CN103627675B - A kind of method improving influenza virus vaccine strain output - Google Patents

Abstract

The present invention provides a method for improving the yield of influenza virus vaccine strains, which is by overexpressing FMRP protein or a functional truncated protein containing KH2 domain in cells used to produce influenza vaccine strains, thereby increasing the production of influenza virus vaccine strains. Yield of virus vaccine strains in cell production. The results show that the replication efficiency of influenza virus can be increased by more than 2 times by overexpressing FMRP protein in the cells used to produce influenza vaccine strains (such as Vero, MDCK cells) in a transient or stable manner, and the harvested virus is prepared into Vaccines to meet market demand for influenza vaccines.

Description

A method for increasing the yield of influenza virus vaccine strains

technical field

The invention relates to the preparation of influenza virus vaccine strains, in particular to a method for increasing the yield of influenza virus vaccine strains.

Background technique

Influenza (flu) is one of the most common infectious diseases in humans. Frequent regional outbreaks and uncertain influenza pandemics seriously endanger human health and cause huge economic losses. Because influenza virus is prone to antigenic mutation, gene recombination, and drug-resistant strains, influenza vaccination has become one of the necessary measures to control seasonal influenza epidemics and influenza pandemics.

Traditional influenza vaccine production is mostly done in fertilized eggs between 9 and 12 days old. In 2007, the European Union approved the marketing of Novartis' influenza vaccine produced in mammalian cells. The production of influenza vaccine in mammalian cells has greater scalability than that in eggs, and can eliminate the allergies caused by the production of influenza vaccine in eggs reactions and other side effects. However, at this stage, due to the limitation of production technology, the yield of using cells to produce influenza vaccines is low, which greatly limits the development and utilization of influenza vaccines, especially affecting the rapid production and vaccination of emergency vaccines during influenza pandemics.

Influenza viruses are single-negative-strand RNA viruses divided into eight segments, encoding 12 viral proteins. Influenza viruses realize the transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA) of the viral genome in the nucleus. The smallest functional unit that performs transcription and replication is the viral nucleocapsid or ribonucleoprotein (RNP), which is composed of viral RNA polymerase complexes (PB1, PB2, PA), viral RNA (vRNA) and multiple Copies of nucleoprotein (NP). After the influenza virus binds to the host cell receptor through its surface glycoprotein HA, removes the outer membrane of the virus through the endocytic fusion of the cell, and releases the viral vRNPs group into the cell cytoplasm, vRNPs are present in host factors (such as With the assistance of nuclear transport protein), the viral RNA is transcribed and replicated and the new chain is transcribed and replicated in the nucleus under the interaction and game with a series of host factors (POLII, transcription factors, splicing factors, etc.) The cutting process of RNA, and then the nascent vRNPs are transported outside the nucleus with the assistance of other viral proteins (NS2, M) and host factors (nuclear export protein, etc.), and in the cytoplasm, viral mRNA is in turn under the action of host factors. It is preferentially translated into proteins, among which some newly synthesized viral proteins, such as the three subunits of the polymerase complex, PB1, PB2, PA, and NP, each require the assistance of different host factors to be transported into the nucleus and assemble the RNA polymerase complex , to continue the transcription and replication of the viral genome. It can be seen that the transcription and replication process of influenza virus is carried out under the close interaction with host protein. Using this characteristic, new technical means have been developed to improve the yield of influenza virus vaccine strains in cell culture production.

Contents of the invention

The purpose of the present invention is to provide a method for improving the yield of influenza virus vaccine strains by utilizing the property of FMRP protein to promote RNP assembly during influenza virus transcription and replication.

In order to achieve the purpose of the present invention, a method of improving the yield of influenza virus vaccine strains of the present invention is by overexpressing FMRP protein or a functional truncated body containing KH2 domain in cells used to produce influenza vaccine strains protein, thereby increasing the yield of influenza virus vaccine strains in cell production. Wherein, the amino acid sequence of the FMRP protein is shown in Seq ID No.1, and the nucleotide sequence encoding the protein is shown in Seq ID No.2. The KH2 domain is located on amino acids 280-422 of the FMRP protein, and its amino acid sequence is shown in Seq ID No.3.

In the aforementioned method, the cells used to produce influenza vaccine strains are Vero or MDCK cells and the like.

In the aforementioned method, it is to carry the gene encoding FMRP protein or the eukaryotic expression vector of the gene encoding the functional truncated body protein containing KH2 domain into the cells used to produce influenza vaccine strains, along with the host Growth of cells to replicate the overexpressed protein (either transiently or by establishing a stably expressing cell line), then infecting the cells with an influenza virus vaccine strain, culturing the cells, and harvesting the cell supernatant, thereby increasing its role in cell production Yield.

In the aforementioned method, the starting vector of the eukaryotic expression vector is pCMV6-entry, pcDNA3.1 or a stable cell line constructed by a lentiviral vector, such as pQCXIP.

In the aforementioned method, the eukaryotic expression vector is pCMV6-entry-FMRP-myc/flag, purchased from Beijing Aurui Dongyuan Biotechnology Co., Ltd. (cat. No RC222699).

Through RNAi screening technology, it was found that FMRP protein is an RNA-binding protein encoded by the FMR1 gene, located in the cytoplasm and nucleus, and can promote the assembly of RNP during the transcription and replication of influenza virus. At the same time, it was also found that the KH2 domain of FMRP protein plays a particularly critical role in the assembly of influenza virus RNP. Experiments have shown that the replication efficiency of influenza virus can be increased by more than 2 times by overexpressing FMRP protein in cells used to produce influenza vaccine strains (such as Vero, MDCK cells) in a transient or stable manner, and the harvested virus can be prepared into Vaccines to meet market demand for influenza vaccines.

Description of drawings

Fig. 1 is the pCMV6-entry-FMRP-myc/flag plasmid map involved in the embodiment of the present invention.

Fig. 2 is the result of detecting FMRP protein expression by Western Blot in the embodiment of the present invention.

Fig. 3 is the yield of influenza virus vaccine strain in Vero cell culture in the embodiment of the present invention.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

Example

1 Plasmid source

The FMRP protein overexpression plasmid pCMV6-entry-FMRP-myc/flag (cat.No RC222699, see Figure 1 for the plasmid map) used in this example was purchased from Beijing Origene Biotechnology Co., Ltd. (www.origene.com. cn), the plasmid clones the human FMR1 cDNA into the eukaryotic expression vector pCMV6-entry, and the restriction sites are sgfI and MluI. During the expression process of the plasmid, myc and flag tags will be introduced at the carboxy terminus of the FMRP protein, which is convenient for subsequent experimental detection.

2 cell culture

In this example, Vero cells were used as cells for producing influenza virus vaccine strains. After the cells grow into a dense monolayer in the T75 culture flask and are basically saturated, suck off the old culture medium in the culture flask, wash the cells with 5 ml of PBS to remove residual serum, and add 0.25% trypsin 4 to the flask. 1 ml, cover the bottom of the bottle, and place it in a 37°C, 5% CO ₂ incubator for 3 minutes. When the cytoplasm shrinks, the cells become round, and the intercellular space increases, add DMEM containing 10% fetal bovine serum to the bottle. (complete medium), pipetting gently and repeatedly to detach the cells from the bottle wall to form a cell suspension. Transfer the cell suspension to a 15ml centrifuge tube and centrifuge at 1000rpm for 5 minutes. Discard the supernatant, add 5 ml of complete medium to the centrifuge tube, break up the cell mass into single cells, adjust the cell concentration, spread the cells in a six-well plate, ⁶ ×105 cells per well, and use the plasmid to be transfected . The remaining cells were transferred to new T75 culture flasks to continue culturing in a 37°C, 5% CO ₂ incubator.

3 cell transfection

16-24 hours after the cells were plated, the plasmid pCMV6-entry-FMRP-myc/flag and the control empty plasmid pCMV6-entry were transfected. Mix 5 microliters of lipo2000 (Invitrogen) into 95 microliters of opti-MEM (Life Technologies), mix gently and leave at room temperature for 5 minutes. Take 3 micrograms of plasmid, mix with 97 microliters of opti-MEM, mix gently, and let stand at room temperature for 5 minutes. The above plasmid mixture was added to the lipo2000 mixture and left at room temperature for 20 minutes. Replace the medium in the six-well plate with fresh DMEM medium, gently add the transfection mixture into the six-well plate, shake gently, and culture in a 37°C, 5% CO ₂ incubator for 30 hours, then infect flu virus.

The expression of FMRP protein was detected by Western Blot. After 30 hours of cell transfection, the cells in one well were scraped off with a cell scraper. After washing twice with PBS, the cells were lysed with 80 microliters of RIPA lysate, placed on ice for 30 minutes, and then centrifuged at 12,000 rpm for 15 minutes. Take 20 microliters of cell lysate supernatant and add 5 microliters of 5×SDS loading buffer, cook at 95°C for 10 minutes, cool to room temperature, and perform SDS-PAGE electrophoresis after brief centrifugation. After the electrophoresis, the protein samples in the gel were electrotransferred to the nitrocellulose membrane with a voltage of 80 volts for 2 hours. 5% skimmed milk powder was blocked for 1 hour, the primary anti-mouse flag antibody (Sigma) was diluted at 1:4000 and incubated for 2 hours, and the secondary antibody was incubated for 1 hour before developing. The results of Western Blot detection are shown in Figure 2.

4 virus infection

The transfected Vero cells were infected at MOI 0.001. Take influenza virus strain A/WSN/1933, according to the titer of the original virus strain ( ^3.6 ×106/ml), add to 1ml virus maintenance solution (containing 0.5% fetal bovine serum, 1 μg/ml TPCK-trypsin DMEM) to add the desired amount of virus (0.33 μl) per well of cells. Aspirate off the medium in the Vero cells and wash twice with PBS. Add 1ml of virus maintenance solution containing the desired virus to each well, and adsorb for 1 hour at room temperature. Aspirate the virus maintenance solution containing the original virus, wash it twice with PBS, and add 2 ml of virus-free virus maintenance solution. Take 200 microliters of cell culture supernatant at 24 hours, 36 hours, 48 hours, 60 hours and 72 hours after virus infection, respectively, for plaque assay (plaque assay) method to detect virus titer, and add 200 microliters at the same time Fresh virus maintenance solution.

5 plaque test

The virus titers in the cell culture supernatants at the above 5 time points were measured. Prepare MDCK cells one day in advance, plate in 12-well plates (about 2×10 ⁵ per well), and the cell density is about 70%-80%. Dilute the virus with the virus maintenance solution according to the concentration gradient of 1-10 ^-6 . Prepare 250 microliters of virus fluid for each dilution. Use a vortex vortex mixer during the dilution process to ensure that the virus is evenly mixed. Aspirate the culture medium of MDCK cells and wash twice with PBS. According to the needs of the experiment, add 250 microliters of virus dilution solution to each well, shake gently, incubate at room temperature for 1 hour, and shake gently every 15 minutes. 2% low melting point agarose (LMT) was melted in a microwave oven and placed in a 37°C water bath to keep warm. Dilute 2% LMT with preheated virus maintenance solution at a volume ratio of 1:1 to make the final concentration 1%. After virus incubation, wash twice with PBS to wash away unadsorbed virus particles. Add 1.5 mL of diluted 1% agar to each well. Leave it at room temperature for 30 minutes. After the agar is completely solidified, put it upside down and put it in the incubator for cultivation. Observe the results after 72 hours. Fix with 4% formaldehyde, put it in ice to help the agar solidify, then remove the agar, stain with crystal violet for 10 minutes, rinse with tap water, and observe the result. Calculate the virus titer of the cell supernatant at each time point. The calculation formula is:

As shown in Figure 3, the titer of influenza virus in the supernatant of Vero cells overexpressing FMRP protein was significantly higher than that in the control cells (transformed into the empty plasmid pCMV6-entry). Through the method of the invention, the yield of the influenza virus vaccine strain in cell culture can be increased by 2 times. The harvested viruses are prepared into vaccines to meet the high demand for influenza vaccines in the market.

In addition, the present invention also verifies that transferring the gene encoding the functional truncated protein containing the KH2 domain into Vero or MDCK cells can also achieve the effect of increasing the yield of influenza virus vaccine strains.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (4)

1. A method for improving the output of influenza virus vaccine strains, characterized in that, by overexpressing FMRP protein in cells for producing influenza vaccine strains, thereby improving the output of influenza virus vaccine strains in cell production; Wherein, the amino acid sequence of the FMRP protein is shown in Seq ID No.1. 2. The method according to claim 1, characterized in that the cells used to produce influenza vaccine strains are Vero or MDCK cells. 3. the method according to claim 1 or 2 is characterized in that, it is that the eukaryotic expression vector that will carry the gene of coding FMRP albumen is transferred in the cell that is used for producing influenza vaccine virus strain, along with the growth of host cell The overexpressed protein is replicated and then infected with the influenza vaccine strain to increase its production in cell production. 4. The method according to claim 3, wherein the starting vector of the eukaryotic expression vector is pCMV6-entry, pcDNA3.1 or pQCXIP.