produces VLPs (Cao et al, 2010), but balancing the expression levels of VP0, VP3, VP1 is often a difficulty.
Among the expression systems currently used for producing FMDV VLPs are mainly prokaryotic expression systems, eukaryotic expression systems and cell-free expression systems. The VP0, VP3 and VP1 structural proteins of FMDV are expressed in the same level and solubility in escherichia coli by adopting a Small ubiquitin-like modifier (SUMO) fusion co-expression technology, and the VLP is formed by enzyme digestion and in vitro assembly. Intracellular self-assembly of VLPs is achieved by co-expression of the capsid protein precursors P12A and 3C protease (3 Cpro) in eukaryotic expression systems. 3Cpro has been reported to have a strong toxic effect on eukaryotic cells, stable strong expression of the P12A and 3Cpro genes with constitutive promoters has not been achieved, and BHK-21 cell lines constitutively expressing the P12A3C gene can only produce very low amounts of FMDV capsid proteins. Currently, eukaryotic expression systems such as baculovirus-insect cell expression systems, mammalian expression systems, and the like can only be used for preparing FMDV VLPs by transient expression. The method for transiently expressing FMDV VLPs by using plasmid transfection mainly uses HEK293 cells as hosts, and although HEK293 cells have strong heterologous protein production capacity, medium-scale transfection of HEK293-6E cells with Polyethyleneimine (PEI) as a transfection reagent is reported to produce FMDV VLPs, but the method is time-consuming and high in use cost, and is unfavorable for large-scale production and application.
Disclosure of Invention
The invention improves the success rate of stably integrating the P12A and 3C genes into the genome of the cell by constructing a PB transposition-tetracycline induction expression system.
In a first aspect, the present invention provides a transposition-induced expression system for foot-and-mouth disease virus-like particles comprising a nucleic acid encoding a transposase, an inverse transactivator, an inducer, a foot-and-mouth disease virus-like particle.
Further, the transposase may be a transposase encoding PiggyBac (PB) and variants thereof.
Further, the transposase is preferably chypase.
Further, the reverse transactivator may be the reverse transactivator itself or a variant thereof.
Further, the reverse transactivator is preferably rtTA-Advanced.
Furthermore, the inducer is tetracycline substances and derivatives thereof.
Further, the inducer is preferably Dox-HCl (doxycycline hydrochloride).
Further, the nucleic acid of the foot-and-mouth disease virus-like particle is an optimized sequence P12A3 Coptiptipti, and is shown as SEQ ID NO. 3.
In a second aspect, the present invention provides a method for constructing a transposition-induced expression system for foot-and-mouth disease virus-like particles, the method comprising the steps of:
s01, constructing PB transposase plasmid;
s02, constructing a transactivator plasmid;
s03, constructing a plasmid of nucleic acid of PB transposition-induced expression foot-and-mouth disease virus-like particles;
s04, co-transfecting the obtained plasmid into host cells to obtain a complete transposition-induced expression system of foot-and-mouth disease virus-like particles.
Further, the expression vector of the PB transposase plasmid is selected from pTT5 or pVAX1.
Further, the reverse transactivator preferably encodes a reverse tetracycline transactivator rtTA.
Further, the nucleic acid of the foot-and-mouth disease virus-like particle is an optimized sequence P12A3 Coptiptipti, and is shown as SEQ ID NO. 3.
Further, the PB transposition-induced expression is preferably PB transposition-tetracycline induced expression.
In a third aspect, the present invention provides an application of a transposition-induced expression system of foot-and-mouth disease virus-like particles in preparing a biological preparation for foot-and-mouth disease.
Further, the biological agent comprises a transposition-induced expression system of the foot-and-mouth disease virus-like particle of the first aspect.
Further, the biological agent may be a foot-and-mouth disease vaccine formulation and/or a composition of a foot-and-mouth disease vaccine formulation.
Further, the vaccine may be an inactivated vaccine, a recombinant protein vaccine, a synthetic peptide vaccine, a nucleic acid vaccine, and an adenovirus vector vaccine.
Further, the composition of the foot-and-mouth disease vaccine preparation is a composition containing the foot-and-mouth disease vaccine and an immunological adjuvant.
Drawings
FIG. 1 shows a double restriction map of the plasmid pTT5-P12A3Cwt, pTT5-P12A3 Coptiopti. ( wt (E-B): plasmid pTT5-P12A3Cwt was digested with EcoRI and BamHI; wt: plasmid pTT5-P12A3Cwt, opti: plasmid pTT5-P12A3Coptiopti, opti (E-B): the plasmid pTT5-P12A3 Coptiptipti was digested with EcoRI and BamHI. )
FIG. 2 identification of PCR amplified Fra1, fra2 fragments
FIG. 3 shows the identification of plasmid pEGFP-N1, PBBSD by double cleavage (BamHI, not I)
Fig. 4, left: amplifying rtTA gene by PCR; right: rtTA gene and plasmid PBBSD double enzyme digestion identification chart
FIG. 5 PCR amplification of the bGHP (A) fragment
FIG. 6 PCR amplification of mCherry Gene
FIG. 7 functional verification of PB transposition-tetracycline inducible expression systems. (A) detecting the transposition activity of the ChuPBase; (B) verification of plasmid PBBSD transposition function; (C) verification of the induction function of the plasmid PBTet; (D) verification of plasmid PBTet transposition function; (E) Amplification of rtTA gene (left), double enzyme digestion identification of rtTA and PBBSD (right); (F) Verification of expression of rtTA protein by plasmid PBBSD-rtTA
FIG. 8 shows the insertion and transient expression of the target gene. (a) double cleavage product analysis; (B) transient test of constitutive expression of the target gene. (C) Transient test of inducible expression target gene
FIG. 9 production of cells expressing the gene of interest. (A) fluorescent observation of cells constitutively expressing the objective gene. (B) fluorescent observation of cells expressing the target gene in an inducible manner. (C) PCR detection of genomic 3C and rtTA genes (D) morphological observations of the induced expression cells after addition of the inducer.
FIG. 10 detection of VLPs expressed by cells. (A) And (5) performing western blot detection on the constitutive expression target gene cells. (B) And (5) detecting the western blot of the inducible target gene expression cell. (C) quantification of protein expression in FIG. 10B. (D) ELISA detection of relative amounts of VLPs (protein expression level detection).
Detailed Description
Transposase: the enzyme for performing the transposition function is usually encoded by a transposon, recognizes specific sequences at both ends of the transposon, can separate the transposon from adjacent sequences, and then inserts the transposon into a new DNA target site without homology requirements.
In one embodiment, the fragment of the chyPBase transposase is synthesized by primers and ligated to the pTT5 vector by Hind III and Not I endonucleases, designated pTT5-chyPBase.
"variants" as described herein: the original amino acid sequence is mutated to form a new amino acid sequence, and the mutation can be substitution, deletion or addition of amino acid.
EXAMPLE 1 construction of hybridization System for PB transposition-tetracycline-inducible expression
Plasmids pTT5 and PiggyBac Dual Promoter (PBDP) used in the invention are purchased from Guangzhou Cuben Biotechnology company, pcDNA6/V5-HisB is purchased from Beijing Wangyang Biotechnology company, pTet-On-Advanced is purchased from Changchun Hui Yi Biotechnology company, and pEGFP-N1 and pCS2+ -mCherry are stored by the foot and mouth disease prevention and control team of the Lanzhou veterinary institute of China academy of agricultural sciences.
BHK-21 cells were stored in the laboratory and cultured in a medium containing 10% FBS (Gibco), 100U/mL penicillin, and 100. Mu.g/mL streptomycin. The Expi293F cells were purchased from sameifei corporation and 293SFM complete medium was purchased from sumac and corporation. Type O FMD pig positive serum was kept from the laboratory. Horseradish peroxidase (HRP) -labeled rabbit anti-pig IgG antibodies, HRP-labeled goat anti-mouse IgG antibodies were purchased from Sigma. Mouse-derived anti-betA-Actin monoclonal antibodies were purchased from century corporation and from Sigma.
A solid powder of linear Polyethylenimine (PEI) was purchased from Polyscience and formulated as a 1mg/mL aqueous solution according to the instructions. High fidelity DNA polymerase, DNA Maker, was purchased from bao bioengineering (da). Protein Maker was purchased from smoio corporation. Plasmid extraction kits were purchased from OMEGA company. DNA homologous recombinase ligase was purchased from Northenan Inc. Puromycin, blasticidin, doxycycline hydrochloride (Dox-HCl) were purchased from beijing claibao.
1.1 Gene and primer Synthesis
The reported highly active PB transposase was codon optimized according to Chinese Hamster Ovary (CHO) cell preference codons. The synthetic PB transposase was named chyPBase. The primers used in the present invention are detailed in Table 1. Gene and primer synthesis was performed by Beijing Optimago.
TABLE 1 primers and sequences
1.2 construction of PB transposition-constitutive expression P12A3C plasmid
1.2.1pTT5-chyPBase plasmid construction
The reported highly active PB transposase was codon optimized according to Chinese Hamster Ovary (CHO) cell preference codons and the synthetic transposase chyPBase fragment was ligated to pTT5 vector by HindIII and NotI endonucleases and named pTT5-chyPBase.
The coding sequence of the PB transposase is the amino acid sequence of the high-activity PB transposase, and the cDNA sequence of the PB transposase is shown as SEQ ID NO. 1 after codon optimization.
SEQ ID NO:1:
ATGGGCAGCAGCCTGGACGACGAGCACATCCTGAGCGCCCTGCTGCAGAGCGACGACGAGCTGGTGGGCGAGGACAGCGACAGCGAGGTGAGCGACCACGTGAGCGAGGACGACGTGCAGAGCGACACCGAGGAGGCCTTCATCGACGAGGTGCACGAGGTGCAGCCCACCAGCAGCGGCAGCGAGATCCTGGACGAGCAGAACGTGATCGAGCAGCCCGGCAGCAGCCTGGCCAGCAACAGGATCCTGACCCTGCCCCAGAGGACCATCAGGGGCAAGAACAAGCACTGCTGGAGCACCAGCAAGCCCACCCGGCGGAGCCGGGTGAGCGCCCTGAACATCGTGCGGAGCCAGCGGGGCCCCACCAGGATGTGCCGGAACATCTACGACCCCCTGCTGTGCTTCAAGCTGTTCTTCACCGACGAGATCATCAGCGAGATCGTGAAGTGGACCAACGCCGAGATCAGCCTGAAGCGGCGGGAGAGCATGACCAGCGCCACCTTCAGGGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTGGTGATGACCGCCGTGCGGAAGGACAACCACATGAGCACCGACGACCTGTTCGACCGGAGCCTGAGCATGGTGTACGTGAGCGTGATGAGCCGGGACCGGTTCGACTTCCTGATCCGGTGCCTGCGGATGGACGACAAGAGCATCAGGCCCACCCTGCGGGAGAACGACGTGTTCACCCCCGTGCGGAAGATCTGGGACCTGTTCATCCACCAGTGCATCCAGAACTACACCCCCGGCGCCCACCTGACCATCGACGAGCAGCTGCTGGGCTTCCGGGGCCGGTGCCCCTTCCGGGTGTACATCCCCAACAAGCCCAGCAAGTACGGCATCAAGATCCTGATGATGTGCGACAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTGGGCCGGGGCACCCAGACCAACGGCGTGCCCCTGGGCGAGTACTACGTGAAGGAGCTGAGCAAGCCCGTGCACGGCAGCTGCCGGAACATCACCTGCGACAACTGGTTCACCAGCATCCCCCTGGCCAAGAACCTGCTGCAGGAGCCCTACAAGCTGACCATCGTGGGCACCGTGCGGAGCAACAAGCGGGAGATCCCCGAGGTGCTGAAGAACAGCCGGAGCCGGCCCGTGGGCACCAGCATGTTCTGCTTCGACGGCCCCCTGACCCTGGTGAGCTACAAGCCCAAGCCCGCCAAGATGGTGTACCTGCTGAGCAGCTGCGACGAGGACGCCAGCATCAACGAGAGCACCGGCAAGCCCCAGATGGTGATGTACTACAACCAGACCAAGGGCGGCGTGGACACCCTGGACCAGATGTGCAGCGTGATGACCTGCAGCCGGAAGACCAACCGGTGGCCCATGGCCCTGCTGTACGGCATGATCAACATCGCCTGCATCAACAGCTTCATCATCTACAGCCACAACGTGAGCAGCAAGGGCGAGAAGGTGCAGAGCCGGAAGAAGTTCATGCGGAACCTGTACATGGGCCTGACCAGCAGCTTCATGCGGAAGCGGCTGGAGGCCCCCACCCTGAAGCGGTACCTGCGGGACAACATCAGCAACATCCTGCCCAAGGAGGTGCCCGGCACCAGCGACGACAGCACCGAGGAGCCCGTGATGAAGAAGCGGACCTACTGCACCTACTGCCCCAGCAAGATCCGGCGGAAGGCCAGCGCCAGCTGCAAGAAGTGCAAGAAGGTGATCTGCCGGGAGCACAACATCGACATGTGCCAGAGCTGCTTC
1.2.2 construction of the plasmids PBDP-P12A3Cwt and PBDP-P12A3Copti
The P12A3Cwt and P12A3Coptiopti genes were synthesized and inserted between HindIII and NotI cleavage sites of plasmid pTT5 to give plasmids pTT5-P12A3Cwt and pTT5-P12A3Copti. The plasmids pTT5-P12A3Cwt and pTT5-P12A3Copti were double digested with the endonucleases EcoRI and BamHI, and a plasmid non-digested control was set. The results of the enzyme digestion identification are shown in figure 1. The excised gene fragment was inserted into EcoRI and BamHI sites of the plasmid PBDP using T4 DNA ligase to obtain plasmids PBDP-P12A3Cwt and PBDP-P12A3Copti.
1.3 construction of plasmid for P12A3C expression by the induction of transposition-tetracycline
1.3.1 construction of plasmid PBBSD-rtTA
(1) The target fragment Fra1 is amplified by taking the plasmid PBDP as a template and P3 and P4 as primers, the target fragment Fra2 is amplified by taking the plasmid pcDNA6/V5-HisB as a template and P1 and P2 as primers, and the fragments Fra1 and Fra2 are recombined by DNA homologous recombination enzymes to obtain the plasmid PBBSD. The PCR amplification of the Fra1 and Fra2 fragments is identified in FIG. 2.
(2) Plasmid pEGFP-N1 and PBBBSD were digested with endonucleases BamHI and NotI, and the eGFP and PBBSD fragments were recovered and ligated with T4 DNA ligase to give PBBSD-eGFP, see FIG. 3.
(3) Amplifying rtTA fragments by using plasmid pTet-On-Advanced as a template and P5 and P6 as primers (see FIG. 4); the rtTA fragment and the plasmid PBBBSD were digested with endonucleases KpnI and BamHI (see FIG. 4), and the rtTA and PBBSD fragments were recovered and ligated with T4 DNA ligase to obtain PBBSD-rtTA.
1.3.2 construction method of inducible plasmid PBTet-P12A3C
(1) The plasmid PBDP is used as a template, the CMV promoter is replaced by the P-SGTRE promoter, pcDNA3.1 (+) is used as a template, P11 and P12 are used as primers to amplify the bGHp (A) fragment (see figure 5), the bGHp (A) fragment is subjected to double digestion by using endonucleases BamHI and Not I, and the plasmid PBTet is obtained by inserting the plasmid into the vector PBDP with the replaced promoter.
(2) The mCherry gene was amplified using pCS2+ -mCherry as a template and P7 and P8 as primers (see FIG. 6) and inserted into EcoRI and BamHI sites of PBTet to obtain plasmid PBTet-mCherry.
(3) The endonucleases EcoRI and BamHI were used to double cleave pTT5-P12A3Cwt and pTT5-P12A3Copti (see FIG. 1) and insert into the corresponding cleavage sites of PBTet, resulting in plasmids PBTet-P12A3Cwt and PBTet-P12A3Copti.
Example 2 functional verification of PB transposition-tetracycline inducible expression System
2.1 verification of the transposable Activity of chyPBase
The plasmids PBDP and pTT5-chyPBase were transfected into BHK-21 cells at a mass ratio of 9:1, and cell selection was performed by adding 10. Mu.g/mL puromycin 1 day after transfection, and fresh medium containing antibiotics was added every 2 days. The number of positive clones was observed under a fluorescence microscope 12 days after screening.
2.2 verification of the transposition function of the plasmid PBBSD
The plasmids PBBSD-eGFP and pTT5-chyPBase were used to transfect BHK-21 cells at a mass ratio of 9:1, 10. Mu.g/mL blasticidin was added 1 day after transfection for cell selection, and fresh medium containing antibiotics was added every 2 days. The number of positive clones was observed under a fluorescence microscope after 10 days of screening.
2.3 verification of the transposition and expression inducing function of the plasmid PBTet
Plasmids PBTet-mCherry and pTet-On-Advanced at 9:1 mass ratio BHK-21 cells were transfected, 2. Mu.g/mL Dox-HCl was added 12 hours later, and red fluorescence was observed under a fluorescence microscope 36 hours after transfection.
Plasmid PBTet-mCherry, PBBSD-rtTA and pTT5-chyPBase were co-transferred into BHK-21 cells at a mass ratio of 8:1:1, and cell selection was performed by adding 10. Mu.g/mL puromycin and 5. Mu.g/mL blasticidin 1 day after transfection, with fresh medium containing antibiotics every 2 days. The number of positive clones was observed under a fluorescence microscope after 10 days of screening.
2.4 experimental results
(1) The transposable activity of PB transposase was examined, and plasmid PBDP was cotransformed with plasmid pTT5-chyPBase, and after 12 days of screening with high concentration puromycin, a large number of positive clones were generated, whereas the control group had almost no positive clones (see FIG. 7A), indicating that the constructed transposase had high activity.
(2) To rapidly detect the transposable element function of plasmid PBBSD, PBBSD-eGFP was co-transformed with pTT5-chyPBase, and cells obtained by blasticidin screening contained a large number of positive clones, whereas control groups had almost no positive clones (see FIG. 7B), demonstrating that plasmid PBBSD was able to act with PB transposase.
(3) To rapidly verify the function of the inducible promoter of plasmid PBTet, PBTet-mCherry was co-transformed with plasmid pTet-On-Advanced and the addition of the inducer Dox-HCl showed intense red fluorescence (FIG. 7C), indicating that the inducible promoter of PBTet was able to interact with rtTA.
(4) The plasmid PBTet and pTT5-chyPBase were co-transformed 10 to rapidly produce target cells (see FIG. 7D), demonstrating that PBTet has a transposition function.
(5) To obtain PB transposable plasmid expressing rtTA with constitutive promoter, rtTA was amplified by PCR using pTet-On-Advanced as template (see left in FIG. 7E), and the product was inserted into plasmid PBBSD by double cleavage (right in FIG. 7E), to obtain plasmid PBBSD-rtTA. Co-transformation experiments of the plasmid PBBSD-rtTA and PBTet-mCherry prove that the plasmid PBBSD-rtTA can correctly express rtTA protein (see FIG. 7F).
Example 3 insertion and transient verification of the P12A3C Gene
The plasmids pTT5-P12A3Copti were digested simultaneously (FIG. 8A) and inserted into the plasmids PBDP and PBTet, respectively, to give plasmids PBDP-P12A3Copti and PBTet-P12A3Copti. In transient experiments, the remaining clones were successful in expressing FMDV capsid proteins (VP 0, VP3, VP 1) except for PBDP-P12A3C wt-2 (FIG. 8B). The inducible expression panel had no FMDV capsid protein expression without Dox-HCl addition, increased FMDV capsid protein expression levels after Dox-HCl addition, and PBTet-P12A3Copti had FMDV capsid protein expression levels much higher than PBTet-P12A3Cwt (FIG. 8C).
PBTet-P12A3Cwt(SEQ ID NO:2):
ATGGGCGCCGGCCAGAGCAGCCCCGCCACCGGCTCCCAGAACCAGTCCGGCAACACCGGCTCCATCATCAACAACTACTACATGCAGCAGTACCAGAACTCCATGGACACCCAGCTGGGCAACAACGCCATCTCCGGCGGCTCCAACGAGGGCTCCACCGACACCACCTCCACCCACACCACCAACACCCAGAACAACGACTGGTTCTCCAAGCTGGCCTCCTCCGCCTTCTCCGGCCTGTTCGGAGCCCTGCTGGCCGACAAAAAGACCGAGGAGACCACACTGCTGGAAGACAGAATCCTGACCACCAGAAACGGACACACCACCTCCACAACCCAGTCCTCCGTGGGCATCACCCACGGCTACGCCACCGCCGAGGACTTCGTGAACGGCCCCAACACCTCCGGCCTGGAGACCAGGGTGGTGCAGGCCGAGCGGTTCTTCAAGACCCACCTGTTCGACTGGGTGACCTCCGACCCCTTCGGCCGGTACTACCTGCTGGAGCTGCCCACCGACCACAAGGGCGTGTACGGCAGCCTGACCGACTCCTACGCCTACATGCGGAACGGCTGGGACGTGGAGGTGACCGCCGTGGGCAACCAGTTCAACGGCGGCTGCCTGCTGGTGGCCATGGTGCCCGAGCTGTGCTCCATCGAGCAGAGGGAGCTGTTCCAGCTGACCCTGTTCCCCCACCAGTTCATCAACCCCCGGACCAACATGACCGCCCACATCAAGGTGCCCTTCGTGGGCGTGAACAGGTACGACCAGTACAAGGTGCACAAGCCCTGGACCCTGGTGGTGATGGTGGTGGCCCCCCTGACCGTGAACACCGAGGGCGCCCCCCAGATCAAGGTGTACGCCAACATCGCCCCCACCAACGTGCACGTGGCCGGCGAGTTCCCCTCCAAGGAGGGCATCTTCCCCGTGGCCTGCTCCGACGGCTACGGCGGCCTGGTGACCACCGACCCCAAGACCGCCGACCCCGTGTACGGCAAGGTGTTCAACCCCCCCCGGAACATGCTGCCCGGCAGGTTCACCAACCTGCTGGACGTGGCCGAGGCC
TGCCCCACCTTCCTGCACTTCGACGGCGACGTGCCCTACGTGACCACCAAGACCGA
CTCCGACCGGGTGCTGGCCCAGTTCGACCTGTCCCTGGCCGCCAAGCACATGTCCA
ACACCTTCCTGGCCGGCCTGGCCCAGTATTACACCCAGTACTCCGGCACCGTGAACC
TGCACTTCATGTTCACCGGCCCCACCGACGCCAAGGCCCGGTACATGATCGCCTAC
GCCCCCCCCGGCATGGAGCCCCCCAAGACCCCCGAGGCCGCCGCCCACTGCATCCA
CGCCGAGTGGGACACCGGCCTGAACTCCAAGTTCACCTTCAGCATCCCCTACCTGTC
CGCCGCCGACTACGCCTACACCGCCTCCGACGCCGCCGAGACCACCAACGTGCAGG
GCTGGGTGTGCCTGTTCCAGATCACCCACGGCAAGGCCGAGGGCGACGCCCTGGTG
GTGCTGGCCTCCGCCGGCAAGGACTTCGAGCTGCGGCTGCCCGTGGACGCCCGGCA
GCAGACCACCTCCACCGGCGAGTCCGCCGACCCCGTGACCGCCACCGTGGAGAACT
ACGGCGGCGAGACCCAGGTGCAGCGACGGCACCACACCGACGTGTCCTTCATCCTG
GACCGGTTCGTGAAGGTGACCCCCAAGGACTCCATCAACGTGCTGGACCTGATGCA
GACCCCCTCCCACACCCTGGTGGGCGCCCTGCTGCGGACCGCCACCTACTACTTCGC
CGACCTGGAGGTGGCCGTGAAGCACAAGGGCGACCTGACCTGGGTGCCCAACGGC
GCCCCCGTGGCCGCCCTGGACAACACCACCAACCCCACCGCCTACCACAAGGCCCC
CCTGACCCGGCTGGCCCTGCCCTACACCGCCCCCCACCGGGTGCTGGCCACCGTGTA
CAACGGCAAGTGCAAGTACGCCGAGGGCTCCCTGCCCAACGTGCGGGGCGACCTGC
AGGTGCTGGCCCAGAAGGCCGCCCGGCCCCTGCCCACCTCCTTCAACTACGGCGCC
ATCAAGGCCACCCGGGTGACCGAGCTGCTGTACCGGATGAAGCGGGCCGAGACCTA
CTGCCCCCGGCCCCTGCTGGCCGTGCACCCCTCCGCCGCCCGGCACAAGCAGAAGA
TCGTGGCCCCCGTGAAGCAGAGCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGAC
GTGGAGTCCAACCCCGGCCCCAGCGGCCGCAGCGGCGCCCCCCCGACCGACTTGCA
GAAGATGGTCATGGGCAACACCAAGCCCGTGGAGCTCATACTCGACGGGAAGACC
GTGGCCATCTGCTGCGCCACCGGCGTGTTCGGCACCGCCTACCTCGTGCCCCGGCAC
CTGTTCGCCGAGAAGTACGACAAGATCATGTTGGACGGCAGGACCATGACCGACAG
CGACTACAGGGTGTTCGAGTTCGAGATCAAGGTGAAGGGCCAGGACATGCTCTCCG
ACGCCGCGCTCATGGTGCTGCACCGGGGGAACCGCGTGAGGGACATCACGAAGCA
CTTCCGGGACACCGCCAGGATGAAGAAGGGCACCCCCGTCGTGGGCGTGATCAACA
ACGCCGACGTCGGGAGGCTGATCTTCTCCGGCGAGGCCCTCACCTACAAGGACATC
GTGGTGTGCATGGACGGCGACACCATGCCGGGCCTGTTCGCCTACAAGGCCGCCAC
CAAGGCCGGCTACTGCGGGGGCGCCGTCCTGGCCAAGGACGGCGCCGACACCTTCA
TCGTGGGCACCCACTCCGCCGGCGGCAACGGCGTGGGGTACTGCTCCTGCGTGTCC
AGGTCCATGCTCCAGAAGATGAAGGCCCACATCGACCCCGAGCCCCACCACGAG
PBTet-P12A3Copti(SEQ ID NO:3):
ATGGGCGCCGGCCAGAGCAGCCCCGCCACCGGCTCCCAGAACCAGTCCGGCAACACCGGCTCCATCATCAACAACTACTACATGCAGCAGTACCAGAACTCCATGGACACCCAGCTGGGCAACAACGCCATCTCCGGCGGCTCCAACGAGGGCTCCACCGACACCACCTCCACCCACACCACCAACACCCAGAACAACGACTGGTTCTCCAAGCTGGCCTCCTCCGCCTTCTCCGGCCTGTTCGGAGCCCTGCTGGCCGACAAAAAGACCGAGGAGACCACACTGCTGGAAGACAGAATCCTGACCACCAGAAACGGACACACCACCTCCACAACCCAGTCCTCCGTGGGCATCACCCACGGCTACGCCACCGCCGAGGACTTCGTGAACGGCCCCAACACCTCCGGCCTGGAGACCAGGGTGGTGCAGGCCGAGCGGTTCTTCAAGACCCACCTGTTCGACTGGGTGACCTCCGACCCCTTCGGCCGGTACTACCTGCTGGAGCTGCCCACCGACCACAAGGGCGTGTACGGCAGCCTGACCGACTCCTACGCCTACATGCGGAACGGCTGGGACGTGGAGGTGACCGCCGTGGGCAACCAGTTCAACGGCGGCTGCCTGCTGGTGGCCATGGTGCCCGAGCTGTGCTCCATCGAGCAGAGGGAGCTGTTCCAGCTGACCCTGTTCCCCCACCAGTTCATCAACCCCCGGACCAACATGACCGCCCACATCAAGGTGCCCTTCGTGGGCGTGAACAGGTACGACCAGTACAAGGTGCACAAGCCCTGGACCCTGGTGGTGATGGTGGTGGCCCCCCTGACCGTGAACACCGAGGGCGCCCCCCAGATCAAGGTGTACGCCAACATCGCCCCCACCAACGTGCACGTGGCCGGCGAGTTCCCCTCCAAGGAGGGCATCTTCCCCGTGGCCTGCTCCGACGGCTACGGCGGCCTGGTGACCACCGACCCCAAGACCGCCGACCCCGTGTACGGCAAGGTGTTCAACCCCCCCCGGAACATGCTGCCCGGCAGGTTCACCAACCTGCTGGACGTGGCCGAGGCCTGCCCCACCTTCCTGCACTTCGACGGCGACGTGCCCTACGTGACCACCAAGACCGACTCCGACCGGGTGCTGGCCCAGTTCGACCTGTCCCTGGCCGCCAAGCACATGTCCAACACCTTCCTGGCCGGCCTGGCCCAGTATTACACCCAGTACTCCGGCACCGTGAACCTGCACTTCATGTTCACCGGCCCCACCGACGCCAAGGCCCGGTACATGATCGCCTACGCCCCCCCCGGCATGGAGCCCCCCAAGACCCCCGAGGCCGCCGCCCACTGCATCCACGCCGAGTGGGACACCGGCCTGAACTCCAAGTTCACCTTCAGCATCCCCTACCTGTCCGCCGCCGACTACGCCTACACCGCCTCCGACGCCGCCGAGACCACCAACGTGCAGGGCTGGGTGTGCCTGTTCCAGATCACCCACGGCAAGGCCGAGGGCGACGCCCTGGTGGTGCTGGCCTCCGCCGGCAAGGACTTCGAGCTGCGGCTGCCCGTGGACGCCCGGCAGCAGACCACCTCCACCGGCGAGTCCGCCGACCCCGTGACCGCCACCGTGGAGAACTACGGCGGCGAGACCCAGGTGCAGCGACGGCACCACACCGACGTGTCCTTCATCCTGGACCGGTTCGTGAAGGTGACCCCCAAGGACTCCATCAACGTGCTGGACCTGATGCAGACCCCCTCCCACACCCTGGTGGGCGCCCTGCTGCGGACCGCCACCTACTACTTCGCCGACCTGGAGGTGGCCGTGAAGCACAAGGGCGACCTGACCTGGGTGCCCAACGGCGCCCCCGTGGCCGCCCTGGACAACACCACCAACCCCACCGCCTACCACAAGGCCCCCCTGACCCGGCTGGCCCTGCCCTACACCGCCCCCCACCGGGTGCTGGCCACCGTGTACAACGGCAAGTGCAAGTACGCCGAGGGCTCCCTGCCCAACGTGCGGGGCGACCTGCAGGTGCTGGCCCAGAAGGCCGCCCGGCCCCTGCCCACCTCCTTCAACTACGGCGCCATCAAGGCCACCCGGGTGACCGAGCTGCTGTACCGGATGAAGCGGGCCGAGACCTACTGCCCCCGGCCCCTGCTGGCCGTGCACCCCTCCGCCGCCCGGCACAAGCAGAAGATCGTGGCCCCCGTGAAGCAGAGCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGTCCAACCCCGGCCCCAGCGGCCGCAGCGGCGCCCCCCCGACCGACTTGCAGAAGATGGTCATGGGCAACACCAAGCCCGTGGAGCTCATACTCGACGGGAAGACCGTGGCCATCTGCTGCGCCACCGGCGTGTTCGGCACCGCCTACCTCGTGCCCCGGCACCTGTTCGCCGAGAAGTACGACAAGATCATGTTGGACGGCAGGACCATGACCGACAGCGACTACAGGGTGTTCGAGTTCGAGATCAAGGTGAAGGGCCAGGACATGCTCTCCGACGCCGCGCTCATGGTGCTGCACCGGGGGAACCGCGTGAGGGACATCACGAAGCACTTCCGGGACACCGCCAGGATGAAGAAGGGCACCCCCGTCGTGGGCGTGATCAACAACGCCGACGTCGGGAGGCCTATCTTCTCCGGCGAGGCCCTCACCTACAAGGACATCGTGGTGTGCATGGACGGCGACACCATGCCGGGCCTGTTCGCCTACAAGGCCGCCACCAAGGCCGGCTACTGCGGGGGCGCCGTCCTGGCCAAGGACGGCGCCGACACCTTCATCGTGGGCACCCACTCCGCCGGCGGCAACGGCGTGGGGTACTGCTCCTGCGTGTCCAGGTCCATGCTCCAGAAGATGAAGGCCCACATCGACCCCGAGCCCCACCACGAG
Example 4 transient test of inducible and constitutive expression of the P12A3C Gene
The plasmid PBDP-P12A3Cwt or PBDP-P12A3Copti and pTT5-chyPBase are co-transferred into BHK-21 cells, and two kinds of cells C-wt and C-opti which are constitutively expressed with P12A3C genes are obtained after puromycin screening. The plasmids PBTet-P12A3Cwt or PBTet-P12A3C opti, PBBSD-rtTA and pTT5-chyPBase are co-transferred to obtain cells which express target genes in an inducible mode and are named as I-wy and I-opti respectively. The stable integration of the gene of interest into the genomes of the above 4 cells was confirmed by fluorescent observation (see FIGS. 9A, 9B) and detection of the gene of interest in the genome (FIG. 9C), and the stable integration of the rtTA gene into the genomes of the cells I-wt and I-opti. In summary, both constitutive and inducible cells expressing the gene of interest were successfully constructed. In addition, upon induction of protein expression, it was found that cell I-wt exhibited severe cell shedding (see FIG. 9D).
EXAMPLE 5 detection of expression of VLPs by cells
(1) And detecting the FMD VLPs expression of the constitutive expression cells C-wt and C-opti and the inducible expression cells I-wt and I-opti respectively.
Cells C-wt and C-opti did not show expression of FMDV capsid protein (FIG. 10A), indicating that these two cells did not produce VLPs.
(2) And respectively collecting adherent cells and abscisic cells of the cells added with the inducer, and detecting the expression of capsid proteins. Wherein 1, 2 and 3 represent cells I-wt without Dox-HCl, with Dox-HCl collected adherent cells, with Dox-HCl collected exfoliated cells, respectively. Reference numerals 4, 5 and 6 indicate cells I-opti without Dox-HCl, adherent cells collected with Dox-HCl, and exfoliated cells collected with Dox-HCl, respectively.
Fig. 10B shows that there was almost no protein expression prior to addition of the inducer, whereas protein expression occurred after addition of the inducer, and that adherent cells and exfoliated cells showed similar levels of capsid protein expression. Cell C-opti showed higher expression levels of capsid proteins. It was demonstrated that optimized P12A3Copti was beneficial for enhancing expression of mature capsid proteins.
(3) The gray scale analysis was performed on fig. 10B using image J software.
FIG. 10C shows that optimized P12A3Copti increased the expression level of FMDV capsid protein 15-30-fold.
(3) To further examine the expression of cellular FMDV VLPs, ELISA assays were performed by lysing the same number of cells and extracting the cell lysates. Consistent with the western bolt results, cell I-opti showed stronger FMDV VLPs expression after induction (fig. 10D).
In conclusion, by constructing a PB transposition-tetracycline induction expression system and expressing the optimized P12A3Copti in the system, the success rate of stably integrating the P12A and 3C genes into a cell genome is improved, and meanwhile, the expression quantity of FMDV capsid protein is increased by 15-30 times.
Claims (10)
1. A transposition-induced expression system for foot-and-mouth disease virus-like particles, the system comprising a nucleic acid encoding a transposase, an inverse transactivator, an inducer, foot-and-mouth disease virus-like particles.
2. The transposition-induced expression system for foot-and-mouth disease virus like particles of claim 1, wherein the transposase may be a transposase encoding PiggyBac (PB) and variants thereof.
3. A transposition-induced expression system for foot-and-mouth disease virus like particles according to claim 1 wherein the reverse transactivating element is a reverse transactivating element itself or a variant thereof.
4. The transposition-induced expression system for foot-and-mouth disease virus like particles according to claim 1, wherein the inducer is a tetracycline or a derivative thereof.
5. A transposition-induced expression system for foot-and-mouth disease virus-like particles according to claim 1, wherein the nucleic acid of the foot-and-mouth disease virus-like particles is an optimized sequence P12A3Copti as shown in SEQ ID No. 3.
6. A method of constructing a transposition-induced expression system for foot-and-mouth disease virus-like particles, the method comprising the steps of:
s01, constructing PB transposase plasmid;
s02, constructing a transactivator plasmid;
s03, constructing a plasmid of nucleic acid of PB transposition-induced expression foot-and-mouth disease virus-like particles;
s04, co-transfecting the obtained plasmid into host cells to obtain a complete transposition-induced expression system of foot-and-mouth disease virus-like particles.
7. The method of constructing a transposition-induced expression system for foot-and-mouth disease virus-like particles according to claim 6, wherein the expression vector of the PB transposase plasmid is selected from the group consisting of pTT5 and pVAX1.
8. Use of a transposition-inducible expression system for foot-and-mouth disease virus-like particles for the preparation of a biological preparation for foot-and-mouth disease comprising a transposition-inducible expression system for foot-and-mouth disease virus-like particles according to claims 1-5.
9. Use of a transposition-induced expression system of foot-and-mouth disease virus like particles according to claim 8 for the preparation of a foot-and-mouth disease biological agent, wherein the biological agent may be a foot-and-mouth disease vaccine formulation and/or a composition of a foot-and-mouth disease vaccine formulation.
10. Use of a transposition-induced expression system of foot-and-mouth disease virus like particles according to claim 9 for the preparation of foot-and-mouth disease biologicals, wherein the vaccine may be inactivated vaccine, recombinant protein vaccine, synthetic peptide vaccine, nucleic acid vaccine and adenovirus vector vaccine.
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