CN107043426B

CN107043426B - Fusion protein 210 and its use in optimizing viral replication

Info

Publication number: CN107043426B
Application number: CN201610081938.XA
Authority: CN
Inventors: 王晓佳; 李翠翠
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2016-02-05
Filing date: 2016-02-05
Publication date: 2020-01-24
Anticipated expiration: 2036-02-05
Also published as: CN107043426A

Abstract

The invention provides a fusion protein 210 and application thereof in optimizing virus replication, wherein the fusion protein 210 is a truncated body of P60 protein, retains the function of promoting the replication of various viruses of P60, and does not produce toxic effect on cells within the concentration range of effective action. Can improve the virus titer by 2 orders of magnitude units in a wider spectrum range (0.0001-0.1MOI), is beneficial to identifying and detecting viruses, can detect the VSV of rhabdovirus family, the HCV of flaviviridae family, the EV71 of parvovirus family and the like, and shortens the detection time by less than half. The fusion protein 210 obtained by the prokaryotic expression system has high expression efficiency and easy purification, the one-step purification efficiency can reach more than 85 percent, and the final concentration of the protein can reach 3 mg/ml. Therefore, the fusion protein 210 can be directly added into a virus culture medium, and a plurality of virus isolates can be detected and cultured under the condition of lower titer, so that the method is simple, convenient and quick, and has great influence and significance in various aspects of production, science, research and the like of virus disciplines.

Description

Fusion protein 210 and its use in optimizing viral replication

Technical Field

The invention relates to the field of virus separation and identification and whole virus vaccine preparation, in particular to a fusion protein 210 and application thereof in optimizing virus replication.

Background

The identification and detection of viruses are a prerequisite for the cultivation of large quantities of viruses, virology experiments and the preparation of vaccines and specific diagnostic reagents. The establishment of a new technique for virus detection should be considered from essentially 3 points of view, namely, the detection of viral antigens, the detection of antibodies produced by viruses, and the detection of viral genes. Detection of viral antigens and viral antibodies typically requires an ELISA detection kit; the detection of viral genes requires identification by laboratory molecular means such as PCR technology and gene chip technology. At present, the two detection methods have certain limitations, the ELISA detection operation is relatively complicated, and the sensitivity needs to be improved; similar situations exist in PCR technology and gene chip technology.

Furthermore, a technical bottleneck often encountered in many virology laboratories is that many viruses (such as enteroviruses and some newly discovered viruses) are difficult or even impossible to propagate in commonly used cell lines, so that many virology tests cannot follow up, and therefore directly affect subsequent studies. Therefore, how to improve the virus replication capacity without genetic modification is one of the major challenges in the field of virus science.

At present, the field of whole virus vaccine preparation also faces similar problems, and the titer of a plurality of strains for vaccines is not high, which directly influences the effect and price of the vaccines. Some research groups at home and abroad try to reduce the defense influence (such as interferon concentration) of cells on viruses by changing liquid for many times during the virus culture process; there have also been attempts to improve adherent cells of different cell lines into suspension cells to increase titer of cell-propagated virus by increasing cell density per volume. The schemes improve the titer of the virus to a certain extent, but all belong to the modification of external factors, the operation is complicated, and the cell regulatory protein beneficial to virus replication is fundamentally discovered and developed without deeply knowing the host factors of virus replication.

The nucleolin P60 is a protein coded by Glioma tumor suppressor candidate gene 2 (GLTSCR 2), can regulate the stability of tumor suppressor P53 and PTEN, and has double identities of tumor suppressor and cancer suppressor roadblock in the field of oncology, namely P60. The unique biochemical characteristics and complex functions of P60 make it a hot spot in scientific research, but it is only rarely reported in virology field.

Early studies find that the P60 protein has a promoting effect on the replication of several viruses (VSV in Rhabdoviridae, HCV in Flaviviridae and EV71 in parvoviridae), but cytotoxicity experiments show that the P60 holoprotein can induce significant apoptosis and has obvious cytotoxicity.

Disclosure of Invention

The invention aims to provide a fusion protein 210 which is a truncated body of P60 protein.

It is another object of the invention to provide the use of fusion protein 210 for optimizing viral replication.

In order to achieve the object of the present invention, the present invention provides a novel fusion protein 210, which has an amino acid sequence shown as Seq ID No.1, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids in the sequence.

The invention also provides a gene for coding the fusion protein 210, and the nucleotide sequence of the gene is shown as Seq ID No. 2.

The invention also provides a vector or engineering bacterium containing the gene for encoding the fusion protein 210.

The preparation method of the fusion protein 210 comprises the following steps:

1) cloning genes: taking glioma cancer suppression candidate gene 2(GenBank: KJ898763.1) as a template, and designing a primer to perform subcloning of a target gene; wherein, the upstream primer: 5'-CGAGGTCTGTCCCACGCCCG-3', downstream primer: 5'-CAGCTCCGAGCTCAGCTGCA-3', respectively;

2) vector construction: cutting the pET-28a vector by using restriction enzyme NdeI/EcoRI, carrying out double enzyme digestion on the target gene obtained in the step 1) and connecting the target gene with the vector to obtain a recombinant vector pET-210 containing the target gene;

3) cell transformation and expression: BL21(DE3) was transformed with the recombinant vector pET-210 and shake-cultured at 37 ℃ to OD₅₉₀Adding inducer IPTG to the final concentration of 1mM, and culturing at 37 deg.C for 4 hr to obtain a culture medium with concentration of 0.8-1.0;

4) protein purification: centrifuging the induced thallus for 10min at 8000r/min, ultrasonically cracking the thallus, centrifuging to obtain a supernatant, passing the supernatant through a nickel affinity chromatographic column balanced by a balance liquid, washing the chromatographic column by 10 column volumes by using a washing solution 1 and a washing solution 2, and eluting by using an eluent with 3 column volumes to obtain a fusion protein solution containing 6 His labels. Then, the protein was concentrated using a protein ultrafiltration tube with a cut-off molecular weight of 10kD to obtain purified fusion protein 210.

The formula of the balance liquid in the step 4) is as follows: 50mM NaH₂PO₄300mM NaCl, 10mM imidazole, 10mM Trisbase;

the formula of the washing solution 1 is as follows: 50mM NaH₂PO₄300mM NaCl,20mM imidazole, 10mM Tris base;

the formula of the washing liquid 2 is as follows: 50mM NaH₂PO₄300mM NaCl,40mM imidazole, 10mM Tris base;

the formula of the eluent is as follows: 50mM NaH₂PO₄300mM NaCl,200mM imidazole, 10mM Tris base.

The invention also provides application of the fusion protein 210 (or the protein 210 with 6 His labels removed) in preparation of inactivated vaccines and/or attenuated live vaccines and optimization of virus replication.

The aforementioned use, which is by overexpressing the gene encoding the fusion protein 210 (or the 6 His-tag-removed protein 210) in a host cell, and inoculating the host cell with a strain for virus, inactivated vaccine and/or attenuated live vaccine production, culturing the cells, and harvesting a virus solution.

The aforementioned application, which is to add the fusion protein 210 (or the protein 210 with 6 removed His tags) to the host cell culture solution for preparing the virus, the inactivated vaccine and/or the live attenuated vaccine to make the concentration of the fusion protein 210 (or the protein 210 with 6 removed His tags) in the host cell culture solution reach 10-20 μ g/ml (preferably 20 μ g/ml), then inoculate the strain for producing the virus, the inactivated vaccine and/or the live attenuated vaccine, and harvest the virus solution after culturing.

Strains for the production of viruses, inactivated vaccines and/or attenuated live vaccines related to the present invention include, but are not limited to, Rhabdoviridae, Herpesviridae, parvoviridae, Coronaviridae, Paramyxoviridae, Orthomyxoviridae, arteriviruses, reoviridae. For example, Vesicular Stomatitis Virus (VSV), Hepatitis C Virus (HCV), enterovirus EV71, and the like.

The invention also provides a medium for optimizing viral replication, comprising fusion protein 210 (or 6 His-tag-removed protein 210) at a concentration of 10-20. mu.g/ml (preferably 20. mu.g/ml).

The invention also provides a cell line that can optimize viral replication, which is a host cell line that overexpresses the gene encoding fusion protein 210 (or 6 His-tag deleted protein 210).

The fusion protein 210 provided by the invention is a truncated body of the P60 protein, retains the function of promoting the replication of various viruses of P60, and does not produce toxic effect on cells within the concentration range of effective action. Can improve the virus titer by 2 orders of magnitude units in a wider spectrum range (0.0001-0.1MOI), is beneficial to identifying and detecting viruses, can detect the VSV of rhabdovirus family, the HCV of flaviviridae family, the EV71 of parvovirus family and the like, and shortens the detection time by less than half. The fusion protein 210 obtained by the prokaryotic expression system has high expression efficiency and easy purification, the one-step purification efficiency can reach more than 85 percent, and the final concentration of the protein can reach 3 mg/ml. Therefore, the fusion protein 210 can be directly added into a virus culture medium, and a plurality of virus isolates can be detected and cultured under the condition of lower titer, so that the method is simple, convenient and quick, and has great influence and significance in various aspects of production, science, research and the like of virus disciplines.

Drawings

FIG. 1 shows the results of SDS-PAGE gel electrophoresis detection of the nuclear expression of purified fusion protein 210 (left panel) and its high-level structural analysis (right panel) in example 1 of the present invention.

FIG. 2 shows the results of the detection of the effect of protein P60 on VSV replication using the cytopathic method (left panel) and the western-blot method (right panel) in example 2 of the present invention.

FIG. 3 shows the results of the plaque assay used in example 2 of the present invention to determine the potency of fusion protein 210 against VSV infection at various times.

FIG. 4 shows the results of the plaque assay used in example 2 of the present invention to determine the effect of fusion protein 210 on VSV infection titer at different titers (left panel) and the comparison of the effect of interferon and fusion protein 210 on viral replication (right panel).

FIG. 5 shows the results of the detection of the effect of the fusion protein 210 on HCV replication in example 2 of the present invention by the cytopathic method (left panel) and the qRT-PCR method (middle panel and right panel).

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory manual,2001), or the conditions suggested by the manufacturer's instructions.

The strains used in the following examples: the VSV virus strain is wild strain Indiana, and the HCV virus strain is HCVcc (JFH-1).

Example 1 prokaryotic expression and purification of fusion protein 210 Gene

The method for expressing the fusion protein 210 by using the escherichia coli engineering bacteria comprises the following steps:

1. cloning genes: taking glioma cancer suppression candidate gene 2(GenBank: KJ898763.1) as a template, and designing a primer to perform subcloning of a target gene; wherein, the upstream primer: 5'-CGAGGTCTGTCCCACGCCCG-3', downstream primer: 5'-CAGCTCCGAGCTCAGCTGCA-3', respectively;

2. vector construction: cutting the pET-28a vector by using restriction enzyme NdeI/EcoRI, carrying out double enzyme digestion on the target gene obtained in the step 1) and connecting the target gene with the vector to obtain a recombinant vector pET-210 containing the target gene;

3. cell transformation and expression: BL21(DE3) was transformed with the recombinant vector pET-210 and shake-cultured at 37 ℃ to OD₅₉₀Adding inducer IPTG to the final concentration of 1mM, and culturing at 37 deg.C for 4 hr to obtain a culture medium with concentration of 0.8-1.0;

4. protein purification: centrifuging the induced thallus for 10min at 8000r/min, ultrasonically cracking the thallus, centrifuging to obtain a supernatant, passing the supernatant through a nickel affinity chromatographic column balanced by a balance liquid, washing the chromatographic column by 10 column volumes by using a washing solution 1 and a washing solution 2, and eluting by using an eluent with 3 column volumes to obtain a fusion protein solution containing 6 His labels. Then, the protein was concentrated using a protein ultrafiltration tube having a cut-off molecular weight of 10KD to obtain a purified fusion protein 210(Seq ID No.1), which was then subjected to 12% SDS-PAGE gel electrophoresis, and the results are shown in the left panel of FIG. 1.

The formula of the balance liquid in the step 4 is as follows: 50mM NaH₂PO₄300mM NaCl, 10mM imidazole, 10mM Trisbase;

Wherein, the fusion protein 210 with 6 removed His tags corresponds to amino acids 330-432 of the P60 protein. The amino acid sequence of the P60 protein is shown in Seq ID No. 3.

5. Protein high-level structure analysis: protein 210 was exposed to 0.005-0.02% Glutaraldehyde (Glutaraldehyde, GA, MP Biomedicals) at 4 ℃ for 15 minutes by chemical crosslinking, and then subjected to SDS-PAGE assay and immunoblot analysis using His antibody, with the results shown in the right panel of fig. 1.

Example 2 immunoblotting, cytopathic assay and qRT-PCR assay to investigate the Effect of fusion protein 210 on viral virulence

Immunoblot (western-blot): washing the virus-infected cells for 3 times by using precooled PBS (pH7.5PBS), scraping the cells, ultrasonically cracking and extracting total cell protein for 3-5 seconds, mixing with 6 xSDS-PAGE loading buffer, boiling for 10min, performing SDS-PAGE electrophoresis, and transferring the protein on the gel onto a PVDF membrane by an electrophoresis apparatus. Lh were blocked with 5% nonfat dry milk in PBST buffer at room temperature and incubated with primary antibody at 4 ℃ for 2h with shaking. Wash membrane 4 times with PBST, 5min each, 1: the HRP-labeled secondary antibodies were diluted 8000 and incubated for 1h at room temperature with shaking, the membranes were washed 4 times for 5min each, and finally developed with SuperSignalWest Pico chemistry HRP Substrate ECL, exposed to light with Kodak film, and finally developed and fixed for observation. The viral protein antibodies were purchased from san Cruz Biotechnology, Inc. The anti-mouse lgG secondary antibody and other secondary antibodies of the fluorescent goat containing Rhodamine Red-x are purchased from Beijing kang, century Biotechnology Co., Ltd. The viral protein antibodies used, including the VSV-G antibody and the actin antibody, were all diluted 1: 1000.

Cells were infected with virus and treated with the purified nuclear expressed protein of example 1, and cell samples were collected, respectively. First, a certain amount of virus is used to infect cells, the maintenance solution (DMEM containing 2% serum and 1% antibiotics) is replaced after 90min, and protein with a certain concentration (0, 10, 20, 30 mug/ml) is added into the maintenance solution and is shaken gently. Viruses were collected at different time points according to experimental objectives. The virus infected cells are harvested by adopting a repeated freeze thawing method, namely, a cell bottle is placed at the temperature of minus 20 ℃ for cryopreservation for 2 hours and then placed at the room temperature until the cells are partially thawed, at the moment, the cell bottle is shaken to ensure that adherent cells are detached from the wall, the cells are placed at the temperature of minus 20 ℃ for cryopreservation again, and the process is repeated for 3 times to ensure that the virus is released from the cells. All samples were frozen at-70 ℃ or in liquid nitrogen if they were not used temporarily. The following cytopathic analysis experiments were then performed.

Cytopathic analysis: diluting 293T cell sample infected by VSV in multiple proportion to obtain cytotoxic suspension, culturing CEF cell with 96-well plate to monolayer, adding 50 μ L/well cytotoxic suspension into each well, performing 12 dilutions and 8 repetitions, standing in 37 deg.C carbon dioxide incubator for 48 hr, observing cytopathy, and calculating TCID₅₀(viral titer to form cytopathic lesions) was repeated 3 times. In addition, immunofluorescence analysis: VSV-infected 293T cell culture medium was aspirated away, washed 3 times with PBS, fixed with 4% formaldehyde, incubated with VSV-G mouse monoclonal antibody for 60min, washed 3 times with PBS, incubated with Rhodamine Red-x-labeled goat anti-mouse secondary antibody for 40min, and washed 3 times with PBS. Virus fluorescence was recorded by observation at 568nm wavelength of immunofluorescence microscopy for virulence determination and calculation of TCID₅₀. The difference between the fusion protein 210 and the negative control (vector pEGFP-N1) was 1-2 lg, which was significant. The results are shown in Table 1, where- -represents addition of virus only and E represents addition of fusion protein 210 to infected cells.

TABLE 1 TCID after different Virus treatments and untreated E₅₀Comparison

FIG. 2 is a bar graph of three different forms representing control group (only virus, Ctrl), transfection of P60 and VSV, transfection of △ P60 and VSV infection, two groups of infection time 36h and 48h, respectively, from which it can be seen that the control group and the overexpression P60 group show significant differences at two time points of virus infection, the right graph is an immunoblot corresponding to the left bar graph, in which P60 represents an antibody to P60, VSV-G represents a virus protein antibody, VSV infection amount is 0.1MOI, △ P60 represents P60 from which the nuclear sequence is deleted, using a cytopathologic method and a western-blot method to examine the effect of P60 on VSV replication and the effect of deletion of the nuclear sequence (LRAARLRHQELFR) on P60.

FIG. 3 is a plaque assay to examine the effect of fusion protein 210 on VSV viral titer at various times of viral infection. 293T cells were subjected to plaque assay by adding protein or PBS (Ctrl) at 0.1MOI titer, and cell supernatants were collected. As shown in fig. 3, the line graphs of the variation of the virus titer of the control group and the experimental group at different time points. As can be seen, the virus titer after the protein is added for 36h is close to the titer of the protein 210 which is not added for 48h, so that the protein 210 can also shorten the VSV virus culture time by about 1/3.

FIG. 4 is a plaque assay to examine the effect of fusion protein 210 on VSV viral replication at different titers of viral infection. 293T cells were either spiked with protein 210 or PBS and infected with VSV at different titers (left panel) or spiked with protein 210 (20. mu.g/ml) and/or interferon (10ng/ml) at 0.1MOI titers (right panel) and plaque experiments were performed. As shown in FIG. 4, the line graphs of the variation of the virus titer between the control group and the experimental group for different virus titers.

Real-time quantitative RT-PCR: samples of the cells infected with the virus and added with the protein of example 2 were collected separately and total RNA was extracted according to the Trizol lysate protocol. cDNA synthesis was performed using a specific downstream primer as a reverse transcription primer. Real-time quantitative PCR amplification was performed using SYBR Green I dye method. 20 μ L Total reaction: 10 μ L of 2 XSSYBR Green I PCRmix, 0.4 μ L (10 μ M) of each of the upstream and downstream primers, and water to a total volume of 20 μ L. The Real-Time PCR System (ViiA 7) sets the reaction program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ and elongation for 45s, 40 cycles set, and finally melting curve analysis was performed. Adopt 2^-△△CTAnd relatively quantifying the target gene, and selecting beta-actin as an internal reference gene. Inoculating virus to cells, transfecting the fusion protein 210 gene, collecting cells at different time after virus inoculation, extracting total RNA in the cells, performing reverse transcription to obtain cDNA, performing qPCR detection on ViiAA 7 fluorescent quantitative PCR instrument by adopting SYBR Green I dye, and performing qPCR detection by using 2^-△△CTThe method calculates the difference of the expression level of the virus mRNA among different treatment groups. Plotting was performed with GraphPad prism 5. The treatment at each time point was normalized to 1, with significant differences (fig. 5).

Designing a primer: according to the gene sequence of the viral protein published in GenBank, such as HCV-NA5A sequence, DNASAR (molecular biology software) is adopted to carry out nucleotide homology analysis, and a gene conserved sequence related to virus replication is selected as a target sequence for PCR amplification. Primers were designed separately using Primer 5.0 software following the principle of fluorescent PCR Primer design (Table 2).

TABLE 2 primer sequence information

FIG. 5 shows the results of detecting the effect of fusion protein 210 on HCV replication using cytopathic effect (left panel) and qRT-PCR method (middle panel and right panel).

Left panel, at different virus titers, PBS, IFN-. beta.s (10ng/ml) or protein 210 (10. mu.g/ml) were added and then the cytopathies were observed, counted in Foci units and counted (using conventional cytopathic methods, only the counting method was different).

In the middle panel, Huh-7.5 cells were cultured in 48-well plates, and after 48 hours, the RNA content of HCV was quantitatively determined by qRT-PCR method, with addition of PBS and infection with HCV, addition of IFN-. beta.and infection with HCV, addition of protein 210 and infection with HCV at 0.1MOI, respectively, from left to right. It can be seen that the virus titer in 24h of protein addition is close to the titer in 48h of protein 210, so that the protein 210 can shorten the HCV virus culture time by about 1/2.

On the right panel, 210, IFN-beta and PBS are added into Huh-7.5 cells respectively, and 0.1MOI HCV is added for 48 hours, and the RNA content of HCV is quantitatively detected by qRT-PCR method.

The results show that the fusion protein 210 can significantly improve the level of RNA in the early stage of HCV virus replication and can up-regulate the expression of viral nucleoprotein within 48h of virus infection.

Example 3 cytotoxicity assay of fusion protein 210

A Lactate Dehydrogenase (LDH) experiment is adopted to study whether polypeptide or protein has toxicity to cells, and the toxicity is detected by using a toxicity detection kit purchased from Roche. To CEF cells cultured to monolayers were added the following final concentrations of fusion protein 210, respectively: 5 μ M, 25 μ M, 50 μ M, 100 μ M, 250 μ M, 500 μ M, 1.0mM, and gently mixed well, 24 hours later according to the kit instructions for determination of toxicity index. The experiment was repeated three times. The results show that fusion protein 210 has no toxic effect on cells at concentrations of 50. mu.M and below.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The fusion protein 210 is characterized in that the amino acid sequence is shown as Seq ID No. 1.

2. A gene encoding the fusion protein 210 of claim 1.

3. A vector or an engineered bacterium comprising the gene of claim 2.

4. Use of the fusion protein 210 of claim 1 for the preparation of an inactivated vaccine and/or an attenuated live vaccine and for optimizing viral replication;

the application comprises the following steps: overexpressing a gene encoding the fusion protein 210 of claim 1 in a host cell, and inoculating the host cell with a virus, inactivated vaccine and/or strain for attenuated live vaccine production, culturing the cells, and harvesting a virus solution; alternatively, the first and second electrodes may be,

adding the fusion protein 210 into a host cell culture solution for preparing the virus, the inactivated vaccine and/or the attenuated live vaccine to ensure that the concentration of the fusion protein 210 in the host cell culture solution reaches 10-20 mu g/ml, then inoculating a strain for producing the virus, the inactivated vaccine and/or the attenuated live vaccine, and harvesting a virus solution after culturing.

5. The use according to claim 4, wherein the virus, inactivated vaccine and/or live attenuated vaccine production strain is vesicular stomatitis virus or hepatitis C virus.

6. A medium optimized for viral replication comprising the fusion protein 210 of claim 1 at a concentration of 10-20 μ g/ml.

7. A cell line that optimizes viral replication, wherein the cell line is a host cell line that overexpresses the gene of claim 2.