CN106377766B - EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as well as preparation method and application thereof - Google Patents
EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as well as preparation method and application thereof Download PDFInfo
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- CN106377766B CN106377766B CN201610892209.2A CN201610892209A CN106377766B CN 106377766 B CN106377766 B CN 106377766B CN 201610892209 A CN201610892209 A CN 201610892209A CN 106377766 B CN106377766 B CN 106377766B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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Abstract
The invention discloses an EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and a preparation method and application thereof. Human podoropathy virus was isolated, whole genome sequencing yielded the EV71 virus VP1 structural protein, which was divided into 20 groups and synthesized into polypeptide fragments. Screening out serum containing EV71 virus antibody, and primarily screening out polypeptides No.2, No.4 and No.8 with higher immune effect through enzyme-linked immunosorbent reaction. In order to further verify whether the preliminarily screened polypeptide has an immune effect in a mouse model, the Balb/c mouse test model is reasonably designed and pathological section HE staining pathological analysis is carried out on the Balb/c mouse test model, the No.8 polypeptide has a high immune effect on the small intestine, skeletal muscle and brain of the Balb/c mouse model, and the No.2 polypeptide has a high immune effect on the skeletal muscle of the Balb/c mouse model. Through the enzyme-linked immunosorbent assay and the pathological examination comprehensive analysis and verification of a model Balb/c mouse, the result shows that the optimized polypeptide No.2 and the optimized polypeptide No.8 have great potential in the development and application of the EV71 virus vaccine.
Description
Technical Field
The invention belongs to the field of immune biomedicine, and particularly relates to an EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and a preparation method and application thereof.
Background
Enterovirus 71 (EV 71) is one of the main pathogens causing hand-foot-and-mouth disease of infants, and at present, no antiviral drug aiming at EV71 exists, and the antiviral drug is mainly used for symptomatic treatment. In the research aspect of antiviral drugs, in vitro experiments prove that pyridyl imidazoline dione derivatives can effectively inhibit the replication of EV71, and are promising candidate drugs at present. The whole virus inactivated vaccine of the EV71 virus uses whole virus as an antigen, but in view of safety, particularly in an individual with poor immunity, it may have a harmful effect on the human body, and therefore, in recent years, a subunit vaccine has been the focus of research.
The polypeptide vaccine is prepared by synthesizing protective polypeptide or epitope of pathogenic microorganism by chemical synthesis or genetic engineering, connecting the protective polypeptide or epitope to macromolecular carrier, and adding adjuvant. Compared with the conventional vaccine and the gene recombinant vaccine, the polypeptide/epitope vaccine does not contain the virus genome information which poses potential threat to animals, does not integrate or recombine virus genes and host cell genes, does not pose potential threat to animals or human beings, and is a safe vaccine. A large number of researches show that the neutralizing antibody generated by EV71 infected people and animal immunity experiments has the effect of resisting EV71 virus and can form a certain protection effect, and in vitro experiments prove that the generated neutralizing antibody has virus neutralizing activity. Most of EV71 vaccines studied at present are virus inactivated vaccines and enter the phase I clinical trial stage. The subunit vaccine is obviously superior to the traditional vaccine in safety, and the search for effective antigen protein is the key for the success of the genetic engineering recombinant vaccine. VP1 is the most studied capsid protein and is considered to be the region of EV71 where neutralizing epitopes are most concentrated.
The invention separates human foot and mouth disease virus, analyzes the whole genome sequence, obtains EV71 virus VP1 structural protein, divides the amino acid into 20 groups, each group is 15 amino acids plus the upstream 5 amino acids, each group is 20 amino acid polypeptide, the last group is 12 amino acids, and synthesizes the polypeptide fragments through chemistry. Collecting serum of healthy children with 2-3 months old. Coating a solid phase carrier ELISA96 pore plate with the synthesized polypeptide fragment, adding the serum of the infant patient for incubation, adding a horseradish peroxidase-labeled secondary antibody for incubation after washing, developing the substrate to determine the absorbance, and screening the polypeptide fragment capable of being immunologically combined with the serum. The screened polypeptide is then used to study the characteristics of the polypeptide antigen through animal in vivo immunization and related pathological examination. The method provides an important basis for screening candidate antigens of polypeptide vaccines in the future for further and deeply understanding the relevant properties of the four capsid proteins of EV 71.
Disclosure of Invention
The invention aims to provide an EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and a preparation method and application thereof.
The invention relates to a polypeptide, the amino acid sequence of which is shown as any sequence in SEQ ID NO.1-20 in a sequence table:
in order to achieve the purpose of the invention, the technical scheme mainly comprises the following steps:
firstly, human foot-mouth disease virus is separated, and is subjected to whole genome sequencing analysis, so that the EV71 virus VP1 structural protein is obtained. The amino acid sequence of the VP1 structural protein obtained by separation is divided into 20 groups, each group is 15 amino acids, 5 amino acids at the upstream are added, each group is polypeptide of 20 amino acids, the last group is 12 amino acids, the polypeptide fragments (synthesized by Gill Biochemical Co., Ltd.) are synthesized by chemistry, and the specific polypeptide sequence is shown in any sequence of SEQ ID NO.1-20 in the sequence table.
Preferably, the serum of healthy children with a recovery period of 2-3 months is collected.
Preferably, the synthesized polypeptide fragment is coated on a solid phase carrier ELISA96 pore plate (purchased from Shanghai Union Biotech Co., Ltd.), the serum of the patient is added for incubation, after washing, horseradish peroxidase (HRP) labeled secondary IgG (rabbit anti-human, purchased from Qingdao Jiekang Biotech Co., Ltd.) is added for incubation, the substrate is developed to measure the absorbance, and the polypeptide fragment capable of being combined with the serum in an immunological manner is screened out.
VP1 structural protein is segmented and synthesized into polypeptide, and the immune effect of the polypeptide is detected by the following specific method:
the isolated EV71 virus VP1 protein is divided into 20 groups of 15 amino acids each, and is added with 5 downstream amino acids, each group is a polypeptide of 20 amino acids, and the last group is 12 amino acids, and a polypeptide fragment (synthesized by Gill Biochemical Co., Ltd.) is directly synthesized.
And further detecting the serum antibody concentration of the convalescent child and preferably selecting the serum with the highest EV71 virus antibody concentration as a primary antibody for subsequent screening of the polypeptide antigen. More specifically, the method comprises the following steps:
clinical infant serum sample collection information was taken from: liu Zhen, Liu Yue, Wu Lin Man, Gaojunhao, Chen Xuan, Zhou Yutong. The serum antibody concentration was detected by using human enterovirus EV71 type IgG ELISA kit (Yaji Biotech Co., Ltd., Shanghai).
On the basis, a polypeptide coated ELISA plate is further synthesized, a primary antibody (Liuyue Yue serum sample) is added, a secondary antibody IgG (rabbit anti-human) is continuously added on the basis of the polypeptide antigen and primary antibody immunoreaction, the immune effect of the secondary antibody IgG is detected, and the polypeptide with better immune effect is preferably selected as a candidate antigen vaccine to carry out the immune test of animals (mice).
Further preferably, animal tests are carried out, an immune animal model is established, the in vivo immune effect of the polypeptide vaccine is detected through pathological examination, and the candidate polypeptide vaccine is further confirmed.
The invention has the beneficial effects that: firstly, VP1 protein is used as a target antigen to develop an EV71 vaccine for resisting hand-foot-and-mouth disease; secondly, the EV71 virus is obtained by separation, and the whole genome sequencing analysis is carried out on the EV71 virus, so that the EV71 virus VP1 structural protein is successfully obtained. Dividing the amino acids into 20 groups, each group comprises 15 amino acids, adding downstream 5 amino acids, each group comprises 20 amino acid polypeptides, and the last group comprises 12 amino acids, and directly synthesizing polypeptide fragments; serum of clinically representative hand-foot-mouth rehabilitation children is collected, and a human enterovirus EV71 type IgG ELISA kit is adopted to detect the concentration of serum antibodies, so that a target primary antibody is screened out; fourthly, the immune effect of the polypeptide is determined through in vitro immune reaction, candidate polypeptide vaccines are primarily screened out, and the polypeptide vaccines which can be popularized and applied to clinical prevention and/or treatment of the hand-foot-and-mouth disease are further optimized through a series of animal models of polypeptide immunity; fifthly, the method provides important basis for further understanding the relevant properties of the four capsid proteins of EV71 and screening candidate antigens of polypeptide vaccines in the future.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:
fig. 1 (group a): the pathological section images of skeletal muscle, small intestine and brain of the mice of the polypeptide group No.2 are shown.
Fig. 2 (group B): the pathological section images of skeletal muscle, small intestine and brain of the mice of the polypeptide group No.4 are shown.
Fig. 3 (group C): the pathological section images of skeletal muscle, small intestine and brain of the mice of the polypeptide group No.8 are shown.
Fig. 4 (group D): pathological section images of skeletal muscle, small intestine and brain of normal rat groups are shown.
Fig. 5 (group E): the pathological section images of skeletal muscle, small intestine and brain of a polypeptide mouse group (a polypeptide mouse group obtained by breeding 2 female mice and 1 male mice in a cage after polypeptide immunization, respectively, without virus administration) are shown.
Fig. 6 (group F): table normal mouse neonatal mouse group (given virus) skeletal muscle, small intestine and brain pathological section images.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.
Example 1 detection of serum antibodies in convalescent infants
Collecting serum sample information of clinical children patients: liu Zhen, Liu Yue, Wu Lin Man, Gaojunhao, Chen Xuan, Zhou Yutong. The method adopts a human enterovirus EV71 IgGELISA kit to detect the concentration of serum antibodies, and comprises the following specific operation method:
the method comprises the following operation steps:
1. dilution and sample loading of standard: setting 10 holes of a standard substance hole on an enzyme-labeled coating plate, respectively adding 100ul of a standard substance into a first hole and a second hole, then adding 50ul of a standard substance diluent into the first hole and the second hole, and uniformly mixing; then 100ul of the standard dilution solution is respectively taken from the first hole and the second hole and respectively added to the third hole and the fourth hole, and then 50ul of the standard dilution solution is respectively added to the third hole and the fourth hole and is uniformly mixed; then 50ul of each sample is taken from the third hole and the fourth hole and discarded, 50ul of each sample is added into the fifth hole and the sixth hole respectively, 50ul of standard product diluent is added into the fifth hole and the sixth hole respectively, and the mixture is mixed evenly; respectively taking 50ul of the diluent from the fifth hole and the sixth hole after uniformly mixing, respectively adding the diluent into the seventh hole and the eighth hole, respectively adding 50ul of the standard substance diluent into the seventh hole and the eighth hole, respectively taking 50ul of the diluent from the seventh hole and the eighth hole after uniformly mixing, respectively adding the diluent into the ninth hole and the tenth hole, respectively adding 50ul of the standard substance diluent into the ninth hole and the tenth hole, respectively taking 50ul of the diluent from the ninth hole and the tenth hole after uniformly mixing, and discarding. (after dilution, the sample loading amount of each well is 50ul, and the concentration is 30ng/L, 20ng/L, 10ng/L, 5ng/L and 2.5ng/L respectively).
2. Sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. 40ul of sample diluent is added into sample holes to be detected on the enzyme-labeled coated plate, and then 10ul of sample to be detected is added (the final dilution of the sample is 5 times). And adding the sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall.
3. And (3) incubation: the plates were sealed with a sealing plate and incubated at 37 ℃ for 30 minutes.
4. Preparing liquid: diluting the 20 times of concentrated washing solution with 20 times of distilled water for later use.
5. Washing: carefully uncovering the unsealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry.
6. Adding an enzyme: 50ul of enzyme-labeled reagent was added to each well, except for blank wells.
7. And (3) incubation: the operation is the same as 3.
8. Washing: the operation is the same as 5.
9. Color development: adding the color developing agent A50ul into each hole, adding the color developing agent B50ul into each hole, shaking gently and mixing uniformly, and developing for 15 minutes at 37 ℃ in a dark place.
10. And (4) terminating: stop solution was added to each well at 50ul to stop the reaction (blue color turned immediately yellow).
11. And (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with the blank well being zeroed.
And (3) calculating the antibody concentration in the serum sample to be detected in a reduced manner through the standard curve, and selecting the clinical sample Liuyue serum as a primary antibody to perform subsequent immune experiments according to the determination result.
Example 2 synthetic polypeptide-coated ELISA plates and evaluation and screening of polypeptide immune Effect
1. The buffer was prepared as follows:
coating liquid: 0.05mol/L carbonate buffer (pH9.6)
0.75g of sodium carbonate, 1.46g of sodium bicarbonate and deionized water are added to make the volume of the mixture reach 500 ml.
② 0.02mol/L phosphate buffer solution (pH7.4)
0.2g of monopotassium phosphate, 2.90g of disodium hydrogen phosphate and 8g of sodium chloride, and deionized water is added to the mixture to reach the volume of 1000 ml.
③ dilution of antibody: 0.02mol/LPBS (pH7.4) +0.2% BSA
0.2g BSA was dissolved in 0.02mol/L phosphate buffer and quantified to 100 g.
Sealing liquid: 0.05mol/L carbonate buffer (pH9.6) +2.0% BSA
2.0g BSA was dissolved in 0.05mol/L carbonate buffer to give a fixed amount of 100g BSA.
Washing liquid: 0.02mol/LPBS (pH7.4) +0.05% Tween-20
50ul of Tween-20 was dissolved in 100ml of 0.02mol/L phosphate buffer, and mixed by shaking.
Color developing liquid: TMB-Urea Hydrogen peroxide solution
Solution a (3, 3 ', 5, 5' -tetramethylbenzidine, TMB): TMB20mg was weighed and dissolved in 10ml of absolute ethanol, and after complete dissolution, double distilled water was added to 100 ml.
Solution B (0.1mol/L citric acid-0.2 mol/L disodium hydrogen phosphate buffer, pH 5.0-5.4): weighing Na2HPO4·12H2O14.34g and citric acid 1.87g are dissolved in 180ml of double distilled water, 0.75% of urea hydrogen peroxide 1.28ml is added, the volume is adjusted to 200ml, and the pH value is adjusted to 5.0-5.4.
Mixing the solution A and the solution B according to the proportion of 1: l, mixing to obtain the TMB-urea hydrogen peroxide application liquid.
Seventh, stop solution: 2mol/LH2SO4Solutions of
10ml of 98% concentrated sulfuric acid is added into 60ml of double distilled water, the volume is constant to 100ml, and the mixture is stored at room temperature.
The second antibody of the enzyme label: HRP-labeled IgG (rabbit anti-human) was diluted 3000-fold with antibody diluent at the time of application
2. The operation steps are as follows:
(ii) antigen coating
The polypeptide is diluted to 0.01mg/ml by coating solution (0.05mol/L carbonate buffer solution, pH9.6), and 100ul of polypeptide No.1-20 is added into each enzyme label plate hole, and each polypeptide has 4 repeat holes. The microplate was placed in a wet box and coated overnight at 4 ℃.
② washing plate
Discarding the coating solution, filling all enzyme-labeled holes with a washing solution, and wrapping and drying the enzyme-labeled holes with gauze and toilet paper.
③ seal
250ul of blocking solution (pH9.6, 2.0% BSA in 0.05mol/L carbonate buffer) was added to each well of the plate, and the plate was placed in a wet box and incubated overnight at 4 ℃. Incubation can also be carried out at 37 ℃ for 2 h.
Fourthly, removing the sealing liquid, and washing for 1 time as above.
Fifthly, adding primary antibody (Liuyue Yue serum sample)
10ul of Liuyue Yue serum was added to 990ul of antibody dilution to make 1:100 antiserum (run in 1.5ml EP tube). And adding 100ul of serum at a ratio of 1:100, 1:200, 1:400 and 1:800 into 4 compound wells of the polypeptide No.1 on the ELISA plate. In the same way, 100ul of diluted antibody serum is added into the wells coated with the No. 2-20 polypeptide on the ELISA plate, and after the addition is finished, the ELISA plate is incubated in a wet box at 37 ℃ for 1 h.
Sixthly, washing is carried out as above.
Seventhly, adding enzyme labeled secondary antibody
100ul enzyme-labeled secondary antibody (rabbit anti-human) with the dilution ratio of 1:3000 is added into each hole, and the mixture is incubated for 1h in a 37 ℃ wet box.
And the method is as above.
Ninthly color development
After 50ul of solution A and solution B is added to each well, the enzyme-linked plate is put into a wet box and kept in the dark for about 30min, and the reaction is stopped when the negative control well develops blue-green color. When the reaction is terminated, 50ul of 2mol/L concentrated sulfuric acid is added into each well, the evaluation result of the polypeptide immune effect is shown in the table 1, NC represents PBS to replace serum control, and KC represents blank plate control.
The determination result shows that the No.2, 4 and 8 polypeptides have good immune effect, so the No.2, 4 and 8 polypeptides are selected as vaccines to immunize mice.
Example 3 in vivo assays to further verify and evaluate the immunological efficacy of polypeptides
1. Immune mouse (Shanghai laboratory animal center of Chinese academy of sciences) model construction
The obtained polypeptide No.2, polypeptide No.4 and polypeptide No.8 were dissolved in 0.9% sodium chloride solution to a final concentration of 1mg/ml, and mixed with the same volume of Freund's complete adjuvant (purchased from Roche pharmaceutical Co., Ltd.) to prepare a polypeptide vaccine. The Balb/c mice (purchased from Shanghai laboratory animal center of Chinese academy of sciences) adopt 12 female Balb/c healthy mice for 6-8 weeks, and are divided into 4 groups (No. 2 polypeptide group, No.4 polypeptide group and No.8 polypeptide group), the polypeptide solution is injected into the abdominal cavity by 0.1ml, the immunization is repeated once after one week, the heat inactivated virus is used as a positive control, and the hepatitis B virus surface antigen is used as a negative control. After immunization, 2 female mice and 1 male mouse were respectively caged for hybridization.
(1) Taking materials
Mice were intraperitoneally inoculated with 5X 107TCID50 of EV71 virus particles on the day of birth, and 1 day later mice were sacrificed to harvest skeletal muscle, small intestine and brain samples.
Obtaining skeletal muscle materials: the neck of a newborn mouse is clamped by a pair of forceps, the neck is cut off, the newborn mouse is killed, the skin of the leg of the mouse is cut, skeletal muscle is exposed to take materials, and one part of the skin is fixed by 4% paraformaldehyde for subsequent detection. One portion was stored at-80 ℃ for subsequent testing.
Obtaining materials of small intestine: the abdominal cavity of the mouse was opened, and the material was taken from the digestive tract pylorus to about 1.5cm of small intestine, and fixed with 4% paraformaldehyde for subsequent detection.
Taking materials for the brain: shearing the skin of the newborn mouse head, shearing the skull and the dura, taking the brain, and storing at-80 ℃ for subsequent detection.
(2) Immune effect of polypeptide
Skeletal muscle result analysis:
the muscle fiber shapes of the normal mice in the group D and the new mice in the polypeptide mice in the group E (without virus) are more consistent, the sizes are uniform, and muscle cells are normal;
secondly, the normal mice of the group F (for the virus group), the polypeptide group A No.2, the polypeptide group B No.4 and the polypeptide group C No.8 all have pathological changes of skeletal muscle inflammation with different degrees, especially, the group F has more inflammation, and part of the mice has muscle cell necrosis, degeneration, inflammatory cell infiltration, fuzzy muscle fiber and atrophy.
Analysis of small intestine tissue results:
comparing the F group (the normal mouse new mouse after being given virus) with the E group (the normal mouse new mouse without being given virus), the focus is more obvious;
② the three administration groups of A (No. 2 polypeptide group), B (No. 4 polypeptide group) and C (No. 8 polypeptide group) have certain focus compared with the normal group D (normal child mouse), but the difference between the three groups is not obvious.
Brain tissue results analysis:
comparing the F group (the normal mouse new mouse after being given virus) with the E group (the normal mouse new mouse without being given virus), the focus is more obvious;
② compared with the normal group D (normal child mouse), the three administration groups of A (No. 2 polypeptide group), B (No. 4 polypeptide group) and C (No. 8 polypeptide group) have no obvious change, and the three groups have no obvious difference.
The detection result of the immune effect in vivo (pathological section HE staining method) shows that the No.2 polypeptide and the No.8 polypeptide have obvious immune effect, the No.8 polypeptide has higher immune effect on the small intestine, skeletal muscle and brain of a model Balb/c mouse, the No.2 polypeptide has higher immune effect on the skeletal muscle of the model Balb/c mouse, and the No.4 polypeptide has obvious immune effect, so the invention proves that the No.2 polypeptide and the No.8 polypeptide can be used as candidate antigens of EV71 virus polypeptide vaccines in future.
Finally, it is noted that the above preferred embodiments are merely intended to illustrate rather than to limit the invention, and although the invention has been described in detail with reference to the above preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
<110> Chongqing Gaosheng biomedical Limited liability company
<120> EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, and preparation method and application thereof
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Claims (7)
1. A preparation method of EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine is characterized by comprising the following steps:
separating EV71 virus from a hand-foot-mouth patient, carrying out whole genome sequencing analysis on the EV71 virus, successfully obtaining EV71 virus VP1 structural protein, dividing amino acids of the EV71 virus VP1 structural protein into 20 groups, wherein each group comprises 15 amino acids, 5 downstream amino acids, polypeptides of 20 amino acids in each group, and the last group comprises 12 amino acids, and directly synthesizing polypeptide fragments, wherein the fragment sequences are shown as SEQID NO. 1-20;
collecting serum of clinically representative hand-foot-and-mouth rehabilitation children, detecting the concentration of a serum antibody by adopting a human enterovirus EV71 type IgG ELISA kit, screening out serum containing an EV71 virus antibody, namely a target primary antibody, further synthesizing a polypeptide coated ELISA plate, adding the target primary antibody, continuously adding rabbit anti-human secondary IgG on the basis of polypeptide antigen and primary anti-immune reaction, and preliminarily screening out polypeptide with a high immune effect by enzyme-linked immunosorbent assay immune reaction;
and (3) in order to further verify whether the preliminarily screened polypeptide still has an immune effect in vivo, constructing an immune mouse model, and carrying out pathological analysis on the immune mouse model by a pathological section HE staining method, wherein the result shows that polypeptide amino acid sequences such as SEQ ID NO.2 and SEQ ID NO.8 in a sequence table have significant significance on the immune effect of the model immune mouse, the polypeptide amino acid sequences such as SEQ ID NO.4 in the sequence table have insignificant immune effect, and the screened polypeptides shown in SEQ ID NO.2 and SEQ ID NO.8 are the polypeptide vaccine.
2. An EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine, characterized in that the polypeptide is prepared by the preparation method of claim 1.
3. The EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as claimed in claim 2, wherein the amino acid sequence of the polypeptide with immune effect preferably determined by enzyme-linked immunosorbent assay in step (2) is shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO.8 in the sequence list.
4. The EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as claimed in claim 2, characterized in that the pathological examination in step (3) further verifies that the final preferred polypeptide amino acid sequence with immune effect is as shown in SEQ ID No.2 and SEQ ID NO.8 of the sequence list.
5. The EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as claimed in claim 4, wherein the polypeptide amino acid sequence SEQ ID NO.8 preferred in step (3) has a high immune effect on small intestine, skeletal muscle and brain of a model immunized mouse.
6. The EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine as claimed in claim 4, wherein the preferred polypeptide amino acid sequence SEQ ID NO.2 in step (3) has a higher immune effect on skeletal muscle of model-immunized mice.
7. Use of the EV71-VP1 hand-foot-and-mouth disease polypeptide vaccine of any of claims 2-6 for the preparation of a medicament for the prevention of hand-foot-and-mouth disease.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893748A (en) * | 2012-12-27 | 2014-07-02 | 上海吉美生物工程有限公司 | Novel vaccine for hand-foot-and-mouth disease and a preparation method thereof |
| CN104650195A (en) * | 2015-03-09 | 2015-05-27 | 盖中涛 | EV71 virus VP1 recombinant antigens as well as monoclonal antibody and application of eV71 virus VP1 recombinant antigens |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103893748A (en) * | 2012-12-27 | 2014-07-02 | 上海吉美生物工程有限公司 | Novel vaccine for hand-foot-and-mouth disease and a preparation method thereof |
| CN104650195A (en) * | 2015-03-09 | 2015-05-27 | 盖中涛 | EV71 virus VP1 recombinant antigens as well as monoclonal antibody and application of eV71 virus VP1 recombinant antigens |
Non-Patent Citations (1)
| Title |
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| Identification of neutralizing linear epitopes from the VP1 capsid protein of Enterovirus 71 using synthetic peptides;Damian Guang Wei Foo et al.;《Virus Research》;20070112;第125卷;第61-68页 * |
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