Recombinant VSV pseudovirus as well as preparation method and application thereof

Abstract

The invention provides a recombinant VSV pseudovirus as well as a preparation method and application thereof, and belongs to the field of pseudovirus synthesis. The method comprises the following steps: constructing packaging cells, helper plasmids and skeleton plasmids, separately transfecting the packaging cells with the helper plasmids and the skeleton plasmids, transfecting only one plasmid each time in the transfection process, transfecting the obtained cells with the next plasmid, and carrying out transfection for multiple times to obtain the recombinant VSV pseudovirus. And further performing pseudovirus amplification by continuously transfecting a cell strain expressing the outer membrane protein of the virus. The transfection process can be completed in the six-hole cell culture plate, the required reagent amount is small, and the cost is not obviously increased; the success rate is high when the delta G-VSV virus is packaged in the later period, the titer of the obtained pseudovirus is high, and the experiment repetition rate is obviously reduced; the intermediate cell strain obtained in the transfection process can be cryopreserved and can be directly used for packaging after being recovered, and the construction process does not need to be repeated.

Description

Technical Field

[0001] The invention belongs to the field of pseudovirus synthesis, relates to VSV pseudovirus, and specifically relates to a recombinant VSV pseudovirus and a preparation method and application thereof. Background Art

[0002] Vesicular Stomatitis Virus (VSV) is a typical non-segmented negative-strand RNA virus belonging to the genus Vesiculovirus, family Rhabdoviridae. VSV is widely used to study viral entry, replication, and assembly due to its broad host range and robust replication in a variety of mammalian and insect cells. One of the notable properties of VSV is that the VSV virus has no particular selectivity for the type of membrane protein that can be incorporated into the viral envelope. Studies of cells co-infected with VSV and other enveloped viruses have shown that VSV readily forms pseudotypes, i.e., pseudoviruses that assemble envelope proteins of heterologous viruses into the VSV membrane. The ability of VSV to form pseudotypes may be due to its budding mechanism, which has been shown not to require the VSV-G protein.

[0003] VSV is a typical envelope protein animal virus. Because of its wide host range and strong replication ability, it is widely used to study the internalization, replication and enrichment of viruses. The study of VSV enrichment promoted the discovery of recombinant stomatitis herpes virus recombinant VSV, the basic principle of which is to replace the G protein gene of the VSV genome with an exogenous functional gene. Recombinant VSV-△G can be used to generate recombinant VSV pseudoviruses containing heterologous envelope proteins. These heterologous proteins can be some viral proteins with high bioprotection requirements. Because the replication ability of VSV-△G pseudovirus is limited to a single replication, this virus required by BSL-4 laboratories can be studied in BSL-2 laboratories. Pseudoviruses not only provide a model for studying pathogens with high bioprotection requirements, but also provide a good platform for evaluating neutralizing antibodies after vaccination. Recombinant VSV vector vaccines are widely used in the study of viral vaccines, post-infection treatment, and even for the treatment of tumors.

[0004] Since the establishment of VSV reverse genetics in 1995, a variety of exogenous genes have been successfully expressed through recombinant VSV. Representative strains of VSV include the Indiana strain and the Chandipure strain. VSV can infect cattle, horses, pigs and other livestock. The main symptoms after infection are blisters and ulcers on the mouth, hooves and nipples. The probability of humans being infected with VSV in the natural state is very small. Generally, there are no obvious symptoms. Very few will have symptoms similar to a mild cold, so the VSV antibody carrying rate in the human body is very low. Those who carry antibodies are mainly concentrated in VSV epidemic areas, scientific researchers who study VSV, and veterinarians who come into contact with sick animals. The low human antibody carrying rate and the lack of effect of VSV infection on the human body provide the basis for the development of VSV as a vaccine vector. Normal VSV particles are bullet-shaped (65*180nm), with a liposome envelope encapsulating a ribonucleoprotein core. The full length of the genome is about 11Kb, encoding 5 major viral proteins, namely nucleoprotein N, phosphoprotein P, matrix protein M, envelope glycoprotein G, and large polymerase protein L. Nucleoprotein N is the most expressed viral protein. It is tightly bound to the genomic RNA to form the viral nucleocapsid. Both replication and translation are based on RNA as a template. Phosphoprotein P and polymerase L constitute the viral RNA polymerase, which can perform transcription and translation functions at the same time. Neither pure RNA nor reverse transcribed cRNA has the ability to infect. The genomic RNA, nucleoprotein N, phosphoprotein P and a small amount of polymerase subunit L are tightly bound to form the minimum replication unit. Glycoprotein G mediates the adsorption of VSV virus particles to the cell membrane surface and enters the cell through endocytosis. Subsequently, the pH value in the endocytic vesicle decreases, and glycoproteins cause the viral envelope to fuse with the cell membrane, and the nucleocapsid (RNP) is released into the cytoplasm. The polymerase complex composed of L, N, and P packaged in the infected virus particles uses the genomic negative-strand RNA as a template to initiate transcriptional expression. As a representative virus of the Rhabdoviridae family, VSV has a wide range of applications in studying the invasion of virus particles into host cells, identifying cell surface receptors that mediate virus infection of cells, screening antiviral drugs, and vaccine development. Reverse genetics is the basis for various applications. As for negative-strand RNA viruses, rescuing infectious viruses from cDNA clones faces great challenges. The RNA genome of positive-strand RNA viruses is naturally infectious, while negative-strand RNA viruses require their genomes to be correctly packaged by the N protein and then recognized and transcribed by RNA polymerase to produce mRNA of viral proteins. Therefore, during the virus rescue process, N, P, and L proteins need to be expressed by transfected plasmids, which are called helper plasmids. In 1992, the Wertz laboratory first established a method for co-transfecting helper plasmids and plasmids carrying a 2.2kb fragment of the VSV genome to rescue infectious virus particles, but due to the lack of functional genes, the virus particles cannot replicate independently.In 1995, the Wertz laboratory and the Rose laboratory independently reported the rescue of independently replicating viruses using plasmids expressing the full-length genome of VSV and auxiliary plasmids. They successfully rescued VSV by transfecting the N, P, L genes and VSV antigenome chain guided by the T7 promoter in the BHK21 cell line, and co-infecting with a poxvirus expressing T7 RNA polymerase to transcribe the antigenome and N, P, L genes of VSV. The virus rescue method was subsequently continuously optimized, such as the establishment of a poxvirus-free rescue system, that is, by using the CMV promoter to guide, or inserting an IRES sequence between the T7 promoter and the gene of the auxiliary plasmid, and transfecting the BHK21 cell line stably expressing T7 RNA polymerase for rescue.

[0005] In 2010, Michael A Whitt's team used the principle of reverse genetics to construct a plasmid and transfect it into mammalian cells to recombinantly produce ΔG-recombinant VSV virus lacking glycoprotein G under the action of helper virus. After infecting cells with high expression of glycoprotein G, the ΔG-recombinant VSV virus will automatically complete the glycoprotein G, forming a recombinant VSV virus with complete structure, wide infection range and easy replication. In this technology, the entire packaging system includes six plasmids, one helper virus and one cell line; the five key plasmids are the plasmid expressing structural protein nucleoprotein N, the plasmid expressing structural protein phosphorylation protein P, the plasmid expressing structural protein RNA polymerase L, the plasmid expressing structural protein glycoprotein G, and the backbone plasmid loaded with VSV virus nucleic acid sequence (glycoprotein G gene deleted and replaced with common reporter genes such as green fluorescent protein gene EGFP, firefly luciferase gene Firefly Luciferase, etc.); the helper virus is vaccinia virus; the cell line is hamster kidney fibroblast BHK-21. When recombinant VSV virus is packaged, five key plasmids need to be co-transfected into BHK-21 cells that have been incubated with auxiliary vaccinia virus for a certain period of time. However, only when the five key plasmids enter the same BHK-21 cells that have been infected with vaccinia virus at the same time, protein expression and assembly may occur under the action of T7 RNA polymerase, and finally budding to generate ΔG-recombinant VSV virus. This ultimately leads to a very low probability of successful packaging of recombinant VSV virus, and the BHK-21 cells that successfully produce virus particles are in poor condition, and the amount of ΔG-recombinant VSV virus produced is extremely low. In order to obtain a sufficient amount of ΔG-recombinant VSV virus, it is necessary to repeat the packaging experiment many times, which is costly and requires a long experimental time (Whitt, Generation of VSV Pseudotypes Using Recombinant ΔG-VSV for Studies on Virus Entry, Identification of Entry Inhibitors, and Immune Responses to Vaccines, 2010).

[0006] Prior art CN113717990A discloses a recombinant VSV pseudovirus and its preparation method. This technology uses a poxvirus (VV-T7) that recombinantly expresses T7 RNA polymerase to infect BHK21 cells, and then uses a mixed reverse genetics plasmid (pVSV-dG related plasmid: pBS-N: pBS-P: pBS-G: pBS-L = 5:3:5:8:1) to transfect BHK21 cells that have been infected with VV-T7, and replaces the medium after 12 hours. After 48 hours, the supernatant (P0) containing the primary generation VSV-dG virus is collected. The P0 generation virus can be used for further amplification to obtain the P1 generation virus. The disadvantage of this technology is that the probability of successful packaging of the recombinant VSV pseudovirus is low, and the pseudovirus titer obtained is low. Summary of the invention

[0007] The present invention aims at the problem of low efficiency of successful packaging of recombinant VSV pseudovirus existing in the prior art, and provides a recombinant VSV pseudovirus and a preparation method and application thereof. The present invention first constructs packaging cells, auxiliary plasmids and backbone plasmids, and then separately transfects the auxiliary plasmids and the backbone plasmids into packaging cells. During the transfection process, only one plasmid is transfected at a time, and the obtained cells are then transfected with the next plasmid. After multiple transfections, the recombinant VSV pseudovirus is harvested, and pseudovirus amplification is performed by continuing to transfect a cell line expressing viral outer membrane protein. The success rate is high when packaging ΔG-VSV virus in the later stage, the obtained pseudovirus has a high titer, and the experimental repetition rate is significantly reduced; the intermediate cell line obtained during the transfection process can be frozen and stored, and directly used for packaging after recovery, without repeating the construction process.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a method for preparing a recombinant VSV pseudovirus, the preparation method comprising sequentially transferring an auxiliary plasmid and a backbone plasmid into a cell line; the auxiliary plasmid comprises: essential proteins N protein, P protein, G protein and L protein for packaging vesicular stomatitis virus, and the backbone plasmid comprises a reporter gene and a vesicular stomatitis virus genome.

[0010] Preferably, the preparation method comprises the following steps:

[0011] S1. Packaging cell construction: construct a cell line expressing bacteriophage T7 RNA polymerase;

[0012] S2. Construction of auxiliary plasmids: construct an auxiliary plasmid containing the gene encoding the viral outer membrane protein, named PX; construct an auxiliary plasmid containing the gene encoding the N protein of the VSV virus, named PN; construct an auxiliary plasmid containing the gene encoding the P protein of the VSV virus, named PP; construct an auxiliary plasmid containing the gene encoding the L protein of the VSV virus, named PL, a total of 4 auxiliary plasmids;

[0013] S3. Construction of backbone plasmid: constructing a backbone plasmid containing a VSV viral genome, wherein the VSV viral genome does not contain a G protein encoding gene;

[0014] S4, preparation of recombinant VSV pseudovirus: the four auxiliary plasmids in step S2 and the backbone plasmid in step S3 are separately transfected into packaging cells, and only one plasmid is transfected each time, and the obtained cells are then transfected with the next plasmid, and the recombinant VSV pseudovirus is harvested after five transfections;

[0015] S5. Amplification of recombinant VSV pseudovirus: The harvested recombinant VSV pseudovirus is transfected into cells that have expressed viral outer membrane protein, and the viral outer membrane protein is the same as the viral outer membrane protein in step S2.

[0016] Preferably, the cell line in step S1 is a mammalian cell line, and the mammalian cell line includes a BHK21 cell line, a HEK293 cell line, a HeLa cell line or a Vero cell line.

[0017] Specifically, the cell line in step S1 is the BHK-21 cell line.

[0018] Preferably, the viral outer membrane protein in step S2 includes VSV virus G protein, HPV virus outer membrane protein, HIV virus outer membrane protein or SARS virus outer membrane protein.

[0019] Specifically, the viral outer membrane protein in step S2 is VSV virus G protein.

[0020] Preferably, the backbone plasmid in step S3 further comprises a reporter gene, and the reporter gene comprises genes of fluorescent protein and luciferase.

[0021] Preferably, the fluorescent protein includes GFP, RFP, YFP or BFP, and the luciferase includes firefly luciferase, Gaussia luciferase, Renilla luciferase or nanoluciferase.

[0022] Specifically, the fluorescent protein is GFP, and the luciferase is firefly luciferase.

[0023] Preferably, the plasmid in step S2 and step S3 further comprises a promoter, and the promoter comprises a T7 promoter, a CMV promoter or a CBV promoter.

[0024] Specifically, the plasmid in step S2 contains a T7 promoter, and the plasmid in step S3 contains a CMV promoter.

[0025] Preferably, step S4 comprises the following steps:

[0026] The packaging cells were transfected with auxiliary plasmid PP to obtain cells Cell-P; the cells Cell-P were transfected with auxiliary plasmid PL to obtain cells Cell-PL; the cells Cell-PL were transfected with auxiliary plasmid PN to obtain cells Cell-PLN; the cells Cell-PLN were transfected with auxiliary plasmid PX to obtain cells Cell-PLNX; the cells Cell-PLNX were transfected with backbone plasmid to obtain recombinant VSV pseudovirus.

[0027] Preferably, the step S5 comprises transfecting the recombinant VSV pseudovirus harvested in step S4 into the above-mentioned cell Cell-PLNX.

[0028] On the other hand, the present invention provides a recombinant VSV pseudovirus prepared by the above preparation method.

[0029] On the other hand, the present invention provides the use of the above-mentioned preparation method or the above-mentioned recombinant VSV pseudovirus in the preparation of vaccines or drugs.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. There is no need to modify common auxiliary plasmids. A cell line can be constructed by transfecting cells with different repeated plasmids. The transfection process can be completed in a six-well cell culture plate. The required plasmids, transfection solvents, transfection reagents and reagents for cell culture are very few, and there is no obvious cost increase.

[0032] 2. The success rate of ΔG-VSV virus packaging in the later stage is high, the titer of the obtained pseudovirus is high, and the experimental repetition rate is significantly reduced;

[0033] 3. The intermediate cell lines obtained during the transfection process can be frozen and directly used for packaging after thawing, without repeating the construction process. The constructed cell lines are also suitable for packaging pseudotyped viruses based on the replacement of viral glycoproteins of VSV virus with outer membrane proteins of other viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 These are pictures of BSR cells; the left picture shows the morphology of BSR cells under bright field; the middle picture shows the morphology of BSR cells under the green fluorescence channel, without green fluorescence interference; the right picture shows the morphology of BSR cells under the red fluorescence channel, without red fluorescence interference.

[0035] Figure 2 This is a diagram of the BSR cell transfection results.

[0036] Figure 3 This is a diagram of the BSR-P cell transfection results.

[0037] Figure 4This is a diagram of the BSR-PL cell transfection results.

[0038] Figure 5 This is a diagram of the BSR-PLN transfection results.

[0039] Figure 6 Results of plasmid transfection in BSR-PLNG cells and BSR cells.

[0040] Figure 7 This is the fluorescence detection result after the recombinant VSV P0 (BSR cell packaging) supernatant infected BSR-PLNG cells.

[0041] Figure 8 This is the fluorescence detection result after the recombinant VSV P0 (BSR-PLNG cell packaging) supernatant infected BSR-PLNG cells.

[0042] Fig. 9 This is the fluorescence detection result after the recombinant VSV P1 (BSR cell packaging) supernatant infected BSR-PLNG cells.

[0043] Fig.10 This is the fluorescence detection result after BSR-PLNG cells were infected with the supernatant of recombinant VSV P1 (packaged in BSR-PLNG cells).

[0044] Fig.11 Comparison of firefly luciferase activity in different treatment groups. DETAILED DESCRIPTION

[0045] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0046] Example 1: Preparation of recombinant VSV pseudovirus

[0047] (1) Material preparation

[0048] 1.1. The four auxiliary plasmids for expressing structural proteins required for packaging were donated by Fudan University. The specific information is shown in Table 1.

[0049] Table 1 Description of structural protein expression plasmid information

[0050]

[0051] SEQ ID NO.1:

[0052]

[0053] SEQ ID NO.2:

[0054] ;

[0055] SEQ ID NO.3:

[0056]

[0057] SEQ ID NO.4:

[0058]

[0059] 1.2. The full-length sequence of VSVFL-ΔG-EGFP-Luc2 was obtained by the method of full gene synthesis and loaded into the vector pVAX1 (purchased from Invitrogen, catalog number V26020) (synthesized, constructed, and sequenced by Tianlin Biotechnology Wuxi Co., Ltd.), which was recorded as VSV full-length plasmid, as shown in Table 2.

[0060] Table 2 Sequence description

[0061]

[0062] SEQ ID NO.5:

[0063]

[0064] 1.3. The full-length sequence of T7 RNA Polymerase was obtained by full gene synthesis method and loaded into the vector pLVX-CMV-MCS (plasmid purchased from Addgene, catalog number 164589) (synthesized, constructed, and sequenced by Tianlin Biotechnology Wuxi Co., Ltd.) through homologous recombination technology, as shown in Table 3.

[0065] Table 3 Sequence description

[0066]

[0067] SEQ ID NO.6:

[0068]

[0069] 1.4. Baby Hamster Kidney Cell Line (BHK-21) was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., catalog number ZQ0130.

[0070] (2) Construction of BSR cell line (BHK-21 cell line with stable and high expression of T7 RNA polymerase)

[0071] 2.1. Plasmid pLVX-CMV-T7 RNA Polymerase-HA and plasmid psPAX2 (purchased from Addgene, catalog number 12260) and pMDG2 (purchased from Addgene, catalog number #12259) were used for 293T (purchased from ATCC, catalog number CRL-3216) cell transfection at a ratio of 4.5 μg: 2.5 μg: 2 μg per d = 10 cm cell culture dish (purchased from NEST Wuxi Nice Life Science Co., Ltd., catalog number 704001). The transfection reagent was 1X PEI (purchased from PolySciences, catalog number 24765-1), and the total amount of plasmid and 1X PEI ratio was 1 μg: 3 μL. After 24 hours of transfection, the culture supernatant was collected and replaced with 2% FBS (Fetal Bovine Serum). Serum, fetal bovine serum, purchased from ExCell, catalog number FSP500) in DMEM culture medium (purchased from BasalMedia Shanghai Yuanpei Biotechnology Co., Ltd., catalog number L110KJ); 48 hours after transfection, the virus supernatant was collected again and the cells were discarded.

[0072] 2.2. The collected viral supernatant was filtered using a 0.22 μm disposable syringe filter (purchased from Millipore, catalog number SLGP033RB). After filtration, 4.5 mL of the viral supernatant was added to BHK-21 cells (d = 10 cm cell culture dish), supplemented with 0.5 mL of FBS, and Synperonic F108 (purchased from Sigma, catalog number 07579-250G-F) was added to a final concentration of 1 μg / mL. After 24 hours of infection, the cells were digested and passaged.

[0073] 2.3. 24 hours after passage, add 10 μL of puromycin (Puromycin, purchased from Solarbio, product number P8230) to the BHK-21 cells infected with the virus, and the final working concentration is 5 μg / mL;

[0074] 2.4. 48 hours after subculturing, the virus-infected BHK-21 cells were digested and subculturated again;

[0075] 2.5. Repeat the above steps 2.4 and 2.5 twice to finally obtain a BSR cell line that highly expresses T7 RNA Polymerase.

[0076] 2.6. Fluorescence detection: A fluorescence inverted microscope (purchased from OLYMPUS, model CKX53) was used to observe and photograph the BSR cell line according to the instrument instructions. The results are as follows: Figure 1 As shown, there is no green fluorescent protein detection signal, that is, no fluorescent protein is expressed.

[0077] (3) Plasmid transfection cells

[0078] 3.1. BSR cells are in the logarithmic growth phase. Add 2 mL PBS (purchased from BasalMedia, catalog number B320KJ), wash once, then add 1 mL 0.25% Tripsin-EDTA (purchased from Gibco, catalog number 25200-056 100 mL), react at 37°C for 1 min, add culture medium containing 10% FBS to terminate digestion, collect all cells in a 15 mL centrifuge tube by pipetting, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

[0079] 3.2. Resuspend the cell pellet in culture medium containing 10% FBS, count the cells using an automatic cell fluorescence analyzer (purchased from CountStar, product model Countstar Mira FL-S), dilute the cell suspension to 2.5E+05 cells / mL, take 2 mL of the cell solution and add it to 2 wells of a six-well cell culture plate (purchased from NEST Wuxi Nice Life Science Co., Ltd., product number 703002), and place it in a 37°C, 5% CO2 cell culture incubator (purchased from Thermo Fisher Scientific, product model 311) for overnight culture.

[0080] 3.3 Transfection of BSR cells

[0081] a) Medium replacement: discard the old culture supernatant, add 800 μL Opti-MEM solution (purchased from Gibco, product number 31985-070) to each well, and continue culturing in a 37° C., 5% CO 2 cell culture incubator.

[0082] b) Preparation of transfection system:

[0083] Tube A: 100 μL Opti-MEM + 1 μg pBS-VSV-P

[0084] Tube B: 100 μL Opti-MEM + 1 μg pCDNA3.1-EGFP (purchased from Miaoling Biotechnology, product number P64303)

[0085] Tube C: 200μL Opti-MEM+4μL Lipo2000 (gently mix in advance and let stand at room temperature for 5 minutes)

[0086] c) Gently pipette 100 μL of the system from tube C into tubes A and B respectively, mix gently, and let stand at room temperature for 20 min.

[0087] d) Gently add all the tube A and tube B systems to the corresponding BSR cell culture wells, place in a 37°C, 5% CO2 cell culture incubator and continue culturing for 4-6 hours.

[0088] e) Remove the transfection solution, add 2 mL of complete culture medium containing 10% FBS to each well, and culture in a 37°C, 5% CO2 cell culture incubator overnight.

[0089] f) Fluorescence detection: After 24 hours, the BSR cell transfection efficiency was detected by fluorescence. The results are as follows: Figure 2 As shown, the efficiency of green fluorescent protein expression of BSR cells transfected with pCDNA3.1-EGFP plasmid (denoted as BSR+pCDNA3.1-EGFP group) under a fluorescence inverted microscope was higher than 90%, and there was no fluorescence detection signal in BSR cells transfected with pBS-VSV-P plasmid (denoted as BSR+pBS-VSV-P group, named BSR-P cells).

[0090] 3.4 Transfection of BSR-P cells

[0091] a) BSR-P cell passaging and plating: Digest BSR-P cells according to step 3.1, centrifuge and add 6 mL of complete culture medium containing 10% FBS to resuspend the cell pellet, take 2 mL of cell solution and add it to 3 wells of a 6-well cell culture plate, and culture it in a 37°C, 5% CO2 cell culture incubator overnight.

[0092] b) BSR-P cells transfected with pBS-VSV-L plasmid: refer to step 3.3 for plasmid transfection, 2 wells of BSR-P cells were transfected with pBS-VSV-L plasmid, and the remaining 1 well of cells was transfected with pCDNA3.1-EGFP plasmid, and the amount of plasmid added to each well was 1 μg; 24 hours later, the green fluorescent protein expression efficiency of the BSR-P cells transfected with pCDNA3.1-EGFP plasmid wells (denoted as BSR-P+pCDNA3.1-EGFP group) was higher than 90%, and there was no fluorescence detection signal in the BSR-P cells transfected with pBS-VSV-L plasmid wells (denoted as BSR-P+pBS-VSV-L group, named BSR-PL cells), the results are shown in Figure 3 .

[0093] 3.5 Transfection of BSR-PL cells

[0094] a) BSR-PL cell passaging and plating: BSR-PL cells were digested according to step 3.1. After centrifugation, 12 mL of complete culture medium containing 10% FBS was added to resuspend the cell pellet. 2 mL of cell solution was added to 6 wells of a 6-well cell culture plate, and the plates were placed in a 37°C, 5% CO2 cell culture incubator for overnight culture.

[0095] b) BSR-PL cells transfected with pBS-VSV-N plasmid: refer to step 3.3 for plasmid transfection, 5 wells of BSR-PL cells were transfected with pBS-VSV-N plasmid, and the remaining 1 well of cells was transfected with pCDNA3.1-EGFP plasmid, and the amount of plasmid added to each well was 1 μg; 24 hours later, the green fluorescent protein expression efficiency of the BSR-PL cells transfected with pCDNA3.1-EGFP plasmid well cells (denoted as BSR-P-L+pCDNA3.1-EGFP group) was higher than 90%, and there was no fluorescence detection signal in the BSR-PL cells transfected with pBS-VSV-N plasmid well cells (denoted as BSR-P-L+pBS-VSV-N group, named BSR-PLN cells), the results are shown in Figure 4 .

[0096] 3.6 Transfection of BSR-PLN cells

[0097] a) BSR-PLN cell passaging, freezing, and plating: First, the BSR-PLN cells were divided into 7 wells; the cells from 4 wells were digested with trypsin, centrifuged, and the cell pellets were resuspended in 2 mL of freezing solution (90% FBS + 10% DMSO), evenly distributed into 2 cryopreservation tubes, temporarily stored in a -80°C medical low temperature box overnight, and then placed in a liquid nitrogen tank for long-term storage; BSR-PLN cells were digested according to step 3.1, centrifuged, and 12 mL of complete culture solution containing 10% FBS was added to resuspend the cell pellet, and 2 mL of cell solution was added to 3 wells of a six-well cell culture plate, and placed in a 37°C, 5% CO2 cell culture incubator overnight.

[0098] b) BSR-PLN cells transfected with pCMV-VSV-G plasmid: refer to step 3.3 for plasmid transfection, 2 wells of BSR-PLN cells were transfected with pCMV-VSV-G plasmid, and the remaining 1 well of cells was transfected with pCDNA3.1-EGFP plasmid, and the amount of plasmid added to each well was 1 μg; 24 hours later, the green fluorescent protein expression efficiency of the BSR-PLN cells transfected with pCDNA3.1-EGFP plasmid well cells (denoted as BSR-PL-N+pCDNA3.1-EGFP group) was higher than 90%, and there was no fluorescence detection signal in the BSR-PLN cells transfected with pCMV-VSV-G plasmid well cells (denoted as BSR-PL-N+pBS-VSV-G group, named BSR-PLNG cells), the results are shown in Figure 5 The obtained BSR-PLNG cells can be passaged, amplified, and packaged for recombinant VSV virus.

[0099] 3.7. Recombinant VSV virus packaging

[0100] a) The BSR-PLNG cells transfected with the capsid glycoprotein plasmid were passaged, digested, centrifuged, and counted according to step 3.1 in the six-well cell culture plate, and the cell pellet was resuspended in 12 mL of complete culture medium containing 10% FBS, and then 2 mL of cell solution was inoculated into the six-well cell culture plate per well, and then the six-well plate was placed in a 37° C., 5% CO2 cell culture incubator for continued culture; a control group was set up, and the BSR cells were digested, centrifuged, and counted, and the same number of cells as BSR-PLNG were resuspended, inoculated into a six-well cell culture plate, and also placed in a 37° C., 5% CO2 cell culture incubator for continued culture.

[0101] b) Add 0.5 mL of VTF7-3 virus culture supernatant (purchased from Sun Yat-sen University) as helper virus to BSR-PLNG cells and control BSR cells that have been cultured for 2-8 h, and continue to culture in a 37°C, 5% CO2 cell culture incubator overnight.

[0102] c) Discard the old culture medium from all six-well plates, add 0.8 mL of Opti-MEM solution to each well, and place the plates in a 37°C, 5% CO2 cell culture incubator for culture.

[0103] d) Prepare transfection system:

[0104] BSR-PLNG cell experimental group:

[0105] Tube A: 6μg VSVFL-ΔG-EGFP-Luc2+Opti-MEM (600μL);

[0106] Tube B: 600 μL Opti-MEM + 12 μL Lipo2000 (Lipo2000: DNA = 2 μL: 1 μg) (gently mix in advance and let stand at room temperature for 5 min);

[0107] Gently add all the contents of tube B into tube A, mix gently, and let stand at room temperature for 20 minutes.

[0108] BSR cell control group:

[0109] Tube A: 0.9μg pBS-VSV-N+1.5μg pBS-VSV-P+0.3μg pBS-VSV-L+2.4μg pCMV-VSV-G

[0110] +1.5μg VSVFL-ΔG-EGFP-Luc2+Opti-MEM (600μL);

[0111] Tube B: 600 μL Opti-MEM + 13.2 μL Lipo2000 (Lipo2000: DNA = 2 μL: 1 μg) (gently mix in advance and let stand at room temperature for 5 min);

[0112] Gently add all the contents of tube B into tube A, mix gently, and let stand at room temperature for 20 minutes.

[0113] e) Take out the six-well plate, slowly add 200 μL of the transfection system to each well of the corresponding six-well plate, lightly draw a "cross" shape to mix, and place the six-well plate in a 37°C, 5% CO2 cell culture incubator for 48h-72h.

[0114] f) Observe the green fluorescent protein expression efficiency under a fluorescent inverted microscope 24h, 48h, and 72h after transfection, and take photos and record the results. Figure 6 , it was found that fluorescence was detected in both the BSR-PLNG cell experimental group and the BSR cell control group, and the fluorescence intensity became stronger with time. Referring to the operation process of the Whitt team, a total amount of 22μg of mixed plasmids needs to be added to each well, and the corresponding amount of transfection reagent Lipo2000 added is 44μL. Statistics show that the total amount of transfection reagent required for the entire cell line construction process (including the transfection positive control group) and the later packaging is also 44μL, so the preparation method has no significant increase in cost and the time is controllable. In order to ensure that the conditions of the control group (BSR cell packaging) and the experimental group (BSR-PLNG cell packaging) are the same, the five plasmids in the control group are reduced accordingly according to the ratio of 3:5:1:8:5.

[0115] g) Collect the supernatant 72 hours after transfection, centrifuge at 10,000 g and 4°C for 5 min, and transfer the supernatant to a new 50 mL centrifuge tube to obtain the P0 generation recombinant VSV virus solution (it is not recommended to filter the virus solution to remove the vTF7-3 virus at this time).

[0116] result:

[0117] 3.8. Amplification of recombinant VSV virus fluid

[0118] a) Take 2 dishes of BSR-PLNG cells (subculture for 24 h), add the P0 generation recombinant VSV virus fluids harvested from the BSR-PLNG cell packaging experimental group and the BSR cell packaging control group (respectively referred to as rVSV P0 (BSR-PLNG) rescue group and rVSV P0 (BSR) rescue group) into the dishes for infection, and mature and amplify in the cells.

[0119] b) Observe under a fluorescent inverted microscope 24h and 48h after infection to see whether there is green fluorescent protein expression and cell fusion. The number of cells expressing green fluorescent protein at the same position increases with time, and take pictures with a fluorescent inverted microscope. If this phenomenon occurs, it means that the recombinant VSV virus is successfully rescued, otherwise it means that the recombinant VSV virus has failed to be rescued.

[0120] Test results see Figure 7 and Figure 8 . Figure 7 This is the fluorescence detection result after the supernatant of the rVSV P0 (BSR) group infected BSR-PLNG cells, showing no fluorescence, indicating that there was no recombinant VSV virus amplification; Figure 8 This is the fluorescence detection result after the supernatant of rVSV P0 (BSR-PLNG) group infected BSR-PLNG cells. The high fluorescence intensity indicates that the recombinant VSV virus was successfully amplified. The specific analysis is as follows:

[0121] Figure 7 The expression of green fluorescent protein after the supernatant of BSR cell reverse rescue was infected with BSR-PLNG cells expressing VSV-G. The results showed that no cells in the dish expressed green fluorescent protein, indicating that the rescue of BSR cells failed and the transfection process needed to be repeated. Figure 8 The figure shows the expression of green fluorescent protein after BSR-PLNG cells were infected with the supernatant of BSR-PLNG cells reversely rescued. It can be clearly seen that there are cells in the dish expressing green fluorescent protein, and as time goes by, the number of cells expressing green fluorescent protein increases, and cell fusion occurs, proving that BSR-PLNG cells successfully rescued the rVSV virus, and the success rate of the preparation method of BSR-PLNG cells is higher than that of BSR.

[0122] c) Collect the cell culture supernatant, filter the supernatant with a 0.22 μm disposable syringe filter, remove the vaccinia virus vTF7-3 (the vaccinia virus is about 360 nm long, 270 nm wide, and 250 nm high, and will be filtered by the filter), and harvest the amplified P1 generation recombinant VSV virus solution (respectively recorded as rVSV P1 (BSR-PLNG) group and rVSV P1 (BSR) group); take 2 mL of the virus solution to infect BSR-PLNG cells (dishes), and observe the green fluorescent protein expression efficiency under a fluorescence inverted microscope. The results are shown in Fig. 9 and Fig.10 . Fig. 9 This is the result of the green fluorescent protein expression test of the rVSV P1 (BSR) amplification group after infecting BSR-PLNG cells. There is no fluorescent signal, indicating that there is no recombinant VSV virus amplification; Fig.10This is the result of the green fluorescent protein expression test of BSR-PLNG cells infected with rVSV P1 (BSR-PLNG) amplification group. The obvious green fluorescent signal appears, indicating that high-titer recombinant VSV virus has been amplified. The specific analysis is as follows:

[0123] The virus content of rVSV virus is low in the early stage of reverse rescue. After removing the vaccinia virus, it needs to infect cells with high expression of VSV-G protein for amplification. Fig. 9 The supernatant of the virus liquid rescued from BSR cells continued to infect BSR-PLNG cells. Because the rescue failed, there was no virus in the supernatant. Therefore, the infected BSR-PLNG cells did not express green fluorescent protein, and no green fluorescent signal was observed under a fluorescence microscope. Fig.10 The supernatant used for reverse rescue of BSR-PLNG cells continued to infect BSR-PLNG cells. After 72 hours, most of the BSR-PLNG cells expressed green fluorescent protein under a fluorescence microscope, proving that the recombinant rVSV virus was successfully obtained through the cell line method and could be efficiently amplified in cells with high expression of VSV-G protein.

[0124] d) After 72 h, the supernatant was collected, filtered through a 0.22 μm disposable syringe filter, packaged into 1 mL tubes, and stored in a -80°C low-temperature medical refrigerator (purchased from Thermo Fisher Scientific, product model ULTS1368).

[0125] e) The recombinant rVSV virus is a dual reporter gene pseudovirus, which contains the green fluorescent protein EGFP and the firefly luciferase LUC2 gene. To verify whether the successfully rescued rVSV virus solution normally expresses luciferase protein, 1E+4 BSR-PLNG cells were plated in BeyoGold black transparent bottom 96-well cell culture plates (purchased from Beyotime Biotechnology, product number FCP965-48pcs), and four groups were set up: virus solution rescued by BSR cells (P0), recorded as rVSV P0 (BSR) rescue; virus solution rescued and amplified by BSR cells (P1), recorded as rVSV P1 (BSR) amplification; virus solution rescued by BSR-PLNG cells (P0), recorded as rVSV P0 (BSR-PLNG) rescue; virus solution rescued and amplified by BSR-PLNG cells (P1), rVSV P1 (BSR-PLNG) amplification; 100 μL virus solution was added to each well, with 3 replicate wells in each group; at the same time, only 2% FBS culture medium was added to the 3 replicate wells as a blank control group.

[0126] f) After the cells were infected with the virus solution for 72 hours, the old culture medium was discarded, and 50 μL of culture medium containing 2% FBS was added in time, and then 50 μL of Bio-Lite Luciferase Assay System (purchased from Vazyme, product number DD1201) was added, and the cells were fully lysed by pipetting several times. The 96-well plate was placed in a multi-function microplate reader (purchased from TECAN, product model INFINITE MPLEX) to detect the expression of firefly luciferase.

[0127] Results Fig.11 , among which the firefly luciferase of the blank control group was 8, indicating that there was no luciferase interference in BSR-PLNG cells; the P0 virus solution rescued by BSR cells infected BSR-PLNG cells expressed 381.67 luciferase, which may be due to the fact that the virus solution was not filtered and some cell impurities remained; after the virus solution rescued by BSR cells was further infected with cells expressing VSV-G protein for amplification, it was found that the firefly luciferase expression of the amplified cells was lower, indicating that BSR cells did not successfully rescue the rVSV virus; the P0 virus solution rescued by BSR-PLNG cells infected BSR-PLNG cells expressed 15610 luciferase, which was significantly higher than that of the BSR cell rescued virus group and its amplification group by 40.9 times, and after continuing to infect cells expressing VSV-G protein for amplification, the firefly luciferase expression of the amplified cells was greatly improved, by 23 times, which was consistent with the expression of green fluorescent protein, indicating that BSR-PLNG cells successfully rescued the rVSV virus expressing dual reporter genes, and the rVSV virus can be efficiently amplified in cells with high expression of VSV-G protein.

[0128] Example 2: Resuscitation of BSR-PLNG cells

[0129] After the BSR-PLNG cells frozen for 3 months in Example 1 were revived, the experiment was carried out according to the same method as steps 3.7 and 3.8 in Example 1, and the results obtained were consistent with the corresponding results of steps 3.7 and 3.8 in Example 1, indicating that the BSR-PLNG cells can be stored for a long time after freezing and directly used for packaging after resuscitation without repeating the construction process.

[0130] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a recombinant VSV pseudovirus, characterized in that: The preparation method comprises the steps of sequentially transferring an auxiliary plasmid and a backbone plasmid into a cell line; the auxiliary plasmid comprises: the necessary proteins N protein, P protein, G protein and L protein for packaging vesicular stomatitis virus; and the backbone plasmid comprises a reporter gene and a vesicular stomatitis virus genome.

2. The method for preparing the recombinant VSV pseudovirus according to claim 1, characterized in that: The following steps are involved: S1. Packaging cell construction: construct a cell line expressing bacteriophage T7 RNA polymerase; S2. Construction of auxiliary plasmids: construct an auxiliary plasmid containing the gene encoding the viral outer membrane protein, named PX; construct an auxiliary plasmid containing the gene encoding the N protein of the VSV virus, named PN; construct an auxiliary plasmid containing the gene encoding the P protein of the VSV virus, named PP; construct an auxiliary plasmid containing the gene encoding the L protein of the VSV virus, named PL, a total of 4 auxiliary plasmids; S3. Construction of backbone plasmid: constructing a backbone plasmid containing a VSV viral genome, wherein the VSV viral genome does not contain a G protein encoding gene; S4, preparation of recombinant VSV pseudovirus: the four auxiliary plasmids in step S2 and the backbone plasmid in step S3 are separately transfected into packaging cells, and only one plasmid is transfected each time, and the obtained cells are then transfected with the next plasmid, and the recombinant VSV pseudovirus is harvested after five transfections; S5. Amplification of recombinant VSV pseudovirus: The harvested recombinant VSV pseudovirus is transfected into cells that have expressed viral outer membrane protein, and the viral outer membrane protein is the same as the viral outer membrane protein in step S2.

3. The preparation method according to claim 2, characterized in that: The cell line in step S1 is a mammalian cell line, and the mammalian cell line includes a BHK21 cell line, a HEK293 cell line, a HeLa cell line or a Vero cell line.

4. The preparation method according to claim 3, characterized in that: The cell line described in step 1 is the BHK-21 cell line.

5. The preparation method according to claim 2, characterized in that: The viral outer membrane protein in step S2 includes VSV virus G protein, HPV virus outer membrane protein, HIV virus outer membrane protein or SARS virus outer membrane protein.

6. The preparation method according to claim 5, characterized in that: The viral outer membrane protein in step S2 is VSV virus G protein.

7. The preparation method according to claim 2, characterized in that: The backbone plasmid in step S3 further comprises a reporter gene, and the reporter gene comprises genes of a fluorescent protein and a luciferase.

8. The preparation method according to claim 2, characterized in that: The step S4 comprises the following steps: The packaging cells were transfected with auxiliary plasmid PP to obtain cells Cell-P; the cells Cell-P were transfected with auxiliary plasmid PL to obtain cells Cell-PL; the cells Cell-PL were transfected with auxiliary plasmid PN to obtain cells Cell-PLN; the cells Cell-PLN were transfected with auxiliary plasmid PX to obtain cells Cell-PLNX; the cells Cell-PLNX were transfected with backbone plasmid to obtain recombinant VSV pseudovirus.

9. The recombinant VSV pseudovirus prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the preparation method according to any one of claims 1 to 8 or the recombinant VSV pseudovirus according to claim 9 in the preparation of vaccines or medicines.

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