CN116162599A - Use of GSK3 beta for enhancing replication of sai virus - Google Patents

Abstract

The invention belongs to the technical field of molecular biology and provides the application of GSK3β in enhancing the replication of Seneca virus. Also provided is a method for increasing the titer of the Seneca virus: (1) constructing a host cell overexpressing GSK3β; (2) cultivating the host cell overexpressing GSK3β and inoculating the Seneca virus. The present invention successfully constructs the expression vector pCMV-Myc-GSK3β of GSK3β by using molecular biology technology; and constructs the BHK-21 cell line overexpressing the GSK3β gene; after the cell line is inoculated with Seneca virus, the virus replication rate is accelerated, The virus titer is significantly increased and can be used for commercial production of Seneca vaccine.

Description

Use of GSK3β in enhancing Seneca virus replication

technical field

The invention belongs to the technical field of molecular biology, and specifically relates to the application of GSK3β in enhancing the replication of Seneca virus.

Background technique

Glycogen synthase kinase 3 (GSK3), a protein with serine/threonine kinase activity encoded by two distinct genes: GSK3α and GSK3β, has more than 100 known substrates and is therefore involved in a variety of cellular signaling pathway, 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 virus, PI3K/ The AKT/GSK3β signaling pathway promotes its replication and survival. In addition, Coxsackievirus infection can stimulate GSK3β activity through a tyrosine kinase-dependent mechanism, which in turn promotes apoptosis by inhibiting β-catenin activity. Taken together, GSK3β may play a key role in the response to viral infection, replication, and pathogenesis.

Seneca virus (Senecavirus A, SVA), once known as Seneca Valley virus (Seneca Valleyvirus), was renamed Senecavinus A by the International Committee on Taxonomy of Viruses (ICTV) in 2016. The only member of the Cavirus genus. Porcine primary vesicular disease broke out in Canada in 2007. The pathogen detection of vesicular disease confirmed that the pathogens such as foot-and-mouth disease and porcine vesicular disease were negative and SVA was positive. Therefore, SVA is the pathogen that causes porcine primary vesicular disease. Initially, the virus was occasionally detected in pig herds, but there have been no reports of the virus causing disease. Therefore, Seneca is considered an emerging zoonotic disease. From November 2014 to early 2015, the disease broke out successively in Brazil and the United States. Since then, SVA has likely become a globally endemic virus, with cases emerging in new locations each year.

For the prevention of SVA, vaccines have become the most important way; however, there is currently no Seneca vaccine on the market in China. SVA can be replicated in BHK-21 (Baby hamster kidney cell) and can be used to produce inactivated vaccine. The BHK-21 cells are more conducive to the replication of Seneca virus, and the Seneca virus with high virus titer is obtained, which is beneficial to the production of Seneca virus inactivated vaccine.

Contents of the invention

In order to improve the replication of Seneca virus in host cells, the invention provides a method for enhancing the replication of Seneca virus by using the GSK3β gene, by constructing the eukaryotic expression vector pCMV-Myc-GSK3β, and transfecting it into the production Seneca High viral titers of Seneca virus were obtained in viral host cells. The method is easy to produce, low in cost, and highly efficient in increasing the number of viruses.

In order to achieve the above object, the present invention adopts the following technical solutions.

A use of GSK3β to enhance Seneca virus replication.

The application is culturing Seneca virus in host cells overexpressing GSK3β.

A method for increasing the titer of Seneca virus, comprising the steps of:

(1) Construction of host cells overexpressing GSK3β;

(2) Culture host cells overexpressing GSK3β and inoculate Seneca virus for further culture.

Preferably, the host cell is BHK-21 cell.

Preferably, the time for continuing the culture is not less than 12 hours. More preferably, the time for the continuation of cultivation is 24h

Further, the steps of constructing BHK-21 cells overexpressing GSK3β include:

i) obtaining the GSK3β gene of BHK-21 cells;

ii) Connect the GSK3β gene to a eukaryotic expression vector to construct a recombinant vector;

iii) Transfect the recombinant vector into BHK-21 cells to obtain BHK-21 cells overexpressing GSK3β.

The present invention has the following advantages:

The present invention successfully constructs the expression vector pCMV-Myc-GSK3β of GSK3β by using molecular biology technology; proves that the GSK3β gene has an obvious effect of promoting the replication of Seneca virus; constructs the BHK-21 cell line overexpressing the GSK3β gene, and inoculates After Seneca virus, the virus replication rate is accelerated, and the virus titer is significantly increased, which can be used for commercial production of Seneca vaccine.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of PCR product, and wherein, Neg is negative control, M is DL2000marker, and 1, 2 are PCR amplification products;

Figure 2 is the Western blot results of GSK3β overexpression cells;

Figure 3 is the results of q-PCR detection of virus replication at different time points;

Figure 4 is the detection results of virus titers at different time points.

Detailed ways

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

Example 1 Construction of BHK-21 cells overexpressing GSK3β

1. Amplification of the GSK3β gene

According to the hamster GSK3β (XM_040750823.1) sequence, use TAKARA’s In-Fusion HD Cloning primer design program, add BamHI and XhoI restriction sites, and design the primers in Table 1 to amplify the entire GSK3β gene primers.

Table 1 GSK3β gene amplification primers (underlined restriction sites)

The RNA of BHK-21 cells was extracted and transcribed into cDNA using a reverse transcription kit. Using cDNA as a template, the GSK3β gene was amplified with 2 × Phanta Flash Master Mix (Dye Plus) high-fidelity enzyme produced by Novizyme. The electropherogram of the amplified product is shown in Figure 1. The target band size is about 1300bp, which is in line with expectations. The target band was cut with E.Z.N.A.Gel Extraction Kit and recovered and purified. The sequence was correct and stored at -20°C for future use.

2. Construction of pCMV-Myc-GSK3β vector

The pCMV-Myc vector and PCR product were double digested with BamH I and Xho I restriction enzymes produced by NEB. The digested products were recovered and purified after electrophoresis, and the purified products were ligated using the In-Fusion Snap AssemblyMaster Mix enzyme kit from TaKaRa Company. The ligation product was transformed into Trelief ^TM 5α competent cells and screened. The positive colonies were picked and sequenced correctly and expanded for culture. The plasmid was extracted with the AxyPrep ^TM Plasmid Miniprep Kit to obtain the recombinant vector pCMV-Myc-GSK3β.

3. Construction of BHK-21 cells overexpressing GSK3β

The obtained pCMV-Myc-GSK3β plasmid was transfected into BHK-21 cells using Novizyme’s transfection reagent, the cells were harvested for 48 hours, and the total protein was extracted after lysing. The Mock transfected with the pCMV-Myc plasmid was used as a control, and the β-action was used as an internal control, and Western blot analysis was performed with mouse anti-GSK3β antibody. The results of Western blot detection are shown in Figure 2: there is basically no difference in the expression of β-action between Mock cells and GSK3β-overexpressing BHK-21 cells, but in cells overexpressing GSK3β, the expression of GSK3β is significantly increased, indicating that overexpressing GSK3β BHK-21 cells The construction of -21 cells was successful.

Example 2 Effect of overexpressing GSK3β cells on SVA replication

1. Detection of SVA RNA content by real-time fluorescent quantitative PCR

BHK-21 cells overexpressing GSK3β were cultured and inoculated with SVA virus. At 12, 24 and 36 h after SVA infection, the virus at different time points was extracted using the Simply P virus DNA/RNA extraction kit produced by Wuhan Bioer Biotechnology Company. After the RNA was reversed into cDNA, the replication amount of Seneca virus in the cells was detected by q-PCR. The primer sequences are shown in Table 2.

Table 2 Real-time fluorescent quantitative PCR primers

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

2. Virus titer detection

The virus titer was determined by tissue half infectious dose (TCID ₅₀ ). After inoculation, the cells were cultured in a 37°C 5% CO ₂ cell incubator, and after 72 hours, the number of cell wells with cytopathic effects caused by each virus in the 96-well cell culture plate was recorded; the virus titer was calculated according to the Reed-Muench method. The results are shown in Figure 4: the virus titer of SVA progeny in the GSK3β overexpression cell line was significantly higher than that of the normal cell group at 24 h and 36 h, but lower than that of the control group at 12 h.

The stage before 12 h belongs to the stage of virus infection, and the mRNA and virus titer of SVA in the GSK3β overexpression cell line did not increase significantly, but it was significantly increased in the replication stage of the virus at 24h and 36h, which shows that GSK3β is in the stage of virus replication. The stage plays a role, and the overexpression of GSK3β in host cells can promote the replication of SVA.

Claims (5)

1. Use of gsk3β for enhancing the replication of an saint card virus.

2. The use according to claim 1, wherein the saint virus is cultured in a host cell overexpressing gsk3β.

3. A method for increasing the titer of a sai card virus comprising the steps of:

(1) Constructing a host cell which overexpresses GSK3 beta;

(2) Culturing host cells which overexpress GSK3 beta and inoculating the saint virus for further culture.

4. The method of claim 3, wherein the host cell is a BHK-21 cell.

5. A method according to claim 3, wherein the time to continue the cultivation is not less than 12 hours.

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