Novel dengue pseudovirus based on recombinant VSV vector and preparation method thereof
By developing a new dengue pseudovirus based on recombinant VSV vector, integrating and expressing the NS1 gene of four serotype dengue viruses, the problems of dengue virus detection and prevention and control in the existing technology were solved, and efficient proteins and Nucleic acid standards are provided to support the detection and research of dengue virus-related diseases.
Patent Information
- Application Number
- CN202310992879.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-08
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively detect and prevent dengue viruses, especially in early diagnosis and research, where reliable protein and nucleic acid standards are lacking.
Develop a new dengue pseudovirus based on recombinant VSV vector. By integrating the NS1 genes of four serotype dengue viruses, a pseudovirus that can efficiently express NS1 protein in host cells is constructed to provide protein and nucleic acid standards. .
It has achieved efficient expression of four dengue virus NS1 proteins, provided positive standards for dengue virus detection and identification, and supported the clinical detection and pathogenic mechanism research of dengue virus-related diseases.
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Figure CN119464225A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the fields of gene engineering technology and molecular biology, and in particular to a novel dengue pseudovirus based on a recombinant VSV vector and a preparation method thereof. Background Art
[0002] Dengue virus (DENV) is a single-stranded positive-strand RNA virus belonging to the genus Flavivirus of the family Flaviviridae. It includes four serotypes, I, II, III and IV, of which type II is the most widely spread. Dengue virus can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome, with a mortality rate of up to 10%, which seriously endangers human health. Among them, dengue fever has the characteristics of rapid spread, high incidence, general susceptibility of the population, and high mortality rate of severe cases. It is considered to be the most concerned infectious disease in the world. Therefore, it is particularly important to do a good job in the detection and prevention and control of dengue virus.
[0003] The DENV genome is about 11kb in length, encoding 3 structural proteins (nucleoprotein C, membrane-bound protein M and envelope protein E) and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, Ns4a, Ns4b and NS5), of which NS1 protein is a highly conservative non-structural glycoprotein of dengue virus, and exists in two forms, membrane-bound and secretory, both of which have strong immunogenicity. NS1 protein is present in the serum of patients in the early stage of dengue fever, and the appearance time is earlier than IgM antibody, which has been proved to be useful for early diagnosis and prognosis evaluation of dengue fever. In addition, NS1 protein also plays an important role in the pathogenicity and immune process of dengue fever.
[0004] In the process of studying the pathogenic mechanism, detection and prevention and control of DENV, given the high infectivity and pathogenicity of DENV, it is urgent to develop a pseudovirus that can express one or more DENV characteristic proteins (such as NS1 protein, etc.) and has no replication ability. It can express NS1 in host cells, which can provide antigen protein standards for clinical rapid diagnosis of DENV-related diseases. Its genomic RNA can also be used as a nucleic acid standard for clinical PCR detection and diagnosis of DENV-related diseases because it integrates the gene sequence of DENV characteristic proteins. It can provide research tools for clinical detection and identification of DENV, the development of DENV rapid diagnosis kits and in-depth research on DENV pathogenicity and immune mechanisms.
[0005] Vesicular stomatitis virus (VSV) belongs to the genus Vesiculovirus of the family Rhabdoviridae, and its virion structure is in the shape of a bullet or rod. Wild-type VSV is an enveloped virus with a single-stranded negative-sense RNA genome that mainly encodes five structural proteins: nucleoprotein (N), phosphorylated protein (P), RNA polymerase (L), matrix protein (M), and glycoprotein (G). VSV has a wide host range and can infect mammals, fish, plants, and insects, and has a wide range of tissue tropism. In addition, it has the advantages of efficient replication, low pathogenicity in humans, a small genome, and easy manipulation. It is a multi-purpose viral vector with development potential. The recombinant VSV viral vector (rVSV-ΔG) developed based on wild-type VSV has the characteristics of no independent replication ability (replication defect), rapid replication in rescued host cells, and high expression of exogenous genes. It is widely used in the preparation of pseudoviruses of some highly infectious and highly pathogenic viruses.
[0006] The present invention utilizes rVSV-ΔG virus vector, selects full genome sequences of typical strains (HQ166036.1, HM181971.1, HM631854.1 and AY858050.1) of four serotypes of dengue virus, namely, type I, II, III and IV, from GenBank, respectively, intercepts NS1 genes of the four viruses, and constructs a recombinant VSV pseudovirus that can be rapidly prepared and efficiently express four dengue virus NS1 proteins in high quantities, which is used for dengue virus detection, identification and related mechanism research. Summary of the invention
[0007] The invention provides a novel pseudovirus capable of detecting and identifying all four serotypes of dengue virus and a preparation method thereof. The pseudovirus has no independent replication activity, high safety, low cost and is convenient for large-scale industrial production.
[0008] A novel dengue pseudovirus based on a recombinant VSV vector and a preparation method thereof. The pseudovirus uses a modified vesicular stomatitis virus as a recombinant virus vector of the dengue virus, the NS1 protein genes of dengue viruses belonging to four serotypes are integrated into its genome, and the respective NS1 proteins can be expressed in host cells infected by the pseudovirus. The viral genome RNA integrated with the NS1 gene and the NS1 protein obtained after expression in the cells can be used as a positive standard in clinical detection and identification of the dengue virus.
[0009] Preferably, the novel dengue pseudovirus based on a recombinant VSV vector and the preparation method, the recombinant virus vector is a recombinant VSV virus vector pVSV-△G-MCS.
[0010] Preferably, the target gene carried and expressed by the novel dengue pseudovirus based on the recombinant VSV vector and the preparation method is the NS1 gene sequence of dengue virus belonging to four serotypes.
[0011] Preferably, the novel dengue pseudovirus and preparation method based on the recombinant VSV vector carries and expresses target genes, namely, the NS1 gene of dengue virus belonging to four serotypes, to the ends of which are added purification and detection protein tag genes FLAG, 6xHis, Strep-tagII and Myc, respectively.
[0012] Preferably, the order of the target genes carried and expressed by the novel dengue pseudovirus and preparation method based on the recombinant VSV vector is any one of the NS1 protein genes of typical dengue viruses belonging to four serotypes after adding protein tags and 2-4 gene fragments combined in any order.
[0013] A novel dengue pseudovirus based on a recombinant VSV vector and a preparation method thereof, comprising the following steps: 1. Selection of NS1 gene fragments of four serotypes of dengue virus.
[0014] 2. Acquisition, amplification and restriction digestion of DNA fragments after adding protein tags to the NS1 genes of four serotypes of dengue viruses.
[0015] 3. Assembly of the above four NS1 gene DNA fragments in the recombinant VSV viral vector (rVSV-ΔG).
[0016] 4. Transformation, amplification and extraction of rVSV-ΔG viral vector plasmid carrying the above four NS1 gene DNA fragments.
[0017] 5. Rescue and packaging of replication-defective rVSV-ΔG pseudovirus carrying the above four NS1 gene DNA fragments.
[0018] 6. Amplification and harvesting of replication-defective rVSV-ΔG pseudoviruses carrying the above four NS1 gene DNA fragments.
[0019] 7. Identification of replication-defective rVSV-ΔG pseudoviruses carrying the above four NS1 gene DNA fragments.
[0020] Preferably, in the novel dengue pseudovirus based on a recombinant VSV vector and preparation method, the dengue virus target gene is selected from any one of the NS1 protein genes of dengue viruses belonging to four serotypes and 2-4 gene fragments combined in any order.
[0021] Preferably, in the novel dengue pseudovirus based on a recombinant VSV vector and preparation method, protein tags (FLAG, 6xHis, Strep-tagII and Myc) are added to the ends of the NS1 protein gene sequences of the dengue viruses belonging to the four serotypes to facilitate identification and purification.
[0022] Preferably, in the novel dengue pseudovirus based on a recombinant VSV vector and the preparation method, during the acquisition, amplification and enzyme digestion of the four NS1 gene DNA fragments, a high-fidelity enzyme is used to perform PCR amplification on the target DNA fragment, a PCR product recovery kit is used to recover the PCR product, the VSV virus vector cloning site is enzyme-digested, and the fragments and vectors are recovered using agarose gel electrophoresis and a DNA recovery kit.
[0023] Preferably, in the novel dengue pseudovirus based on a recombinant VSV vector and preparation method, the method for connecting the four NS1 gene DNA fragments with the viral vector is to cis-splice the target DNA fragments together in the form of 5'-- 3'>5'--3'>5'-- 3'>5'-- 3', and connect them to the multiple cloning site region (MCS) between genes N and L in the VSV viral vector by homologous recombination using the homologous arm sequences reserved at both ends of the spliced fragments.
[0024] Preferably, the novel dengue pseudovirus based on the recombinant VSV vector and the preparation method thereof comprises the steps of transforming, amplifying and extracting the viral vector plasmids of the four NS1 gene DNA fragments, transforming the ligation product of the DNA fragments after the viral vector and the gene combination into DH5α Escherichia coli, and coating the Kana + The cells were cultured on LB plates with resistance at 35-37°C overnight, and a single colony was picked and cloned on the Kana + The cells were cultured overnight in 5 ml LB liquid medium at 200-250 rpm and 35-37°C, and then plasmid DNA was extracted using an endotoxin-free alkaline lysis plasmid extraction kit.
[0025] Preferably, in the novel dengue pseudovirus based on a recombinant VSV vector and the preparation method thereof, in the rescue and packaging step of the replication-deficient VSV virus carrying four NS1 genes, 293T cells need to be first infected with a tool poxvirus that can express T7 polymerase in 293T cells, and a serum-free medium is used to mix the VSV recombinant packaging plasmid and the auxiliary plasmids pVSV-P, pVSV-N, pVSV-L and pVSV-G of the VSV virus structural proteins. In addition, a serum-free medium is also used to mix an appropriate amount of Lipo2000, which is then mixed with the previously prepared plasmid mixture and incubated at room temperature to transfect 293T cells and achieve transcriptional expression of VSV structural proteins and packaging and extracellular secretion of recombinant VSV pseudoviruses therein, and finally, the novel dengue pseudovirus primary virus is harvested after filtering the cell culture to remove the tool poxvirus.
[0026] Preferably, the novel dengue pseudovirus based on a recombinant VSV vector and the preparation method, wherein the replication-deficient VSV primary virus carrying the NS1 gene fragments of four serotypes of dengue viruses prepared by the method is a pseudovirus with single cell infectivity and non-replicability.
[0027] Preferably, in the amplification and harvesting steps of the replication-defective VSV virus carrying four NS1 genes, the pVSV-G plasmid is first transfected into 293T cells using Lipo2000, and 4 hours later, the primary virus harvested previously is used to infect the 293T cells transfected with pVSV-G, and the recombinant VSV pseudovirus in the culture supernatant is harvested 24 hours and 48 hours after infection with the primary virus, respectively.
[0028] The advantages are as follows: (1) The present invention provides a novel dengue pseudovirus based on a recombinant VSV vector and a preparation method thereof. The dengue pseudovirus prepared by the method comprises a non-replicative (ΔG) VSV backbone vector. The pseudovirus has the characteristics of no replication activity after a single infection of a host cell, high level of exogenous gene expression, high biosafety, high titer of produced virus and strong stability, and is conducive to industrial large-scale production.
[0029] (2) The present invention provides a novel dengue pseudovirus based on a recombinant VSV vector and a preparation method thereof. The dengue pseudovirus prepared by the method comprises the non-structural protein NS1 gene of four serotypes of dengue virus. The pseudovirus can provide positive standards of proteins and nucleic acids required for clinical detection and identification of dengue virus, respectively, and provide a research tool for detection and identification, control of transmission and mechanism research of dengue virus-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1Modification and combination sequence of four dengue virus NS1 antigen proteins in the recombinant VSV vector (example) Figure 2 Schematic diagram of the structure of the recombinant VSV vector rVSV-△G-FLAG-HQ166036.1-NS1 Figure 3 Schematic diagram of the structure of the recombinant VSV vector rVSV-△G-HM181971.1-NS1-6xHis Figure 4 Schematic diagram of the structure of the recombinant VSV vector rVSV-△G-Hm631854.1-NS1-Strep-TagII Figure 5 Schematic diagram of the structure of the recombinant VSV vector rVSV-△G-AY858050-NS1-Myc Figure 6 Schematic diagram of the structure of the recombinant VSV vector rVSV-△G-4DENV-NS1-tags Figure 7 Expression of NS1 of four dengue virus serotypes in 293T cells infected with novel dengue pseudovirus and in culture supernatant Figure 8 Amplification curve of the new dengue pseudovirus treated with different concentrations of RNase A as QPCR template Fig. 9 Amplification curves of QPCR templates with different concentrations of novel dengue pseudovirus Implementation
[0031] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the embodiment of the present invention. The embodiment described in this section is a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative premises belong to the scope of protection of the present invention.
[0032] Example 1 Preparation of novel dengue pseudovirus (1) Selection of dengue pseudovirus target genes The present invention obtains the full genome sequences of typical strains (HQ166036.1, HM181971.1, HM631854.1 and AY858050.1) of four serotypes (types I, II, III and IV) of dengue virus from GenBank, and then respectively extracts the coding region sequences (CDS) of the NS1 gene in the above four virus genome sequences for the following operations.
[0033] (2) Obtaining, amplifying and digesting the target gene fragment.
[0034] The modified (protein tag sequence added to the end of the gene) fragments of the above four NS1 genes were commercially synthesized, and then primers were designed to assemble the modified fragments of the four NS1 genes into a DNA fragment using the overlap extension PCR method ( Figure 1 ), with homology arm sequences reserved at both ends for recombination.
[0035] Amplification system: 5ul 10xQ5 buffer, 5ul dNTP, 2.5ul each of forward and reverse primers, 2ul of various synthetic templates, 5ul high-fidelity enzyme, 28ul clean deionized water, forming a 50ul reaction system; Optimized amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 5 min; 30 cycles; and final extension at 72°C for 10 min.
[0036] Use restriction enzymes Mlu I and Nhe I double-digested the recombinant VSV viral vector pVSV-△G-MCS, subjected the digested products to agarose gel electrophoresis, and used a DNA gel recovery kit to recover the linearized digested fragments.
[0037] Optimized enzyme digestion system: 3ul recombinant vector plasmid, 2ul 10x buffer, restriction endonuclease Mlu I and Nhe I 1.5ul each, 12ul clean deionized water, gently mix and react in a 37℃ water bath for 2h.
[0038] The digested products and PCR products were subjected to agarose gel electrophoresis (concentration of 0.8%). After running at 150 V for 30 min, the target band was cut under ultraviolet light, and the target fragment was recovered using a DNA recovery kit (Axygen), and the respective concentrations were determined.
[0039] (3) Connection between the target gene fragment and the viral vector.
[0040] The PCR product recovered in the previous step and the plasmid linearized fragment were recombined and ligated to construct the pVSV-△G-DENV-NS1 plasmid (structure see Figure 2-6 ).
[0041] The optimized recombination ligation system: 4ul 5xIn-fusion HD Enzyme Premix, 2ul linearized vector, 4ul purified PCR fragment and 10ul clean deionized water constitute a 20ul reaction system. After incubating in a 50°C water bath for 10min, the ligation product was immediately transformed using Tiangen's DH5α competent bacteria.
[0042] (4) Transformation, amplification and extraction of viral vector plasmids containing target gene fragments.
[0043] According to the traditional transformation procedure, DH5α competent bacteria (Tiangen Company) were used to transform the recombinant product in the previous step, and the transformed bacterial mixture was spread on the Kana + The cells were cultured on LB plates with resistance and incubated overnight at 35-37°C. Since the recombinant plasmid carries the kanamycin resistance gene, the colonies grown on the plates should be the target clones after successful recombination.
[0044] Select the relatively dispersed clones with clear edges in the center of the plate and place them in 5 ml LB liquid culture medium with an appropriate amount of kanamycin. Culture them overnight at 250 rpm and 35-37°C. The next day, use the QIAGEN endotoxin-free plasmid extraction kit to extract the plasmid. Finally, sequence the recombination site and the inserted gene to confirm.
[0045] (5) Rescue and packaging of replication-defective VSV virus carrying the target gene.
[0046] One day before virus packaging, 293T cells in the logarithmic growth phase were digested with 0.25% trypsin, prepared into a single cell suspension and inoculated in a 6-well plate. The number of cells inoculated in each well was 1x10 6 2 ml of DMEM complete medium (10% fetal bovine serum) was added. The next day, the cell confluence in the 6-well plate was about 70-90%. 293T cells were first infected with a poxvirus expressing T7 polymerase, and the subsequent plasmid transfection was performed 1 hour after virus infection.
[0047] Preparation of 293T cell transfection mixture: prepare two sterile 1.5ml centrifuge tubes, add 50ul Opti-MEM medium to each, add pVSV-△G-DENV-NS1, pVSV-P, pVSV-N, pVSV-L and pVSV-G to one centrifuge tube, mix evenly with the medium, add an appropriate amount of transfection reagent Lipo2000 to another centrifuge tube, and mix evenly with the medium. After 5 minutes, combine the solutions in the two tubes, mix gently and place at room temperature for 15 minutes. Replace the medium in the well to be transfected with Opti-MEM medium, then gently drop into the mixture, gently shake cross-mix and place in a 37℃, 5% CO2 incubator for culture, and replace with fresh complete medium 4h after transfection. Harvest the cell supernatant 48h after transfection, centrifuge at 3000rpm for 5 minutes, and filter the supernatant with a 0.45um syringe filter. The filtrate is the pseudovirus primary virus, which is stored at -80℃.
[0048] (6) Amplification and harvesting of replication-defective VSV virus carrying the target gene.
[0049] One day before virus amplification, 293T cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension. 293T cells were inoculated in 10 cm dishes, with an inoculation number of about 5x10 6 The volume of DMEM complete medium was 10 ml, and the cell confluence was about 80-90% on the second day (12-18 h after cell inoculation).
[0050] Preparation of 293T cell transfection mixture: prepare two clean and sterile 1.5ml centrifuge tubes, add 100ulOpti-MEM culture medium to each, add pVSV-G plasmid to one centrifuge tube, mix evenly with the culture medium, add an appropriate amount of transfection reagent Lipo2000 to another centrifuge tube, and mix evenly with the culture medium. After 5 minutes, combine the solutions in the two tubes, mix gently, and place at room temperature for 15 minutes. Replace the culture medium in the 10cm dish with Opti-MEM culture medium, then gently drop the transfection mixture into the dish, gently shake it crosswise to mix, and place it in a 37℃, 5% CO2 incubator for culture. 4h after transfection, replace the Opti-MEM culture medium with 10ml complete DMEM complete culture medium, and then add the primary virus harvested in the previous step for infection. The culture supernatant was harvested 24 h after transfection, supplemented with 10 ml of complete medium and cultured for another 24 h, and the culture supernatant was harvested again. After centrifugation at 3000 rpm for 5 min, the supernatant was filtered using a 0.45 um syringe filter. The filtrate was the virus amplification product, containing the corresponding pseudovirus particles, which was stored at -80 °C.
[0051] Example 2 Expression of NS1 antigen protein carried by novel dengue pseudovirus in 293T cells One day before the experiment, 293T cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, which was then inoculated in a 6-well plate at a seeding number of 3x10 per well. 6 On the second day (12-18 hours later), the cell confluence was about 80%, and an appropriate amount of the novel dengue pseudovirus was added for infection. After 72 hours of infection, the culture supernatant and cells were harvested, and the expression of NS1 antigen protein in the harvested samples was detected by WB ( Figure 3 ).
[0052] Example 3 Verification of RNase Resistance of Novel Dengue Pseudovirus as Nucleic Acid Template Standard for Clinical Testing The QPCR probes and primers were designed for the NS1 gene sequences of the four selected dengue viruses. The novel dengue pseudoviruses of the present invention were treated with RNases of different concentrations (0ug / ml, 1ug / ml and 10ug / ml) and then the viral genomic RNA was extracted as a template for QPCR detection. The results are shown in Figure 2. Figure 4 As shown, there is no significant difference in the test results, which proves that the new dengue pseudovirus has good anti-RNase effect and the product is stable.
[0053] Example 4 Linear analysis of novel dengue pseudovirus as a nucleic acid standard for clinical testing The novel dengue pseudovirus of the present invention is treated according to 10 6 copies / ml, 10 5 copies / ml, 10 4 copies / ml and 10 3 After a series of gradient dilutions at a concentration of 100 copies / ml, the samples were used as templates for QPCR reaction. The probes and primers designed in Example 3 for the NS1 gene sequences of four dengue viruses were used for QPCR detection. The results were as follows: Figure 5 As shown, the gradient dilution sample of the novel dengue pseudovirus has good linearity as a template in QPCR detection and is suitable as a nucleic acid positive standard for clinical detection.
[0054] In summary, the novel dengue pseudovirus involved in the present invention can express NS1 proteins of four serotypes of dengue viruses after infecting host cells, and the ends of the four NS1 proteins are respectively embedded with FLAG, 6xHis, Strep TagII and Myc protein tags, which are convenient for detection, recovery and purification; the NS1 gene sequences of four serotypes of dengue viruses are integrated into the genome of the pseudovirus, and the pseudovirus is resistant to RNase, and has good linearity when used as a PCR template for QPCR detection, can be used as a positive standard for dengue virus detection to truly simulate the clinical detection process of dengue virus from RNA extraction to nucleic acid amplification detection, and has broad application prospects and important research value in clinical detection and identification of DENV, development of DENV rapid diagnosis kits, and in-depth research on the pathogenicity and immune mechanisms of DENV.
Claims
1. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same, characterized in that: The pseudovirus uses replication-deficient vesicular stomatitis virus (VSV) as a recombinant virus vector of the dengue virus-specific antigen protein NS1, carries 1 to 4 characteristic antigen protein NS1 genes of different serotypes of dengue viruses, and can be used as a protein and nucleic acid positive standard for dengue virus detection and identification.
2. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 1, characterized in that: The recombinant viral vector is a non-replicable VSV viral vector rVSV-△G-MCS.
3. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 1, characterized in that: The characteristic antigen protein gene of dengue virus carried in the pseudovirus genome is its non-structural protein NS1: Seq-1 or / and Seq-3 or / and Seq-5 or / and Seq-7 gene sequence.
4. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 3, characterized in that: The characteristic antigen protein gene of the dengue virus carried in the recombinant VSV virus genome is a target gene formed by a combination of any one of the NS1 genes (Seq-1 or / and Seq-3 or / and Seq-5 or / and Seq-7 gene sequences) of dengue viruses belonging to 1-4 serotypes and NS1 genes of 2-4 different serotypes of dengue viruses.
5. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in any one of claims 1 to 4, characterized in that: The following steps are included: (1) Selection of NS1 genes of four dengue virus serotypes; (2) Obtaining, amplifying and digesting the NS1 gene DNA fragments of four serotypes of dengue virus; (3) connecting the above four NS1 gene DNA fragments to the viral vector; (4) Transformation, amplification and extraction of viral vector plasmids containing four NS1 gene DNA fragments; (5) Rescue and packaging of replication-defective VSV viruses carrying four NS1 gene DNA fragments; (6) Amplification and harvesting of replication-defective VSV viruses carrying four NS1 gene DNA fragments; (7) Identification of replication-defective VSV viruses carrying four NS1 gene DNA fragments.
6. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 5, characterized in that: The selected characteristic antigenic protein NS1 of dengue virus is a non-structural protein of the dengue virus to which it belongs. It exists in two forms, membrane-bound and secreted, with a molecular weight of approximately 41KD. All DNA fragments are artificially synthesized. The synthesized DNA fragments are assembled in pUC plasmids for preservation. At the same time, high-fidelity PCR amplification and enzyme digestion are performed using a plasmid carrying the NS1 gene DNA fragment, and the NS1 gene DNA target fragment and the linearized viral vector are recovered using an agarose DNA recovery kit.
7. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 5, characterized in that: The method for connecting the NS1 gene DNA fragment with the linearized viral vector is as follows: the viral vector is linearized by enzyme digestion and recovered by agarose DNA electrophoresis, and then the target gene is positively inserted into the viral vector using recombination technology.
8. A novel dengue pseudovirus based on a recombinant VSV vector and a method for preparing the same as claimed in claim 5, characterized in that: The replication-defective pseudovirus was obtained by virus rescue and packaging using the recombinant VSV virus vector rVSV-△G-MCS.