CN113322279A - Recombinant vaccinia virus vector with D8L gene knocked out - Google Patents
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Abstract
The invention relates to the technical field of molecular biology and immunology, in particular to a recombinant vaccinia virus vector with a knocked-out D8L gene; the recombinant vaccinia virus vector has a D8L homologous recombination arm sequence, and is used for constructing a vaccinia virus vector inserted with an exogenous gene in a D8L gene region of the vaccinia virus; the invention provides a recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibodies existing in vivo, a D8L encoding gene is knocked out or damaged and cannot normally express a D8L gene, and the vaccinia virus vector can be used as a tumor vaccine or a gene therapy vector and can be used for preventing or treating various tumors.
Description
Technical Field
The invention relates to the technical field of molecular biology and immunology, in particular to a recombinant vaccinia virus vector with a knocked-out D8L gene.
Background
Vaccinia Virus (Vaccinia Virus) belongs to the genus Orthomyxovirus of the family Poxviridae and has been widely used worldwide as a vaccine for the prevention of smallpox infection, making smallpox the first malignant infectious disease to be completely eradicated by humans (Riva, G.et. al., 1979. Amilestrone in the history of infection. eradication of smallpox. MMW Munch Meachenschr, 121(50), 1669-70). In the last two decades, with the rapid development of genetic engineering techniques, vaccinia viruses have been used in research to design gene expression vectors, or in the development of recombinant live virus vaccines (Moss, B.1996.genetic engineering viruses for recombinant gene expression, vaccination, and safety. ProcNatl Acad Sci USA 93(21), 11341-8; Paoletti, E.et al, 1996.Applications of pox viruses vectors to vaccination: an update. Proc Natl Acad Sci USA 93(21), 11349-53). Various recombinant vaccinia virus vaccines have been used successfully to express rabies virus, Japanese encephalitis virus, and other viral antigens and to control animal infections (Kieny, M.P. et al, 1984.expression of viruses from a recombinant vaccine virus. Nature312(5990), 163-6; Konishi, E.et al, 1992.A high expressed family-restricted vaccine strain, NYVAC, encoding the prM, E, and NS1genes of Japanese infectious virus precursors JEV virus strain 190(1), 454-8). There have also been many important advances in human recombinant live virus vaccines, including preliminary human immune observations including measles, EBV, HAV, HBV, HPV, HIV and rabies viruses (Guofei et al, 2001, The construction of expression vectors for non-replicating recombinant vaccinia virus Tiantan strain C-K deletion region and The study of biological properties of recombinant viruses. viral acta 17(1), 24-28; McMichael, A.et al, 2002.The request for AIDS vaccine: is The CD8+ T-cell ap pro plasmid Nat Rev Immunol 2(4), 283-91; Moss, B.1996.genetic engineering for gene expression vector expression, vaccination, and safety. Proc. Natl Acad Sci 93(21), 11341-8; ZJ.J.J.65. vaccine, see 141. 7. and 141. biological genes).
The vaccinia virus Tiantan strain is a peculiar vaccinia virus strain in China, has been used for a long time in a large number of people in China as a vaccine strain for preventing smallpox infection, has the characteristics of relatively weak toxicity and safe use, and is an ideal strain for developing a genetic engineering virus vector and a recombinant live vaccine suitable for China. The sequencing of the whole genome of the Chinese vaccinia virus Tiantan strain was completed in 1997, which provides important background data for the research and elucidation of the unique biological characteristics of the vaccinia virus Tiantan strain and the genetic engineering modification (King et al, 1997, analysis of the whole genome structural characteristics of the vaccinia virus Tiantan strain, China science (C edition) 27(6), 562 and 567).
With the development of molecular biology, some viruses have been widely researched and developed as recombinant viral vectors due to their characteristics of good safety, strong immunogenicity, and convenience for gene manipulation. At present, the method is mainly applied to the medical fields of vaccines, gene therapy and the like. The poxvirus vector is large, the genome of the poxvirus vector is about 180k, and the poxvirus vector can accommodate large exogenous gene segments, so that the poxvirus vector can be used as an excellent viral vector for mediating the delivery of target proteins or coding genes thereof in vivo.
However, vaccinia virus, as a recombinant viral vector, has a strong vector response that can be activated to produce a strong immune response against vaccinia virus itself, including a strong neutralizing antibody response. Moreover, the response is long lasting and the neutralizing antibody response is provided for the lifetime of the individual vaccinated with the vaccinia virus vector. Thus, the presence of high levels of neutralizing antibodies in vivo will greatly affect reinfection with the recombinant vaccinia virus vector of interest, thereby affecting the effectiveness of viral vector drug delivery. This feature also limits the repeated or multiple applications of vaccinia virus vectors.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a recombinant vaccinia virus vector for knocking out the D8L gene, which selects a main neutralizing antigen of the vaccinia virus as a target spot, and researches whether knocking out or damaging the target spot can escape from an existing neutralizing antibody in vivo, thereby increasing the use frequency of the recombinant vaccinia virus vector in vivo.
For the purposes of the present invention, the following terms are defined below.
The invention relates to a gene knockout or a knockout technology, which is a genetic engineering technology and refers to a technology or a method for replacing an endogenous normal homologous gene by using an exogenous mutated gene through a homologous recombination method, so that the endogenous gene is inactivated to express the character of a mutant. In the invention, the D8L knockout is that a normal D8L gene endogenous to vaccinia virus is replaced by homologous recombination by introducing mutation into the D8L gene, so that the D8L gene cannot be normally expressed.
The "Recombinant virus vector" or "Recombinant virus" as referred to herein is a vector produced by a DNA recombination technique and used for producing a viral vaccine or producing a gene therapy.
The term "Recombinant vaccinia virus vector" or "Recombinant vaccinia virus" as used herein refers to a vaccinia virus vector or vaccinia virus produced by Recombinant DNA techniques, and can be used for producing viral vaccines or vectors for gene therapy. In the present invention, a "recombinant vaccinia virus vector" or "recombinant vaccinia virus" is produced by homologous recombination of a shuttle vector having a homologous arm sequence of a target gene with vaccinia virus in a target cell, and then purified by selection of a reporter gene or a resistance gene carried by the recombinant vaccinia virus.
The term "shuttle plasmid vector" or "shuttle vector" as used herein refers to an intermediate plasmid vector for introducing a gene sequence of interest into the genome of another species. In the present invention, the "shuttle plasmid vector" or "shuttle vector" refers to an intermediate plasmid vector for introducing a target gene sequence into a vaccinia virus vector by homologous recombination, characterized in that the shuttle vector contains two homologous recombination arms having the same sequence as a partial sequence of a vaccinia virus genome.
The term "homologous recombination arm" or "homology arm" as used herein refers to a nucleic acid sequence that is identical to a vaccinia virus endogenous gene.
A recombinant vaccinia virus vector with a knocked-out D8L gene, wherein a D8L encoding gene of the recombinant vaccinia virus vector is knocked out, and the recombinant vaccinia virus vector cannot normally express a D8L gene.
The invention is further configured to: the vaccinia virus vector is a replicative vaccinia virus vector or a non-replicative vaccinia virus vector.
The invention is further configured to: the D8L knockout refers to the replacement of the endogenous normal D8L gene of vaccinia virus by homologous recombination through introducing mutation in the D8L gene, so that the D8L gene cannot be normally expressed.
The invention is further configured to: the D8L knockout was performed by mutating the D8L gene by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of D8L, and further comprising one or more foreign genes other than the reporter gene or the resistance gene.
The invention is further configured to: the vaccinia virus promoter element is P7.5 or PE/L.
The invention is further configured to: the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene.
The invention is further configured to: the resistance gene is a neomycin encoding gene, and when the resistance gene is used for purifying a recombinant vaccinia virus vector, selective pressure is applied.
The invention is further configured to: the exogenous gene is a non-vaccinia virus-derived gene other than the reporter gene or the resistance gene.
The invention is further configured to: the nucleic acid sequences at two ends of the D8L are a D8L-L homologous recombination arm and a D8L-R homologous recombination arm respectively.
The invention is further configured to: the recombinant vaccinia virus vector with the knockout D8L has a nucleotide sequence inserted into the homologous recombination arm sequence of D8L.
The invention is further configured to: the recombinant vaccinia virus vector knocked out by D8L is rvv-delta D8L/G.
The invention is further configured to: the recombinant vaccinia virus vector capable of escaping the existing anti-vaccinia virus neutralizing antibody existing in vivo is a recombinant vaccinia virus vector with a D8L gene knockout function.
The invention is further configured to: the recombinant vaccinia virus vector with the knocked-out D8L gene comprises a D8L-L homologous recombination arm sequence, and the recombinant vaccinia virus vector with the knocked-out D8L gene further comprises a D8L-R homologous recombination arm sequence.
A shuttle vector for constructing a recombinant vaccinia virus vector with a D8L gene knockout, wherein the shuttle vector comprises a D8L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector contains a D8L-L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector also contains a D8L-R homologous recombination arm sequence.
The invention is further configured to: the shuttle vector also inserts a nucleic acid sequence between the homologous recombination arm sequences of D8L, wherein the nucleic acid sequence comprises at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, and the nucleic acid sequence also comprises one or more exogenous genes besides the reporter gene or the resistance gene.
The invention is further configured to: the vaccinia virus promoter element is P7.5 or PE/L.
The invention is further configured to: the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene.
The invention is further configured to: the resistance gene is a neomycin encoding gene, and when the resistance gene is used for purifying a recombinant vaccinia virus vector, selective pressure is applied.
The invention is further configured to: the exogenous gene is a non-vaccinia virus-derived gene other than the reporter gene or the resistance gene.
The invention is further configured to: the nucleic acid sequences at two ends of the D8L are a D8L-L homologous recombination arm and a D8L-R homologous recombination arm respectively.
The invention is further configured to: the shuttle vector also has a nucleic acid sequence inserted into the homologous recombination arm sequence of D8L.
The invention is further configured to: the shuttle vector is p delta D8L-GFP.
A preparation method for constructing a recombinant vaccinia virus vector with a D8L gene knockout function comprises the following steps:
s1, designing primers, and respectively amplifying two homologous recombination arms of D8L by PCR;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing a D8L homologous recombination arm sequence;
s3, transfecting the shuttle vector into a vaccinia virus infected cell;
s4, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
Advantageous effects
Compared with the known public technology, the technical scheme provided by the invention has the following beneficial effects:
the invention selects the main neutralizing antigen of the vaccinia virus as a target spot, and researches whether knocking out or destroying the target spot can escape the existing neutralizing antibody in vivo, thereby increasing the using times of the recombinant vaccinia vector in vivo, knocking out or destroying the D8L encoding gene, and not normally expressing the D8L gene.
Drawings
FIG. 1 is a plasmid map of shuttle vector p.DELTA.D8L-GFP with homologous recombination arm sequences D8L-L and D8L-R;
FIG. 2 is a diagram showing the double restriction enzyme identification of the shuttle vector p.DELTA.D8L-GFP with homologous recombination arm sequences of D8L-L and D8L-R;
FIG. 3 is a diagram showing the identification of recombinant vaccinia virus vector rvv- Δ D8L/G by PCR amplification;
FIG. 4 shows an expression identification chart of recombinant vaccinia virus vector rvv- Δ D8L/G;
FIG. 5 is also an expression signature of recombinant vaccinia virus vector rvv- Δ D8L/G;
FIG. 6 shows the results of detection of neutralizing antibodies in example 5;
FIG. 7 shows the results of the kinetics of viral replication in example 6, including the viral dose in infected cells and the viral dose in the infection supernatant.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The present invention will be further described with reference to the following examples.
Referring to FIGS. 1-7: the invention aims to provide a recombinant vaccinia virus vector with a knocked-out D8L gene, wherein a D8L encoding gene of the recombinant vaccinia virus vector is knocked out, and the D8L gene cannot be normally expressed.
In one embodiment of the invention, the Vaccinia virus vector is a replicating Vaccinia virus vector, such as a Vaccinia virus Tiantan strain, e.g., strain 752-1, or a non-replicating Vaccinia virus vector, such as a Vaccinia virus attenuated vaccine Ankara strain (Modified Vaccinia Ankara, MVA).
In one embodiment of the invention, the D8L knockout refers to replacement of the endogenous normal D8L gene of vaccinia virus by homologous recombination by introducing a mutation inside the D8L gene, so that the D8L gene cannot be normally expressed.
In one embodiment of the invention, the D8L knockout is performed by mutating the D8L gene by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of D8L, the nucleic acid sequence further comprising one or more exogenous genes other than the reporter gene or the resistance gene.
In one embodiment of the invention, the vaccinia virus promoter element is P7.5 or PE/L, preferably, the nucleic acid sequence of P7.5 is shown as SEQ ID NO. 13 and the nucleic acid sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment of the present invention, the reporter gene is a fluorescent protein coding gene or a galactosidase coding gene (Lac Z), preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene, a yellow fluorescent protein coding gene, a red fluorescent protein coding gene or a blue fluorescent protein coding gene, more preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene (EGFP), and the nucleic acid coding sequence thereof is shown in SEQ ID NO. 15.
In one embodiment of the invention, the resistance gene is a neomycin encoding gene and selective pressure is applied when the resistance gene is used in the purification of recombinant vaccinia virus vectors.
In one embodiment of the present invention, the foreign gene is a gene derived from non-vaccinia virus itself except for a reporter gene or a resistance gene, and the target antigen or immunogen-encoding gene selected according to the purpose of use may be any one or more immunogens of interest, preferably, the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other intact microorganism, and further, the target antigen or immunogen is a tumor antigen or an infection-associated antigen.
In one embodiment of the invention, the nucleic acid sequences at the two ends of D8L are respectively a D8L-L homologous recombination arm and a D8L-R homologous recombination arm, preferably, the nucleic acid sequences of the D8L-L homologous recombination arm are shown as SEQ ID NO. 1, and the nucleic acid sequences of the D8L-R homologous recombination arm are shown as SEQ ID NO. 2.
In one embodiment of the invention, the recombinant vaccinia virus vector knockout of D8L further comprises a nucleic acid sequence inserted into the homologous recombination arm sequence of D8L, wherein the nucleic acid sequence is shown in SEQ ID NO. 16.
In one embodiment of the invention, the recombinant vaccinia virus vector for the D8L knockout was rvv-vD 8L/G.
In one embodiment of the invention, the recombinant vaccinia virus vector from which the existing anti-vaccinia virus neutralizing antibody can escape is a recombinant vaccinia virus vector with the D8L gene knockout.
In one embodiment of the invention, the recombinant vaccinia virus vector with the knocked-out D8L gene comprises a D8L-L homologous recombination arm sequence, and the recombinant vaccinia virus vector with the knocked-out D8L gene further comprises a D8L-R homologous recombination arm sequence, preferably, the nucleotide sequence of the D8L-L homologous recombination arm is shown as SEQ ID NO. 1, and the nucleotide sequence of the D8L-R homologous recombination arm is shown as SEQ ID NO. 2.
The invention also provides a shuttle vector for constructing the recombinant vaccinia virus vector knocked out by the D8L gene, wherein the shuttle vector contains a D8L homologous recombination arm sequence.
In one embodiment of the invention, the shuttle vector comprises a D8L-L homologous recombination arm sequence, preferably, the D8L-L homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises a D8L-R homologous recombination arm sequence, preferably, the D8L-R homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further comprises a nucleic acid sequence inserted between the homologous recombination arm sequences of D8L, wherein the nucleic acid sequence comprises at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, and the nucleic acid sequence further comprises one or more exogenous genes other than the reporter gene or the resistance gene.
In one embodiment of the invention, the promoter element of vaccinia virus is P7.5 or PE/L, the sequence of P7.5 is shown as SEQ ID NO. 13, and the sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment of the present invention, the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene (Lac Z), preferably, the fluorescent protein-encoding gene is a green fluorescent protein-encoding gene, a yellow fluorescent protein-encoding gene, a red fluorescent protein-encoding gene or a blue fluorescent protein-encoding gene, more preferably, the fluorescent protein-encoding gene is a green fluorescent protein-Encoding Gene (EGFP), and the nucleic acid-encoding sequence thereof is shown in SEQ ID NO. 15.
In one embodiment of the invention, the resistance gene is a neomycin encoding gene and selective pressure is applied when the resistance gene is used in the purification of recombinant vaccinia virus vectors.
In one embodiment of the present invention, the foreign gene is a gene derived from non-vaccinia virus itself except for a reporter gene or a resistance gene, and the target antigen or immunogen-encoding gene selected according to the purpose of use may be any one or more immunogens of interest, preferably, the target immunogen is a peptide, an antigen, a hapten, a carbohydrate, a protein, a nucleic acid, an allergen, a virus or a part of a virus, a bacterium, a parasite or other intact microorganism, and further, the target antigen or immunogen is a tumor antigen or an infection-associated antigen.
In one embodiment of the invention, the nucleic acid sequences at the two ends of D8L are respectively a D8L-L homologous recombination arm and a D8L-R homologous recombination arm, preferably, the nucleic acid sequences of the D8L-L homologous recombination arm are shown as SEQ ID NO. 1, and the nucleic acid sequences of the D8L-R homologous recombination arm are shown as SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further has a nucleic acid sequence inserted into the homologous recombination arm sequence of D8L, and the nucleic acid sequence is shown as SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is p shuttle D8L-GFP, and its nucleic acid sequence is shown in SEQ ID NO. 17.
The invention also provides a preparation method for constructing the recombinant vaccinia virus vector with the knocked-out D8L gene, which comprises the following steps:
step one, designing primers, and amplifying two homologous recombination arms of D8L by PCR respectively.
And step two, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the D8L homologous recombination arm sequence.
And step three, transfecting the shuttle vector into the vaccinia virus infected cell.
And step four, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
Wherein, the primer is a primer sequence which can be combined with two ends of D8L, and is preferably a primer of SEQ ID NO. 3D8L-LC-F, 4D8L-LC-R, 5D8L-RC-F or 6D 8L-RC-R.
The shuttle vector is characterized by containing the D8L homologous recombination arm sequence.
In one embodiment of the invention, the shuttle vector contains the D8L-L homologous recombination arm sequence. Preferably, the nucleotide sequence of the D8L-L homologous recombination arm is shown as SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises a D8L-R homologous recombination arm sequence. Preferably, the nucleotide sequence of the D8L-R homologous recombination arm is shown as SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further comprises a nucleic acid sequence inserted between the sequences of the homologous recombination arms of D8L. Preferably, the nucleic acid sequence comprises at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene. In embodiments of the invention, the nucleic acid sequence may also include one or more additional exogenous genes in addition to the reporter gene or the resistance gene.
In one embodiment, the vaccinia virus promoter element is P7.5 or PE/L. Preferably, the nucleic acid sequence of P7.5 is shown as SEQ ID NO. 13, and the nucleic acid sequence of PE/L is shown as SEQ ID NO. 14.
In one embodiment, the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene (Lac Z). Preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene, a yellow fluorescent protein coding gene, a red fluorescent protein coding gene or a blue fluorescent protein coding gene. More preferably, the fluorescent protein coding gene is a green fluorescent protein coding gene (EGFP), and the nucleic acid coding sequence of the fluorescent protein coding gene is shown in SEQ ID NO. 15.
In the present invention, the resistance gene is, for example, a Neomycin (Neomycin) encoding gene, and can be used for applying selective pressure when purifying a recombinant vaccinia virus vector.
In the present invention, the other foreign gene is a gene derived from a non-vaccinia virus itself in addition to the reporter gene or the resistance gene. The target antigen or immunogen-encoding gene may be selected according to the purpose of use. The immunogen of interest is any one or more immunogens. Preferably, the immunogen of interest is a peptide, antigen, hapten, carbohydrate, protein, nucleic acid, allergen, virus or part of a virus, bacterium, parasite or other whole microorganism. In addition, the antigen or immunogen of interest is a tumor antigen or an infection-associated antigen.
In one embodiment of the present invention, the shuttle vector further has a nucleic acid sequence inserted into the homologous recombination arm sequence of D8L, and the nucleic acid sequence is shown as SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is pDELTA D8L-GFP, and its nucleic acid sequence is shown in SEQ ID NO: 17.
Among them, the Vaccinia virus is preferably a replicative Vaccinia virus vector such as Vaccinia virus Tiantan strain, e.g., 752-1 strain, or a non-replicative Vaccinia virus vector such as Vaccinia virus attenuated vaccine Ankara strain (Modified Vaccinia Ankara, MVA).
The cells are eukaryotic cell lines, preferably mammalian cell lines, such as 293T cells, 143B cells, Vero cells or BHK-21 cell lines.
Transfection is a technique well known in the art, i.e., delivery of the plasmid DNA of interest into cells, and includes, but is not limited to, physical methods (e.g., electroporation, cell extrusion, nanoparticles, and magnetic transfer), or chemical methods (e.g., calcium phosphate transfection, lipofection, DEAE-dextran or polyethyleneimine transfection), preferably, transfection is polyethyleneimine transfection, such as Turbofect (Thermo Fisher Scientific, R0531) transfection reagent.
For a better description of the invention, see examples 1-6:
example 1 p.DELTA.D 8L-GFP shuttle vector construction.
By designing primers (Table 1) on the genomic sequence of the Tiantan strain poxvirus TP5 clone (Genbank: KC207811), using the genomic DNA of vaccinia virus wild-type rvv-WT (provided by Beijing biologicals) (preparation method reference example 3) as a template (to amplify D8L-L (SEQ ID NO:1) and D8L-R (SEQ ID NO:2) fragments, respectively, wherein the D8L-LC-F and D8L-LC-R primer pair was used to amplify the D8L-L fragment, and the D8L-RC-F and D8L-RC-R primer pair was used to amplify the D8L-R fragment.
Table 1: primers in example 1
In step 1, an intermediate vector p delta D8L-L with a D8L-L homologous recombination arm is constructed. First, a linearized vector of about 5kbp was PCR-amplified using pSC65-GFP vector (obtained by replacing LacZ expressing galactosidase on pSC65 vector with EGFP gene expressing green fluorescent protein, supplied by Soviken Tokyo Biotech Co., Ltd.) modified from pSC65 shuttle vector (addge, cat # 30327) as a template, and TKL-F and TKL-R (Table 1) primers. The linearized vector was then subjected to homologous recombination with the D8L-L fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. The vector p.DELTA.D 8L-L with the homologous arm sequence of D8L-L was then constructed using recombinant transformation methods well known in the art.
In step 2, a shuttle vector p.DELTA.D 8L-GFP was constructed. Using the intermediate vector p.DELTA.D 8L-L constructed in step 1 as a template, a linearized vector of about 5kbp was amplified using TKR-F and TKR-R primer set (Table 1). The linearized vector was then subjected to homologous recombination with the D8L-R fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. Then, a vector p delta D8L-GFP (a plasmid map is shown in a figure 1) with a D8L-L and D8L-R homologous arm sequence is constructed by a recombinant transformation method well known in the art, and is stored in a warehouse after being sequenced and identified correctly. The vector p.DELTA.D 8L-GFP (restriction system shown in Table 2) was identified by using restriction endonucleases Not I and Kpn I, and its cleavage verification map is shown in FIG. 2.
Table 2: plasmid p.DELTA.D 8L-GFP restriction enzyme identification System (restriction enzyme for 2 hours at 37 ℃ C.)
| Enzyme digestion system | Volume of |
| Plasmid p.DELTA.D 8L- |
3 μ L, about 1 μ g |
| Not I (Baoyuan, goods number 1166A) | 1μL |
| Kpn I (Baozi, cat No. 1068A) | 1μL |
| Enzyme digestion buffer solution | 1μL |
| ddH2O | Make up to 10 mu L |
Example 2 recombinant vaccinia virus vector rvv- Δ D8L/G was constructed.
Recombinant vaccinia virus vectors were obtained in 293T cells as follows. On day 1, 293T cells were plated in 6-well cell culture plates (JET, TCP-010-6Well, incubated overnight at 37 ℃ in a carbon dioxide cell incubator. The following day, at 0.05MOI (i.e., 5X 10)4PFU (plaque forming Unit)/well) was added vaccinia virus wild strain rvv-WT (supplied by Beijing biologicals) and incubated in a 37 ℃ carbon dioxide cell incubator for two hours during which the shuttle vector/transfection reagent complex was prepared. Wherein the shuttle vector is the p-derived complex obtained in example 1. The transfection reagent is Turbofect (Thermo Fisher Scientific, R0531), and the transfection reagent specification can be referred to the transfection reagent. After completion of the multiplex system, 293T cell supernatants were changed to 2 mL/well DMEM maintenance medium containing 2% Fetal Bovine Serum (FBS) followed by addition of shuttle vector/transferStaining the reagent complex. 48 hours after transfection, supernatant was removed, cells were harvested and resuspended in 0.5mL maintenance medium, freeze-thawed three times repeatedly, and recombinant cell lysate was then inoculated into Vero cells (R: (R) (R))
CCL-81), 37, incubation for 1 to 2 days. During which cytopathic effects are observed and single plaque purification is performed when the appropriate number of viral plaques are present (less than 20 plaques/well).
Single-spot purification:
and (3) observing virus plaques expressing green fluorescence under a fluorescence microscope, and marking.
Remove the supernatant, pick several well-dispersed green fluorescent plaques per well, and transfer to centrifuge tubes containing 0.5mL of maintenance medium, respectively.
Shaking and mixing the centrifuge tube containing the virus, repeatedly freezing and thawing for three times (about 5 minutes at minus 80 ℃ in a refrigerator and about 2 minutes at room temperature), finally shaking and mixing the mixture, and freezing and storing the mixture at minus 80 ℃.
Vero cells (T) were prepared on 6-well cell culture plates (JET, TCP-010-
CCL-81), virus was then removed from the-80 ℃ freezer, seeded onto Vero cells in 10 μ L each, and incubated at 37 ℃ for 2 to 3 days. During which cytopathic effects are observed and the next round of single plaque purification is performed when the appropriate number of viral plaques are present (less than 20 plaques/well).
At least six rounds of single-spot purification were repeated until the purity reached 100%.
Example 3 identification of recombinant vaccinia virus vector rvv- Δ D8L/G.
The purified poxvirus vector rvv- Δ D8L/G was seeded onto Vero cells (1X 10)7Cells) were observed after 48 hours, and when 80% or more of the cells were infected, the cells were collected, centrifuged at 1800g for 5 minutes, and the supernatant was removed. Then extracting the poxvirus genomic DNA by using a genomic DNA extraction kit (TRAN, EE101-02), wherein the specific method refers to the kit specification, and finally obtaining 100 mu L of genomic DNA and freezing and storing at-20 ℃.
The poxvirus genomic DNA was PCR amplified using primers from both ends of the D8L gene (Table 3), and the results are shown in FIG. 3. the recombinant poxvirus vector rvv- Δ D8L/G genomic DNA amplified a 1846bp band that was significantly larger than the negative control (808bp) amplified using rvv-WT genome. And (3) sending a PCR amplification product sample to Jinzhi corporation for sequencing, wherein the sequence result is in line with the expectation, and the D8L fragment is successfully knocked out.
Table 3: primers for identification in example 3
Similarly, the purified vaccinia virus vector rvv- Δ D8L/G was inoculated into Vero cells (1X 10)7Cells) were observed after 48 hours, and when 80% or more of the cells were infected, the cells were collected, centrifuged at 1800g for 5 minutes, and the supernatant was removed. SDS-PAGE samples were then prepared and used for immunoblot hybridization experiments, and primary antibodies were anti-GFP monoclonal antibody-HRP (Santacruz, sc-9996 HRP). As shown in FIG. 4, there is a specific band around 20kD, which is consistent with the expected molecular weight position, while the control sample (rvv-WT) has no band at the corresponding molecular weight position, indicating that rvv- Δ D8L/G is constructed to correctly express the inserted foreign gene GFP target antigen. And simultaneously, the cells were incubated with an anti-D8L-antibody (LA5, virology.2011Jan 20; 409(2):271-9.) and a rabbit anti-human-HRP (Boster, BA1070) antibody, and the results are shown in FIG. 5, wherein the Vero cell control and the recombinant vaccinia virus vector rvv-delta D8L/G have no D8L expression, while the vaccinia virus wild-type rvv-WT has obvious D8L expression at 35kD, indicating that the D8L gene is knocked out.
Example 4 recombinant vaccinia virus vector rvv- Δ D8L/G amplification preparation and titration.
The recombinant vaccinia virus vector rvv- Δ D8L/G and the wild strain of vaccinia virus (rvv-WT) constructed in example 2 were amplified on Vero cells, respectively, as follows:
on the previous day, Vero monolayers (1X 10) with 100% confluency were prepared7Cells/dish) for a total of 10 dishes.
The supernatant was removed and replaced with maintenance medium, the poxvirus to be amplified was inoculated onto the cells (0.01 PFU/cell) and incubated in an incubator at 37 ℃ for 2-3 days, and the lesions were observed.
The cells were scraped and collected, 1800g centrifuged for 5 minutes and the supernatant removed.
Resuspending with 5mL of maintenance medium, and sonicating with a sonicator cell disruptor on ice under the following conditions: 50 watts, 5 seconds sonication/5 seconds interval for 15 minutes.
Repeatedly freezing and thawing twice (about 5 minutes at 80 ℃ in a refrigerator and about 2 minutes at room temperature), and finally oscillating and uniformly mixing; subpackaging in a secondary biological safety cabinet into 1.5mL centrifuge tubes, and freezing at-80 ℃ for 1 mL/tube.
The infectious titer of the amplified vaccinia virus is titrated on Vero cells by the specific method as follows:
vero cells, 3X 10, with a 100% confluency were prepared in 24-well plates on the previous day5A hole.
Remove supernatant and add 200 μ L of maintenance medium per well to prevent cells from drying out.
Adding 100 mu L poxvirus to be detected into 900 mu L maintenance medium, ten-fold diluting, and continuously diluting 10 times1，102，103… …, up to 109And (4) doubling. Note that: when dilution is performed, the tip should be replaced every time dilution is performed from high concentration to low concentration.
From small to large virus concentration (10)9，108，……104) Added to a 24-well plate at 400. mu.L dilution per well, and repeated two times, and the dilution was measured at 6 times in series. The added 24-well plate was incubated in a 37 ℃ cell incubator for 2 days.
The number of viral plaques, more than 20, was counted microscopically and scored as 20 +. Two replicate wells with a measurable number of 20 spots (including 20) were averaged by a dilution of 2.5 (1000. mu.L/400. mu.L). times.corresponding wells to obtain the recombinant virus titer (PFU/mL). The titer of vaccinia virus vectors was titrated as shown in Table 4.
Table 4: vaccinia Virus vector titer titration
| Vaccinia virus | Potency (PFU/mL) |
| Vaccinia virus wild type rvv- |
1×108 |
| Recombinant vaccinia virus rvv-delta D8L/ |
1×108 |
Example 5 detection of neutralizing antibodies.
The detection method of the neutralizing antibody comprises the following steps: a96-well flat-bottom plate was prepared by adding 100. mu.L of DMEM cell culture medium to all wells in column 1 (cell control) and 50. mu.L of LDMEM cell culture medium to all wells in columns 2-12 (column 2 will be used as virus control). mu.L of DMEM cell culture medium (1: 10 initial dilution, 2-fold gradient dilution) was added to wells H3-H12. 20 mu L of human plasma samples to be tested are respectively added into H3-H12 wells. Mix all samples in row H and then transfer 50. mu.L to row G. Mixing and transfer were repeated until row a (2-fold gradient dilution). After the final transfer and mixing was complete, 50. mu.L was discarded from row A, columns 3-12. The amount of vaccinia virus was calculated and 40 PFU/well was used as the infectious dose to thaw sufficient viral stocks. When the thawing was complete, the virus was diluted to 800PFU/mL with DMEM cell culture medium. 50 μ L of virus dilution was added to all wells in 2-12 columns in a 96 well plate, the plate was covered, and incubated for 1 hour in a cell incubator. Preparing Vero cells, digesting Vero cells with pancreatin/EDTA to obtain cell suspension, and maintaining culture medium with 2% DMEM to adjust cell concentration to 5 × 105mL, then added to a 96 well cell culture plate at 100. mu.L/well, i.e., 5X 104A hole. After 48 hours, the supernatant was carefully aspirated, 0.1% crystal violet was added, 50 μ L/well, and stained for 10 minutes at room temperature. Photograph, count the number of plaques in each well and divide the number of plaques in the experimental well by the mean of the plaques in the virus control (column 2)And then the percent inhibition of the tested sample is obtained by 1 reduction and then multiplication by 100. IC50 titers were determined as percent inhibition, and neutralizing antibody IC50 titers were expressed as percent inhibition>The reciprocal of the highest dilution of 50, the IC50 can also be calculated from a percentage inhibition fit curve.
As shown in FIG. 6, human plasma showed higher neutralizing antibody levels against vaccinia virus wild type (rvv-WT) (mean value of GMT 113.1), while the neutralizing antibody levels against D8L defective strain rvv- Δ D8L/G were significantly reduced (mean value of GMT 22.5, p < 0.0001). The result shows that the strain after the D8L knockout generates escape to the existing antibody in human body, increases the chance of reinfection and improves the application possibility of the vaccinia virus vector.
Example 6 virus replication kinetics.
Vero cells were prepared in 6-well plates and vaccinia virus was inoculated to each well (1.5X 10) at 0.1MOI5PFU), supernatants and cells were then taken at various time points post infection, frozen and thawed 3 times and titrated on Vero cells (see method for virus titration in example 4).
As a result, as shown in FIG. 7, the wild-type vaccinia virus rvv-WT had a strong replication ability, and the virus was mainly present in the cells and contained a small amount of supernatant, which was about 2 orders of magnitude lower than that of the cells. Compared with a wild strain, the D8L defective strain rvv-delta D8L/G has slightly weaker replication capacity, the replication kinetic curves are consistent, peaks are reached at 24 hours, and then the peaks are maintained at 48 hours, and the two strains of viruses have no significant difference. The D8L knockout was shown not to affect the replication capacity of vaccinia virus.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (16)
1. A recombinant vaccinia virus vector with a knocked-out D8L gene, wherein a D8L encoding gene of the recombinant vaccinia virus vector is knocked out, and the D8L gene cannot be normally expressed.
2.The recombinant vaccinia virus vector with a knocked-out D8L gene of claim 1, wherein the vaccinia virus vector is a replicating vaccinia virus vector or a non-replicating vaccinia virus vector.
3. The recombinant vaccinia virus vector with a knocked-out D8L gene of claim 2, wherein the knocked-out D8L is achieved by inserting a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene into the nucleic acid sequence at both ends of D8L, so that the D8L gene is mutated, and the nucleic acid sequence further comprises one or more exogenous genes besides the reporter gene or the resistance gene.
4. The recombinant vaccinia virus vector with a knocked-out D8L gene according to claim 3, wherein the vaccinia virus promoter element is P7.5 or PE/L, preferably, the nucleic acid sequence of P7.5 is SEQ ID NO. 13, and the nucleic acid sequence of PE/L is SEQ ID NO. 14.
5. The recombinant vaccinia virus vector with a knocked-out D8L gene of claim 4, wherein the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene.
6. The recombinant vaccinia virus vector of knocking out D8L gene according to claim 5, wherein the nucleic acid sequences at two ends of D8L are D8L-L homologous recombination arm or D8L-R homologous recombination arm, respectively, wherein the nucleic acid sequence of the D8L-L homologous recombination arm is SEQ ID NO. 1, and the nucleic acid sequence of the D8L-R homologous recombination arm is SEQ ID NO. 2.
7. The recombinant vaccinia virus vector with a knocked-out D8L gene of claim 6, wherein the recombinant vaccinia virus vector with a knocked-out D8L is rvv-vv-group vaccinia.
8. A shuttle vector for constructing a recombinant vaccinia virus vector with a knocked-out D8L gene, wherein the shuttle vector comprises a homologous recombination arm sequence of D8L.
9. Shuttle vector according to claim 8, wherein the shuttle vector comprises the D8L-L homologous recombination arm sequence, wherein the D8L-L homologous recombination arm nucleic acid sequence is SEQ ID NO 1.
10. The shuttle vector according to claim 9, further comprising a D8L-R homologous recombination arm sequence, wherein the D8L-R homologous recombination arm nucleic acid sequence is SEQ ID No. 2.
11. A shuttle vector according to claim 10 wherein a nucleic acid sequence comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, is inserted between the homologous recombination arm sequences of D8L, and wherein the nucleic acid sequence further comprises one or more foreign genes other than the reporter gene or the resistance gene.
12. The shuttle vector for constructing the recombinant vaccinia virus vector for D8L gene knockout, according to claim 11, wherein the vaccinia virus promoter element is P7.5 or PE/L, preferably the nucleic acid sequence of P7.5 is SEQ ID NO. 13, and the nucleic acid sequence of PE/L is SEQ ID NO. 14.
13. The shuttle vector of claim 12, wherein the reporter gene is a fluorescent protein-encoding gene or a galactosidase-encoding gene.
14. The shuttle vector of claim 13, wherein the shuttle vector is pDELTA D8L-GFP and has the nucleic acid sequence of SEQ ID NO. 17, for use in the construction of a recombinant vaccinia virus vector with a D8L knock-out gene.
15. A preparation method for constructing a recombinant vaccinia virus vector with a D8L gene knockout function is characterized by comprising the following steps:
s1, designing primers, and respectively amplifying two homologous recombination arms of D8L by PCR;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing a D8L homologous recombination arm sequence;
s3, transfecting the shuttle vector into a vaccinia virus infected cell;
s4, collecting the recombinant vaccinia virus vector from the cells, and performing single-spot purification on the new cells.
16. The method according to claim 15, wherein the primer is a primer sequence capable of binding to both ends of D8L, and preferably comprises the following nucleic acid sequence:
3D8L-LC-F primer of SEQ ID NO
TATAACCAGACCGTTCAGGGGTACTGTATCATTTAGCG
4D8L-LC-R primer
ATTAATTTTTATCGATCTATTTTATCTAGACAATTTGCTG
Primer SEQ ID NO 5D8L-RC-F
GCGGCCGCGGATCACTGAATCAAACGGTGCAGA
6D8L-RC-R primer
TTAATAATCTCATTTCAGATTGTAATTCCCATACTAAGAGC。
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| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| CN109810953A (en) * | 2019-01-07 | 2019-05-28 | 西安彤盛生物科技有限公司 | The recombination the Temple of Heaven strain oncolytic vaccinia virus of removal TK gene and its preparation and application |
| WO2020086423A1 (en) * | 2018-10-22 | 2020-04-30 | Icell Kealex Therapeutics | Mutant vaccinia viruses and use thereof |
-
2021
- 2021-06-18 CN CN202110679465.4A patent/CN113322279A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861558A (en) * | 2016-06-24 | 2016-08-17 | 西安医学院 | Vaccinia virus shuttle vector and preparation method and application thereof |
| WO2020086423A1 (en) * | 2018-10-22 | 2020-04-30 | Icell Kealex Therapeutics | Mutant vaccinia viruses and use thereof |
| CN109810953A (en) * | 2019-01-07 | 2019-05-28 | 西安彤盛生物科技有限公司 | The recombination the Temple of Heaven strain oncolytic vaccinia virus of removal TK gene and its preparation and application |
Non-Patent Citations (1)
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| KEVIN SONG等: "Design and Engineering of Deimmunized Vaccinia Viral Vectors", 《BIOMEDICINES》 * |
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