CN116036258A

CN116036258A - Baculovirus live vector vaccine based on SARS-CoV-2S protein and application thereof

Info

Publication number: CN116036258A
Application number: CN202211569557.8A
Authority: CN
Inventors: 芦宝静; 李报; 王倩云; 刘雪娟; 彭晖; 朱彬彬
Original assignee: Anhui Medical University
Current assignee: Anhui Medical University
Priority date: 2022-12-08
Filing date: 2022-12-08
Publication date: 2023-05-02

Abstract

The invention relates to the technical field of gene recombination and vaccine, in particular to a construction method and application of a recombinant baculovirus expressing SARS-CoV-2S gene. In the invention, a CMV promoter is constructed to replace a baculovirus promoter and two baculoviruses with non-modified promoters to obtain recombinant viruses vAc-S and vAc-S-CMV, so that the mode of introducing S genes through baculoviruses and expressing S proteins on the surfaces of the baculoviruses is studied, and cellular immunity and humoral immunity reactions in mice are induced after the two recombinant baculoviruses are injected into the mice, thereby providing a new idea for developing SARS-CoV-2 vaccine.

Description

Baculovirus live vector vaccine based on SARS-CoV-2S protein and application thereof

Technical Field

The invention relates to the technical field of gene recombination and vaccine, in particular to a recombinant baculovirus expressing SARS-CoV-2S gene, a construction method and application thereof.

Background

Baculovirus (BV), an enveloped virus with a circular double-stranded DNA genome, is currently most studied in three species, namely a baculovirus polyhedra baculovirus (Autographa californica multiple nuclear polyhedrosis virus, acMNPV) AcMNPV, which is widely used mainly because of its advantages: 1) Can correctly translate and modify; 2) Insertion of multiple genes or one large fragment can be accommodated; 3) Safety, no infection to human beings; 4) With the P10 and polyhedrin promoters. Baculovirus can infect a variety of mammalian cells except blood cell lines, making it a backup army for gene therapy. Because baculovirus has the characteristics of no toxicity to human and mammal cells, high transduction efficiency and the like, the expression vector constructed by using the baculovirus has great advantages in the aspects of vaccine development, foreign protein expression, gene therapy and the like.

The invention mainly aims at the structural protein S protein of SARS-CoV-2 to carry out immune research, and uses the modified and unmodified baculovirus as a vector to express the S protein in a mouse body so as to research the immune response of the baculovirus in the mouse body, thereby providing a theoretical basis for clinical research of vaccines.

In the invention, we construct CMV promoter to replace baculovirus promoter and two baculovirus expression human SARS-CoV-2S genes with unmodified promoter to obtain recombinant viruses vAc-S and vAc-S-CMV, and discuss the cell immunity and humoral immunity reaction in mice caused by directly injecting the recombinant baculovirus constructed in the two ways into the mice.

Disclosure of Invention

The main purpose of the invention is to construct safe and efficient vaccine targeting SARS-CoV-2, and the modified and unmodified baculovirus is used as a vector to express S protein in mice, which is expected to become a candidate way for preparing severe acute respiratory distress syndrome 2 vaccine. Providing a recombinant transfer vector pFB-S-CMV, pFB-S. Provides SARS-CoV-2 bacterium artificial chromosome recombinant plasmid cAcFB-S, cAcFB-S-CMV. Recombinant baculovirus vAc-S-CMV, vAc-S, is provided. A method for constructing recombinant virus SARS-CoV-2S is provided. Provides the application of recombinant SARS-CoV-2 virus. The aim of the invention is realized by the following technical scheme:

the full length of SARS-CoV-2 genome is about 29.9kbp. Comprising a 5 'cap structure, ORF1a and ORF1b, pp1a, pp1ab encoded by ORF1a and ORF1b, which was further hydrolysed to the remaining ORF region of 16 nonstructural proteins (nonstructural proteins, nsps) encoding 8 helper proteins (3 a,3b, p6,7a,7b,8b,9b and ORF 14) and four structural proteins S, E, M, N,3' poly (A) tails. S protein is type I fusion membrane protein, its main function is to recognize receptor, and mediate virus to enter host cell. The S protein of SARS-CoV-2 consists of two subunits, S1 and S2, furin cleaves the S protein into the S1 and S2 subunits, S1 contains a Receptor Binding Domain (RBD) that binds to the cellular receptor responsible for the binding of the virus to the receptor angiotensin converting enzyme 2 (ACE 2). The S protein has the characteristic of high conservation, so that the S protein becomes a target point for vaccine development.

The invention has the beneficial effects that: in the invention, recombinant viruses vAc-S and vAc-S-CMV are obtained by constructing a non-modified baculovirus and a modified mammalian promoter element CMV promoter to replace two baculovirus expression human SARS-CoV-2S genes modified by the original promoter, and the vaccine has high virus inhibition rate and good safety performance, and provides a new thought and new method for preventing and treating new coronaviruses.

Drawings

FIG. 1 is a schematic diagram of recombinant baculovirus vector construction.

FIG. 2 shows fragments obtained by double cleavage of recombinant plasmids KpnI and Xhol I.

FIG. 3 transformation of recombinant plasmids into DH10Bac competent cells, colony PCR after blue-white screening identified recombinant cAc-S/cAc-S-CMV bacids.

FIG. 4 recombinant cAc-S/cAc-S-CMV bacmides transfected into sf9 cells and the expression of S protein was detected by western-blot of viral transduction BHK cells.

FIG. 5 ELISA detected IgG from SARS CoV-2 anti-S protein in mouse serum at

day

0, 14, 28, 42, 52 after immunization of mice. Wherein the PBS group-specific antibodies remained at a lower level, and the vAc-S and vAc-S-CMV groups had a significant rise in antibodies 28 days after immunization. On day 52, the antibody level of the vAc-S and vAc-S-CMV immune groups was 30-fold higher than that of the PBS control group, and there was no significant difference between the groups. The antibody level of vAc-S was slightly lower, but increased after the second boost. The empty vector control group vAc-WT was immunized for 28 days, after 42 days, a 3.2-fold difference in antibody level was detected compared to the PBS group, and after 52 days, a 1.4-fold difference was detected compared to the immunized group.

FIG. 6vAc-S-CMV immune group CD4+ positive cells produced IFN-gamma levels higher than IL-4.

FIG. 7vAc-S-CMV immune group CD8+ positive cells produced IFN-gamma levels higher than IL-4.

FIG. 8, 10 days after the last immunization, shows that the sera of mice immunized with PBS, vAc-WT, and vAc-S, vAc-S-CMV all produced effective neutralizing antibodies by using a pseudo-viral system, and that the neutralizing antibody titer produced by vAc-S immunized mice was higher than that produced by vAc-S-CMV immunized mice.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following examples are provided for the sole purpose of illustrating the principles of the present invention; they are in no way intended to limit or narrow the scope of the present invention.

EXAMPLE 1 modification of the transfer vector pFastBac Dual (FIG. 1)

pFastBac Dual was digested with BamHI and SmalI, and the recovered fragment was digested with the same digested pT-CMV vector fragment overnight at 4℃to yield pFB-CMV, which was transformed into E.coli DH 5. Alpha. And then digested and identified to yield a positive clone designated pFB-CMV. The recombinant transfer vector pFB-S-CMV was constructed by cloning the S gene into the pFB-CMV plasmid using KpnI, xhol I site.

The transfer vector pFB-S was obtained by cloning the S gene into the pP10 promoter of the pFastBac Dual vector using KpnI, xholI double cleavage sites.

Example 2 double restriction identification of recombinant plasmids (FIG. 2)

And (3) performing agarose nucleic acid electrophoresis gel running verification after 2h of action at 37 ℃. 0.3g agarose is dissolved in 30ml1xTAE, heated by high fire in a microwave oven for 1min until no visible particles in the solution are poured into a nucleic acid electrophoresis tank, a comb is inserted, and the solution is left for 30min until solidification is carried out for sample detection. After the double enzyme digestion reaction is finished, 10x loading buffer,90V voltage electrophoresis is added for 30min, and the gel is put into EB dye to be soaked for 30min, and then the result is observed.

EXAMPLE 3 construction of recombinant baculoviruses (FIG. 3)

The recombinant plasmid successfully identified by double enzyme digestion is transformed into DH10Bac cells to construct recombinant Bacmids, and the method is briefly described as follows: 2 competent cells were removed from-80℃and inserted into ice until they had thawed. pFB-S, pFB-S-CMV (1 ug) plasmids to be transformed are respectively added into competent cells, the competent cells are flicked, an EP tube is placed on ice for reaction for 30min, then the EP tube is placed into a water bath kettle at 42 ℃ for heat activation for 90S, the EP tube is immediately placed into ice for standing for 2-3min, 900ul of preheated SOC culture medium is respectively added into the EP tube for uniform mixing, shaking table 220rpm at 37 ℃ for 4h is then carried out for 12000rpm for centrifugation for 5min, 800ul of supernatant is discarded, and the sediment is respectively coated on solid culture medium containing 100ug/ml X-gal, 40ug/ml IPTG, 7ug/ml Gen+, 50ug Kan+, 10ug Tet+ after being placed for 30min at 37 ℃, and the culture dish is inverted for culture overnight at 37 ℃. pFB-S picks up 4 white spots, pFB-S-CMV bacteria picks up 5 white spots and 1 blue spot, and adds them into LB culture medium containing Gen+, kan+ resistance to shake bacteria at 37 ℃, and M13 universal primer and gene specific primer for daily use are used for PCR identification, the system is as follows:

PCR reaction system

The successfully identified recombinant virus plasmid is transfected into sf9 cells, and after culturing for 4-5 days at 26 ℃ to obtain recombinant baculovirus, the virus is amplified, and the virus is purified by ultracentrifugation, 100000g and 4 ℃ for 4 hours. Purified viruses were titered using TCID50 and stored for detoxification.

EXAMPLE 4 recombinant baculovirus transduced BHK cells Western blot detection of recombinant baculovirus transduced BHK cells S protein expression (FIG. 4)

A suitable amount of BHK cells was plated on 35mm cell culture dishes, incubated overnight at 37℃until the density reached 65%, the medium was aspirated, 100MOI of recombinant baculovirus was added and 500ul was made up with Grace' and adsorbed for 2 hours at 37 ℃. The virus solution was aspirated, washed once with DMEM, 2ml of DMEM+10% FBS was added, 10mM sodium butyrate was added, and the result was observed after incubation at 37℃for 24 hours. BHK cells were collected 24h after transduction by the method described in section 2.4.6 of the first section. After transfer, the membrane was blocked for 2h at room temperature in TBST containing 5% nonfat milk powder, then incubated overnight at 4℃in S monoclonal antibody of SARS CoV-2 (1:1000 dilution), washed and incubated on a shaker in goat anti-rabbit anti-IgG (1:10000, zhonghua gold bridge) for 2h, and finally developed by chemiluminescence.

EXAMPLE 5 immunization of animals (FIG. 5)

BALB/c female mice were purchased from university animal experiment center of Anhui medical science at 8-12 weeks. They were randomly divided into four groups of 6. vAc-S or vAc-S-CMV virus, vAc-WT and PBS were injected as controls, respectively. The total injection of 7.5×106 viruses is carried out by subcutaneous and intraperitoneal injection, and the total injection is carried out for 4 times every 2 weeks; the inner canthus of the mice was bled before each injection and the neutralizing antibody titer and serum IgG levels were measured.

EXAMPLE 6 spleen lymphocyte isolation culture

After 10 days of final immunization, the spleen of the mice is taken out under aseptic condition, 4ml of lymphocyte separation liquid is added into a 35mm cell culture dish, the spleen is homogenized on a 200-mesh nylon net by a sterile syringe piston, then the spleen is added into a 15ml aseptic centrifuge tube, 500 mu l of RPM I1640 culture liquid is added on the surface layer of the separation liquid, the horizontal rotor is centrifuged for 30min at 800g, cells on the layer where the lymphocytes are positioned are sucked into the 15ml centrifuge tube, 10ml of RPM I1640 are added for uniform mixing, the horizontal rotor is centrifuged for 10min at 250g, the supernatant is discarded and then resuspended in 1ml of RPMI 1640 containing 10% FBS, and a blood cell counting plate is counted for standby.

Example 7 flow cytometry detection (FIGS. 6, 7)

After counting the isolated splenic lymphocyte resuspension, 1X 106 cells were added to a 24-well plate per well, and a mixture of the corresponding specific antigen (S antigen 2. Mu.g/ml) and human CD28 antibody (5. Mu.g/well) was added as a co-stimulator for 5h incubation. According to the instructions of Cytofix/Cytoperm W/Golgi Stop Kit, golgi Stop (0.5 μl/ml) was added for 4h blocking at 37℃and 1ul of FITC-CD4 and 1.25ul of APC-CD8a were added, after light-resistant staining at 4℃for 30min PBS washing, fixation, membrane rupture and intracellular PE-IFN-gamma and PE/Cy7-IL-4 staining for 30min, 500ul of BD 1XPerm/wash buffer was added for resuspension, centrifugation at 1000rpm for 5min and supernatant was discarded for resuspension in Flow cytometry Staining buffer for on-machine detection (Beckman, EPICS ALTRA II, USA).

Example 8 neutralization assay (FIG. 8)

293T-ACE2 cells were plated at 4X 105/ml in 96 well plates at 100ul per well and incubated at 37℃until a density of 40-50% was reached for use. Inactivating the serum of a mouse to be detected at 56 ℃ for 30min, diluting the serum by a DMEM (medium-concentration electron beam) from 1:4 to 1:128 in a multiple ratio, and adding pseudovirus (the final dose is 1ul per hole) into the diluted serum to react for 1h at 37 ℃; absorbing and discarding the cell culture supernatant of the 96-well plate, adding the neutralized mixed solution, and absorbing for 2 hours at 37 ℃; the supernatant was removed, 100 ul/well of DMEM+10% FBS complete medium was added, incubated in an incubator containing 5% CO2 at 37℃for 48 hours, and then operated according to the Biyundian kit, 100ul Bright-Lumi II firefly luciferase assay reagent was added to each well, and after 5min of room temperature reaction, luciferase activity was detected by using a multifunctional microplate assay system (BioTek, CYTATION 5) to calculate the neutralizing antibody titer at the highest antibody sparsity for 50% inhibition of viral infection.

Claims

1. A baculovirus expression SARS-CoV-2 live virus vector vaccine, which is characterized by comprising recombinant transfer vectors pFB-S-CMV, pFB-S; SARS-CoV-2 bacterium artificial chromosome recombinant plasmid cAcFB-S, cAcFB-S-CMV; recombinant baculovirus vAc-S-CMV, vAc-S.

2. Use of a baculovirus-expressed SARS-CoV-2 live viral vector vaccine as defined in claim 1 in the field of novel coronavirus prophylaxis.