CN103083656A - Conjugate of influenza A virus conservative peptides M2e and virus-like particles, and application thereof - Google Patents
Conjugate of influenza A virus conservative peptides M2e and virus-like particles, and application thereof Download PDFInfo
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- CN103083656A CN103083656A CN2011103308932A CN201110330893A CN103083656A CN 103083656 A CN103083656 A CN 103083656A CN 2011103308932 A CN2011103308932 A CN 2011103308932A CN 201110330893 A CN201110330893 A CN 201110330893A CN 103083656 A CN103083656 A CN 103083656A
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Abstract
The invention relates to a conjugate of virus-like particles (VLP) of oligopeptide vector Q[beta] bacteriophage with target immunotherapy and conservative peptides M2e of human influenza virus M2 protein. The conjugate is mainly used for preventing human influenza A. The conjugate is coupled by a Sulfo-SMCC method which comprises a first step of preparing the virus-like particles (VLP) of the Q[beta] bacteriophage; a second step of preparing the virus-like particles (VLP) activated by Sulfo-SMCC; and a third step of preparing the conjugate of M2e-VLP. The conjugate can produce high titer protective antibody in mice and cross-protect the attack of various subtypes of the influenza A viruses.
Description
Technical field
The invention belongs to biological technical field, be specifically related to immunology with the conjugate of immune targeted therapeutic carrier and the conservative small peptide M2e coupling formation of influenza A virus.The attack of described each hypotype influenza A virus of conjugate energy cross protection.
Background technology
Preparing general influenza vaccines is focuses of present influenza vaccines research.Influenza is a kind of Acute respiratory infectious disease of serious harm human health, and the inoculation influenza vaccines are the most effective preventive means.The influenza vaccines that produce at present are mainly for inducing the surface glycoprotein hemagglutinin (Hemagglutinin that produces neutralizing antibody, HA) and neuraminidase (Neuraminidase, NA) still have two factors to restrict the application of seasonal current influenza vaccine in the prevention and control flu outbreak: one is the problem whether vaccine strain and epidemic isolates mate, because the seasonal current influenza vaccine mainly plays immunoprotection by the antibody that brings out hemagglutinin HA and neuraminidase NA, strain does not mate the protection effect that just not has; Another is from determining that vaccine strain is to the required longer cycle of final acquisition vaccine with to the dependence of Embryo Gallus domesticus.Therefore, utilize the conservative antigen of influenza A virus, have the broad spectrum influenza vaccine of cross-protection between the development different subtype, just become the important content of influenza vaccines research.Influenza A virus stromatin 2 (Mat rixprotein 2, M2) be the 3rd outer membrane protein except HA and NA, extracellular region (the M2ectodomain of M2, M2e) be 23 aminoacid of M2 albumen n end, M2e is at influenza A virus high conservative between the influenza A virus different subtype in people source particularly, and M2 especially M2e has become target antigen commonly used in broad spectrum influenza vaccine research.At present, existing four kinds of vaccines based on M2e enter I phase clinical research, and the result of study of these vaccines provides a large amount of experimental datas for the broad spectrum influenza vaccine of succeeding in developing early the prevention and control flu outbreak.
Virus-like particle (VLP) is the hollow shell structure that does not contain viral nucleic acid that the capsid protein by virus is assembled into, the virulent profile of tool and immunogenicity, can be by inducing body to produce take humoral immunization as main reaction with the approach of viral infection, thereby induce generation antibody, because VLP lacks hereditary material nucleic acid, therefore avoided the risk of viral infection.VLP can make foreign epitope present with orderly, array way, can also increase stability and the immunogenicity of antigen, be applied to the M2e-VLP vaccine research as carrier as VLP such as the HBc of escherichia coli expression, papaya mosaic virus (PapMV), human papillomavirus (HPV), phage Q β.Phage V LP a kind ofly can be presented on polypeptide antigen the carrier of particle surface.Q β-V LP can be used as the nasal cavity immunity carrier, is conducive to bring out local and systemic immunoreation.Prepared at present the IIa clinical experiment that Q phagus beta VLP virus-like particle is used for the blood pressure lowering vaccine abroad.And the VLP of existing M2e chemical coupling or gene fusion enters the clinical I phase and tests.At present existing 4 companies have reported the clinical experiment result based on the influenza vaccines of M2e of I phase, and this four company is Acambis Inc. (Britain) respectively, Cytos Biotechnology (Switzerland), Merck ﹠amp; Co Inc. (U.S.), and VaxInnate Corp. (U.S.).Have based on the M2e-HBcVLP and the M2e-flagellin that merge, M2e-OMPC and the M2e-AP205VLP of chemical coupling also arranged.Q phagus beta and AP205 phage all belong to RNA viruses, its profile globulate, and the virion that is comprised of capsid protein is suitable as vaccine carrier very much.
Summary of the invention
The present invention seeks to build the general influenza vaccines of virus-like-particle conjugates (M2e) n-Q β-VLP of a kind of conserved sequence M2e based on human influenza A matrix protein 2, the method for coupling can be chemical coupling or gene fusion.The M2e peptide section of coupling can connect for M2e-M2e or (M2e) n wherein n be 1,2,3,4,5,6,7,8.
For achieving the above object, the technical solution used in the present invention is as follows:
1. the preparation of virus-like particle:
The present invention has utilized the invention disclosed patent, and (number of patent application: 201010028904.7) Q phagus beta VLP is as the coupling carrier.The VLP of this sudden change has following characteristics: the 72nd of (1) Q phagus beta CP extended proteins gene inserts coding lysine and leucic nucleotide sequence AAGCTT (perhaps inserting the single-gene sequence of coding M2e or the M2e gene order that repeats to connect) between 73 bit codons.And CP protein gene termination codon is sported the GGA rear clone to prokaryotic expression carrier by TGA.(2) being inserted in the 72nd and 73 amino acids is that lysine and leucine are Q phagus beta capsid protein furcella tops, is the main department of body immune system identification, i.e. the immunodominance determining area of virus-like particle.(3) this VLP is expressed by bacterium coli solubility.
The concrete steps of preparation phage virus-like particle Q β-VLP: strain bacillus coli DH 5 alpha/pETQ β-CP is inoculated into LB culture medium culturing to suitable bacterium liquid and reaches the OD value, after adding derivant IPTG to induce a few hours, collect bacterium liquid, use the ultrasonication cell, after cracking, the centrifugal upward cleer and peaceful precipitation of collecting respectively.To the polymer that forms, use Sepharose CL-4B chromatographic column purification.
2. influenza virus A hominis's matrix protein 2 conserved sequence M2e peptide sections is synthetic:
Adopt solid-phase synthesis to utilize the aminoacid sequence of the conservative peptide section M2e of the automatic influenza virus A hominis's matrix protein 2 of Peptide synthesizer to be Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys Arg Cys Asn AspSer SerAsp Cys (GenBank:ACA49877.1), wherein the C-terminal of M2e adds cysteine Cys so that Q β-VLP coupling.The M2e peptide section of coupling can connect for M2e-M2e or (M2e) n wherein n be 1,2,3,4,5,6,7,8.
Peptide section M2e respectively with phage virus-like particle Q β-VLP, keyhole limpet hemocyanin (KLH) coupling:
Step: get Q β-VLP of 50mg and the Sulfo-SMCC10ml of 5mg/mL and mix, room temperature reaction 2 hours, and lasting stirring at low speed.Use the PBS dialysed overnight of 0.1MpH7.2, change dialysis solution next day, continue dialysis and be used for coupling after 4 hours.Get the 100mgM2e peptide and be dissolved in 5ml coupling buffer (83mM sodium phosphate buffer, 0.1MEDTA, 0.9MNaCl, 0.02%NaN3, pH7.2) in, after it is mixed with Q β-VLP in conjunction with Sulfo-SMCC, after room temperature reaction 2 hours, the PBS dialysed overnight is changed dialysis solution next day, continue dialysis-80 ℃ of preservations after 4 hours, this is the M2e-Q β VLP for preparing.
The invention has the advantages that:
1. the conjugate of the conservative peptide M2e of influenza A virus and virus-like particle has the ability of the attack of each hypotype influenza A virus of cross protection, for the developments of universal influenza vaccines lays the first stone.
2. by the chemical method coupling, preparation time is short, and is easy to large-scale production with virus-like particle for the conservative peptide M2e of influenza A virus, overcome traditional with Embryo Gallus domesticus produce influenza vaccines develop, deficiency aspect the production technology route.
Description of drawings
Fig. 1 embodiment two small mouse antiserum titre curves.
The specific embodiment
The invention will be further described below in conjunction with drawings and Examples:
The preparation of embodiment one, M2e-Q β-VLP conjugate and reference substance M2e-keyhole limpet hemocyanin (KLH) conjugate
The preparation of step 1, phage virus-like particle Q β-VLP: strain bacillus coli DH 5 alpha/pETQ β-CP is inoculated into LB culture medium culturing to suitable bacterium liquid reaches the OD value, after adding derivant IPTG to induce a few hours, collect bacterium liquid, use the ultrasonication cell, after cracking, the centrifugal upward cleer and peaceful precipitation of collecting respectively.To the polymer that forms, use Sepharose CL-4B chromatographic column purification.
Synthesizing of step 2, M2e polypeptide: adopt solid-phase synthesis to utilize the Peptide synthesizer automatic pressing to become influenza virus A hominis M2 albumen conserved sequence M2e
Step 3, peptide section M2e respectively with phage virus-like particle Q β-VLP and keyhole limpet hemocyanin (KLH) coupling.
Get respectively Q β-VLP and the KLH of 50mg, and the Sulfo-SMCC10ml of 5mg/mL mixing, room temperature reaction 2 hours, and lasting stirring at low speed.Use the PBS dialysed overnight of 0.1MpH7.2, change dialysis solution next day, continue dialysis and be used for coupling after 4 hours.Get the 100mgM2e peptide and be dissolved in 5ml coupling buffer (83mM sodium phosphate buffer, 0.1MEDTA, 0.9MNaCl, 0.02%NaN3, pH7.2) in, after it is mixed with Q β-VLP and KLH in conjunction with Sulfo-SMCC respectively, after room temperature reaction 2 hours, the PBS dialysed overnight is changed dialysis solution next day, continue dialysis-80 ℃ of preservations after 4 hours, this is M2e-Q β VLP and the M2e-KLH for preparing.M2e-KLH is for detecting M2e-Q β-antigenic reference substance of VLP conjugate.
Embodiment two, ELISA method detect the mice serum antibody titer
The 6 female Mus of week C57BL/6 in age, 20 are divided into 4 groups at random, every group average 5.Group is divided into M2e polypeptide group, M2e-Q β VLP group and M2e-KLH group and matched group.Immunization method, dosage, interval, number of times see the following form 1.
Table 1: immunization strategy
| Time | Number of times | Dosage | Immunization method |
| 0 week | The 1st time | 50ug | Subcutaneous multiple spot |
| 2 weeks | The 2nd time | 50ug | Subcutaneous multiple spot |
| 4 weeks | The 3rd time | 50ug | Subcutaneous multiple spot |
Gather mice serum 100ul before each immunity.-20 ℃ of preservations.
Be dissolved in respectively with M2e polypeptide, VLP, KLH in the carbonate buffer solution of 0.1mM (pH9.6), be made into the antigen coated liquid of 20ug/mL, with coated 96 orifice plates of every hole 100ul, 4 ℃ are spent the night coated.Then use 1%BSA (bovine serum albumin) in 37 ℃ of sealing 2h.Then dilution (1: 1000 to 1: 80000) serum sample adds in the ELISA Plate reacting hole by a certain percentage, every sample 3 holes, and every hole 100ul is placed in 37 ℃ of incubator 60min.After PBST cleaning mixture washing 5 times, add ELIAS secondary antibody, react 30min under similarity condition.After PBST cleaning mixture washing 5 times, add nitrite ion and stop buffer, measure the OD value in the Imark of bio-rad type microplate reader with the 450nm wavelength.
Result is judged: [(sample OD value-blank OD value)/(negative control OD value-blank OD value)] 〉=2.1 can judge that antibody test is positive.The antiserum titre adopts meansigma methods ± standard deviation to represent, analyzes with the SPSS10.0 statistical software, and statistics mapping employing excel, group difference relatively adopts variance analysis, has significant difference with P＜0.05.Result shows: have significant difference, P＜0.05 between the antibody group for M2e in the Mice Body after immunity for the third time.M2e-Q β VLP immune group M2e specific antibody titre is significantly higher than M2e-KLH, and this is to be listed in Q phagus beta virus-like particle immunodominance district because M2e is as polypeptide antigen, can cause rapidly like this immunoreation in body.Obviously M2e-KLH does not have such advantage.The antibody titer that also significantly produces lower than M2e of the antibody titer that produces of the immunoreation that causes of VLP self in addition, P＜0.05.M2e group M2e immune mouse produces antibody hardly, and the antibody minuent is extremely low, significantly lower than M2e-Q β VLP immune group, P＜0.01.Data results is seen Fig. 1.
Embodiment three, mice protective effect
The 8 female Mus of week C57BL/6 in age, 56 are divided into 4 groups at random, every group average 14.Group is divided into placebo group, M2e-Q β VLP group and M2e-KLH group, M2e group.Immunization method, dosage, interval, number of times see the following form 2.
The plan of table 2:C57BL/6 mouse immune
| Time | Number of times | Dosage | The immunity position |
| 0 day | For the first time | 50ug | Subcutaneous inoculation |
| The 14th day | For the second time | 50ug | Subcutaneous inoculation |
The 28th day to every group of mice wherein 7 the inoculation 4 times of half lethal doses influenza A poison strain (A/PR/8/34[PR8, H1N1] the PR8 strain, the influenza A poison NIB-64A/Perth/16/2009[H3N2 of 4 times of half lethal doses of other 7 inoculations] strain, subsequently every day the close observation mice survival condition, continue to observe 28 days.Statistics mouse survival quantity.Experimental result sees Table 3.Experimental result shows, M2e-Q β VLP group 50ug immunity 2 times, and the antibody of generation is enough to resist the attack of 4 times of PR8 strains, and protective rate is 100%, and M2e-KLH also has 85.7% Protection in addition, and matched group and M2e polypeptide group mice are all dead.
Table 3: the mouse survival statistical table is attacked in influenza virus PR8 strain
| Group | The total number of elements of mice | Dead number of elements | The survival number of elements |
| M2e-Q β VLP group | 7 | 0 | 7 |
| The M2e-KLH group | 7 | 1 | 6 |
| M2e polypeptide group | 7 | 7 | 0 |
| Placebo group | 7 | 7 | 0 |
Table 4: the mouse survival statistical table is attacked in influenza virus NIB-64A strain
| Group | The total number of elements of mice | Dead number of elements | The survival number of elements |
| M2e-Q β VLP group | 7 | 0 | 7 |
| The M2e-KLH group | 7 | 1 | 6 |
| M2e polypeptide group | 7 | 7 | 0 |
| Placebo group | 7 | 7 | 0 |
Claims (4)
1. influenza A virus is guarded the conjugate of peptide M2e and virus-like particle, it is characterized in that, adopt the extracellular region M2e polypeptide of influenza virus A hominis's matrix protein 2 as hapten, with (the number of patent application: 201010028904.7) Q phagus beta virus-like particle (Q β-VLP) as carrier of invention disclosed patent, 1 M2e or a plurality of M2e concatermer are combined with Q β-VLP are prepared into conjugate (M2e) n-Q β-VLP, n is 1 to 8 natural number.
2. influenza A virus according to claim 1 is guarded the conjugate of peptide M2e and virus-like particle, it is characterized in that, described M2e polypeptide comes from influenza virus A hominis's strain matrix protein 2 extracellular part, and described M2e peptide sequence is as shown in SEQ No.1.
3. influenza A virus according to claim 1 is guarded the conjugate of peptide M2e and virus-like particle, it is characterized in that, (what Q β-VLP) adopted is invention disclosed patent (number of patent application: 201010028904.7) the Q phagus beta virus-like particle (technology of preparing of Q β-VLP) to carrier part.
4. the conjugate of the conservative peptide M2e of influenza A virus according to claim 1 and virus-like particle, is characterized in that, described conjugate can be for the protection of different subtype influenza virus A hominis's attack.
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| CN107344969A (en) * | 2016-05-05 | 2017-11-14 | 中国科学院武汉病毒研究所 | A kind of nanometer influenza vaccines and construction method and application |
| CN113801205A (en) * | 2021-09-13 | 2021-12-17 | 湖南大学 | Modification method for improving antigenicity of influenza A virus H1 tripolymer protein |
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Cited By (5)
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