CN113105529B - Nucleic acid sequence, enterovirus containing nucleic acid sequence and application of enterovirus - Google Patents
Nucleic acid sequence, enterovirus containing nucleic acid sequence and application of enterovirus Download PDFInfo
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- CN113105529B CN113105529B CN202010026635.4A CN202010026635A CN113105529B CN 113105529 B CN113105529 B CN 113105529B CN 202010026635 A CN202010026635 A CN 202010026635A CN 113105529 B CN113105529 B CN 113105529B
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Abstract
The invention relates to a nucleic acid sequence, an enterovirus containing the sequence and application thereof. The virus containing the nucleic acid sequence disclosed by the invention can be used for preparing vaccines and preventing intestinal diseases. The antibody produced by the virus immunization can be used for preparing a diagnostic kit for diagnosing enterovirus.
Description
Technical Field
The invention relates to a nucleic acid sequence, enterovirus containing the sequence and application thereof.
Background
A group of enteroviruses of the family picornaviridae, which are important pathogens that can cause high-incidence and new-onset infections in humans, include Enterovirus (Enterovirus species-A, B, C, D, more than 120 serotypes) and rhinoviruses A-C (Rhinovirus species-A, B, C, at least 200 serological types), hereinafter collectively referred to as Enteroviruses (EV). Although the members of the enterovirus genus have similar biological properties, there is no effective cross-protection among serotypes and the clinical manifestations are complex and diverse, and the latter may be caused by different receptors of enterovirus and different distribution of enterovirus in tissues and organs. Human enteroviruses are mainly transmitted through a feces-oral route, have strong infectivity and can cause about billions of people to infect and attack every year; mild cases are self-limiting, and severe clinical manifestations involve the neuro-muscular system, such as aseptic meningitis, meningoencephalitis, poliomyelitis, and acute delayed paralysis (etiologies include poliovirus, PV for short, and non-poliovirus, such as EV-a71, EV-D68, EV B6, and B11, etc.), severe myocarditis and dilated cardiomyopathy (Coxsackievirus B3, CVB 3), type I diabetes (CVB 1-5, echovirus 3,4,6,9,16,30, etc.), severe pneumonia (EV-D68), severe asthma (rhinovirus, RV), which are important lethal infections in children under 5 years of age. In China, about 200 ten thousand reported cases of hand-foot-and-mouth diseases are caused by enteroviruses such as EV-A71 and CVA16 in recent years, and the severe phenomenon is aggravated by nervous system symptoms of infants suffering from severe hand-foot-and-mouth diseases. In addition, an EV-D68 epidemic outbreak in the united states in 2014 caused more than 1000 patients with severe respiratory disease, with some of the children also presenting with symptoms of acute relaxant myelitis, which are very similar to those of poliovirus and EV-a 71-induced neurological damage. EV-D68 sporadic case reports are continuously generated in 2006 in China, and actual cases may far exceed reported cases due to limits of infectious disease report systems. Recent studies have shown that the EV-D68 virus is an important pathogen that causes severe respiratory and nervous system diseases following EVA-71. Furthermore, recent increasing data indicate that enterovirus serotypes, which have not been considered before, can also cause the above-mentioned severe clinical symptoms. Due to the enormous quantity of serotypes of enteroviruses infecting human beings, along with the increase of the prevalence of infection of people and the continuous evolution of viruses, the severe epidemic situation of high-incidence and new infectious diseases caused by the enteroviruses in the global scope must be regarded as high.
The enterovirus genome structure and protein function are highly conserved, the genome is single positive strand RNA, about 7.4kb, two sides are respectively 5 '-and 3' -non-coding regions (UTR), and the middle is 1 open reading frame. The 5' -UTR contains a specific genetic element, internal Ribosome Entry Site (IRES), which is a cis-element IRES essential for viral initiation of protein translation, and mediates viral protein translation. Based on the IRES translation mechanism, the viral genome encodes a precursor protein of about 2700 amino acids, which is converted by the viral proteolytic enzyme 2A pro And 3C pro After cleavage 11 mature viral proteins were formed. These proteins have different functions and play an irreplaceable role in protecting the vital activities of viruses. Such as enterovirus 2A pro Cleavage of eIF4GI in trans, resulting in a sinkThe translation mechanism of the cap of the main cell is closed, which is beneficial to starting the translation mechanism of the IRES of the virus in the initial stage of virus replication and provides effective guarantee for virus proliferation. Human enteroviruses are a group of important pathogens causing new and high-incidence infectious diseases of human beings, but severe infections caused by most enteroviruses lack specific vaccines and antiviral drug control, and cause direct and indirect economic losses in hundreds of millions each year. If the technology is industrialized, corresponding direct and indirect market benefits and social benefits can be generated.
Disclosure of Invention
The present invention has been made in order to provide a useful vaccine and diagnostic agent for preventing or diagnosing enterovirus-induced related diseases.
In one aspect, the present invention relates to a nucleic acid sequence having an amino acid sequence encoding: gly Ile Val Ala Thr Gly Gly wherein Ile can be replaced by Leu; val may be replaced by Ile or Leu; thr may be replaced by any amino acid, preferably by Met, ala, gly or Val.
As one of the specific embodiments of the present invention, the nucleic acid sequence is SEQ ID No:1 and synonymous sequences thereof.
The synonymous sequences are nucleic acid sequences that encode homologous amino acid sequences with the sequences.
As another aspect of the invention, it relates to an amino acid sequence which is Gly Ile Val Ala Thr Gly, wherein Ile can be replaced by Leu; val may be replaced by Ile or Leu; thr can be replaced by any amino acid, preferably by Met, ala, gly or Val.
As a further aspect of the present invention, it relates to a virus comprising the above nucleic acid sequence. In one embodiment of the present invention, the virus is an enterovirus. The invention also relates to the application of the virus containing the nucleic acid sequence in preparing vaccine, the prepared vaccine, the antibody produced by the virus immunization and a diagnostic kit containing the antibody.
As a further aspect of the invention, it relates to a virus encoding the above amino acid sequence. In one embodiment of the present invention, the virus is an enterovirus. The invention also relates to the application of the virus for coding the amino acid sequence in the preparation of vaccines, the prepared vaccines, antibodies generated by the immunization of the virus and a diagnostic kit containing the antibodies.
As a further aspect of the invention, it relates to the use of the above vaccine in the prevention of disease.
As one aspect of the present invention, it relates to the use of the above-mentioned antibodies and corresponding diagnostic kits for the diagnosis of diseases.
Based on the content of the disclosure, the prepared vaccine can be used for preventing and diagnosing related diseases caused by enteroviruses, and has great economic value and obvious social benefit.
Drawings
FIG. 1: muscle tissue isolation virus nucleic acid sequencing result graph. The results are based on using clinically isolated and plaque experimental cloning of purified EV-A71 ABV331 strain, intraperitoneal injection of ICR suckling mice infected with birth for 1 day, observation of clinical expression of suckling mice and extraction of viral nucleic acid from the muscle tissue of suckling mice for sequencing. The results show that 4 suckling mice have obvious clinical symptoms, and are manifested as hindlimb weakness, tremor or paralysis, and the 4 suckling mice muscle tissues are separated and purified to obtain the virus nucleic acid sequence shown in SEQ ID No. 3, and the coded amino acid sequence is shown in SEQ ID No:4, the sequence is consistent with the sequence of the virus injected in the abdominal cavity; no obvious clinical symptoms are found in 1 suckling mouse, and the virus nucleic acid sequence obtained by separating and purifying the muscle tissue of the suckling mouse is SEQ ID No:1, and the coded amino acid sequence is SEQ ID No:2, namely serine (Ser) at the original position to alanine (Ala).
FIG. 2 is a schematic diagram: A71-A-mutated (Ser mutation Ala) attenuation result graph. The results are based on the construction of an infectious clone of EV-A71 (encoding the amino acid sequence shown in SEQ ID No: 4) and Ala (sequence of SEQ ID No: 2) mutated from Ser, and the verification of whether Ala mutated from Ser causes the change of virus pathogenicity. The infectious clones were transcribed in vitro, transfected with cells, and the rescued progeny virus EV-A71 ABV331 (A71-WT) and Ala-mutated progeny virus (A71-A-mutated) were harvested. The progeny virus was titrated separately and the RD cells were infected separately with 1MOI of virus and the experiment was repeated 3 times. The results show that the cytopathic effect caused by A71-A-mutated is obviously weaker than that of A71-WT group, the cutting of the eukaryotic translation initiation factor of host protein is obviously weaker than that of A71-WT group, and the gray value of the virus capsid protein VP1 is obviously weaker than that of A71-WT group. Indicating that A71-A-mutated has attenuated virus virulence at the cellular level.
FIG. 3: alignment results graph. The result is obtained by analyzing the amino acid sequences of 15 types of enteroviruses EV-A, EV-B, EV-C, EV-D, EV-E, EV-F, EV-G, EV-H, EV-I, EV-J, EV-K, EV-L, RV-A, RV-B and RV-C. The result shows that the amino acid sequence is Gly Ile (Leu) Leu (Ile/Val) Thr (Ser) Met (Ala/Gly/Val) Gly Gly similar to the amino acid sequence Gly Ile Val Ser Thr Gly of EV-A71 virus, the sequence is relatively conserved, but the virulence site Ser is not a typical conserved site and can also be threonine (Thr).
FIG. 4 is a schematic view of: B3-A-mutated (Thr mutation Ala) attenuation result graph. This result is based on Alignment results, where the Ser site is not conserved, and can also be Thr. A mutation of Thr to Ala may not cause a decrease in virus virulence. In the experiment, infectious clones of CVB3 wooddruff (encoding Gly Leu Ile Thr Met Gly Gly Gly) and Ala (Gly Leu Ile Ala Met Gly Gly) mutated from Thr are established, RNA is obtained through in vitro transcription, cells are transfected, and rescued progeny viruses CVB3 wooddruff variant (B3-WT) and Ala mutated progeny viruses (B3-A-mutated) are harvested. The progeny virus was titrated separately and the RD cells were infected with 1MOI of virus separately and the experiment was repeated 3 times. The IFA fluorescence method shows that the fluorescence intensity of the virus capsid protein of the B3-A-mutated infected group is obviously weaker than that of the B3-WT group, namely the proliferation capacity of the B3-A-mutated is obviously weakened. That is to say, the virulence of virus infected cells is weakened by the fact that the enterovirus contains Gly Leu Ile Thr Met Gly Gly and Thr in the enterovirus is mutated into Ala.
FIG. 5: immunoblot results of Ser/Thr mutation to Ala. The results are based on whether the EV-a71 virus Ser mutation to Ala results in reduced virulence, the CVB3 virus T mutation to a results in reduced virulence, and then the Ser/Thr mutation to Ala attenuation in enteroviruses is a universal mechanism? The plasmid with Ala mutated Ser of coxsackievirus A5 Swartz (CVA 5 Swartz, coded Gly Ile Val Ser Thr Gly Gly) and coxsackievirus A12 Texas-12 (CVA 12 Texas-12, coded Gly Ile Val Ser Thr Gly Gly Gly Gly) is constructed, experiments show that the cutting effect of the protein coded by the plasmid on host translation initiation factor eIF4GI is weakened, the propagation of the virus in cells is not facilitated, and the universality of Ala attenuation caused by Ser mutation of enterovirus species is proved; plasmids were constructed in which Thr of poliovirus Mahoney (PV Mahoney, encoding Gly Ile Ile Thr Ala Gly), enterovirus D68 Fermon (EV-D68 Fermon, encoding Gly Leu Leu Thr Ala Gly), and rhinovirus ATCC VR-1559 (RV ATCC VR-1559, encoding Gly Ile Leu Thr Ala Gly) was mutated to Ala. The result shows that the protein coded by the plasmid weakens the cutting effect of host translation initiation factor eIF4GI, proves the universality of attenuation of Thr mutation to Ala in enterovirus, and the experiment is repeated for 3 times. From this, it can be concluded that, among enteroviruses, a virus encoding an amino acid sequence having the following characteristics has a reduced virulence. The amino acid sequence is characterized in that: gly X YAla Z Gly (X: ile or Leu; Y: ile or Val or Leu; Z: thr or Met or Ala or Gly or Val). In conclusion, the virus virulence will be reduced as long as the mutated amino acid encoding the amino acid sequence is Ala.
FIG. 6: sequencing result graph of S mutation Asp. This result is based on a reduction in virulence of Ser/Thr site mutation to Ala; is it mutated to a non-Ala amino acid, is virulence still reduced? In the experiment, EV-A71 virus is used as a model, infectious clone which codes Ser mutated into aspartic acid (Asp) is constructed, the amino acid sequence coded by the nucleic acid sequence is Gly Ile Val Asp Thr Gly Gly, and progeny virus (A71-D-mutated) of the infectious clone is harvested for observation. Equal amounts of three viruses A71-WT, A71-A-mutated and A71-D-mutated are respectively infected into RD cells, and the result shows that the cytopathic effect of the A71-A-mutated is obviously weaker than that of the cells infected with the A71-WT; interestingly, the lesion effect of the RD cells infected with A71-D-mutated was unstable, some did not show significant lesions, and some were consistent with the lesions of the cells infected with A71-WT. Collecting cell supernatant, extracting virus RNA for sequencing, and displaying that the virus genome infected with A71-WT shows that the site is Ser (100%); A71-A-mutated shows that this site is Ala (100%); the genome of a virus infected with a71-D-mutated showed instability at this site and other types of mutations in the encoded amino acids, such as Gly (glycine, 26%), asn (asparagine, 7%), ser (serine, 5%), tyr (tyrosine, 4%), were repeated 112 times. In conclusion, we can conclude that: only if the mutation is Ala, the obtained virus is stably inherited, and the toxicity of the virus is obviously reduced.
FIG. 7: figure of experimental results of ICR infected mice. This result is to verify that the virus with the Ala mutation is significantly less pathogenic to animals. A71-A-mutated virus (10) was used in this experiment 7 TCID 50 /ml), ICR mice infected with the first 1 day of birth were injected intraperitoneally. Because EV-A71 has neurotoxicity, aseptic encephalitis, delayed muscular paralysis and other symptoms can be caused, and the pathogenic capability of the virus can be judged by observing the survival state, weight change and clinical manifestations (hind limb weakness/tremor/paralysis) of ICR mice infected with the virus within 2 weeks. The experimental results showed that both A71-WT and A71-Amutated groups were 100% viable; there was no significant difference in body weight. Clinical symptoms of A71-WT were significantly heavier than those of the A71-A-muted group, and were most severe on day 4 of infection, after which the symptoms slowly subsided. Section (3 μm) HE staining of muscle and brain tissue of ICR mice showed that the tissue infected with A71-WT had a stronger inflammatory response than the tissue infected with A71-A-mutated: the infiltration of muscle interstitial cells by lymphocytes can be seen in muscle tissues; lymphocyte infiltration is seen in the cortex of brain tissue. Meanwhile, the virus load of the muscle tissue and the brain tissue infected with A71-WT is obviously higher than that of the A71-A-mutated tissue. In conclusion, our results fully demonstrate that A71-A-mutated virus has a significantly reduced pathogenic capacity in mice.
FIG. 8: graph of the results of the antibody neutralization experiment. ICR mice infected with A71-A-mutated antibodies were immunized, blood was collected from the eyeball 2 weeks later, left overnight at 4 ℃ and centrifuged to obtain serum. And (3) detecting the neutralizing effect of the antibody in the serum on the EV-A71 virus. Experimental results show that CPE appears when the negative control serogroup is diluted to 2^ 3 to 8 times, and weak CPE appears when the antibody serogroup is diluted to 2^ 12 to 4096 times, which indicates that the antibody has good neutralization protection effect on EV-A71 infection.
Detailed Description
Unless otherwise indicated, all experimental methods used in the present disclosure are routine experimental methods in the art, and the experimental procedures of the present disclosure can be fully implemented by those skilled in the art based on the present disclosure. The use of the viral strains used in the present disclosure to practice the present invention is available to applicants. For convenience of description, the description of amino acids in this disclosure sometimes uses three-dimensional letters and sometimes uses one letter, whether three-dimensional or one letter, following standards in the art, and one skilled in the art can correspondingly determine the respective corresponding amino acids. The present disclosure relates to an EV-a71 virus which is a positive strand RNA virus, and since a nucleic acid sequence in a sequence database such as GenBank is often included as a DNA sequence, those skilled in the art can understand that although a base T and a base U may be used in combination when describing a nucleic acid sequence, those skilled in the art can accurately understand the corresponding sequence based on the context.
The inventors considered that mutation at this site may be a cause of reduction in toxicity, and carried out verification. The results showed that A71-A-mutated induced CPE significantly weaker than the A71-WT group, see FIG. 2A; the fluorescence intensity of the virus VP1 protein in the A71-A-mutated infected group is significantly weaker than that in the A71-WT group, as shown in FIG. 2B; the cleavage of the host protein eukaryotic translation initiation factor (eIF 4G) by the virus was also significantly weaker than in the A71-WT group, and little viral capsid protein VP1 expression was observed, see FIG. 2C. Specific operations can be referred to as follows: the inventor adopts a Gibson gene recombination principle and a single-chain primer bridging strategy, and constructs an infectious clone containing EV-A71 complete sequence by taking an enterovirus strain EV-A71 ABV331 separated in a laboratory as a template; on the basis, an EV-A71 ABV331 mutant strain infectious clone which codes a Gly Ile Val Ala Thr Gly Gly sequence is constructed by adopting a point mutation method. Firstly, linearizing DNA fragments required for constructing infectious clones, adding a terminal repetitive sequence at the tail ends of the DNA fragments by utilizing primer design, then connecting the DNA fragments together through Gibson assembly, preserving heat for 30min at 50 ℃, and treating and digesting methylated template plasmids by using DMT enzyme so as to avoid the generation of false positive clones; then, the ligation product is transformed and plated to obtain monoclonal bacteria, and the monoclonal bacteria are sequenced to identify whether the obtained monoclonal bacteria are positive clones. The infectious clone plasmid is linearized by NsiI restriction endonuclease, and the size of the band is determined to be correct. Individual colonies identified above as positive for the EV-A71 infectious clone were picked for culture and quality enhancement. Transcribing the clone in vitro to obtain RNA transcript of the mutant virus, transfecting 2 mu g of mutant virus RNA to RD cells when the cell fusion degree is about 80%, adding cells with the same amount of transfection reagent as a blank control group, adding cells infected by 1MOI EV-A71 wild strain as a positive control group, observing cytopathic effect (CPE) and harvesting progeny virus; continuously passaging the progeny virus until the virulence of the progeny virus is stable; the progeny virus was harvested and stored at-80 ℃ until use. A progeny virus identical in sequence to the laboratory wild-type EV-A71 ABV331 gene (Gly Ile Val Ser Thr Gly Gly Gly Gly Gly) was designated A71-WT, and an EV-A71 mutant encoding Gly Ile Val Ala Thr Gly Gly was designated A71-A-mutated. A71-WT and A71-A-mutated viruses were titrated, and RD cells were infected with I MOI virus, respectively, and it was found that CPE caused by A71-A-mutated was significantly weaker than that of A71-WT group. The fluorescence intensity of the virus capsid protein VP1 is observed by an immunofluorescence assay (IFA) to judge the proliferation level of the virus. The method comprises the following steps: RD cell cells are inoculated in a 24-well plate, A71-WT and A71-A-mutated cells are respectively inoculated when the cell density is 80%, PBS is washed for 3 times after 2 hours of infection, and DMEM is added for continuous culture for 2 days. DMEM was discarded, PBS was washed 3 times, 4% paraformaldehyde solution was added, cells were fixed at room temperature for 10min, and then PBS was used to wash the cells 2 times. 0.1% Triton X-100 was added in an amount of about 200. Mu.l per well, incubated at room temperature for 5min, and the cells were washed 1 time with PBS. Add 200. Mu.l of IFA blocking solution (10% donkey serum +5% BSA in PBS) to each well and incubate for 1h at room temperature. Mu.l of a dilution of an antibody against the viral capsid protein VP1 (1. Mu.l Alexa Fluor488 donkey anti-mouse IgG secondary antibody dilution (1, 500) was added to each well and incubated for 1h at room temperature in the absence of light. PBS wash 3 times for 10min each. Mu.l of DAPI stain was added to each well, incubated at room temperature for 10min, washed with PBS for 10min. Add 100. Mu.l PBS to each well and place under a fluorescence microscope for observation. The experimental result shows that the fluorescence intensity of the virus VP1 protein of the A71-A-mutated infected group is obviously weaker than that of the A71-WT group, and the proliferation capability of the A71-A-mutated in cells is weakened, namely the virulence of the virus is weakened. Separately, 2. Mu.g of RNA was transfected into RD cells, the cells were harvested in 48 hours, and the lysate was added to extract proteins, and the protein concentration was measured. After the sample concentration is obtained, the volume of the required sample is calculated according to the protein loading amount required by the experiment, and the total volume of each group is filled by using loading buffer solution and PBS. The sample was denatured in 100 ℃ water for 5min and then iced for 5min. Short rapid centrifugation ensures that the sample is concentrated at the bottom of the tube. Electrophoresis is carried out for 1h at a voltage of 70V, and the voltage is adjusted to 120V 1h after the electrophoresis is carried out on the gel. Film transfer: sequentially placing a fiber pad, two pieces of filter paper, gel, a PVDF (polyvinylidene fluoride) membrane, two pieces of filter paper and a fiber pad from the cathode to the anode; placing the PVDF film, and removing air bubbles between the PVDF film and the gel; note the positive and negative poles of the electrode bath. And (3) sealing: and after the electric membrane conversion is finished, taking out the PVDF membrane, putting a cleaning solution, slightly shaking for 5min on a shaking table, then soaking the PVDF membrane into 5% milk sealing solution, and sealing for 1-2 h at room temperature or standing overnight at 4 ℃. Antibody hybridization: taking out the PVDF membrane, shaking and washing the PVDF membrane in a cleaning solution for three times at room temperature, wherein each time lasts for 10min; then soaking the PVDF membrane in primary-antibody working solution, and shaking the PVDF membrane overnight at 4 ℃; after the first-time hybridization, taking out the PVDF membrane, putting the PVDF membrane into a cleaning solution, and shaking and washing the PVDF membrane for three times at room temperature for 10min each time; transferring the PVDF membrane into a secondary antibody working solution, and reacting for 1-2 h at room temperature; after labeling the secondary antibody, the PVDF membrane was placed in a washing solution and shaken at room temperature for 5min three times. And obtaining an experimental result by chemiluminescence, darkroom exposure or development of an immunoblotting instrument. The cutting effect of the virus on the host protein eukaryotic translation initiation factor (eIF 4G) is obviously weaker than that of the A71-WT group, virus capsid protein VP1 expression is hardly observed, the experiment is repeated for 3 times, and the results are consistent. Our experiments prove that the Ser site in the Gly Ile Val Ser Thr Gly Gly Gly is an important virulence site of the virus.
In general, the virulence sites of the virus are conserved. Therefore, based on the above verification experiments the inventors believe that the Ser site in Gly Ile Val Ser Thr Gly Gly Gly should be conserved. To further verify that this site is conserved in the Enterovirus genus, the inventors obtained the whole genome sequences of 15 Species (specifices) of Enteroviruses EV-A, EV-B, EV-C, EV-D, EV-E, EV-E, EV-E, EV-E, EV-E, EV-E, RV-A, RV-B and RV-C from GenBank, analyzed using MEGA5.0, and the results of Alignment (see FIG. 3) found that the amino acid at the corresponding site of S can also be threonine (T); that is, the site is not typically conserved.
To verify the effect of threonine (Thr) at this site on the virulence of the virus, the inventors mutated this site to alanine (Ala) in viruses with threonine (Thr) at this site to observe changes in the virulence of the virus. The verification process is performed using coxsackievirus (CVB 3) as an example. Specific operations can be referred to as: the inventors constructed wild type CVB3 and infectious CVB3 clones in which Thr at this site was mutated to Ala. Like the construction of EV-A71 infectious clone described above, the inventors constructed a CVB3 wooddruff variant (Gly Leu Ile Thr Met Gly Gly) whole genome sequence infectious clone by using Gibson's assembly ligation principle and a strategy of bridging by combining single-stranded primers and using an enterovirus strain CVB3 wooddruff variant as a template, and constructed an infectious clone of a CVB3 wooddruff variant mutant strain encoding a Gly Leu Ile Ala Gly Gly Gly Gly sequence by using a point mutation method. Firstly, linearizing DNA fragments required for constructing infectious clones, adding a terminal repetitive sequence at the tail ends of the DNA fragments by utilizing the design of primers, then assembling through Gibson, connecting the DNA fragments together, preserving the temperature for 30min at 50 ℃, and treating and digesting a template plasmid containing methylation by using DMT enzyme so as to avoid the generation of false positive clones; and then, after the ligation product is transformed and plated, harvesting the monoclonal bacteria and sending the monoclonal bacteria to a sequencing machine, and identifying the whole virus genome sequence of the harvested monoclonal bacteria. The infectious clone plasmid is linearized by NsiI restriction endonuclease, and the size of the band is determined to be correct. Individual colonies identified above as positive for CVB3 infectious clones were picked for culture and quality enhancement. Transcribing the clone in vitro to obtain RNA transcript of the mutant virus, transfecting 2 mu g of mutant virus RNA to RD cells when the cell fusion degree is about 80%, adding cells with the same quantity of transfection reagent as a blank control group, adding cells infected by the 1MOI CVB3 wild strain as a positive control group, observing CPE and harvesting progeny virus; continuously passaging the progeny virus until the virulence of the progeny virus is stable; harvesting progeny virus, at-80 ℃ for use. Progeny viruses with a sequence identical to the laboratory wild-type CVB3 wooddruff variant gene sequence (Gly Leu Ile Thr Met Gly Gly Gly Gly) were designated B3-WT, and the CVB3 wooddruff variant mutant encoding Gly Leu Ile Ala Gly Gly Gly Gly was designated B3-A-mutated. And (3) titrating the progeny virus, infecting RD cells with the wild strain and the mutant virus of 1MOI respectively, observing the virus proliferation condition, and repeating the experiment for 3 times to obtain consistent results. The fluorescence intensity of the virus capsid protein VP1 is observed by an immunofluorescence assay (IFA) to judge the proliferation level of the virus. The method comprises the following steps: RD cell cells are inoculated in a 24-well plate, B3-WT and B3-A-mutated cells are respectively inoculated when the cell density is 80%, PBS is washed for 3 times after infection for 2 hours, and DMEM is added for continuous culture for 2 days. DMEM was discarded, PBS was washed 3 times, 4% paraformaldehyde solution was added, cells were fixed at room temperature for 10min, and then PBS was used to wash the cells 2 times. Adding about 200 μ l of 0.1% Triton X-100 per well, incubating for 5min at room temperature, washing cells 1 time with PBS. Add 200. Mu.l of IFA blocking solution (10% donkey serum +5% BSA in PBS) per well and incubate for 1h at room temperature. Mu.l of a dilution of an antibody against the viral capsid protein VP1 (1. Mu.l Alexa Fluor488 donkey anti-mouse IgG secondary antibody dilution (1, 500) was added to each well and incubated for 1h at room temperature in the absence of light. PBS wash 3 times for 10min each. Mu.l of DAPI stain was added to each well, incubated at room temperature for 10min and washed with PBS for 10min. Add 100. Mu.l PBS to each well and place under a fluorescence microscope for observation. The experimental result shows that the fluorescence intensity of the virus VP1 protein of the B3-A-mutated infected group is obviously weaker than that of the B3-WT group, which shows that the proliferation capability of the B3-A-mutated in cells is weakened, namely the virus virulence is weakened, and the figure is shown in figure 4. The experimental result shows that Thr of the site is mutated into Ala, and the virus toxicity is weakened.
Based on the above experimental results, the inventors believe that this Ser/Thr to Ala mutation in this amino acid sequence may be a broad-spectrum mechanism leading to attenuated virulence in enteroviruses. To test this hypothesis, the inventors constructed plasmids in which CVA5 Swartz (encoding Gly Ile Val Ser Thr Gly Gly Gly Gly) and CVA12 Texas-12 (encoding Gly Ile Val Ser Thr Gly Gly Gly Gly) were mutated from Ser to Ala, and the cleavage of host translation initiation factor eIF4GI by the protein encoded by the mutated plasmids was reduced, which resulted in a reduction in the virus' ability to proliferate. The attenuation effect of mutating Ser to Ala at the site in enterovirus is proved to have universality; we have constructed plasmids in which the Thr of PV Mahoney (encoding Gle Ile Thr Ala Gly Gly), EV-D68 Fermon (encoding Gly Leu Leu Thr Ala Gly Gly Gly), and RV ATCC VR-1559 (encoding Gly Ile Leu Thr Ala Gly Gly Gly Gly Gly) is mutated to Ala, and the cleavage of host translation initiation factor eIF4GI by the protein encoded by the mutated plasmids is reduced. This also demonstrates the universality of attenuation of the Thr to Ala mutation at this site in Enterovirus. This part of the experiment was repeated 3 times and the results were consistent, see figure 5. Thus, in conjunction with the Alignment results, the inventors concluded that: in enteroviruses, viruses that encode an amino acid sequence characterized by a reduced virulence are: gly X Y Ala Z Gly (X is Ile or Leu; Y is Ile, val or Leu; Z is any amino acid, preferably Thr or Met or Ala or Gly or Val). As described above, if the mutant amino acid containing the amino acid sequence is Ala, the virus has a reduced virulence. The specific experimental operations were as follows: using 2A plasmid of EV-A71 as a template, adding a primer for introducing a mutation site, obtaining a fragment through PCR, adding recombinase, reacting for 1h at 50 ℃, adding Dpn1, reacting for 30min at 37 ℃, and digesting the template. The recombinant system was transformed into DH 5. Alpha. Competence. Ice-bath 30min, heat shock at 42 ℃ for 45s, immediately placing on ice for 2min, adding 700 mul of non-resistant LB culture medium, shaking at 37 ℃ for 180rpm 1h, taking 100 mul of bacterial liquid coated plate (containing corresponding resistant LB culture plate such as Amp), inverting at 37 ℃, overnight culturing, picking up single colony sister from an aseptic gun head, finally adding 10ml of liquid culture medium added with antibiotics, shaking at 37 ℃,200rpm, 169h, remaining 5ml, sequencing the rest, selecting the right sequencing person, and amplifying and culturing to extract plasmids. Each of the cells was transfected with 2. Mu.g of plasmid into RD cells, the cells were harvested in 48 hours, and then lysate was added to extract proteins and the protein concentration was measured. After the sample concentration is obtained, the volume of the required sample is calculated according to the protein loading amount required by the experiment, and the total volume of each group is filled by using loading buffer solution and PBS. The sample was denatured in 100 ℃ water for 5min and then iced for 5min. Short rapid centrifugation ensures that the sample is concentrated at the bottom of the tube. Electrophoresis is carried out for 1h at a voltage of 70V, and the voltage is adjusted to 120V 1h after the electrophoresis is carried out on the gel. Film transfer: sequentially placing a fiber pad, two pieces of filter paper, gel, a PVDF (polyvinylidene fluoride) membrane, two pieces of filter paper and a fiber pad from the cathode to the anode; placing the PVDF membrane and removing air bubbles between the PVDF membrane and the gel; note the positive and negative poles of the electrode bath. And (3) sealing: and after the electric membrane conversion is finished, taking out the PVDF membrane, putting a cleaning solution, slightly shaking for 5min on a shaking table, then soaking the PVDF membrane into 5% milk sealing solution, and sealing for 1-2 h at room temperature or standing overnight at 4 ℃. Antibody hybridization: taking out the PVDF membrane, and shaking and washing the PVDF membrane in a cleaning solution for three times at room temperature, wherein each time is 10min; then soaking the PVDF membrane into primary-resistant working solution, and shaking the PVDF membrane at 4 ℃ overnight; after the first-time hybridization, taking out the PVDF membrane, putting the PVDF membrane into a cleaning solution, and shaking and washing the PVDF membrane for three times at room temperature for 10min each time; transferring the PVDF membrane into a secondary antibody working solution, and reacting for 1-2 hours at room temperature; after labeling with the secondary antibody, the PVDF membrane was put into a washing solution and shaken at room temperature for 5min three times. And carrying out chemiluminescence, darkroom exposure or development by an immunoblotting instrument to obtain an experimental result. In conclusion, the inventors believe that a Ser/Thr to Ala mutation in this amino acid sequence results in a virus of the Enterovirus genus which is attenuated.
The above experiments were carried out by the inventors with strains of EV-A71 ABV331 (1), CVA5 Swartz (2), CVA12 Texas-12 (3), CVB3 wood druff variant (4), PV Mahoney (5), EV-D68 Fermon (6) and RV ATCC VR-1559 (7), respectively, and the results were consistent with each other. These strains correspond to enteroviruses EV-A sites (1-3), EV-B sites (4), EV-C sites (5), EV-D sites (6) and RV-A sites (7), respectively, and thus the site should be Sup>A site playing Sup>A key role in virus virulence in all enteroviruses.
Based on the above experimental results, it appears that the virulence of the virus is reduced as long as the Ser/Thr site is mutated. To verify this conclusion, the inventors modeled EV-A71, constructed an infectious clone at this site that mutated the codon encoding Ser to the codon encoding aspartic acid (Asp), and harvested their progeny virus (A71-D-mutated) for observation. The experimental results show that in RD cells infected with three viruses of A71-WT, A71-A-mutated and A71-D-mutated in equal amounts, the pathological effect of RD cells infected with A71-A-mutated group is obviously weaker than that of RD cells infected with A71-WT; however, the pathological effects of A71-D-mutated-infected RD cells were inconsistent, and some were not observed for significant CPE, but some were consistent with A71-WT-infected CPE. To investigate the cause of CPE instability in A71-D-mutated RD cells, the inventors collected the virus in the supernatant, extracted the viral RNA using a viral RNA kit, reverse-transcribed into cDNA, and then designed primer sequencing to observe the changes in the gene sequences of A71-WT, A71-A-mutated and A71-D-mutated. The sequencing result shows that: in the sequencing result of the virus genome infected with A71-A-mutated, the site is still A; in the sequencing result of the virus genome infected with A71-D-mutated, the site is unstable, and other types of amino acid mutations occur, such as: glycine (Gly), asparagine (Asn), serine (Ser) or tyrosine (Tyr), see fig. 6. In conclusion, the inventors reached the conclusion that: the virus is not necessarily reduced in virulence and stably inherited as long as the site is mutated to Ala, but the virus is stably inherited only when the site is mutated to Ala, and the virulence of the virus is obviously reduced.
After demonstrating a significant reduction in virulence of the strain with the Ala mutation at this site, the inventors investigated whether its pathogenic ability in infected animals would also be significantly reduced. The inventors selected 3 litters of ICR suckling mice just born for 1 day, and 12 mice in each group. The first group was injected intraperitoneally and intracranially with DMEM as a negative control, and the second group with A71-WT (10) 7 TCID 50 /ml), third group with A71-A-muted (10) 7 TCID 50 In ml). The changes in body weight and limb movements of the suckling mice were recorded daily. Taking hind limb muscle tissue and brain tissue of suckling mice with the most serious clinical symptoms 4-5 days after virus injection, observing histopathological changes through HE staining, measuring virus load, and judging the pathogenic intensity of the virus. EV-A71 has neurotoxicity, and can cause symptoms such as aseptic encephalitis, delayed muscular paralysis and the like. Therefore, the pathogenic ability of the virus was judged by observing the survival status, weight change, clinical manifestations (hind limb weakness/tremor/paralysis) of ICR mice within 2 weeks of infection. The experimental results showed that both A71-WT and A71-Amutated groups were 100% viable, and from day 7, the body weight of the wild type group was lower than that of the attenuated strain group, as shown in FIG. 7A. Clinical symptoms were evident in A71-WT compared to the A71-A-mutated group, with symptoms most severe on day 4 of infection, followed by a gradual reduction in symptoms, as shown in FIG. 7B. Fixing hind limb muscle tissue and brain tissue of suckling mouse with paraformaldehyde, dehydrating and embedding into wax block, making into 3 μm tissue slice, and performing HE staining. The staining result shows that compared with the A71-A-mutated group, the muscle tissue of the mice infected with A71-WT has stronger inflammatory reaction, and the lymphocytes can wrap or infiltrate the muscle interstitial cells; in brain tissue of mice infected with A71-WT, there was lymphocyte infiltration in the cortex, as shown in FIG. 7C. Grinding muscle tissue and brain tissue by liquid nitrogen, digesting the tissue by trizol, extracting RNA, performing reverse transcription to form cDNA, then performing absolute quantitative qPCR, and quantitatively determining the virus load in the muscle tissue and the brain tissue. The viral load was significantly higher in both muscle and brain tissues infected with A71-WT than in A71-A-mutated tissues, as shown in FIGS. 7D and 7E. This result is a good indication that the pathogenic capacity of the virus strain with the Ala mutation at this site is significantly reduced.
Since it can encode Gly X YThe mutant strain of Ala Z Gly sequence can effectively reduce the pathogenic capability of the virus and has no obvious clinical pathogenic expression, and the virus infected ICR mouse shows that the mutant strain has better safety. Whether the mutated virus produces protective antibodies or whether the virus can be used as an attenuated vaccine? To verify this hypothesis, the inventors re-immunized an A71-A-mutated ICR mouse, bled the eyeball 2 weeks later, and left the resulting blood at 4 ℃ overnight at 2000rpm for 30min to obtain serum. Serum from the DMEM-injected group was also used as a negative control. Plating, when the cell fusion degree is 80%, diluting the serum according to the gradient of 2^ 2 to 2^ 12, and mixing the serum with the virus at 37 ℃ for 1h; the cells were infected with the mixture for 2h, the solution was changed, and cytopathic effect (CPE) was observed after 48 h. Neutralizing effect of the antibody in the serum on EV-A71 virus is judged by CPE. The experimental results show that the negative control serogroup is diluted 2 -3 I.e. 8-fold virus-induced CPE was present, whereas the antibody serogroup was diluted to 2 -12 Namely 4096 times, weak CPE appears, which indicates that the antibody has good neutralizing effect on EV-A71. On the basis, the blank serum and the antibody serum are assembled to be made into a diagnostic kit for diagnosing EV-A71 virus infection, and the diagnostic kit is used for diagnosing the EV-A71 virus.
Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
Sequence listing
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<120> nucleic acid sequence, enterovirus containing the same and application thereof
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Claims (3)
1. An engineered enterovirus comprising a nucleic acid sequence encoding an amino acid sequence shown as Gly X YAla Z Gly, wherein X is selected from Ile or Leu; y is selected from Ile or Val or Leu; z is selected from Thr or Met or Ala or Gly or Val; the enterovirus is EV-A71 ABV331, CVA5 Swartz, CVA12 Texas-12, CVB3 wood druff variant, PV Mahoney, EV-D68 Fermon or RV ATCC VR-1559.
2. Use of an engineered enterovirus of claim 1 in the preparation of a vaccine.
3. A vaccine comprising the modified enterovirus of claim 1.
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| CN104805061A (en) * | 2014-01-24 | 2015-07-29 | 中国科学院上海巴斯德研究所 | Virus adaptive strain capable of infecting Chinese hamster ovary cell and application thereof |
| CN106138030A (en) * | 2015-04-17 | 2016-11-23 | 中国科学院上海巴斯德研究所 | New Enterovirus 71 strain and formononetin or the application in Enterovirus 71 of its salt |
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| CN104805061A (en) * | 2014-01-24 | 2015-07-29 | 中国科学院上海巴斯德研究所 | Virus adaptive strain capable of infecting Chinese hamster ovary cell and application thereof |
| CN106138030A (en) * | 2015-04-17 | 2016-11-23 | 中国科学院上海巴斯德研究所 | New Enterovirus 71 strain and formononetin or the application in Enterovirus 71 of its salt |
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