Preparation method and application of rabies subtype pseudovirus

Abstract

The present invention provides a preparation method and application of a rabies subtype pseudovirus, belonging to the field of biotechnology. The present invention prepares a PV-VSVG-3FLAG synthetic gene having a sequence as shown in SEQ ID NO.1. The synthetic gene carries the extracellular region and transmembrane region of the capsid glycoprotein G of the rabies virus subtype Pasteur strain (PV strain), and the C-terminus is replaced by the intracellular region of VSV-G, with a wide targeting range. Among them, the G protein mutates L231P and S436N, the purpose is to improve the expression of the G protein and enhance the infection efficiency; the H113Q, P254S, and V164I are mutated, the purpose is to improve the neutralization sensitivity. The rabies subtype pseudovirus prepared by the preparation method of the present invention can load RNA more efficiently, has significant targeting, and can replace the wild-type rabies virus for neutralizing antibody detection experiments.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and specifically relates to a preparation method and application of a rabies subtype pseudovirus. Background Art

[0002] Rabies is a zoonotic infectious disease caused by the rabies virus (RABV) of the Rhabdoviridae family and the genus Lyssavirus. It is mainly transmitted through saliva after being bitten by infected animals. Rabies virus can infect nerve cells and transfer to the central nervous system (CNS), with a very high mortality rate. Therefore, rabies post-exposure prophylaxis (PEP), host vaccination and human pre-exposure prophylaxis (PREP) are crucial for controlling and alleviating the disease.

[0003] Rabies virus (RABV) is an enveloped single-stranded negative-sense RNA virus with a genome size of about 12kb, encoding nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and polymerase protein (RNA dependent RNA polymerase or Large protein, L). Among them, G protein is a type I transmembrane protein that allows the virus to enter the host cell by interacting with the receptor and can induce the production of virus neutralizing antibodies (VNAs).

[0004] Virus neutralizing antibody detection tests are useful for determining acquired immune responses after infection and the effectiveness of vaccination. The gold standard for virus neutralizing antibody detection tests is the rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVN). These VNA tests are not easy to standardize and require the use of live viruses and are performed in laboratories with biosafety level 3 or above.

[0005] Therefore, it is necessary to develop alternative technologies to optimize and improve rabies neutralizing antibody detection. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for preparing and using a rabies subtype pseudovirus. The present invention prepares a PV-VSVG-3FLAG synthetic gene having a sequence as shown in SEQ ID NO.1. The synthetic gene carries the extracellular region and transmembrane region of the capsid glycoprotein G of the rabies virus subtype Pasteur strain (PV strain), and the C-terminus is replaced with the intracellular region of VSV-G, with a wide targeting range. Among them, the G protein mutates L231P and S436N, the purpose is to improve the expression of the G protein and enhance the infection efficiency; the H113Q, P254S, and V164I are mutated, the purpose is to improve the neutralization sensitivity. The rabies subtype pseudovirus prepared by the preparation method of the present invention can load RNA more efficiently, has significant targeting, and can replace the wild-type rabies virus for neutralizing antibody detection experiments.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a method for preparing a rabies subtype pseudovirus, the method comprising preparing a synthetic gene (1), wherein the synthetic gene (1) has a sequence shown in SEQ ID NO.1.

[0009] Specifically, the synthetic gene (1) is PV-VSVG-3FLAG.

[0010] Specifically, the synthetic gene (1) includes the extracellular region and transmembrane region of the capsid glycoprotein G of the rabies virus subtype PV strain, and the C-terminus is replaced with the VSV-G intracellular region sequence.

[0011] Preferably, the glycoprotein G is mutated with L231P, S436N, H113Q, P254S, and V164I.

[0012] Specifically, the preparation method further comprises the following steps:

[0013] S1. Clone the synthetic gene (1) shown in SEQ ID NO.1 into a linearized vector to obtain a plasmid (1);

[0014] S2, cloning the synthetic gene (2) shown in SEQ ID NO.3 into a linearized vector to obtain a plasmid (2);

[0015] S3. Transfect cells to obtain rabies subtype pseudovirus.

[0016] Preferably, the plasmid (1) in step S1 is pCAGGS-PV-VSVG-3FLAG.

[0017] Further preferably, the pCAGGS-PV-VSVG-3FLAG has the sequence shown in SEQ ID NO.4.

[0018] Preferably, the plasmid (2) in step S2 is Lenti-ZsGreen-IRES-Luc2.

[0019] Further preferably, the Lenti-ZsGreen-IRES-Luc2 has a sequence as shown in SEQ ID NO.5.

[0020] Preferably, the linearized vectors described in steps S1 and S2 are obtained by digesting pCAGGS with BamHI / EcoRI.

[0021] Preferably, the cells in step S3 are 293T cells.

[0022] More specifically, the plasmid transfected into cells in step S3 is a mixed transfection plasmid.

[0023] Preferably, the mixed transfection plasmid comprises plasmid (1), plasmid (2) and psPAX2.

[0024] On the other hand, the present invention provides a rabies subtype pseudovirus prepared by the above preparation method.

[0025] In another aspect, the present invention provides the use of the above-mentioned rabies subtype pseudovirus in any of the following:

[0026] (1) Application in the detection or screening of rabies virus neutralizing antibodies for non-disease diagnosis purposes;

[0027] (2) Use in the preparation of reagents for detecting neutralizing antibodies against rabies virus;

[0028] (3) Use in the preparation of reagents for detecting neutralizing antibodies against rabies virus;

[0029] (4) Use in the preparation of reagents for diagnosing diseases caused by rabies virus infection.

[0030] The positive and beneficial effects of the present invention are:

[0031] The present invention prepares a PV-VSVG-3FLAG synthetic gene having a sequence as shown in SEQ ID NO.1. The synthetic gene carries the extracellular region and transmembrane region of the capsid glycoprotein G of the rabies virus subtype Pasteur strain (PV strain), and the C-terminus is replaced by the intracellular region of VSV-G, with a wide targeting range. Among them, the G protein mutates L231P and S436N, the purpose is to improve the expression of the G protein and enhance the infection efficiency; the H113Q, P254S, and V164I are mutated, the purpose is to improve the neutralization sensitivity. The rabies subtype pseudovirus prepared by the preparation method of the present invention can load RNA more efficiently, has significant targeting, and can replace the wild-type rabies virus for neutralizing antibody detection experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the packaging, transfection and purification process of rabies subtype pseudovirus.

[0033] Figure 2 This is the plasmid map of pCAGGS-PV-VSVG-3FLAG.

[0034] Figure 3 This is the plasmid map of pCAGGS-PV-WT-3FLAG.

[0035] Figure 4 This is the plasmid map of psPAX2.

[0036] Figure 5 This is the Lenti-ZsGreen-IRES-Luc2 plasmid map.

[0037] Figure 6 This is an agarose gel image of the plasmid that constructs the rabies subtype pseudovirus.

[0038] Figure 7 This is a bar graph of the copy number of rabies subtype pseudovirus detected by qPCR.

[0039] Figure 8 These are fluorescence images of 293T cell lines, BHK-21 cell lines and HT22 cell lines infected with rabies subtype pseudovirus.

[0040] Fig. 9 This is a gel imaging image of rabies subtype pseudovirus protein.

[0041] Fig.10 Bar graph for luciferase assay data analysis of rabies subtype pseudoviruses.

[0042] Fig.11 This is a line graph for the data analysis of rabies subtype pseudovirus neutralizing antibody detection. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0044] Example 1 Construction of rabies subtype pseudovirus-related plasmids

[0045] 1. The sequence obtained by whole genome synthesis is shown in Table 1

[0046] Table 1 Synthetic sequence information

[0047]

[0048] 2. Plasmid pCAGGS was used as a vector, and restriction enzymes BamHI and EcoRI were used to obtain a linearized vector. The linearized pCAGGS vector was connected to the synthesized PV-VSVG-3FLAG by homologous recombination to obtain a pCAGGS-PV-VSVG-3FLAG plasmid. The amino acid sequence is shown in SEQ ID NO.4, and the map is shown in Figure 2 shown.

[0049] 3. Plasmid pCAGGS was used as a vector, and the linearized vector was obtained by restriction endonuclease BamHI and EcoRI. The linearized pCAGGS vector was connected with the synthetic PV-WT-3FLAG by homologous recombination to obtain the pCAGGS-PV-WT-3FLAG plasmid. The amino acid sequence is shown in SEQ ID NO.5, and the map is shown in Figure 3 shown.

[0050] 4. Based on the lentiviral vector, the restriction endonucleases BamHI and EcoRI were used to obtain a linearized vector. The linearized lentiviral vector was connected with the synthesized ZsGreen-IRES-Luc2 through homologous recombination to obtain the Lenti-ZsGreen-IRES-Luc2 plasmid. The map is shown in Figure 5 shown.

[0051] 5. The constructed pCAGGS-PV-VSVG-3FLAG plasmid and pCAGGS-PV-WT-3FLAG plasmid were identified by agarose gel electrophoresis and first-generation gene sequencing. The results are shown in Figure 6 .

[0052] Example 2 Rabies subtype pseudovirus packaging experiment

[0053] 1. Before transfection, observe the state of 293T cells under a microscope to make sure that the cells are in good condition, with a density of about 90% and no mycoplasma, and that cell transfection can be performed;

[0054] 2. Before transfection, replace the medium of each 10cm dish with 10mL of DMEM medium containing 2% FBS serum to reduce the effect of serum on plasmid transfection;

[0055] 3. Experimental groups and plasmid dosage are shown in Table 2:

[0056] Table 2 Experimental groups and plasmid dosage

[0057]

[0058] 4. Add the required amount of plasmid and 1xPEI in the above table into the corresponding 1.5mL EP tube containing 1mL Opti-MEM / dish, add the plasmid first and then add 1xPEI to mix well, and let it stand at room temperature for 15 minutes to obtain the transfection system;

[0059] 5. Slowly add the transfection system to a 10 cm dish. Replace the medium with 10 mL of DMEM medium containing 2% FBS serum 6-8 hours after transfection.

[0060] 6. 72 hours after transfection, collect the cell culture supernatant, centrifuge at 50,000 x g for 2 hours, resuspend in 150 μL 1xPBS, filter and sterilize after centrifugation to obtain purified rabies pseudovirus, and freeze in a -80°C refrigerator.

[0061] Example 3 Detection of rabies subtype pseudovirus RNA load

[0062] 1. Use the Viral Nucleic Acid Extraction Kit (Beaverbio, Cat. NO. 70406) to extract RNA from the viral supernatant and purified solution respectively. The specific extraction method is carried out according to the instructions;

[0063] 2. After the viral RNA is extracted, a one-step RT-qPCR kit (HiScript II One StepqRT-PCR SYBR Green Kit, Catalog No. Q221-01) is used for qPCR detection. This kit can simultaneously complete RT-PCR to convert RNA into cDNA and QPCR real-time quantitative detection;

[0064] 3. The qPCR primer sequences for detecting dual Luciferase mRNA are shown in Table 3:

[0065] Table 3 qPCR primer sequences

[0066]

[0067] 4. The 20 μL reaction system for RT-qPCR is shown in Table 4:

[0068] Table 4 20 μL reaction system for RT-qPCR

[0069]

[0070] Prepare the system on ice according to the reaction system in the table above and mix well.

[0071] 5. The reaction procedure of RT-qPCR is shown in Table 5:

[0072] Table 5 RT-qPCR reaction procedure

[0073]

[0074] Set up the qPCR reaction program according to the procedure in the table above to perform qPCR reaction and export the data for analysis.

[0075] The analysis results are as follows Figure 7 As shown, the RNA loaded by the virus was relatively quantified by the QPCR method. The results showed that the rabies subtype pseudovirus prepared by Group 1, i.e., pCAGGS-PV-VSVG-3FLAG as the capsid protein, could load RNA more efficiently than other groups.

[0076] Example 4 Targeted Detection of Rabies Subtype Pseudovirus Infection

[0077] 1. Cell line culture:

[0078] (1) 293T cell line (human embryonic kidney cells) was cultured in DMEM medium containing 10% serum;

[0079] (2) BHK-21 cell line (hamster kidney fibroblasts) was cultured in DMEM medium containing 10% serum;

[0080] (3) HT22 cell line (mouse hippocampal neuronal cells) was cultured in DMEM medium containing 10% serum.

[0081] 2. After the cells have been cultured for more than three generations, observe the cell growth status. When the cells are in good growth condition, inoculate the cells into a 6-well plate with a density of 20%-30% and culture at 37°C and 5% CO2.

[0082] 3. After 24 hours of culture, the 293T cells in the 6-well plate were infected with pseudovirus at MOI=1 without adding co-infection reagent; at MOI=10, 100x co-infection reagent dilution was added and mixed with pseudovirus to infect BHK-21 cells and HT22 cells in the 6-well plate; at the same time, a group of empty cells without virus-like particle infection were set up.

[0083] 4. After 12-16 hours of infection, the culture medium was replaced with complete medium for cultivation. Fluorescence microscope images were taken 24 and 72 hours after infection. The fluorescence images of rabies subtype pseudovirus are shown in the following figure. Figure 8 shown.

[0084] Rabies subtype pseudovirus was used to infect 293T cell lines, BHK-21 cell lines and HT22 cell lines for targeted detection. At 24 hours, it was observed that rabies subtype pseudovirus (Group1) was highly infectious to 293T cell lines, and the infection efficiency was significantly improved compared with the wild type group (Group2) and VSVG control group (CON1). After 72 hours, it was observed that rabies subtype pseudovirus also had significant targeting to BHK-21 cell lines and HT22 cell lines.

[0085] Example 5 Identification of rabies subtype pseudovirus proteins (WB)

[0086] 1. Take 60 μL of the purified virus solution, add 20 μL of 4x SDS loading buffer, mix well, boil at 100°C for 10 min for denaturation, and centrifuge at 12000 rpm for 2 min to remove impurities;

[0087] 2. Load 15 μL of sample for polyacrylamide gel electrophoresis at 120 V for 75 min.

[0088] 3. After electrophoresis, transfer to the membrane using PVDF membrane, with a transfer voltage of 100 mA for 90 minutes;

[0089] 4. After the transfer, the PVDF membrane was blocked with 5% skimmed milk powder and incubated on a 60 rpm horizontal shaker for 1 hour;

[0090] 5. After the incubation, the PVDF was washed with 1xPBST and incubated with 3FLAG-TAG primary antibody at 4°C overnight. The dilution ratio of 3FLAG tag antibody was 1:1000.

[0091] 6. After the primary antibody incubation, the PVDF membrane was washed with 1xPBST for 3 times, each time in a 60 rpm horizontal shaker for 5 minutes;

[0092] 7. Add the corresponding secondary antibody to the cleaned PVDF membrane for incubation, place it on a 60rpm horizontal shaker for incubation for 1 hour, and the dilution ratio of the secondary antibody is 1:10000;

[0093] 8. After the secondary antibody incubation, the PVDF membrane was washed with 1xPBST for 3 times, each time in a 60 rpm horizontal shaker for 5 minutes;

[0094] 9. After washing, add developer evenly to the PVDF membrane and place it in a protein gel imager for luminescence photography. The protein gel imaging is shown in the figure below. Fig. 9 shown.

[0095] WB detected the expression of related coat proteins through 3FLAG-TAG. The results showed that the position of the G protein band was consistent with the actual situation, proving that it was successfully expressed and had stronger expression compared with the wild-type group (Group2).

[0096] Example 6 Determination of the expression level of rabies subtype pseudovirus

[0097] 1. 293T cell lines were cultured in DMEM medium containing 10% serum.

[0098] 2. After the cells have been cultured for more than three generations, observe the cell growth status. When the cells are in good growth condition, inoculate the cells into a 6-well plate with a density of 20%-30% and culture at 37°C and 5% CO2.

[0099] 3. After culturing for 24 hours, use pseudovirus to infect 293T cells in 96-well plates at an MOI of 1 without adding co-infection reagents; at the same time, set up a group of empty cells without virus-like particle infection.

[0100] 4. After 12-16 hours of infection, the complete medium was replaced for culture. After 72 hours of infection, the cells were collected and centrifuged at 1000rpm for 5 minutes. After centrifugation, the supernatant was discarded. The cell pellet was lysed with 150μL lysis buffer in the luciferase detection kit for each sample, and three sets of wells were set. The plate was transferred to the ELISA plate, placed in the ELISA instrument, and the program was set for detection. A bar graph was drawn for analysis based on the detection data. The bar graph is shown in Fig.10 shown.

[0101] The infected cells were collected for 72 hours to detect the expression of luciferase. The results showed that the rabies subtype pseudovirus prepared by using Group 1, i.e. pCAGGS-PV-VSVG-3FLAG as the capsid protein, could express stronger fluorescence than the wild-type group (Group 2) and the VSVG control group (CON1).

[0102] Example 7 Detection of Neutralizing Antibodies to Rabies Subtype Pseudovirus

[0103] 1. Cell plating: Take 293T cells in the logarithmic growth phase and digest them with trypsin. After counting, make 1x10 5 cells / mL cell suspension, inoculate 100 µL of cell suspension into each well of a 96-well plate, and continue culturing in a 37°C, 5% CO2 cell culture incubator.

[0104] 2. Serum or neutralizing antibody preparation:

[0105] (1) Serum or neutralizing antibody samples were heated in a 56°C water bath for 30 min to remove complement interference;

[0106] (2) Dilution: Use DMEM medium to dilute the serum or neutralizing antibody samples, perform a series of gradient dilutions, and use them as independent samples for neutralizing antibody detection.

[0107] 3. Pseudovirus dilution: Take out the pseudovirus from the -80℃ ultra-low temperature freezer and place it on ice or at 4℃ to melt. Centrifuge briefly after it is completely melted to allow the virus liquid to accumulate at the bottom of the storage tube. Perform serial dilutions according to the pseudovirus titer (1TCID50=1 fluorescent spot).

[0108] 4. Pre-incubation of the test sample and pseudovirus dilution: Mix the diluted test sample and pseudovirus dilution in equal volumes, set up cell control (DMEM culture medium, 100µL / well) and pseudovirus control (pseudovirus dilution and DMEM culture medium are mixed in equal volumes), and incubate in a 37°C, 5% CO2 incubator for 1 hour.

[0109] 5. Infect cells: After the incubation, take 100 µL of the mixed solution (including cell control and pseudovirus control) and slowly add it to the corresponding wells of the 96-well plate that has been inoculated with cells. Gently draw a "cross" shape on the work surface to mix well, and place it in a 37°C, 5% CO2 incubator for 72 hours.

[0110] 6. Infection detection: 72 hours after the virus infected the cells, take out the 96-well plate and place it under a fluorescent inverted microscope to observe the expression efficiency of green fluorescent protein, and take pictures for record; discard 100µL of supernatant from the well plate, add 50µL of luciferase detection reagent equilibrated at room temperature, mix thoroughly by pipetting, then transfer the reaction solution from each well to the corresponding well position in the ELISA plate, and use an ELISA reader to detect its luminescence value.

[0111] 7. Result analysis: Calculate the neutralizing antibody inhibition rate based on the luciferase test data and draw a line graph for analysis. The line graph is as follows: Fig.11 shown.

[0112] After incubating two rabies virus antibody samples with rabies subtype pseudoviruses, the cells were added to the well plate for 72 hours to detect the expression of luciferase and calculate the effective inhibition rate of the antibody. The results showed that rabies subtype pseudoviruses can replace wild-type rabies virus for neutralizing antibody detection experiments.

[0113] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A method for preparing a rabies subtype pseudovirus, characterized in that: The preparation method comprises the following steps: S1. Clone the synthetic gene (1) shown in SEQ ID NO.1 into a linearized vector to obtain a plasmid (1); S2, cloning the synthetic gene (2) shown in SEQ ID NO.3 into a linearized vector to obtain a plasmid (2); S3, transfect cells to obtain rabies subtype pseudovirus; The plasmids used to transfect cells in step S3 are mixed transfection plasmids, and the mixed transfection plasmids include plasmid (1), plasmid (2) and psPAX2.

2. The preparation method according to claim 1, characterized in that: The linearized vectors described in steps S1 and S2 were obtained by digesting pCAGGS with BamHI / EcoRI.

3. The preparation method according to claim 1, characterized in that: The cells described in step S3 are 293T cells.

4. The rabies subtype pseudovirus prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the rabies subtype pseudovirus according to claim 4 in the preparation of a reagent for detecting neutralizing antibodies against rabies virus.

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