Preparation and application of forest encephalitis virus (TBEV) pseudovirus particles

Abstract

According to the invention, a Chinese forest encephalitis virus inactivated vaccine is adopted to produce a strain'senzheng 'strain gene sequence, a structural protein preM-E gene sequence of the strain is amplified, an HIV-1 lentivirus packaging system is utilized, preM-E is used as a pseudovirus particle envelope protein, green fluorescent protein (EGFP) or luciferase reporter gene (Luciferae) is used as a detection indication signal to prepare the pseudovirus, and a BHK21 cell is used as a target cell; the pseudovirus-BHK21 cell system is used for replacing live virus operation experiments such as vaccine potency evaluation, neutralizing antibody detection (animal method or plaque reduction neutralization test) and the like, the biosafety operation environment is degraded from BSL-3 to BSL-2 by using pseudoviruses, the detection period is shortened, and animal experiments are reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of forest encephalitis virus pseudovirus particle research, and particularly relates to a preparation of forest encephalitis virus (TBEV) pseudovirus particle and application thereof. BACKGROUND

[0002] Forest encephalitis virus is transmitted by ticks and is also called tick-borne encephalitis virus (TBEV), which belongs to the Flavivirus genus of the Flaviviridae family and includes five serotypes. The main epidemic strain in China is the Far East serotype. TBEV has strong neurotropic toxicity, and the mortality rate of severe infection is extremely high, and the irreversible sequelae are extremely serious. In the list of pathogenic microorganisms that can be transmitted between humans, the degree of harm is ranked as the most serious first class, and virus culture, animal challenge, and waste material treatment must be operated in a BSL-3 laboratory. At present, there is no specific treatment for forest encephalitis in the clinic, and vaccination is the most effective preventive measure.

[0003] At present, the global forest encephalitis vaccine is a whole virus inactivated vaccine, and only one vaccine production enterprise in China uses live virus for evaluation in the vaccine production process, such as vaccine potency evaluation and neutralizing antibody detection. The operation environment, detection period, and technical quality of personnel are extremely high.

[0004] At present, there are two methods for forest encephalitis vaccine potency evaluation and neutralizing antibody detection.

[0005] 1. In China, the potency of forest encephalitis vaccine is determined (i.e., potency evaluation) according to the third part of the People's Republic of China Pharmacopoeia (2025 edition), the animal protection law is used to calculate the animal protection index, and the specific steps are as follows: vaccine immunization of animals---virus challenge---calculation of survival rate---conversion of vaccine immunization protection index (should be greater than 105).

[0006] 2. Plaque reduction neutralization test (PRNT), by observing the change in the number of plaques formed by antibodies in cell culture, the neutralizing effect of antibodies on viruses is quantitatively analyzed to evaluate the immunogenicity of vaccines and the detection of neutralizing antibody titers.

[0007] At present, pseudovirus neutralization test is used for antibody detection of new crown and Zika viruses. There is no such method applied to forest encephalitis virus or related research.

[0008] Both the existing animal method and the PRNT method need to use live viruses. First, the use of live viruses in the detection of amplification, calibration, management, supervision, destruction and other aspects is not safe, time-consuming and labor-intensive. Second, the antibody determination operation has high requirements for the environment and personnel skills, and the detection time is long. Finally, the animal challenge protection experiment is prone to distortion of the challenge model, and the experimental challenge dose is often much higher than the natural infection in humans, resulting in excessive pathological damage. The sensitivity of the cell plaque reduction neutralization experiment depends on the plaque size, and there is a large difference between operators, with low consistency. SUMMARY

[0009] The present application designs and develops a preparation of forest encephalitis virus pseudovirus particles and its application. The present application uses an HIV-1 four-plasmid co-transfection system to prepare forest encephalitis pseudovirus, uses preM-E as a pseudovirus particle envelope protein, uses green fluorescent protein (EGFP) or luciferase reporter gene (Luciference) as a detection indicator signal to prepare a pseudovirus, and uses BHK21 cells as target cells for TBEV neutralizing antibody detection. The purpose is to solve the problems of not using live viruses, reducing safety, quantitatively detecting indicators, not using animals, and being widely used.

[0010] The technical scheme provided by the present application is as follows:

[0011] A preparation of forest encephalitis virus pseudovirus particles comprises the following steps:

[0012] Step one: using a lentivirus packaging system containing a virus packaging auxiliary plasmid and a tool vector plasmid carrying a target gene to transfect HEK293T cells;

[0013] Step two: after transfection, the supernatant is collected for the first time to remove cell debris, and then the second centrifugation is performed, complete DMEM medium is added for resuspension, filtration is performed, and the forest encephalitis virus pseudovirus particles are obtained by aliquot freezing storage. BHK21 cells are used to determine the virus TCID50.

[0014] Preferably, in the step one, the virus packaging auxiliary plasmid includes pRSV-Rev, pMDLg / pRRE, and

[0015] pCDH-EGFP or pCDH-luci.

[0016] Preferably, in the step one, the tool vector plasmid carrying the target gene is pCMV-E, pCMV-preM-E, or pCMV-preM-E-NS1.

[0017] Preferably, in the step two, the first centrifugation condition is 6000r / min centrifugation for 30min at 6℃.

[0018] Preferably, in the step two, the second centrifugation condition is 20000r / min centrifugation for 2h at 6℃.

[0019] Preferably, in the step two, the freezing temperature is -80℃.

[0020] Preferably, in the step two, the supernatant is collected after 48h transfection.

[0021] Preferably, in the step two, the freezing temperature is -80℃.

[0022] A preparation of forest encephalitis virus pseudovirus particles is used in a forest encephalitis virus neutralizing antibody assay.

[0023] Preferably, the method comprises the following steps:

[0024] A 96-well plate is used, the BHK21 cell density is 1X10 5 / ml, the pseudovirus using titer is 100TCID50 / ml, the neutralization temperature is 37℃, the neutralization time is 1h, and the BHK21 cell infection time is 48-72h.

[0025] The beneficial effects of the present application are:

[0026] The TBEV pseudovirus neutralization experiment method constructed in the present study has the advantages of safe operation, low cost, and friendly to experimental animals, and is expected to be applied to the immunogenicity evaluation link of TBEV inactivated vaccine in the future, thereby helping vaccine production enterprises to reduce production cost and reduce the operation risk of vaccine quality inspection personnel. DETAILED DESCRIPTION

[0027] The present application will be further described in detail below, so that those skilled in the art can implement it according to the description.

[0028] The present application discloses a preparation of forest encephalitis virus (TBEV) pseudovirus particles, which adopts the gene sequence of "Sen Zhang" strain of Chinese forest encephalitis virus inactivated vaccine production strain, amplifies the structural protein preM-E gene sequence, uses HIV-1 lentivirus packaging system, takes preM-E as a pseudovirus particle envelope protein, takes green fluorescent protein (EGFP) or luciferase reporter gene (Luciference) as a detection indicating signal to prepare a pseudovirus, and takes BHK21 cells as target cells; specifically including:

[0029] The application adopts an HIV-1 four-plasmid co-transfection system (pRSV-Rev, pMDLg / pRRE, pCDH-EGFP or pCDH-luci, pCMV-E, or pCMV-preM-E, or pCMV-preM-E-NS1), transfects HEK293T cells, and packages to prepare three kinds of TBEV pseudovirus particles with EGFP or Luciference protein genes and different TBEV envelope proteins, and screens suitable envelope proteins, and the three kinds of envelope protein genes are cloned into pCMV plasmids, including: (1) a pseudovirus with only E protein in the envelope (hereinafter referred to as E); (2) a pseudovirus with prM protein and E protein in the envelope (hereinafter referred to as preM-E); and (3) a pseudovirus with prM protein, E protein and NS1 protein in the envelope (hereinafter referred to as preM-E-NS1).

[0030] The EGFP or Luciference gene is cloned into the pCDH-CMV-MCS-EF1-puro plasmid as an overexpression detection indicator signal.

[0031] The TBEV pseudovirus prepared in the application is identified by Western Blot and electron microscopy observation, and the three kinds of envelope protein pseudoviruses are respectively infected into BHK-21 cells, Vero cells and 293T cells to screen sensitive cell lines.

[0032] The application determines that the preM-E envelope pseudovirus is a detection virus, and BHK21 is a detection cell.

[0033] The application determines virus titer, detection procedures and other operation links.

[0034] The application method has higher correlation compared with the animal method and PRNT.

[0035] The application further discloses a preparation and application of a forest encephalitis virus (TBEV) pseudovirus particle, a pseudovirus-BHK21 cell system, which is used for replacing vaccine efficacy evaluation, neutralizing antibody detection (animal method or plaque reduction neutralization test) and other live virus operation experiments, using the pseudovirus to degrade the biological safety operation environment from BSL-3 to BSL-2, reduce the detection period and reduce animal experiments.

[0036] Embodiment

[0037] The application provides a preparation of a forest encephalitis virus pseudovirus particle, including the following steps:

[0038] Step one, pseudovirus packaging: adopt the virus packaging auxiliary plasmid pRSV-Rev, pMDLg / pRRE, pCDH-luciferase plasmid and tool vector plasmid pCMV-prM-E plasmid carrying target gene to be introduced into HEK293T cells by using a lentivirus packaging system;

[0039] Step two, pseudovirus preparation and calibration: collect the supernatant after transfection for 48h, centrifuge at 6000r / min for 30min at 6℃ to remove cell debris, adopt ultracentrifugation, centrifuge at 20000r / min for 2h at 6℃, discard the supernatant, resuspend with complete DMEM medium, filter through a 0.22μM filter, then distribute, store at-80℃, and determine the virus TCID50 by using BHK21 cells.

[0040] The application also provides a preparation of forest encephalitis virus pseudovirus particles, which comprises: neutralizing antibody determination: 96-well plates, BHK21 cell density is 1X10 5 / ml, the pseudovirus is used at a titer of 100TCID50 / ml, the neutralization condition is 37℃, 1h, and the BHK21 cell infection time is 48-72h.

[0041] The application first establishes a forest encephalitis virus pseudovirus detection method, and no such method for the virus has been reported; the virus strain "Sen Zhang" strain for producing vaccines in China is first applied to prepare the pseudovirus; the TBEV preM-E fusion protein is first determined as the pseudovirus envelope protein; the pseudovirus detection system is first applied to TBEV neutralizing antibody determination; the forest encephalitis pseudovirus is first used to establish an in vitro neutralization experiment method, and the consistency and correlation between the method and the current forest encephalitis inactivated vaccine evaluation method are successfully verified.

[0042] The application replaces the current vaccine evaluation and neutralizing antibody detection method: no live virus is used, and the safety is high; the detection index is quantified, comparable and standardizable by observing or reading the EGFP or luciference reading; the repeatability is good, and the method is easy to popularize and inter-laboratory evaluation; no animals are used, and no animal ethics is involved.

[0043] Although the embodiments of the application have been disclosed as above, it is not limited to the application listed in the specification and the embodiments, and can be fully applied to various fields suitable for the application, and additional modifications can be easily realized by those skilled in the art, and therefore the application is not limited to specific details under the general concept defined by the claims and the equivalent scope.

Claims

1. Preparation of pseudovirus particles of tick-borne encephalitis virus, characterized in that, Includes the following steps: Step 1: Transfect HEK293T cells into a lentiviral packaging system containing a viral packaging helper plasmid and a tool vector plasmid carrying the target gene; Step 2: After transfection, the supernatant was collected and centrifuged for the first time to remove cell debris. Then, a second centrifugation was performed, and the cells were resuspended in complete DMEM medium. After filtration, the cells were aliquoted and cryopreserved to obtain tick-borne encephalitis virus pseudovirus particles. The viral TCID50 was measured using BHK21 cells.

2. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 1, characterized in that, In step one, the virus-containing packaging helper plasmid includes pRSV-Rev, pMDLg / pRRE, and pCDH-EGFP or pCDH-luci.

3. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 1 or 2, characterized in that, In step one, the tool vector plasmid carrying the target gene is pCMV-E, pCMV-preM-E, or pCMV-preM-E-NS1.

4. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 3, characterized in that, In step two, the first centrifugation condition is 6000 r / min for 30 min at 6℃.

5. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 3, characterized in that, In step two, the second centrifugation conditions are: centrifugation at 20000 r / min for 2 hours at 6°C.

6. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 4 or 5, characterized in that, In step two, the sample is filtered using a 0.22μM filter and then aliquoted and frozen for storage.

7. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 6, characterized in that, In step two, the supernatant is collected 48 hours after transfection.

8. The preparation of the pseudovirus particles of tick-borne encephalitis virus as described in claim 6, characterized in that, In step two, the freezing temperature is -80°C.

9. The application of a preparation of tick-borne encephalitis virus pseudovirus particles in the determination of tick-borne encephalitis virus neutralizing antibodies, characterized in that, Preparation of tick-borne encephalitis virus pseudovirus particles as described in claims 1-8.

10. The application of the preparation of tick-borne encephalitis virus pseudovirus particles as described in claim 9 in the determination of tick-borne encephalitis virus neutralizing antibodies, characterized in that, Includes the following steps: Using 96-well plates, BHK21 cells were spaced at a density of 1 x 10⁻⁶. 5 / ml, the pseudovirus titer was 100 TCID50 / ml, the neutralization temperature was 37℃, the neutralization time was 1h, and the BHK21 cell infection time was 48-72 hours.