CN117051040A - Preparation and application of VSV delta G replication defective virus - Google Patents
Preparation and application of VSV delta G replication defective virus Download PDFInfo
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Abstract
The application relates to the technical field of biology, in particular to a preparation method of VSV delta G replication defective seed virus and application thereof in pseudovirus preparation. The method has the characteristics of easy material acquisition, simple and practical process, high safety and the like, and simultaneously has the advantages of fast signal generation, high strength and the like.
Description
Technical Field
The application relates to the technical field of immunity, in particular to a method for obtaining VSV delta G replication defective seed virus and application thereof in pseudovirus preparation.
Background
Replication-defective pseudoviruses have been used in recent years to evaluate neutralizing antibody levels, and have accelerated the development of infectious disease vaccines, drugs, and the like. VSV viruses are useful for packaging pseudoviruses of a variety of viral envelope proteins because of their characteristic independence from the nature of the envelope proteins. The VSV delta G virus, namely the VSV virus with the G protein encoding gene defect, has replication defect and can be used for packaging various replication-defective pseudoviruses. The VSV Δg seed virus can replicate with the aid of a gene encoding an exogenous G protein.
Michael A.Whitt invented a method for preparing VSV.DELTA.G seed virus in 2010. First, eukaryotic cells in which T7 phage RNA polymerase can work are constructed, specifically, vaccinia virus expressing T7RNA polymerase is constructed, and BHK cells are infected with the virus. Subsequently, 5 plasmids were co-transfected, including 1 each of the plasmids containing the VSV N, P, L, G protein-encoding genes under the control of the T7 promoter, and 1 each of the VSV genomic plasmids defective in the G protein-encoding genes under the control of the T7 promoter. After a certain period of time, the cell culture supernatant contained vsvΔg seed virus.
However, mRNA transcribed by T7RNA polymerase does not contain a 5' cap structure and therefore cannot be translated normally in eukaryotic cells, resulting in lower efficiency. Thus, the help of vaccinia virus (containing capping enzymes) is needed. However, vaccinia virus seeds are difficult to obtain, modification and amplification of the virus are complicated, and laboratory needs to apply for the qualification of the virus, which makes the acquisition of VSV Δg replication defective seed virus very complex and difficult. Yuan Zhigang is equal to 2005, it was proposed that eukaryotic cells operable to construct T7 phage RNA polymerase could theoretically also be constructed with the aid of IRES sequences from encephalomyocarditis virus (EMCV) and the like, which provides a new idea for bypassing vaccinia virus, but how to achieve this is technically specific and is not described in the prior art.
In view of this, the present application has been proposed.
Summary of The Invention
Aiming at the technical problems, the application discovers that the VSV delta G replication defective seed virus prepared by the method has obvious technical advantages in the field of pseudovirus preparation.
Specifically, the application provides the following technical scheme:
the application firstly provides a preparation method of VSV delta G replication defective seed virus, which comprises the following steps:
1) Packaging cell construction: constructing a cell line expressing phage T7RNA polymerase;
2) Helper plasmid construction: respectively constructing expression vectors of the T7 promoter connected with VSV virus N, P, L or G protein coding genes;
3) Construction of genome plasmid: constructing a carrier of which a T7 promoter is connected with a VSV virus genome, wherein a G protein coding gene in the VSV virus genome is replaced;
4) Seed virus preparation: introducing helper plasmids and genome plasmids into the packaging cells prepared in step 1), and harvesting the VSV delta G replication defective seed virus.
Further, the method further comprises the following steps:
5) Seed virus amplification: infecting the original cell strain again with the VSV delta G replication defective seed virus prepared in the step 4), and simultaneously adding a VSV virus G protein expression plasmid;
further, in step 1), the cell strain is a mammal, including but not limited to HEK 293 cell strain, heLa cell strain, vero cell strain;
preferably, the means of construction include, but are not limited to, transient transfection of the T7RNA polymerase gene by plasmid or viral vector introduction into the cell line;
further preferred, the T7RNA polymerase gene is linked to a promoter, including but not limited to CMV or CBV promoters;
still more preferably, the specific steps of the construction are: the CMV or CBV promoter is connected with the CDS sequence of the T7RNA polymerase gene and is introduced into HEK 293 cell strain, heLa cell strain or Vero cell strain through plasmid transient transfection or lentiviral vector.
Further, in the step 2),
an Internal Ribosome Entry Site (IRES) sequence is connected between the T7 promoter and the protein coding gene;
preferably, the IRES is operably linked after the T7 promoter sequence.
Further, in the step 3),
the substitution is to replace the G protein encoding gene in the VSV virus genome with a reporter gene comprising a firefly luciferase reporter gene
Further, in the step 4),
the harvesting is as follows: after 1-2 days of culture, the cell culture supernatant was harvested for VSV.DELTA.G replication defective seed virus.
Further, in step 5), the primary cell strain includes, but is not limited to, HEK 293 cell strain, heLa cell strain or Vero cell strain; the VSV virus G protein expression plasmid is an expression plasmid with eukaryotic promoter connected with VSV virus G protein coding gene.
The application also provides a preparation method of the VSV pseudovirus, comprising the method of any one of the above claims, and further comprising the following steps:
6) The heterologous viral envelope protein expression plasmid was introduced into HEK 293 cells, and the cells were infected with the prepared VSV.DELTA.G virus, and pseudoviruses were harvested after 1-2 days.
The application also provides a helper plasmid for constructing the VSV delta G virus, wherein the helper plasmid comprises a T7 promoter sequence, a VSV virus N, P, L or G protein coding gene sequence and an Internal Ribosome Entry Site (IRES) sequence;
further, the IRES is operably linked after the T7 promoter sequence.
The application also provides a VSV delta G virus construction system, which comprises the helper plasmid.
Further, the system further comprises: a cell line expressing bacteriophage T7RNA polymerase, a VSV virus genome plasmid and/or a VSV virus G protein expression plasmid;
further preferably, the VSV virus genome plasmid is a vector of a T7 promoter connected with the VSV virus genome;
still further preferred, the VSV genomic G protein encoding gene is replaced;
furthermore, the VSV virus G protein expression plasmid is an expression plasmid with eukaryotic promoter connected with VSV virus G protein coding gene.
The application also provides application of any auxiliary plasmid or virus construction system in preparation of VSV delta G replication defective seed virus.
The application also provides application of any auxiliary plasmid or virus construction system in preparation of VSV skeleton heterologous pseudoviruses.
It will be appreciated that the pseudoviruses obtained by the method of the application are replication-incompetent and non-pathogenic, and that the mechanism of invasion of cells is mainly determined by the "heterologous viral envelope proteins", so that the process of "heterologous viral" invasion of cells can be simulated, irrespective of the VSV viral G protein. Therefore, the pseudovirus can be used in the fields of vaccines, medicaments and the like to safely carry out the related research on the invasion cells of the heterologous virus.
Therefore, the application also provides the application of the helper plasmid or virus construction system in the preparation of vaccine and medicine fields.
The beneficial technical effects of the application are as follows:
1) In the conventional preparation process of the VSV virus vector, vaccinia virus is needed (the vaccinia virus expresses capping enzyme and can assist T7 RNAP to work in eukaryotic cells) and is difficult to remove, the VSV delta G virus is not constructed by using vaccinia virus, a T7RNA polymerase eukaryotic system is skillfully fused with a VSV framework, the VSV delta G virus is prepared, and conventional BHK cells such as HEK 293 and the like can be replaced by the conventional cells. Therefore, compared with the traditional method, the method has the advantages of at least easy material acquisition, simple process, practicability and the like, and the laboratory does not need special application of vaccinia virus use qualification, so that the construction of the VSV delta G virus is simpler and more popular.
2) The VSV virus vector platform has the advantage of strong universality, and can be used for preparing various pseudoviruses, including but not limited to novel coronaviruses, fever with platelet syndrome (novel bunyas), ebola, nipa and other viruses. In addition, the pseudovirus infection effect and the reporter gene expression level prepared by the method have the advantages of rapid signal generation, high intensity and the like compared with the traditional method, namely the pseudovirus based on the lentiviral skeleton (for example, the S protein pseudovirus signal of the novel coronavirus XBB.1 mutant strain prepared in implementation is about 1 order of magnitude higher than that of the pseudovirus of the traditional lentiviral skeleton, and the experimental period is shortened from 48-72 hours to 24 hours).
3) The application has good safety, the VSV virus vector does not integrate the host cell genome, does not influence the stability of the cell genome and does not cause long-term influence; the virus envelope protein gene is not contained, and the related protein gene is introduced for amplification.
Drawings
FIG. 1 is a general flow of the VSV ΔG replication-defective seed virus acquisition and packaging of heterologous pseudoviruses of the present application.
FIG. 2 shows comparison of the expression effect of reporter genes (green fluorescent protein coding genes) controlled by T7 promoters, whether different eukaryotic cell lines, different promoters and IRES sequences are inserted or not.
FIG. 3 is a graph comparing cell status during VSV.DELTA.G virus amplification in control and experimental groups.
FIG. 4 is a graph comparing signal values of control and experimental groups 24h after infection of cells.
FIG. 5 is a graph comparing fluorescent signals of a lentiviral backbone-constructed heterologous pseudovirus and a VSV backbone-constructed heterologous pseudovirus.
FIG. 6 is a graph of pseudovirus signal intensity for multiple species packaged with VSV ΔG seed viruses according to the application.
Detailed Description
The present application discloses a method for obtaining VSV delta G replication defective seed virus and its application in pseudovirus preparation, and those skilled in the art can refer to the present disclosure to realize its application, and it should be especially pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and are considered to be included in the present application. While the present application has been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations and modifications can be made in the methods and applications of the present application, and that the techniques of the application can be implemented and practiced without departing from the spirit and scope of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The following terms or definitions are provided solely to aid in the understanding of the application. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.
The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.
The terms "about" and "substantially" in this application mean the range of accuracy that one skilled in the art can understand yet still guarantee the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.
The terms "or more", "at least", "exceeding", etc., such as "at least one" should be understood to include, but not be limited to, values of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 400, 600, 700, 900, or 5000. But also any larger numbers or scores therebetween.
Conversely, the term "no more than" includes every value that is less than the recited value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 and 0 nucleotides. But also any smaller numbers or scores therebetween.
The terms "plurality," "at least two," "two or more," "at least a second," and the like should be understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or 200, 300, 600, 700, 900, or more, 5000, or more. But also any larger numbers or scores therebetween.
Method or technical platform aspects
The preparation method of the VSV delta G replication defective seed virus of the present application is shown in FIG. 1, and generally includes the steps of packaging cell construction, helper plasmid construction, genome plasmid construction, seed virus preparation and seed virus amplification. In summary, the application bypasses the traditional vaccinia virus capping process, develops a preparation platform with a brand new idea, and overcomes the problems of difficult acquisition of vaccinia virus seeds, complicated virus transformation and amplification, laboratory qualification requirements and the like.
In some embodiments, the steps of the technical platform implementation include the following:
1) Packaging cell construction: constructing a cell line expressing phage T7RNA polymerase;
2) Helper plasmid construction: respectively constructing expression vectors of the T7 promoter connected with VSV virus N, P, L or G protein coding genes;
3) Construction of genome plasmid: constructing a carrier of which a T7 promoter is connected with a VSV virus genome, wherein a G protein coding gene in the VSV virus genome is replaced;
4) Seed virus preparation: introducing helper plasmids and genome plasmids into the packaging cells prepared in step 1), and harvesting the VSV delta G replication defective seed virus.
The above is a core step of the present technology, and of course, in practice, for better application to the preparation of pseudoviruses, further amplification of seed viruses is required to meet the requirement of batch preparation, and thus, in some embodiments, the following steps are further included:
5) Seed virus amplification: infecting the original cell strain again with the VSV delta G replication defective seed virus prepared in the step 4), and simultaneously adding a VSV virus G protein expression plasmid;
as is evident from the evaluation of example 3 of the present application, the cell lines in step 1) have general applicability, and preferably are mammalian, and may include, but are not limited to, HEK 293 cell lines, heLa cell lines, vero cell lines, and the like.
Packaging cell construction may be accomplished by a variety of methods well known in the art, and in some embodiments, by transient transfection of the T7RNA polymerase gene into the cell line; in other embodiments, the construction may be performed by introducing the T7RNA polymerase gene into the cell line via a viral vector, which may be used for the purpose of construction.
In some embodiments, the T7RNA polymerase gene is linked to a promoter, including but not limited to a CMV or CBV promoter;
illustratively, in some of the more specific embodiments of the application, the specific steps of the construction may be: connecting a CMV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing the CMV promoter into an HEK 293 cell strain through plasmid transient transfection; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting a CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing into an HEK 293 cell strain through plasmid transient transfection; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting a CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing into an HEK 293 cell strain through plasmid transient transfection; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting a CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing the CDS sequence into an HEK 293 cell strain through a lentiviral vector; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting a CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing the CBV promoter into a HeLa cell strain through a cytoplasmic lentiviral vector; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting CMV with CDS sequence of T7RNA polymerase gene, and introducing into HeLa cell strain via lentiviral vector; in other more specific embodiments of the present application, the specific steps of the construction may be: connecting a CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing the CBV promoter into a Vero cell strain through a lentiviral vector; in some more specific embodiments of the present application, the specific steps of the construction may be: CMV was ligated to the CDS sequence of the T7RNA polymerase gene and introduced into HEK Vero cell line by plasmid transient transfection.
In step 2), the T7 promoter, the protein encoding gene, and the Internal Ribosome Entry Site (IRES) sequence are in the same reading frame, and in some embodiments, the Internal Ribosome Entry Site (IRES) sequence is linked between the T7 promoter and the protein encoding gene; in some more specific embodiments, the IRES is operably linked after a T7 promoter sequence.
In step 3), the substitution is a substitution of the G protein encoding gene in the VSV viral genome with a reporter gene, which is not limited herein, any gene having a reporter function satisfying the needs of the present application, and in some embodiments, the reporter gene is a firefly luciferase reporter gene.
In step 4), the harvesting is: after 1-2 days of culture, the cell culture supernatant is harvested and the VSV.DELTA.G replication defective seed virus is harvested.
In step 5), the primary cell strain includes, but is not limited to, HEK 293 cell strain, heLa cell strain or Vero cell strain; the VSV virus G protein expression plasmid is eukaryotic promoter-initiated or linked VSV virus G protein coding gene expression plasmid.
The method for preparing the VSV pseudovirus of the present application comprises the above steps, and further comprises introducing a heterologous viral envelope protein expression plasmid into, for example, HEK 293 cells, while infecting the cells with the prepared VSV delta G virus, harvesting the pseudovirus after culturing for a certain period of time, and in some embodiments, harvesting the pseudovirus after culturing for 1-2 days.
Product aspect
The helper plasmid for constructing the VSV delta G virus is a helper plasmid, and the helper plasmid comprises a T7 promoter sequence, a VSV virus N, P, L or G protein coding gene sequence and an Internal Ribosome Entry Site (IRES) sequence; in some embodiments, an Internal Ribosome Entry Site (IRES) sequence is linked between the T7 promoter and the protein-encoding gene; in some more specific embodiments, the IRES is operably linked after a T7 promoter sequence.
The VSV delta G virus construction system comprises the key auxiliary plasmid; preferably further comprising: a cell line expressing bacteriophage T7RNA polymerase, a VSV virus genome plasmid and/or a VSV virus G protein expression plasmid; in some specific embodiments, the VSV viral genome plasmid is a T7 promoter plus VSV viral genome vector; in some more specific embodiments, the VSV genomic G protein encoding gene is replaced; in some specific embodiments, the VSV viral G protein expression plasmid is an expression plasmid in which a eukaryotic promoter is linked to a VSV viral G protein encoding gene.
Application aspect
It will be appreciated that where the structural composition of the helper plasmids or viral construction systems described above are known, based on the description of the method of the present application, it will be appreciated that these helper plasmids or viral construction systems can be used in the preparation of VSV Δg replication defective seed viruses, as well as in the preparation of VSV backbone heterologous pseudoviruses.
Furthermore, considering that the pseudoviruses obtained by the method of the present application are replication-free and non-pathogenic, the mechanism of invasion into cells is mainly determined by the "heterologous viral envelope proteins", so that the process of invasion into cells by the "heterologous viruses" can be simulated irrespective of VSV viral G proteins. Therefore, the pseudovirus can be used in the fields of vaccines, medicaments and the like to safely carry out the related research on the invasion cells of the heterologous virus. Thus, the helper plasmid or virus construction system of the present application is more suitable for use in vaccine and pharmaceutical field preparation.
The technical solutions of the present application will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Examples
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Experiment field: biosafety class 2 laboratories should be qualified to handle non-replicating VSV viruses. More preferably, the laboratory is qualified to handle non-replicating lentiviral vectors. In particular, laboratories need not be qualified to handle vaccinia virus.
Instrument apparatus: class AII or BII biosafety cabinet, carbon dioxide cell incubator, water bath, centrifuge, microplate reader for detecting chemiluminescence, cell counter, microscope, refrigerator, ultra-low Wen Bingxiang, liquid nitrogen tank, autoclave, pipette, pipettor, etc.; ultra-clean bench, bacteria constant temperature incubator, bacteria constant temperature culture shaking table, PCR instrument, electrophoresis apparatus, electrophoresis tank, microwave oven, gel imager, alcohol lamp, etc.
Consumable: sterile cell culture container, sterile pipette tip, sterile whole white 96-well cell culture plate, sterile syringe, sterile 0.45 μm needle filter, sterile centrifuge tube, high temperature resistant garbage bag, high temperature resistant glass bottle, sterilization indicator tape, etc.; bacterial culture plates, erlenmeyer flasks, test tubes, spreaders, and the like.
Reagent: HEK 293 and other cells, high-sugar DMEM culture medium, fetal bovine serum, penicillin-streptomycin, transfection reagent, firefly luciferase detection reagent, disinfectant and the like; a seamless cloning kit, a bacterial culture medium, a bacterial screening antibiotic, a high-fidelity PCR premix, agarose gel, an electrophoresis buffer, competent cells, a DNA recovery kit, a plasmid extraction kit and the like.
Example 1 exploration and establishment of the inventive method platform
In order to solve the problems mentioned in the background art, the application develops a preparation platform of VSV pseudovirus (VSV delta G virus) containing a reporter gene (such as firefly luciferase reporter gene) with G protein gene defect, wherein the whole flow of the platform is shown in figure 1, and the specific steps are as follows:
firstly, 1 expression vector of CMV or CBV promoter connected with T7RNA polymerase gene CDS is constructed; 1 expression vector of T7 promoter connected with IRES sequence and then connected with VSV virus N, P, L or G protein gene CDS is constructed, 4 expression vectors are respectively; 1 vector of T7 promoter connected with VSV virus genome is constructed, wherein the G protein gene expression frame of VSV genome is replaced by firefly luciferase gene CDS sequence.
Secondly, constructing a cell strain which overexpresses T7RNA polymerase, connecting a CMV or CBV promoter with a CDS sequence of a T7RNA polymerase gene, and introducing the cell strain into HEK 293 or other cell strains through plasmid transient transfection or lentiviral vector.
Subsequently, VSV virus N, P, L or G protein gene expression vector and genome vector are introduced into the above cells by means of plasmid transient transfection; after 24-48 hours, the cell culture supernatant was harvested to contain VSV.DELTA.G seed virus.
In view of the limited virus content of the VSV delta G seed, the virus suspension can be used for infecting HEK 293 and other cells, and simultaneously transiently transfecting VSV G protein expression plasmids controlled by a recognizable promoter of eukaryotes. After 24-48 hours, more VSV.DELTA.G virus was obtained by collecting the cell culture supernatant. This step may be repeated one to several times until the VSV Δg virus titer reaches the expected.
Finally, cells such as HEK 293 and the like are infected with the obtained VSV delta G virus, and after about 24 hours, signal values are measured by using a firefly luciferase detection kit microplate reader to evaluate the virus effect or calculate the virus titer.
Example 2 preparation of heterologous pseudoviruses
In order to obtain a heterologous pseudovirus, which is used for safely evaluating the neutralization activity of a sample in the development process of vaccines, medicines and the like, the application provides a method for constructing the heterologous pseudovirus based on the VSV delta G virus obtained in the example 1.
First, an envelope protein vector of a heterologous virus is constructed, and if necessary, the expression sequence can be appropriately edited. The vector plasmid was introduced into HEK 293 cells by transient transfection of the plasmid.
Next, the cells were infected with VSV.DELTA.G virus and after 6 hours fresh medium was changed, during which step the cells were rinsed appropriately to remove residual plasmid and VSV.DELTA.G virus. After 24h transfection, the cell culture supernatant is filtered and harvested to obtain heterologous pseudovirus particles containing infectivity, which can be stored at-80℃after sub-packaging (avoiding freeze thawing), or subjected to necessary purification.
Example 3 evaluation of Effect
1) Construction and Effect evaluation of T7RNA polymerase working System
In this example, T7RNA polymerase expression sequences were introduced into HEK 293, heLa and Vero cells by a lentivirus or plasmid transient transfection method, respectively, and the corresponding promoters were selected from CMV or CBV, respectively, while examining the effect of insertion of IRES sequences, and the results are shown in FIG. 2.
It is found that, when the vector with the T7 promoter connected with the green fluorescent protein coding gene is transferred into HEK 293 cells expressing T7RNA polymerase, almost no detectable green fluorescent signal exists, which indicates that the T7 expression system has poor working effect in eukaryotic cells. And the vector of the IRES sequence connected with the T7 promoter and the green fluorescent protein coding gene is transferred into HEK 293 cells expressing T7RNA polymerase, obvious green fluorescent signals can be observed, and the IRES sequence is suggested to enable the T7 expression system to work normally in eukaryotic cells. Meanwhile, the expression of T7RNA polymerase by using different promoters (CMV or CBV), the introduction of T7RNA polymerase into cells (lentiviral infection, plasmid transient transfection) by using different methods, and the like do not affect the conclusion, and the expression control element and the gene element introduction mode of the T7RNA polymerase are not key factors for the normal operation of the system. In addition, in different cell models, after the carrier of the T7 promoter connected IRES sequence and the green fluorescent protein coding gene is transferred, a green fluorescent signal is generated, which indicates that the cell type is not a key factor of the normal operation of the system.
The above results indicate that, in order to express a target gene controlled by a T7 promoter in eukaryotic cells containing T7RNA polymerase without using vaccinia virus, it is necessary to insert an IRES sequence before the target gene, and that a specific mode and cell type for expression of T7RNA polymerase have a large choice.
2) Evaluation of cell State after infection with acquired VSV ΔG Virus
After the first acquisition of the VSV delta G virus, the cell status of the control and experimental groups was evaluated 24 hours after the virus addition during the amplification by HEK 293 cells. Wherein, the control group is: when the virus is first obtained, introducing the relevant plasmid into cells over-expressing T7RNA polymerase, but replacing the VSV genome plasmid with a firefly luciferase expression plasmid; the experimental group is: when the virus is obtained for the first time, all relevant plasmids are introduced into cells over-expressing T7RNA polymerase, namely, the T7 promoter is connected with a VSV virus N, P, L or an expression vector of a G protein coding gene, and the T7 promoter is externally connected with a vector of a VSV virus genome (G protein coding gene defect).
As shown in FIG. 3, the control group has good cell status, the experimental group has obvious abnormal cell morphology (rounding), virus particles enter the cells, and after entering the cells, the virus particles utilize the resources in the host cells to complete the process of partial life cycle of the virus particles, which can cause damage to the host cells. The apparent abnormal status of the experimental group relative to the control group indicates successful production of VSV Δg.
3) Evaluation of post-infection acquired VSV ΔG virus fluorescent signals
The VSV delta G virus was first obtained and amplified in one round, and then infected with different cells, and the signal value after 24 hours was evaluated. The results are shown in FIG. 4, where 3. Mu.L of the control or experimental suspension was added per well, there was no significant increase in signal values for the control versus the cell control (no virus suspension added) and an order of magnitude increase in signal values for the experimental (containing VSV. DELTA.G virus).
From a combination of the results of FIGS. 3 and 4, it can be seen that VSV.DELTA.G seed viruses can still be successfully produced by the method of the present application without using vaccinia virus.
4) Heterogeneous pseudovirus advantage based on VSV framework construction
The culture supernatants were harvested at 24h (VSV) and 72h (lentivirus), respectively, and 3. Mu.L of each of the infected Vero cells was used to detect fluorescent signals at 24h (VSV, lentivirus), 48h (lentivirus), 72h (lentivirus), using equivalent amounts of heterologous viral envelope protein expression plasmid and HEK 293 cell packaging lentivirus backbone and novel coronavirus XBB.1 mutant spike protein pseudovirus of VSV backbone.
As a result, as shown in FIG. 5, the pseudo-virus of VSV backbone can generate a higher signal value in a very short time (24 h), while the pseudo-virus of lentiviral backbone requires a longer time (48 h) to generate a signal value, and the signal value is lower, with an order of magnitude difference in Vero cells relative to the pseudo-virus of VSV backbone.
The results show that the heterologous virus (exemplified by novel coronavirus) prepared based on the VSV virus skeleton has the advantages of strong signal, quick response and the like compared with the traditional lentivirus skeleton, and provides more convenience for the research of related diseases.
5) VSV delta G replication defective seed viruses can be used to construct a variety of heterologous pseudoviruses
HEK 293 cells are transfected by using heterologous virus envelope protein expression plasmids of different species, VSV delta G viruses are infected at the same time, the culture solution is discarded after 6 hours, the HEK 293 cells are rinsed by using the HEK 293 cells complete culture solution, the new HEK 293 cells complete culture solution is replaced, cell supernatants are collected after 24 hours, and cell fragments are removed by centrifugation and filtration, so that the VSV framework heterologous pseudoviruses of different species are obtained. Then, using the obtained pseudoviruses, cells susceptible to pseudoviruses of the corresponding species were infected, and fluorescent signals were detected after 24 hours.
As shown in FIG. 6, various heterologous pseudoviruses of VSV frameworks can efficiently infect target cells, and various heterologous pseudoviruses including Nipah virus, ebola virus and the like can enable respective target cells to generate high-intensity fluorescent signals, so that VSV delta G replication defective seed viruses can be used for constructing various heterologous pseudoviruses and further applied to related researches of different diseases.
In summary, compared with the existing VSV delta G virus obtained based on vaccinia virus characteristics, the VSV delta G virus obtained by the application has equivalent functions, and can be used for infecting cells or constructing heterologous pseudoviruses. Compared with the heterogeneous pseudovirus constructed based on the slow virus skeleton, the heterogeneous pseudovirus of the VSV delta G virus constructed based on the application has the characteristics of wide range, strong signal, high safety, short experimental period and the like.
The foregoing descriptions of specific exemplary embodiments of the present application are presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the application and its practical application to thereby enable one skilled in the art to make and utilize the application in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the application be defined by the claims and their equivalents.
Claims (10)
1. A method for preparing a VSV Δg replication defective seed virus, the method comprising the steps of:
1) Packaging cell construction: constructing a cell line expressing phage T7RNA polymerase;
2) Helper plasmid construction: respectively constructing expression vectors of the T7 promoter connected with VSV virus N, P, L or G protein coding genes;
3) Construction of genome plasmid: constructing a carrier of which a T7 promoter is connected with a VSV virus genome, wherein a G protein coding gene in the VSV virus genome is replaced;
4) Seed virus preparation: helper plasmids and genomic plasmids were introduced into packaging cells, and VSV.DELTA.G replication defective seed viruses were harvested.
Preferably, the method further comprises the steps of:
5) Seed virus amplification: and 4) infecting the original cell strain again by the VSV delta G replication defective seed virus prepared in the step 4), and simultaneously adding a VSV virus G protein expression plasmid.
2. The process according to claim 1, wherein in step 1),
the cell strain is a mammalian cell strain including, but not limited to, HEK 293 cell strain, heLa cell strain or Vero cell strain;
preferably, the construction method includes, but is not limited to: introducing a T7RNA polymerase gene plasmid into a cell strain by transient transfection or viral vector;
more preferably, the T7RNA polymerase gene is linked to a promoter, including but not limited to CMV, CBV promoters;
further preferably, the specific steps of the construction are as follows: the CMV or CBV promoter is connected with the CDS sequence of the T7RNA polymerase gene and is introduced into HEK 293 cell strain, heLa cell strain or Vero cell strain through plasmid transient transfection or lentiviral vector.
3. The process according to any one of claims 1 to 2, wherein in step 2),
an Internal Ribosome Entry Site (IRES) sequence is connected between the T7 promoter and the protein coding gene;
preferably, the IRES is operably linked after the T7 promoter sequence.
4. A process according to any one of claims 1 to 3, wherein in step 3),
the substitution is a substitution of a G protein encoding gene in the VSV viral genome with a reporter gene, including but not limited to a firefly luciferase reporter gene.
5. The method of any one of claims 1-4, wherein in step 5), the primary cell strain includes, but is not limited to, HEK 293 cell strain, heLa cell strain, or Vero cell strain; the VSV virus G protein expression plasmid is an expression plasmid with eukaryotic promoter connected with VSV virus G protein coding gene.
6. A method of producing a VSV pseudovirus comprising the method of any one of claims 1-5, and further comprising the steps of:
6) Introducing a heterologous viral envelope protein expression plasmid into a cell line, and harvesting pseudoviruses after infecting the cells with the vsvΔg virus prepared according to any one of claims 1-5;
the cell line is a mammalian cell line including, but not limited to, HEK 293 cell line, heLa cell line or Vero cell line.
7. A helper plasmid for construction of VSV Δg virus, characterized in that the helper plasmid comprises a T7 promoter sequence, a VSV virus N, P, L or G protein encoding gene sequence, and an Internal Ribosome Entry Site (IRES) sequence; preferably, the IRES is operably linked after the T7 promoter sequence.
8. A VSV Δg virus construction system, comprising the helper plasmid of claim 7.
9. The virus construction system according to claim 8, wherein the system further comprises: a cell line expressing bacteriophage T7RNA polymerase, a VSV virus genome plasmid and/or a VSV virus G protein expression plasmid; preferably, the VSV virus genome plasmid is a vector of which the T7 promoter is connected with the VSV virus genome; more preferably, the VSV genomic G protein encoding gene is replaced; further preferably, the VSV virus G protein expression plasmid is an expression plasmid in which a eukaryotic promoter is connected with a VSV virus G protein coding gene.
10. Use of a helper plasmid or virus construction system according to any of claims 7-9 for any of the following:
1) Application in preparation of VSV delta G replication defective seed virus;
2) Application in the preparation of VSV skeleton heterologous pseudoviruses;
3) The application in the field of vaccine or medicine preparation.
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| CN1934260A (en) * | 2004-01-22 | 2007-03-21 | 株式会社载体研究所 | Method of producing minus strand rna virus vector with the use of hybrid promoter containing cytomegalovirus enhancer and avian beta-actin promoter |
| US20090162321A1 (en) * | 2007-12-20 | 2009-06-25 | Wyeth | Methods for packaging propagation-defective vesicular stomatitis virus vectors using a stable cell line that expresses g protein |
| CN115725657A (en) * | 2022-09-16 | 2023-03-03 | 中国科学院广州生物医药与健康研究院 | Segmented vesicular stomatitis virus vector and preparation method and application thereof |
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| CN1934260A (en) * | 2004-01-22 | 2007-03-21 | 株式会社载体研究所 | Method of producing minus strand rna virus vector with the use of hybrid promoter containing cytomegalovirus enhancer and avian beta-actin promoter |
| US20090162321A1 (en) * | 2007-12-20 | 2009-06-25 | Wyeth | Methods for packaging propagation-defective vesicular stomatitis virus vectors using a stable cell line that expresses g protein |
| CN115725657A (en) * | 2022-09-16 | 2023-03-03 | 中国科学院广州生物医药与健康研究院 | Segmented vesicular stomatitis virus vector and preparation method and application thereof |
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