Use of GSK3β in enhancing Seneca virus replication for vaccine preparation

Abstract

The present invention belongs to the field of molecular biology technology, and provides the use of GSK3β in enhancing Seneca virus replication for the preparation of vaccines. A method for increasing the titer of Seneca virus in host cells for vaccine preparation is also provided: (1) constructing a host cell overexpressing GSK3β; (2) culturing the host cell overexpressing GSK3β and inoculating Seneca virus. The present invention successfully constructs the expression vector pCMVMyc‑GSK3β of GSK3β by molecular biology technology; and constructs a BHK‑21 cell line overexpressing the GSK3β gene; after inoculation of Seneca virus, the cell line has an accelerated viral replication rate and a significantly increased viral titer, and can be used for the commercial production of Seneca vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and specifically relates to the use of GSK3β in enhancing Seneca virus replication for preparing vaccines. Background Art

[0002] Glycogen synthase kinase 3 (GSK3) is a protein with serine / threonine kinase activity, encoded by two different genes: GSK3α and GSK3β. It has more than 100 known substrates and is therefore involved in a variety of cell signaling pathways, but it is a little-known signaling molecule in the field of virus research. However, recent studies have shown that GSK3β, as a substrate of AKT, can affect the process of influenza virus entry into cells, while inhibiting the upstream signaling molecule PI3K of GSK3β can enhance the replication of porcine epidemic diarrhea virus; for herpes viruses, the PI3K / AKT / GSK3β signaling pathway can be used to promote their replication and survival. In addition, coxsackievirus infection can stimulate GSK3β activity through a tyrosine kinase-dependent mechanism, which in turn promotes cell apoptosis by inhibiting the activity of β-catenin. In summary, GSK3β may play a key role in the response to viral infection, replication, and pathogenesis.

[0003] Senecavirus A (SVA), formerly known as Seneca Valleyvirus, was renamed Senecavinus A by the International Committee on Taxonomy of Viruses (ICTV) in 2016. It is the only member of the Senecavirus genus of the Picornaviridae family. In 2007, an outbreak of primary vesicular disease in pigs occurred in Canada. Pathogen testing for vesicular disease determined that pathogens such as foot-and-mouth disease and swine vesicular disease were negative, while SVA was positive. Therefore, SVA is the pathogen causing primary vesicular disease in pigs. Initially, the virus was occasionally detected in pigs, but there were no reports of the virus causing disease. Therefore, Seneca is considered to be an emerging zoonotic disease. From November 2014 to early 2015, the disease broke out in Brazil and the United States. Since then, with cases appearing in new locations every year, SVA may become a globally prevalent virus.

[0004] Vaccines are the main method for preventing SVA; however, there is no Seneca vaccine available in China. SVA can replicate in baby hamster kidney cells BHK-21 (Baby hamster kidney cell) and can be used to produce inactivated vaccines. Making BHK-21 cells more conducive to Seneca virus replication and obtaining Seneca virus with high virus titer is conducive to the production of Seneca virus inactivated vaccines. Summary of the invention

[0005] In order to improve the replication of Seneca virus in the host cells for vaccine production, the present invention provides a method for preparing a vaccine by using the GSK3β gene to enhance the replication of Seneca virus, by constructing a eukaryotic expression vector pCMV-Myc-GSK3β, and transfecting it into the host cells for producing Seneca virus to obtain Seneca virus with high virus titer. The method is easy to produce, low in cost, and high in efficiency in increasing the number of viruses.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A use of GSK3β in enhancing Seneca virus replication for preparing a vaccine.

[0008] The application is to culture Seneca virus in host cells overexpressing GSK3β.

[0009] A method for increasing the titer of Seneca virus in host cells for vaccine preparation comprises the following steps:

[0010] (1) Construction of host cells overexpressing GSK3β;

[0011] (2) Cultivating host cells that overexpress GSK3β and inoculating Seneca virus for further cultivation.

[0012] Preferably, the host cell is a BHK-21 cell.

[0013] Preferably, the continued culture time is not less than 12 hours. More preferably, the continued culture time is 24 hours.

[0014] Furthermore, the steps of constructing BHK-21 cells overexpressing GSK3β include:

[0015] i) Obtain the GSK3β gene of BHK-21 cells;

[0016] ii) connecting the GSK3β gene to a eukaryotic expression vector to construct a recombinant vector;

[0017] iii) The recombinant vector was transfected into BHK-21 cells to obtain BHK-21 cells overexpressing GSK3β.

[0018] The present invention has the following advantages:

[0019] The present invention successfully constructs the expression vector pCMV-Myc-GSK3β of GSK3β by using molecular biology technology; proves that the GSK3β gene has a significant effect of promoting the replication of Seneca virus; constructs a BHK-21 cell line overexpressing the GSK3β gene, and after inoculation with Seneca virus, the virus replication rate is accelerated and the virus titer is significantly improved, and the cell line can be used for the commercial production of Seneca vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the agarose gel electrophoresis diagram of PCR products, where Neg is the negative control, M is the DL2000 marker, and 1 and 2 are PCR amplification products;

[0021] Figure 2 Western blot results of GSK3β-overexpressing cells;

[0022] Figure 3 The results of q-PCR detection of viral replication at different time points;

[0023] Figure 4 The figures are the results of virus titer detection at different time points. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0025] Example 1 Construction of BHK-21 cells overexpressing GSK3β

[0026] 1. Amplification of GSK3β gene

[0027] According to the hamster GSK3β (XM_040750823.1) sequence, the primers in Table 1 were designed to amplify the entire GSK3β gene by adding two restriction sites, BamHI and XhoI, using the In-Fusion HD Cloning primer design program of TAKARA.

[0028] Table 1 GSK3β gene amplification primers (underlined are restriction sites)

[0029]

[0030] RNA was extracted from BHK-21 cells and transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, the GSK3β gene was amplified using 2×Phanta Flash Master Mix (Dye Plus) high-fidelity enzyme produced by Novazonics.

[0031] The electrophoresis of the amplified product is shown in Figure 1 As shown, the target band size is about 1300bp, which is in line with expectations. The target band was cut and recovered and purified using the EZNA Gel Extraction Kit. The sequencing was correct and stored at -20°C for future use.

[0032] 2. Construction of pCMV-Myc-GSK3β vector

[0033] The pCMV-Myc vector and PCR product were double-digested with BamH I and Xho I restriction endonucleases produced by NEB. The digested product was recovered and purified after electrophoresis, and the purified product was ligated using the In-Fusion Snap Assembly Master Mix enzyme kit from TaKaRa. The ligated product was transformed into Trelief TM 5α competent cells were screened, positive colonies were selected and expanded after sequencing, and AxyPrep TM Plasmid was extracted using Plasmid Miniprep Kit to obtain the recombinant vector pCMV-Myc-GSK3β.

[0034] 3. Construction of BHK-21 cells overexpressing GSK3β

[0035] The obtained pCMV-Myc-GSK3β plasmid was transfected into BHK-21 cells using the transfection reagent of Norvegic. After 48 hours, the cells were harvested and lysed to extract the total protein. The Mock transfected with pCMV-Myc plasmid was used as a control and β-action was used as an internal reference. Western blot analysis was performed using mouse anti-GSK3β antibody. The results of Western blot detection are shown in Figure 2. Figure 2 As shown: There is basically no difference in the expression of β-action between Mock cells and BHK-21 cells overexpressing GSK3β, but in cells overexpressing GSK3β, the expression level of GSK3β increased significantly, indicating that the construction of BHK-21 cells overexpressing GSK3β was successful.

[0036] Example 2 Effect of cells overexpressing GSK3β on SVA replication

[0037] 1. Real-time fluorescence quantitative PCR detection of SVA RNA content

[0038] BHK-21 cells overexpressing GSK3β were cultured and inoculated with SVA virus. At 12, 24, and 36 h after SVA infection, viral RNA at different infection time points was extracted using the Simply P Virus DNA / RNA Extraction Kit produced by Wuhan Bio-Technology Co., Ltd. After being reversed into cDNA, q-PCR was used to detect the replication amount of Seneca virus in the cells. The primer sequences are shown in Table 2.

[0039] Table 2 Real-time fluorescence quantitative PCR primers

[0040]

[0041] The results are as follows Figure 3As shown: Compared with normal BHK-21 cells, SVA mRNA in GSK3β-overexpressing cells was significantly increased at 24 h and 36 h, but was lower than that in the control group at 12 h.

[0042] 2. Virus titer detection

[0043] The virus titer was determined using the tissue half-infectious dose (TCID50). After the cells were inoculated, they were cultured in a 37°C 5% CO2 cell culture incubator. After 72 hours, the number of cell wells in the 96-well cell culture plate that produced cytopathic effects for each virus was recorded; the virus titer was calculated using the Reed-Muench method. The results are shown in Figure 4 As shown: The SVA progeny virus titer in the GSK3β-overexpressing cell line was significantly higher than that in the normal cell group at 24 h and 36 h, but was lower than that in the control group at 12 h.

[0044] The stage before 12 h belongs to the virus infection stage, and the mRNA and virus titer of SVA in the GSK3β-overexpressing cell line did not increase significantly, but increased significantly during the virus replication stage at 24 h and 36 h. This indicates that GSK3β plays a role in the virus replication stage, and overexpression of GSK3β in host cells can promote the replication of SVA.

Claims

1. Application of GSK3β to enhance Seneca virus replication in the preparation of Seneca virus vaccine, characterized in that: Senecavirus was cultured in host cells overexpressing GSK3β; The host cell is a BHK-21 cell; The culturing time is not less than 24 hours.

2. A method for increasing the titer of Seneca virus for preparing a Seneca virus vaccine, characterized in that: The following steps are involved: (1) Construction of host cells overexpressing GSK3β; (2) Cultivating host cells that overexpress GSK3β and inoculating them with Seneca virus for further cultivation; The host cell is a BHK-21 cell; The continued culturing time is no less than 24 hours.

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