CN113481241A - Recombinant vaccinia virus vector with H3L 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 H3L gene knocked out; the recombinant vaccinia virus vector has an H3L homologous recombination arm sequence, and is used for constructing a vaccinia virus vector inserted with an exogenous gene in a vaccinia virus H3L gene region; the invention provides a recombinant vaccinia virus vector capable of escaping existing anti-vaccinia virus neutralizing antibodies existing in vivo, wherein the H3L encoding gene of the recombinant vaccinia virus vector is knocked out or damaged and cannot normally express the H3L 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 H3L gene knocked out.
Background
Vaccinia Virus (Vaccinia Virus), belonging to the genus Orthomyxovirus of the family Poxviridae, 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.A milestone in the history of infection. eradiation of smallpox. MMW Munch Meachhenschr, 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 H3L gene, which selects main neutralizing antigen of vaccinia virus as a target spot, and researches whether knocking out or destroying the target spot can escape from the existing neutralizing antibody in vivo, thereby increasing the use frequency of the recombinant vaccinia virus vector in vivo.
In order to achieve the purpose, the invention provides the following technical scheme:
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 H3L knockout is that a mutation is introduced into the H3L gene, and the endogenous normal H3L gene of the vaccinia virus is replaced by means of homologous recombination, so that the H3L 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 H3L gene knocked out, wherein a gene encoding H3L of the recombinant vaccinia virus vector is knocked out, and the H3L gene cannot be normally expressed.
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 H3L knockout refers to that the endogenous normal H3L gene of the vaccinia virus is replaced by homologous recombination through introducing mutation in the H3L gene, so that the H3L gene cannot be normally expressed.
The invention is further configured to: the H3L knockout is performed by mutating the H3L 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 H3L, 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 H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm.
The invention is further configured to: the H3L knockout recombinant vaccinia virus vector further has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence.
The invention is further configured to: the H3L knockout recombinant vaccinia virus vector is rvv-delta H3L/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 an H3L gene knockout.
The invention is further configured to: the H3L gene knockout recombinant vaccinia virus vector contains an H3L-L homologous recombination arm sequence, and the H3L gene knockout recombinant vaccinia virus vector also contains an H3L-R homologous recombination arm sequence.
A shuttle vector for constructing a recombinant vaccinia virus vector with an H3L gene knockout, wherein the shuttle vector comprises an H3L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector contains H3L-L homologous recombination arm sequences.
The invention is further configured to: the shuttle vector also contains the H3L-R homologous recombination arm sequence.
The invention is further configured to: the shuttle vector also inserts a nucleic acid sequence between the H3L homologous recombination arm sequences, 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 H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm.
The invention is further configured to: the shuttle vector also has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence.
The invention is further configured to: the shuttle vector is p.DELTA.H 3L-GFP.
A preparation method for constructing a recombinant vaccinia virus vector with an H3L gene knockout function comprises the following steps:
s1, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L 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 H3L encoding gene of the recombinant vaccinia vector, and not normally expressing the H3L 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.
Drawings
FIG. 1 is a plasmid map of shuttle vector p.DELTA.H3L-GFP with H3L-L and H3L-R homologous recombination arm sequences;
FIG. 2 is a diagram showing the double restriction enzyme identification of the shuttle vector p.DELTA.H3L-GFP with H3L-L and H3L-R homologous recombination arm sequences;
FIG. 3 is a diagram showing the identification of recombinant vaccinia virus vector rvv- Δ H3L/G by PCR amplification;
FIG. 4 shows an expression identification chart of recombinant vaccinia virus vector rvv- Δ H3L/G;
FIG. 5 shows the results of detection of neutralizing antibodies in example 5.
FIG. 6 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. 1-6: the invention aims to provide a recombinant vaccinia virus vector with a H3L gene knocked out, wherein a H3L encoding gene of the recombinant vaccinia virus vector is knocked out, and the H3L 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, H3L knockout refers to replacement of the endogenous normal H3L gene of vaccinia virus by homologous recombination by introducing a mutation inside the H3L gene, such that the H3L gene is not normally expressed.
In one embodiment of the invention, the H3L knockout is performed by mutating the H3L 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 H3L, 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 H3L are respectively H3L-L homologous recombination arm and H3L-R homologous recombination arm, preferably, the nucleic acid sequence of the H3L-L homologous recombination arm is shown as SEQ ID NO. 1, and the nucleic acid sequence of the H3L-R homologous recombination arm is shown as SEQ ID NO. 2.
In one embodiment of the invention, the H3L knockout recombinant vaccinia virus vector further comprises a nucleic acid sequence as shown in SEQ ID NO. 16 inserted into the H3L homologous recombination arm sequence.
In one embodiment of the invention, the H3L knock-out recombinant vaccinia virus vector is rvv- Δ H3L/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 an H3L gene knockout.
In one embodiment of the invention, the H3L gene knockout recombinant vaccinia virus vector comprises an H3L-L homologous recombination arm sequence, and the H3L gene knockout recombinant vaccinia virus vector further comprises an H3L-R homologous recombination arm sequence, preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 1, and the H3L-R homologous recombination arm nucleic acid sequence is shown as SEQ ID NO. 2.
The invention also provides a shuttle vector for constructing the H3L gene knockout recombinant vaccinia virus vector, wherein the shuttle vector contains the H3L homologous recombination arm sequence.
In one embodiment of the invention, the shuttle vector comprises the H3L-L homologous recombination arm sequence, preferably the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises an H3L-R homologous recombination arm sequence, preferably, the H3L-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 comprising at least one vaccinia virus promoter element, or at least one reporter gene or one resistance gene, and one or more foreign genes other than the reporter gene or the resistance gene, inserted between the H3L homologous recombination arm sequences.
In one embodiment of the invention, the vaccinia virus promoter element is P7.5 or PE/L, 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-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 H3L are respectively H3L-L homologous recombination arm and H3L-R homologous recombination arm, preferably, the sequence of the H3L-L homologous recombination arm is shown as SEQ ID NO. 1, and the sequence of the H3L-R homologous recombination arm is 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 H3L homologous recombination arm sequence, and the nucleic acid sequence is shown in SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is p.DELTA.H 3L-GFP, the nucleic acid sequence of which is shown in SEQ ID NO: 17.
The invention also provides a preparation method for constructing the H3L gene knockout recombinant vaccinia virus vector, which comprises the following steps:
step one, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively.
And step two, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L 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 H3L, and is preferably SEQ ID NO. 3H3L-LC-F primer, SEQ ID NO. 4H3L-LC-R primer, SEQ ID NO. 5H3L-RC-F primer or SEQ ID NO. 6H3L-RC-R primer.
The shuttle vector is characterized by containing H3L homologous recombination arm sequences.
In one embodiment of the invention, the shuttle vector contains the H3L-L homologous recombination arm sequence. Preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1.
In one embodiment of the invention, the shuttle vector further comprises an H3L-R homologous recombination arm sequence. Preferably, the H3L-R homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 2.
In one embodiment of the invention, the shuttle vector further comprises a nucleic acid sequence inserted between the sequences of the H3L homologous recombination arms. 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 the invention, the nucleic acid sequences at two ends of H3L are respectively an H3L-L homologous recombination arm and an H3L-R homologous recombination arm. Preferably, the H3L-L homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 1. Preferably, the H3L-R homologous recombination arm nucleic acid sequence is shown in SEQ ID NO. 2.
In one embodiment of the present invention, the shuttle vector further has a nucleic acid sequence inserted into the H3L homologous recombination arm sequence, the sequence is shown in SEQ ID NO. 16.
In one embodiment of the present invention, the shuttle vector is p.DELTA.H 3L-GFP, the nucleic acid sequence of which 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.H 3L-GFP shuttle vector construction.
H3L-L (SEQ ID NO:1) and H3L-R (SEQ ID NO:2) fragments were amplified respectively by designing primers (Table 1) on the genomic DNA (preparation method reference example 3) of vaccinia virus wild-type rvv-WT (provided by Beijing biologicals) on the sequence of the cloned genome of the Techn strain poxvirus TP5, wherein the H3L-LC-F and H3L-LC-R primer pairs were used to amplify the H3L-L fragment, and the H3L-RC-F and H3L-RC-R primer pairs were used to amplify the H3L-R fragment.
Table 1: primers in example 1
In step 1, an intermediate vector p delta H3L-L-GFP with H3L-L homologous recombination arms is constructed. First, a linearized vector of about 5kbp was PCR-amplified using a pSC65-GFP vector (obtained by replacing LacZ expressing galactosidase on the pSC65 vector with EGFP gene expressing green fluorescent protein, supplied by Soviken Korea Tokyo Biotech Co., Ltd.) modified from pSC65 shuttle vector (addge, cat # 30327) (plasmid map shown in FIG. 1) as a template, and TKL-F and TKL-R (Table 1) primers. The linearized vector was then subjected to homologous recombination with the H3L-L fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. The vector p.DELTA.H 3L-L-GFP with the H3L-L homology arm sequence was then constructed using recombinant transformation methods well known in the art.
In step 2, a shuttle vector p.DELTA.H 3L-GFP was constructed. Using the intermediate vector p.DELTA.H 3L-L-GFP 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 H3L-R fragment using the EasyGeno Rapid cloning kit (Tiangen, VI201), methods referenced in the kit instructions. Then, a vector p delta H3L-GFP (a plasmid map is shown in a figure 1) with H3L-L and H3L-R homologous arm sequences is constructed by a recombinant transformation method well known in the art, and is stored after being sequenced and identified correctly. The vector p.DELTA.H 3L-GFP (the digestion system is shown in Table 2) was identified by using restriction enzymes EcoR V and Kpn I, and the digestion verification map is shown in FIG. 2.
Table 2: plasmid p.DELTA.H 3L-GFP restriction enzyme identification system (restriction enzyme for 2 hours at 37 ℃ C.)
Enzyme digestion system | Volume of |
Plasmid p.DELTA.H 3L- |
4 μ L, about 1 μ g |
EcoR V (Baoyuan, goods number 1042A) | 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 H3L/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 was p.DELTA.H 3L-GFP obtained in example 1, the transfection reagent was Turbofect (Thermo Fisher Scientific,r0531), the transfection dosage and the compounding method can be seen in the specification of the transfection reagent. After completion of the complexing system, the 293T cell supernatant was changed to 2 mL/well DMEM maintenance medium containing 2% Fetal Bovine Serum (FBS), and then the shuttle vector/transfection reagent complex was added. 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) and incubated at 37 ℃ 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 H3L/G.
The purified vaccinia virus vector rvv-. DELTA.H 3L/G was inoculated 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 using the genomeDNA extraction kit (TRAN, EE101-02) for extracting vaccinia virus genome DNA, wherein the specific method refers to kit specification, and finally 100 μ L of genome DNA is obtained and frozen at-20 ℃.
The result of PCR amplification of poxvirus genomic DNA using primers at both ends of H3L gene is shown in FIG. 3, the target band with size of 567bp can be amplified by using primer pair H3L-JF and EGPV 62-R (Table 3) in recombinant vaccinia virus vector rvv- Δ H3L/G genomic DNA, the target band with size of 1910bp can be amplified by using primer pair P7.5-F and H3L-RC-R (Table 3), the PCR amplification product sample is sent to Jinzhi corporation for sequencing, the sequence result is in line with the expectation, which indicates that H3L fragment is successfully knocked out.
Table 3: primers for identification in example 3
Similarly, the purified poxvirus vector rvv-. DELTA.H 3L/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. SDS-PAGE samples were then prepared and immunoblot hybridization experiments were performed, and incubated with anti-H3L-antibody (D67, see U.S. Pat. No. 9447172) and rabbit anti-human-HRP (Boster, BA1070) as a secondary antibody, as shown in FIG. 4, vaccinia virus wild-type rvv-WT showed significant H3L expression at 35kD, consistent with the expected molecular weight location, while Vero cell control and recombinant vaccinia virus vector rvv-. DELTA.H 3L/G showed no H3L expression, indicating that the H3L gene was knocked out.
Example 4 recombinant vaccinia virus vector rvv- Δ H3L/G amplification preparation and titration.
The recombinant vaccinia virus vector rvv- Δ H3L/G and the vaccinia virus wild strain (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 H3L/ |
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, divide the number of plaques in the experimental well by the mean of the plaques in the virus control (column 2), and thenThe percent inhibition of the sample was determined by subtracting 1 and then multiplying 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. 5, human plasma showed a high level of neutralizing antibodies against vaccinia virus wild type (rvv-WT) (mean value of GMT 113.1), while the level of neutralizing antibodies against H3L defective strain rvv-. DELTA.H 3L/G was significantly reduced (mean value of GMT 31.8, p < 0.001). The result shows that the strain subjected to H3L knockout generates escape for existing antibodies in human bodies, so that the chance of reinfection is increased, and the application possibility of the vaccinia virus vector is improved.
Example 6 kinetics of viral replication
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. 6, 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 the wild-type strain, the H3L defective strain rvv-delta H3L/G has weak replication capacity and consistent trend of replication kinetic curves, all reach a peak at 48 hours and are about 2 orders of magnitude lower than the wild-type rvv-WT, and then the difference gradually reduces at a later time point. The H3L knock-out was shown 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 H3L gene knocked out, wherein a gene encoding H3L of the recombinant vaccinia virus vector is knocked out, and the H3L gene cannot be normally expressed.
2.The recombinant vaccinia virus vector with H3L knockout function according to 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 knockout of H3L gene of claim 2, wherein the H3L knockout is performed 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 H3L, so that the H3L 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 for knocking out H3L 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 knockout of H3L 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 knockout of H3L gene of claim 3, wherein the nucleic acid sequences at both ends of H3L are H3L-L homologous recombination arm or H3L-R homologous recombination arm, respectively, wherein the nucleic acid sequence of the H3L-L homologous recombination arm is SEQ ID NO. 1, and the nucleic acid sequence of the H3L-R homologous recombination arm is SEQ ID NO. 2.
7. The recombinant vaccinia virus vector with a knockout of H3L gene of claim 6, wherein the recombinant vaccinia virus vector with a knockout of H3L is rvv- Δ H3L/G.
8. A shuttle vector for constructing a recombinant vaccinia virus vector with an H3L gene knockout, wherein the shuttle vector comprises an H3L homologous recombination arm sequence.
9. The shuttle vector of claim 8, wherein the shuttle vector comprises an H3L-L homologous recombination arm sequence, wherein the H3L-L homologous recombination arm nucleic acid sequence is SEQ ID No. 1.
10. The shuttle vector of claim 9, further comprising an H3L-R homologous recombination arm sequence, wherein the H3L-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 H3L homologous recombination arm sequences, 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 of 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 pΔ H3L-GFP, and the nucleic acid sequence is SEQ ID NO 17.
15. A preparation method for constructing a recombinant vaccinia virus vector with an H3L gene knockout function is characterized by comprising the following steps:
s1, designing primers, and amplifying two homologous recombination arms of H3L by PCR respectively;
s2, inserting the two homologous recombination arms into a target shuttle vector to construct a shuttle vector containing the H3L 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 H3L, and preferably comprises the following nucleic acid sequence:
3H3L-LC-F primer of SEQ ID NO
TATAACCAGACCGTTCAGACTCTTGTCATTATAGTGGGA
Primer SEQ ID NO. 4H3L-LC-R
ATTAATTTTTATCGATCTGCCAAATATGTAGAACACGA
Primer SEQ ID NO 5H3L-RC-F
GTTATGCGGCCGCGGATCTGGCATAACTTCGTTGTCC
6H3L-RC-R primer
TTAATAATCTCATTTCAGAACGGTAGAAGAATCCACTC。
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Application publication date: 20211008 |