CN117143204A - Construction and application of enterovirus71 type VP1 protein 75 th amino acid mutant virus strain - Google Patents
Construction and application of enterovirus71 type VP1 protein 75 th amino acid mutant virus strain Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, in particular to construction and application of an enterovirus71 type VP1 protein 75 th amino acid mutant virus strain. The amino acid sequence of the enterovirus71 VP1 protein mutant provided by the invention is shown as SEQ ID NO. 1. The VP1 protein mutant and the corresponding encoding nucleic acid molecules thereof can reduce the proliferation capacity of the enterovirus71, but enhance the in vivo toxicity of mice, can be used for constructing enterovirus71 mutant virus strains, and provide effective biological materials for preparing medicines for preventing and treating enterovirus71 infection and screening and quality control of the medicines.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to construction and application of an enterovirus71 type VP1 protein 75 th amino acid mutant virus strain.
Background
Enterovirus (Enterovirus) type 71 (EV 71) belongs to the family Picornaviridae, genus Enterovirus. The genome is single-stranded positive strand RNA, which is about 7400 nucleotides in length and encodes a 2193 amino acid-containing polyprotein. EV71 is mainly infected by children under 5 years old, and is transmitted through the faeces-oral route or spray, and the infection mainly causes hand-foot-and-mouth disease (HFMD) which is difficult to be distinguished clinically from hand-foot-and-mouth disease caused by Coxsackie virus A16 infection. However, severe EV71 infection can cause aseptic encephalitis, meningoencephalitis, poliomyelitis-like paralysis, neurogenic myocarditis, and pulmonary edema, leading to severe infections and even death cases.
The genomic encoded polyprotein of EV71 comprises structural protein P1 regions (VP 4, VP2, VP3, VP 1), non-structural protein P2 regions (2A, 2B, 2C) and P3 regions (3A, 3B, 3C, 3D). Among them, VP1 protein is exposed on the surface of viral capsid, is the main neutralizing protein, and contains a large amount of antigenic determinants. VP1 protein is also primarily involved in constituting epitopes that bind to cellular receptors and play a key role in the viral adsorption process. In addition, VP1 has been reported to play an important role in the stability and immunoregulation of EV71 virus structure. Thus, VP1 protein may determine host tropism and pathogenicity of EV71 virus.
The VP1 protein of EV71 consists of 297 amino acids, and some studies have revealed that some of these sites may be potential sites of viral virulence. McMinn et al, by comparing VP1 gene sequences of Australian EV 71-epidemic strains in 1999, found that the main difference between strains with clinical symptoms of hand-foot-and-mouth disease and strains with neurotoxicity is the substitution of amino acid from alanine to valine at position 170 of VP1 protein. The change in this site may result in a change in the spatial structure of the VP1 protein, thereby altering the binding capacity of the virus to the host cell and the virulence of the virus. Yorihiro et al found that amino acid 145 of VP1 protein was able to regulate binding of EV71 virus to PSGL-1 receptor on leukocyte surface. When glycine (G) or glutamine (Q) is at position 145, the virus is able to bind to PSGL-1, but after mutation to negatively charged glutamic acid (E), the ability to bind PSGL-1 is lost. Whereas when human SCARB2 transgenic mice and macaques are infected, the VP1-145E mutant instead exhibits greater virulence, which may be related to the regulation of adsorption of the site to different receptors and the sensitivity of the antibody neutralization.
At present, no specific medicine for treating EV71 infection exists, and most of clinical symptomatic support treatment is carried out. Therefore, the development of protein mutants and mutant virus strains that affect the virulence and proliferation capacity of EV71 is of great importance for the development of drugs for the prevention or treatment of EV71 infection, screening and quality control, and the prevention and treatment of EV71 infection.
Disclosure of Invention
The invention provides enterovirus71 VP1 protein mutant, mutant virus strain and application thereof.
The applicant constructs cDNA infectious clone of EV71 virus by reverse genetics technology in early research, and can easily realize modification of RNA virus genome by genetic engineering technology means. Years of researches prove that a trans-type high-conservation site of the VP1 protein of the EV71 virus is likely to be a potential virulence determining site, and site-directed mutation screening is carried out on the VP1 protein. Through continuous screening and verification, the invention discovers that after the 75 th amino acid site of VP1 protein is mutated from threonine (T) to alanine (A), the maturation and shearing of VP0 protein in the EV71 packaging process can be obviously influenced, so that the proliferation capacity of viruses in cells is influenced. However, the mutation at this site again enhances the virulence of the virus in mice after infection of 2-day-old rats by intraperitoneal injection. The mutation site, the corresponding protein mutant and the mutant virus strain thereof lay an important foundation for developing a new anti-EV 71 drug and obtaining a new EV71 vaccine candidate strain, and provide a basis for elucidating the pathogenic mechanism of the virus and locating the virulence determining site of the virus.
Specifically, the invention provides the following technical scheme:
the invention provides an enterovirus71 VP1 protein mutant, and the amino acid sequence of the VP1 protein mutant is shown as SEQ ID NO. 1.
Compared with the wild enterovirus71 VP1 protein, the amino acid sequence shown in SEQ ID NO.1 has mutation from threonine (T) to alanine (A) at 75 th amino acid.
The VP1 protein mutant can obviously reduce the proliferation capacity of EV71 and obviously enhance the toxicity.
The invention provides a nucleic acid molecule which codes for the enterovirus71 VP1 protein mutant.
Based on the amino acid sequence and codon regularity of the VP1 protein mutants provided above, the skilled person will be able to obtain the nucleotide sequence of the nucleic acid molecule encoding the VP1 protein mutants. The nucleotide sequence of the nucleic acid molecule is not unique based on the degeneracy of the codons, but all nucleic acid molecules capable of encoding the VP1 protein mutants described above are within the scope of the invention.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is one in which the codon corresponding to amino acid 75 is mutated to an alanine codon based on the gene encoding the VP1 protein of the EV71 wild-type strain.
In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 2.
The invention provides a biological material, which comprises the nucleic acid molecules or expresses the enterovirus71 VP1 protein mutant;
the biological material is an expression cassette, a vector or a host cell.
Wherein the expression cassette may be a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with transcriptional and/or translational regulatory elements.
Such vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, and the like.
The host cell includes a microbial cell or an animal cell, and the microorganism includes a bacterium (e.g., E.coli), a fungus (e.g., yeast), or a virus (e.g., enterovirus type 71). The animal cells may be animal cells commonly used for culturing viruses (e.g., vero cells, etc.).
The invention provides an enterovirus71 type mutant virus strain, which comprises the nucleic acid molecules or expresses the enterovirus71 type VP1 protein mutant.
The enterovirus71 mutant strain can be any enterovirus71 strain serving as a starting strain, and the starting strain is modified so as to contain the nucleic acid molecules or express the enterovirus71 VP1 protein mutants.
In some embodiments of the invention, the mutant strain is obtained by mutating the genome of an enterovirus71 type wild-type strain such that the 75 th amino acid of its VP1 protein (preferably threonine) is mutated to alanine.
In some embodiments of the invention, the mutant strain is obtained by mutating the nucleotide position 2660 of the viral genome of enterovirus71 type wild-type strain HP (Zhang YX, huang YM, li QJ, et al A highly conserved amino acid in VP1 regulates maturation of enterovirus [ J ]. PLoS Pathogens,2017,13 (9): e1006625.DOI: 10.1371/journ. Ppat. 1006625.) from A to G.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule or the biological material in construction of enterovirus71 mutant virus strains.
Preferably, the enterovirus 71-type mutant strain has increased virulence and/or reduced proliferative capacity.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule or the biological material in reducing the proliferation capacity of the enterovirus71 and/or enhancing the virulence of the enterovirus 71.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule, the biological material or the enterovirus71 strain in preparation of medicines for preventing and/or treating enterovirus71 infection.
Preferably, the drug comprises a therapeutic drug (e.g., an antibody, etc.), a vaccine, etc.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule, the biological material or the enterovirus71 strain in preparation of products for detecting whether enterovirus71 exists in a sample or the existence level of the enterovirus71 in the sample.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule, the biological material or the enterovirus71 strain in screening and/or quality control of medicines for preventing and/or treating enterovirus71 infection.
Preferably, the drug comprises a therapeutic drug (e.g., an antibody, etc.), a vaccine, etc.
The invention provides application of the enterovirus71 VP1 protein mutant, the nucleic acid molecule, the biological material or the enterovirus71 virus strain in preparation of an enterovirus71 infected animal model.
The present invention provides a method for reducing the proliferative capacity of enterovirus type 71 and/or enhancing the virulence of enterovirus type 71, the method comprising: the genome of enterovirus type 71 is mutated so that it encodes amino acid 75 of the VP1 protein (preferably threonine) into alanine.
The beneficial effects of the invention at least comprise: the enterovirus71 VP1 protein mutant and the corresponding encoding nucleic acid molecule thereof provided by the invention can reduce the proliferation capacity of the enterovirus71, enhance the toxicity, can be used for constructing enterovirus71 mutant virus strains, and provide effective biological materials for preparing medicines for preventing and treating enterovirus71 infection and screening and quality control of the medicines.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the result of sequencing verification of mutation sites in example 1 of the present invention.
FIG. 2 shows the result of immunoblotting in example 4 of the present invention, wherein Ctrl represents a control, EV71-HP represents wild-type strain HP, and EV71-T75A represents HP-T75A mutant.
FIG. 3 shows the results of the proliferation phenotype test of the HP-T75A mutant in example 5 of the present invention on Vero cells, wherein Ctrl represents the control, FY represents the wild-type strain HP, and T75A represents the HP-T75A mutant.
FIG. 4 is a graph showing the results of the infectious titer test of the virus in example 5 of the present invention, wherein the upper graph shows the results of the inoculum size MOI=10, and the lower graph shows the results of the inoculum size MOI=1; EV71-HP represents the wild strain HP, and T75A represents the HP-T75A mutant.
FIG. 5 shows the results of detection of protein fractions and RNAs of virus particles purified by density gradient centrifugation with different sedimentation coefficients in example 6 of the present invention, wherein EV71-HP represents wild-type strain HP and T75A represents HP-T75A mutant strain.
FIG. 6 shows the results of toxicity detection of the HP-T75A mutant in BalB/C mice in example 7 of the present invention, wherein A is the degree of hind limb paralysis of the mice, B is the weight change and mortality, and C is the tissue distribution of qPCR-determined virus; MOCK represents control, EV71-HP represents wild strain HP, T75A represents HP-T75A mutant.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 construction of an infectious clone of the cDNA of the 75 th amino acid position mutant (HP-T75A) of the enterovirus71 type HP strain VP1
The infectious cloning plasmid of wild strain virus (EV 71-HP strain) is used as a template, and the 75 th amino acid site of VP1 protein is mutated from threonine residue to alanine residue (namely, the 2660 th nucleotide site of virus genome is mutated from A to G) by a site-directed mutagenesis method. First, a plasmid with a mutation site and an nick was amplified using primers 75-F (5'-GTGTTCTTAACTCGC ACAGTG CAGCTGAGACCACTCTTGAT-3') and 75-R (5'-ATCAAGAGTGGTCTCAGCTGCACTGTGCGA GTTAAGAACAC-3') with a mutation base. After the PCR reaction, the methylated original plasmid template was digested with restriction enzyme DpnI. And (3) transforming the treated PCR product into escherichia coli DH5 alpha competent cells, and repairing and amplifying the mutant plasmid carrying the deficiency in DH5 alpha. Finally, the mutation site is successfully introduced by sequencing verification (figure 1), and FY-T75A cDNA infectious clone is obtained.
EXAMPLE 2 rescue of EV71 HP-T75A mutant Virus by infectious clones
In vitro transcription: the 3' -end of FY-T75A cDNA infectious clone was digested with restriction enzyme HindIII to obtain linearized DNA template, which was purified using gel recovery purification kit (cat# CW2301M, available from CWBIO Co.), and full-length viral genomic RNA was transcribed in vitro using T7 RNA polymerase in vitro transcription kit MEGAscript High Yield Transcription Kit (cat# ON-040, available from HONGENE BIOTECH Co.), followed by purification of the RNA using lithium chloride.
Cell transfection: vero cells were cultured in DMEM medium containing 10% FBS, and after 24h inoculation of six well plates, they were changed to serum-free DMEM medium and transfected with RNAiMAX transfection reagent (cat# 13778150, available from ThermoFisher Scientific) at 2. Mu.g RNA per well. The pathological effect (cytopathic effect, CPE) of the cells was observed daily after transfection, and virus-infected cells and culture supernatants were harvested for identification and passaging after the cells were round down, shed over 75% or 7d after transfection.
EXAMPLE 3RT-PCR identification of the VP1 sequence of the rescued mutant Virus strain HP-T75A
Genomic RNA of the mutant virus was extracted using Trizol LS as a template, and the viral genome nucleotide sequence (comprising the coding sequence for VP1 protein) was amplified by RT-PCR reaction using the general primers EV71-qPCR-F (5'-GCAGCCCAAAAGAACTTCACTATG AAA-3') and EV71-qPCR-R (5'-GAGTGGCAAGATGTCGGTTG-3'). The rescued mutant strains were identified by sequencing analysis and showed that the mutant viruses rescued by the HP-T75A infectious clone contained mutation sites consistent with their infectious clone.
EXAMPLE 4 Western blotting (Western blotting) detection of expression of viral proteins of HP-T75A mutant
After 24h inoculation of Vero cells in six well plates, 2 μg of viral genomic RNA was transfected per well. After 48h transfection, cells were collected with 1mL of pre-chilled PBS, centrifuged at 800rpm for 10min, PBS was discarded, 1X SDS loading buffer was added, the metal bath was set at 100deg.C for 30 min, and the samples were subjected to electrophoresis. The expression of viral structural proteins VP1, VP2 and VP0 was detected by Western Blotting, respectively, using specific antibodies. The primary antibodies were respectively anti-EV 71 VP1 murine monoclonal antibody (cat# MAB1255-M08, available from Abnova), anti-EV 71 VP0/2 murine monoclonal antibody (cat# MAB979, available from Millipore), and horseradish peroxidase (HRP) -labeled goat anti-murine IgG secondary antibody (cat# ZB-2305, available from China fir gold bridge). Immunoblotting showed that the expression of specific viral proteins was detected in both the HP strain and the cells transfected with the mutant strain HP-T75A, and that the VP1, VP2 and VP0 proteins of the mutant strain HP-T75A were expressed in significantly lower amounts than in the wild-type strain (FIG. 2).
EXAMPLE 5 proliferation phenotype of HP-T75A mutant on Vero cells
2 mug of viral genome RNA is transfected into each six-hole plate of Vero cells, CPE progress of the cells is observed every day, and when 90% -100% of the cells are diseased, the supernatant is collected, and the corresponding viruses are obtained through rescue. CPE of cells was recorded microscopically (fig. 3). After transfection of the HP strain genomic RNA into the cells, the proliferation of the virus and the progression of the cellular CPE were significantly faster than for the HP-T75A strain. After 1d transfection, both strains produced sporadic cytopathy. After 3d transfection, more than 80% of CPE was produced by the HP strain, while no significant progress was made by the HP-T75A strain. After 7d transfection, CPE produced by HP-T75A strain could not reach more than 80% (FIG. 3), indicating that mutation of amino acid position 75 of VP1 protein can significantly affect the proliferation phenotype of the virus.
The infectious titer of the virus was determined by microtitration. Vero cells were seeded in 96-well plates with 1X 10 cells per well 4 Vero cells were used. After the cells grow into a monolayer on the next day, the virus liquid to be tested is diluted by a 10-time serial gradient of DMEM culture medium, and 100 mu L of diluted virus liquid is inoculated in each hole. Daily observations record CPE progression for each well of cells until day 5 post inoculation, were terminated. Calculating TCID of the virus according to Reed-Muench formula 50 . In addition, HP-T75A mutant and wild-type strain viruses were inoculated into Vero cells at high infection dose (moi=10) and low infection dose (moi=0.1), respectively, and cultured at 37 ℃, and samples were collected 0h,2h,4h,8h,12h,24h, 36h, 48h and 72h after infection after high infection dose inoculation, and samples were collected 1d,2d and 3d after low infection dose inoculation, respectively. The virus titer of the supernatant was determined by a microtiter method, and the proliferation kinetics of the corresponding virus strain was plotted. The results showed that after inoculation of Vero cells with moi=10, the titer of both strains gradually increased with the extension of the incubation time, but the titer of the HP-T75A mutant was always lower than that of the wild strain within 72h of inoculation. When two strains of virus were inoculated with Vero cells at moi=1, the virus titer of the HP-T75A mutant was much greater than that of the wild strain and was not substantially leveled to the wild strain until 72h after infection (fig. 4).
EXAMPLE 6 VP0 precursor protein of HP-T75A mutant is defective during maturation cleavage
When progeny EV71 virus particles are packaged, firstly, a hollow protein capsid is assembled by precursor proteins VP0 and VP1 and VP3, then genome RNA of the virus enters the capsid, and the precursor proteins VP0 undergo spontaneous hydrolysis and are sheared into VP4 and VP2 proteins due to the change of protein conformation at the same time, so that mature virus particles are generated, and therefore, the shearing process of VP0 is also called maturation shearing.
To investigate whether the T75A mutation would affect the maturation shear of EV71, virus particles with different sedimentation coefficients were purified by density gradient centrifugation, and their protein fraction and RNA were detected separately. Supernatants of EV71-HP and HP-T75A mutant viruses were thawed by three freeze-thawing cycles, centrifuged at 3000rpm for 10min to obtain supernatants, and cell debris was removed by filtration through a 0.22 μm filter. The collected virus solution was concentrated by super-ionization with 15% sucrose cushion by centrifugation at 37000rpm for 2.5h (Beckman SW41 Ti) at 4 ℃. The concentrated virus samples were then centrifuged at 37000rpm for 70 minutes (Beckman SW41 Ti) at a 15% -35% continuous sucrose density gradient to separate virus particles with different sedimentation coefficients. After centrifugation, samples were taken from the top in an amount of 0.5 mL/serving. Half of each sample was assayed for viral protein components by western blot analysis after RNA extraction with Trizol LS and quantification of viral genomic RNA copy number by RT-qPCR, and the other half by super-concentration. The experimental results showed (FIG. 5) that the RNA-containing virions had two peaks at the 8 to 11 (135S) and 13 to 15 (160S) samples, corresponding to the EV 71A particle and mature virions, respectively. Notably, in 160S mature particles, the VP0 precursor protein of the HP-T75A strain was significantly more than that of the EV71-HP strain, indicating that this site mutation could affect maturation cleavage of VP0 protein. And the protein of the HP-T75A strain 135S virus particle is obviously less than that of the EV71-HP strain, which indicates that the capsid removal process of the HP-T75A strain virus particle is also affected.
EXAMPLE 7 virulence enhancement of HP-T75A mutant in BalB/c milk mice
Inoculation by way of intraperitoneal inoculation (100. Mu.L/min.) 10 7 lgTCID 50 In 2-day-old BALB/C lactating mice (5/group) were subjected to continuous observation for 16 days with the wild strain as a control, and the hind limb paralysis degree (A in FIG. 6), weight change and mortality (B in FIG. 6) of the lactating mice were recorded daily, spleen, stomach and muscle tissues of the mice were dissected after the completion of the observation, and the supernatant was ground and assayed for viral tissue distribution by qPCR (C in FIG. 6). The results show that the weight of mice infected by the HP-T75A mutant strain is reduced more severely and the lethality is higher than that of the wild strain, which indicates that the mutation of the 75 th amino acid site of VP1 protein can enhance the toxicity of EV71 in the mice.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The enterovirus71 VP1 protein mutant is characterized in that the amino acid sequence of the VP1 protein mutant is shown as SEQ ID NO. 1.
2. A nucleic acid molecule encoding the enterovirus71 VP1 protein mutant of claim 1.
3. A biological material comprising the nucleic acid molecule of claim 2 or a VP1 protein mutant of enterovirus71 of claim 1;
the biological material is an expression cassette, a vector or a host cell.
4. An enterovirus71 type mutant virus strain, wherein the enterovirus71 type mutant virus strain comprises the nucleic acid molecule of claim 2 or expresses the enterovirus71 type VP1 protein mutant of claim 1.
5. The enterovirus 71-type mutant virus strain according to claim 4, wherein the mutant virus strain is obtained by mutating the genome of an enterovirus 71-type wild-type virus strain such that the 75 th amino acid of its VP1 protein is mutated to alanine.
6. Use of the enterovirus71 type VP1 protein mutant of claim 1 or the nucleic acid molecule of claim 2 or the biological material of claim 3 in the construction of an enterovirus71 type mutant virus strain.
7. Use of the enterovirus71 type VP1 protein mutant of claim 1 or the nucleic acid molecule of claim 2 or the biological material of claim 3 for reducing the proliferative capacity of enterovirus71 and/or enhancing the virulence of enterovirus 71.
8. Use of an enterovirus71 type VP1 protein mutant of claim 1 or a nucleic acid molecule of claim 2 or a biological material of claim 3 or an enterovirus71 type strain of claim 4 or 5 in the manufacture of a medicament for preventing and/or treating an enterovirus71 type infection.
9. Use of an enterovirus71 type VP1 protein mutant of claim 1 or a nucleic acid molecule of claim 2 or a biological material of claim 3 or an enterovirus71 type virus strain of claim 4 or 5 for screening and/or quality control of a medicament for preventing and/or treating an enterovirus71 type infection.
10. A method of reducing the proliferative capacity of enterovirus type 71 and/or enhancing the virulence of enterovirus type 71, the method comprising: the genome of enterovirus type 71 was mutated so that it encodes amino acid 75 of VP1 protein into alanine.
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