CN103724413B - Trichina paramyosin B cell antigen epi-position 8A1 and application thereof - Google Patents
Trichina paramyosin B cell antigen epi-position 8A1 and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- 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
Abstract
The invention belongs to field of immunobiology, relating to for preventing and treating trichinous epitope, its composition and purposes.Particularly, the present invention relates to a kind of epitope, is its aminoacid sequence as SEQ? ID? shown in any one of NO:1-4.Epitope of the present invention has effective trichina reduction rate, thus has the potentiality as the medicine (such as vaccine) preventing and treating trichinzation or its associated diseases.
Description
The divisional application of the present invention's to be application number be female case of 201110448420.2, the applying date of this female case is on December 29th, 2011, and denomination of invention is " trichina paramyosin B cell antigen epi-position, its composition and purposes ".
Technical field
The invention belongs to field of immunobiology, relating to for preventing and treating trichinous epitope, its composition and purposes.
Background technology
Trichonematosis is a kind of parasitic zoonoses in global distribution.The infection of people is mainly derived from highly pathogenic trichina(Trichinella spiralis) (PozioE.WorlddistributionofTrichinellaspp.infectionsinani malsandhumans. [J] .VetParasitol, 2007,149 (1-2): 3-21.).Because trichonematosis clinical symptom is complicated, comprise diarrhoea, heating, myalgia, oedema etc., make it be difficult to correct diagnosis, cause certain difficulty to pharmacological agent timely and pharmacological agent can not solve the repeated infection problem (GottsteinB of this worm, PozioE, NocklerK.Epidemiology, diagnosis, treatment, andcontroloftrichinellosis. [J] .ClinMicrobiolRev, 2009,22 (1): 127-145.).Trichinous popular, not only directly threaten the healthy of the mankind, and cause heavy economic losses to livestock industry, meat product industry and foreign export etc., is China's the Eleventh Five-Year Plan period one of emphasis food-borne parasitic disease of capturing.
Paramyosin (paramyosin, Pmy) be the main structural protein of multiple invertebrates, be persistent expression in the different developmental phases of parasitic worm, the main component (EpsteinHF of the thick filament participating in Muscle contraction, MillerDR, OrtizI, etal.Myosinandparamyosinareorganizedaboutanewlyidentifie dcorestructure [J] .JCellBiol, 1985,100 (3): 904-915.).At the non-muscle sites of some parasitic worm, as body surface, digestive tube and reproductive tract also have the distribution (ZhaoQP of paramyosin, MoonSU, NaBK, etal.Paragonimuswestermani:biochemicalandimmunologicalch aracterizationsofparamyosin. [J] .ExpParasitol, 2007,115 (1): 9-18.MatsumotoY, PerryG, LevineRJ, etal.Paramyosinandactininschistosomalteguments [J] .Nature, 1988,333 (6168): 76-78.).Paramyosin is also immune modulatory molecules simultaneously, critical function (LoukasA is played in the interaction of parasitic worm and host, JonesMK, KingLT, etal.ReceptorforFconthesurfacesofschistosomes. [J] .InfectImmun, 2001,69 (6): 3646-3651.DengJ, GoldD, LoverdePT, etal.InhibitionofthecomplementmembraneattackcomplexbySch istosomamansoniparamyosin. [J] .InfectImmun, 2003,71 (11): 6402-6410.).Paramyosin gene (Ts-PmyGenBankaccessionNo.:EF429310) total length of Trichinella spiralis is 2996bp, its open reading frame (ORF) 2655bp, and 885 amino acid of encoding, theoretical molecular is 102kDa.
But at present the research of the epitope of trichina paramyosin is still goed deep into.Use the serum of infection by Onchocerca volvulus patient to the epitope mapping research display of heart worm paramyosin, infect the aminoterminal (SteelC that serum mainly identifies paramyosin molecule, LimbergerRJ, McreynoldsLA, etal.Bcellresponsestoparamyosin.Isotypicanalysisandepito pemappingoffilarialparamyosininpatientswithonchocerciasi s. [J] .JImmunol, 1990, 145 (11): 3917-3923), and show in the correlative study of Schistosoma japonicum and taeniasis suis, infect the carboxyl terminal (Vazquez-TalaveraJ that serum mainly identifies albumen, SolisCF, Medina-EscutiaE, etal.HumanTandBcellepitopemappingofTaeniasoliumparamyosi n. [J] .ParasiteImmunol, 2001, 23 (11): 575-579.NaraT, TanabeK, MahakunkijcharoenY, etal.TheBcellepitopeofparamyosinrecognizedbyaprotectivem onoclonalIgEantibodytoSchistosomajaponicum [J] .Vaccine, 1997, 15 (1): 79-84.).Why identifying different positions, may be because the distribution of the paramyosin Dominant Epitopes of nematoda and fluke and Cestoda is different.
So, still need and will develop new antibody and epitope, to effectively preventing and treating trichinzation and associated diseases thereof.
Summary of the invention
The present inventor is through deep research and performing creative labour, Ts-Pmy is found to divide three segment tables to reach partly overlapping protein fragments (rTs-Pmy-N1-322aa, rTs-Pmy-M286-600aa and rTs-Pmy-C571-885aa), identify with the mouse pooled serum infecting latter 27-45 days, that discovery wherein mainly identifies paramyosin is N end fragment (rTs-Pmy-N1-322aa).The present inventor carries out the research of paramyosin antigen molecule epi-position based on this N end fragment, obtains epitope.The present inventor is surprised to find, and the epitope obtained has effective trichina reduction rate, thus has the potentiality as the medicine (such as vaccine) preventing and treating trichinzation or its associated diseases.Thus provide following invention:
One aspect of the present invention relates to a kind of epitope, and it has the aminoacid sequence according to any one of SEQIDNO:1-4:
ALSTPTFSTLPA（SEQIDNO：1）
LPWHFKSRHRYQ（SEQIDNO：2）
SHWNSHSTPARA（SEQIDNO：3）
LSTPYSKSQAST（SEQIDNO：4）。
In one embodiment of the invention, the aminoacid sequence of described epitope is shown in any one of SEQIDNO:1-4.
The mode that described epitope can be synthesized by artificial chemistry obtains, and also can other biological chemistry of knowing of those skilled in the art or molecular biological method obtain.
Of the present inventionly also relate to a peptide species, it has the aminoacid sequence according to any one of SEQIDNO:1-4.
Another aspect of the invention relates to a kind of nucleotide sequence, its epitope described in any one of code book invention; Particularly, described nucleotide sequence is shown in respectively any one of SEQIDNO:8-11:
GCGCTGAGTACTCCGACTTTTTCGACTCTGCCTGCG（SEQIDNO：8）
CTTCCGTGGCATTTTAAGTCGCGTCATACGTATCAG（SEQIDNO：9）
TCTCATTGGAATAGTCATTCGACTCCTGCGCGTGCG（SEQIDNO：10）
CTGTCGACGCCTTATTCGAAGTCTCAGGCGTCGACT（SEQIDNO：11）。
Another aspect of the present invention relates to a kind of antigen, and it has the epitope described in any one of the present invention; Particularly, the aminoacid sequence of described antigen is as shown in SEQIDNO:16.
MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETERELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESEDSLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAAEKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQLEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKSKFDAEVSLHQEE（SEQIDNO：16）
The nucleotide sequence also relating in one aspect to antigen of the present invention of encoding of the present invention; Particularly, the coding nucleotide sequence of described antigen is as shown in SEQIDNO:15.
ATGTCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCATGCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCAACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGATTTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCAACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACGCCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTGGAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCAGCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGCAGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCGGAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCTGGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATTTGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAGACTCAACTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCAAATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAGAGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCCCAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAA（SEQIDNO：15）
Another aspect of the invention relates to a kind of recombinant vectors, and it contains the nucleotide sequence described in any one of the present invention.In one embodiment of the invention, described recombinant vectors is recombinant expression vector.
The Nucleotide of any one of the present invention and regulating and controlling sequence can be linked together and prepare recombinant expression vector, this carrier can comprise 1 or multiple restriction site easily, to insert or to replace the nucleotide sequence of coded polypeptide in these sites.Or, nucleotide sequence of the present invention can be expressed by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted suitable expression vector.When preparing expression vector, encoding sequence can be made to be arranged in carrier to be operatively connected with suitable expression regulation sequence.
Wherein, term " regulating and controlling sequence " be defined as comprise express epitope of the present invention institute must or favourable all components.Each regulating and controlling sequence can be natural that contain or external for the nucleotide sequence of coded polypeptide (epitope).These regulating and controlling sequences include, but not limited to leader sequence, polyadenylation sequence, propeptide sequence, promotor, signal sequence and transcription terminator.Bottom line, the termination signal that regulating and controlling sequence will comprise promotor and transcribe and translate.In order to import specific restriction site to be connected the coding region of regulating and controlling sequence with the nucleotide sequence of coded polypeptide, the regulating and controlling sequence of belt lacing can be provided.Term " is operatively connected " and is defined as so a kind of conformation in the text, and wherein regulating and controlling sequence is positioned at the appropriate location of the encoding sequence of relative DNA sequence dna, with the expression making regulating and controlling sequence instruct polypeptide.
Recombinant expression vector can be that any being convenient to carries out recombinant DNA operation and the carrier (such as plasmid or virus) of express nucleic acid sequence.Carrier and its consistency of host cell that will import are depended in the selection of carrier usually.Carrier can be linear or closed circular form plasmid.
Another aspect of the invention relates to a kind of recombinant host cell, and it contains the recombinant vectors described in any one of the present invention.
The recombinant vectors comprising the nucleotide sequence of the present invention can be imported host cell, thus this carrier is maintained with the outer carrier format of karyomit(e) of above-mentioned chromosomal integrant or self-replacation.Any offspring different from parental cell due to the sudden change occurred between replicative phase contained in term " host cell ".Polypeptide coding genes and source thereof are depended in the selection of host cell to a great extent.
Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, such as bacterium or yeast cell.Can by technology well known to those skilled in the art by vector introduction host cell.
Another aspect of the invention relates to a kind of epitope conjugate, comprise epitope and coupling moiety, wherein, described epitope is the epitope described in any one of the present invention, described coupling moiety be selected from radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H one or more; Particularly, described coupling moiety is carrier proteins BSA or KLH.
Another aspect of the invention relates to a kind of composition, and it comprises one or more epitopes of the present invention, and/or one or more antigens of the present invention, and/or one or more epitope conjugates of the present invention; Particularly, described composition is vaccine composition.
Another aspect of the invention relates to the epitope described in any one of the present invention or the antigen described in any one of the present invention or the epitope conjugate described in any one of the present invention and is preparing the purposes treated and/or prevented in the medicine of trichinzation.
The data presentation of embodiment 6, epitope of the present invention has effective trichina reduction rate, effectively can prevent and treat trichinzation or trichinzation associated diseases, there are the potentiality as the medicine (such as vaccine) preventing and treating trichinzation or its associated diseases.
Another aspect of the invention relates to a kind of antibody, and it can specifically in conjunction with the epitope described in any one of the present invention or the antigen described in any one of the present invention or the epitope conjugate described in any one of the present invention.
In the present invention, term " specific binding " has immunologic general sense, such as, combination between antigen-antibody.
Another aspect of the invention relates to a kind of antibody coupling matter, comprise antibody moiety and coupling moiety, wherein, described antibody moiety is the antibody described in any one of the present invention, described coupling moiety be selected from radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H one or more.
Another aspect of the invention relates to a kind of pharmaceutical composition, and it comprises the antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention; Alternatively, described pharmaceutical composition also comprises pharmaceutically acceptable carrier or auxiliary material.
Another aspect of the invention relates to a kind of test kit, and it comprises the antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention.In one embodiment of the invention, described test kit is detection or the diagnostic kit of trichinzation or trichinzation associated diseases.
Another aspect of the invention relates to the antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention is preparing the purposes treated and/or prevented in the medicine of trichinzation.
The beneficial effect of the invention
The effective trichina reduction rate of epitope tool of the present invention, thus there are the potentiality as the medicine (such as vaccine) preventing and treating trichinzation or its associated diseases.
Accompanying drawing explanation
The ascites 8F12mAb of Fig. 1: SDS-PAGE purification Identification.1, low molecular weight protein (LMWP) marker; The non-purifying ascites of 2,8F12 strain; Antibody after 3,8F12 strain column purification.Note: the assorted band at 65kDa place should be the position of Human Serum Albumin, content is very large, so be difficult in purifying remove completely.
Fig. 2: 8F12 affinity of antibody constant measuring.Note: C.For antibody starting point concentration, C is concentration after antibody dilution.
Fig. 3: mAb8F12 and the passive immune protection of the anti-trichinzation of natural infection serum.The muscle larvae number that muscle larvae worm lotus (LPG) is every gram of muscle.Represent with mean ± standard deviation (n=6).Note:
*compare with PBS control group, p<0.01.
Fig. 4: mAb8F12 and the specific recognition (ELISA method is identified) of positive phage clones.Using wild phage M13 as negative control, BSA wraps by hole in order to get rid of cross reaction.
Fig. 5: mAb8F12 and the specific recognition (Westernblot) of positive phage clones
Swimming lane 1: phage clone 8JJ; Swimming lane 2: phage clone 8A1;
Swimming lane 3: phage clone 8A9; Swimming lane 4: phage clone 8F1;
Swimming lane 5: phage clone 8F6; Swimming lane 6: phage clone 8F7;
Swimming lane 7: phage clone 8F10; The wild phage clone of swimming lane 8:M13.
Fig. 6: the ELISA of improvement on synthesis and mAb8F12 specific binding detects (polypeptide coupling carrier BSA bag quilt).Note: mAb5A3 contrasts as irrelevant ascites.
Fig. 7: the muscle larvae number of each group immune mouse.Note: compare with KLH control group, * * p<0.01, * p<0.05.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, (such as show with reference to J. Pehanorm Brooker etc. according to the technology described by the document in this area or condition, " Molecular Cloning: A Laboratory guide " that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
the preparation of embodiment 1:rTs-Pmy-N1-322aa albumen
As shown in step 1-6 order below.
The amplification of 1.Ts-Pmy-N1-322aa gene fragment
According to paramyosin gene (Ts-PmyGenBankaccessionNo.:EF429310) reading frame sequence, the nucleotide sequence of Ts-Pmy-N1-322aa can be obtained, as follows:
ATGTCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCATGCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCAACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGATTTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCAACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACGCCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTGGAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCAGCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGCAGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCGGAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCTGGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATTTGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAGACTCAACTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCAAATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAGAGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCCCAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAA（SEQIDNO：15）
The aminoacid sequence of its coding is as follows:
MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETERELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESEDSLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAAEKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQLEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKSKFDAEVSLHQEE（SEQIDNO：16）
Ts-Pmy-N1-322aa gene fragment can be obtained by synthetic according to SEQIDNO:15, and the method for pcr amplification below also can be adopted to obtain.
Design of primers: as shown in Table 1 below.Primer synthesis completes by Shanghai Ying Jun company with sequencing.
Table 1: primer data
Amplification condition: 95 DEG C of 5min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 2min, 35 circulations; 72 DEG C of 10min; PCR primer carries out 1% agarose gel electrophoresis, observes amplification.Reaction system:
Note: Ts-Pmy plasmid bacterial solution is the intestinal bacteria that conversion has containing paramyosin gene (Ts-PmyGenBankaccessionNo.:EF429310) reading frame total length plasmid, to be equal to publication number be CN100999737A(application number is 200710000018.1) patent application in the conversion prepared of embodiment 2 have the e. coli bl21 of paramyosin gene (Ts86cDNA), its preparation method can also with reference to this patent application.
The structure of 2.pET28a (+)/Ts-Pmy-N1-322aa recombinant plasmid
Plasmid and bacterial strain: select pET28a (+)/BL21(DE3) prokaryotic expression system.BL21 competence bacteria is purchased from TIANGEN company.
(1) PCR primer of Ts-Pmy-N1-322aa gene fragment and the enzyme of pET-28a (+) plasmid are cut
Enzyme tangent condition: 37 DEG C, water-bath 2h.Digestion products carries out the agarose gel electrophoresis detection of 1%, and endonuclease bamhi test kit reclaims.Reaction system is as follows:
(2) fast purifying of DNA reclaims (also can reference reagent box specification sheets)
1) cut the sepharose (100-300mg) containing Ts-Pmy-N1-322aa gene fragment or pET-28a (+) plasmid, smash to pieces, by weight, volume ratio 1: 3(DNA fragment: solution B) ratio add solution B;
2) 50 DEG C of water-bath 10min, until glue dissolves completely, period vortex oscillation 3 times;
3) solution is added in centrifugal column, high speed centrifugation 1min after standing 2min;
4) Xiang Zhuzhong adds 500 μ l solution C (diluting with dehydrated alcohol 1: 1), and high speed centrifugation, repeats this step once;
5) high speed centrifugation 1min, to dry remaining liq;
6) centrifugal column is placed in a new centrifuge tube, adds 20 μ l solution D of 50 DEG C of preheatings, leave standstill 2min, collected by centrifugation DNA;
7) agarose gel electrophoresis observes recovering effect, and-20 DEG C save backup.
(3) the ligation condition of Ts-Pmy-N1-322aa gene fragment and pET-28a (+) carrier digestion products: 4 DEG C are spent the night, and linked system is as follows:
3.pET28a the protokaryon of (+)/Ts-Pmy-N1-322aa recombinant plasmid transforms
(1) get the above-mentioned connection product of 5 μ l, be added in 100 μ l competence intestinal bacteria (Top10), mix gently, be placed in 30min on ice;
(2) 42 DEG C of heat-shocked 90sec;
(3) centrifuge tube is placed on ice fast, makes cell cool 2min;
(4) add 900 μ lLB substratum, 37 DEG C, 50rpm, 45min, make bacteria resuscitation;
(5) get on LB solid medium that the above-mentioned bacterium of 100 μ l coats containing kantlex and IPTG, X-gal, cultivate 12-16h for 37 DEG C;
The qualification of the plasmid-bearing strains of Ts-Pmy-N1-322aa gene fragment is carried after conversion:
In flat board after transforming, picking list bacterium colony is in containing in the LB liquid nutrient medium of kantlex, extracts plasmid and carry out PCR and enzyme cuts qualification after incubated overnight.Or carry out order-checking qualification.Result is correct.
The prokaryotic expression of 4.pET28a (+)/rTs-pmy-N1-322aa
(1) picking has transformed the mono-bacterium colony of BL21 of pET28a (+)/Ts-Pmy-N1-322aa recombinant plasmid, adds 5ml containing in the LB nutrient solution of kantlex (30 μ g/ml).37 DEG C, 150rpm shaking culture is spent the night;
(2) next day, 5ml culture is added 500ml containing kantlex LB substratum in.37 DEG C, 300rpm, continues shaking culture until OD
600≈ 0.6;
(3) adding IPTG to final concentration is 1mM abduction delivering, 37 DEG C, 150rpm, continues to cultivate 4h;
(4) collected by centrifugation thalline, 200ml20mMTris-HCl(pH7.9) abundant suspended bacterial precipitation;
(5) fully ultrasonic on ice, ultrasonic output intensity 40%, each 8sec, interval 5sec, totally 10 times;
(6) centrifugally supernatant is abandoned, 100ml20mMTris-HCl(pH7.9) suspend precipitation;
(7) add freshly prepared N,O-Diacetylmuramidase (100 μ g/ml), put 30min on ice, stirring and evenly mixing for several times therebetween;
(8) again ultrasonic, centrifugally abandon supernatant, throw out is inclusion body and cell debris;
(9) add 50ml containing 1 × bindingbuffer of 6M Guanidinium hydrochloride, ultrasonic precipitation is fully suspended, dissolve, 4 DEG C are spent the night.
5. affinity chromatography operation steps (His-Bind purification kit test kit, purchased from Novagen company, can reference reagent box description operation)
(1) preparation of affinity column: take out His-BindResin(binding resin), concussion suspends.In affinity column, add 2.5mlHis-BindResin, flick cylinder to remove bubble;
(2) add 15mlDDW successively, 12ml1 × ChargeBuffer, 9ml1 × BindingBuffer(is containing 6M Guanidinium hydrochloride) balance chromatography column;
(3) the inclusion body solution centrifugal be dissolved in containing in 1 × BindingBuffer of 6M Guanidinium hydrochloride is got supernatant, slowly add in post after 45 μm of frit;
(4) add 12ml1 × BindingBuffer(more successively containing 6M Guanidinium hydrochloride), 15ml20mM imidazole buffer (11ml1 × BindingBuffer, 4ml1 × WashBuffer, all containing 6M Guanidinium hydrochloride) clean chromatography column, 12ml1 × EluteBuffer(is containing 6M Guanidinium hydrochloride) wash-out target protein;
(5) dialyse: by the collection liquid impouring dialysis tubing of 1 × EluteBuffer wash-out (molecular weight cut-off is 12-14kDa), put into the 20mMTris-HCl(pH7.9 of 50 times of volumes), 4 DEG C of dialysis more than 3h.
(6) proceed in the dialyzate of same volume, 4 DEG C of dialysed overnight;
(7) in dialysis tubing, deactivated protein precipitation is separated out, and by the solution impouring centrifuge tube containing albumen precipitation, centrifugal collecting precipitation is the recombinant protein of purifying.
6. protein renaturation operation steps (protein renaturation test kit, purchased from Novagen company, can reference reagent box specification sheets)
(1) prepare 1 appropriate × IBSolubilizationBuffer(to prepare from 10 × storage liquid.10 × IBSolubilizationBuffer comprises 200mMTis-HCl, pH7.5,100mMEDTA, 10%TritonX-100), and add 0.3%N-lauroylsarcosine and 1mMDTT;
(2) with the concentration suspension albumen of 1-2mg/ml, room temperature leaves standstill 10min, and albumen is fully dissolved;
(3) after centrifugal, supernatant is proceeded in dialysis tubing;
(4) put into 50 times of volumes, containing 0.1mMDTT 1 × DialysisBuffer(by 50 × DialysisBuffer dilute and obtain, wherein composition is 1MTris-HCl, pH8.5), in dialyzate, 4 DEG C of dialysis more than 3h, then change same dialyzate and continue dialysis 3h;
(5) dialyzate of 1 × DialysisBuffe that put into 50 times of volumes, that do not contain DTT, 4 DEG C of dialysis 3h, change same dialyzate and continue dialysed overnight;
Next day, collect recombinant protein liquid, obtain rTs-Pmy-N1-322aa albumen (solution).
Protein concentration is surveyed ,-20 DEG C of preservations by BCA method.
embodiment 2: the foundation of hybridoma cell strain
By rTs-Pmy-N1-322aa albumen (solution) immune BALB/c mouse prepared by embodiment 1, through 4 immunity, after final immunization, mouse antibodies is tired and is reached 1: 128000.By the SP2/0 myeloma cell fusion of immune mouse spleen cell and logarithmic phase, select to cultivate through HAT nutrient solution.In 96 well culture plates, visible fused cell growth, when cell colony grows to 1/3 hole, by indirect ELISA and Westernblot qualification, pick out in culture supernatant and have anti-rTs-Pmy-N1-322aa antibody to exist, and can with the positive hybridoma cell strain of rTs-Pmy-N1-322aa and the identification of larva polypide albumen.Through 3 subclones by screening acquisition one strain of hybridoma strain called after 8F12, the antibody of its secretion belongs to the IgG1 Subclass Antibodies of secretion Th2 type, cultivated for 20 generations through continuous passage, get F5, F10, F15 and F20 wherein respectively for the recovery cells and supernatant after cells and supernatant and Liquid nitrogen storage half a year, measure antibody titer with indirect ELISA method, the results are shown in Table 2.Hybridoma antibody-secreting after continuous passage and cryopreservation resuscitation is stablized, and selects use for further study.
Table 2: the ELISA qualification of hybridoma cell strain 8F12 stability
| Passage number | 8F12 (A value) |
| F5 | 2.096 |
| F10 | 1.886 |
| F15 | 1.973 |
| F20 | 1.896 |
| Cryopreservation resuscitation cell | 1.796 |
the qualification of the monoclonal antibody (mAb) of embodiment 3:8F12 cell strain secretion
ELISA detects the titre of mAb in the cell culture supernatant of the 8F12 cell strain in embodiment 2 and ascites, is respectively 1: 3200 and 1: 64000.The Subclass of antibody of secretion is IgG1 subclass κ type (table 3).Monoclonal antibody ascites carries out SDS-PAGE electrophoresis after HiTraprProteinA column purification, has band to manifest respectively at 50kDa and 25kDa place, meets the position (Fig. 1) of IgG antibody heavy chain and light chain.
The qualification of table 3: monoclonal antibody 8F12
For clear and definite mAb under certain antigen concentration with antigen in conjunction with time relative affinity size, adopt indirect non-competing ELISA method.With the logarithmic value of extension rate and lg(C
0/ C) be X-coordinate (C
0for mAb starting point concentration, C is concentration after mAb dilution), with A value for ordinate zou, draw the relative affinity curve (Fig. 2) of two strain mAb.Be 9.1 × 10 by the antibody affinity costant Ka value of formulae discovery 8F12mAb
7m
-(table 3).
embodiment 4: monoclonal antibody (mAb) passive immune protection is tested
1. laboratory sample:
The monoclonal antibody (mAb) of 8F12 cell strain secretion.
2. laboratory animal and grouping:
6-8 BALB/c female mice in age in week is divided into 3 groups at random, is respectively monoclonal antibody group, natural infection serologic group and PBS control group, often organizes 6.
3. experimental technique:
Use the method passive immunization mouse of tail vein injection antibody, the antibody purification (being dissolved in 100 μ lPBS) of monoclonal antibody group every injected in mice 500 μ g; The BALB/c mouse of natural infection serologic group every injected in mice 100 μ l infects serum (serum titer is 1: 3000); The PBS of PBS control group every injected in mice 100 μ l.After passive immunization, 2 hours every mouse attack worm 400.Within the 4th day after attacking worm, distinguish booster immunization once with same dosage.Within after attacking worm the 45th day, cut open and kill mouse, count the muscle larvae number of every mouse, evaluate the effect of passive immunization.
4. experimental result:
As shown in table 4.
Passive immunization mAb8F12, obtains the immune protective efficiency of anti-trichinzation in BALB/c mouse body.The experimental group of injection mAb8F12 compared with the control group of injection PBS, obtains the muscle larvae worm reduction rate (p<0.01) of 25.6% and 24.6% with natural infection serologic group mouse respectively.But muscle larvae number (LPG) difference of monoclonal antibody group and natural infection serologic group is without significance (Fig. 3).Confirm mAb8F12 tool passive immune protection.Can be used for the B cell antigen epi-position of follow-up screening tool immune protective.
Table 4: the muscle larvae check result of each group immune mouse
Note:
*through statistical test, there is significant differences (p<0.01) with PBS control group and simple adjuvant group.
embodiment 5: the screening of monoclonal antibody 8F12 epitope
This experiment is carried out with reference to NEB phage dodecapeptide storehouse specification sheets, and test-results is as follows:
1. the enrichment of phage
For observing the validity of phage display peptide library, calculate the rate of recovery of often taking turns phage.Phage virus of often taking turns input is 2 × 10
11, mensuration often takes turns the titre of the phage under wash-out, calculates the rate of recovery, result following (table 5):
Table 5: the change of the three-wheel phage display peptide library pnagus medius rate of recovery
| Screening number of times | The phage added | The phage of wash-out | The phage rate of recovery |
| 1 | 1.5×10 11 | 1×10 3 | 6.6×10 －9 |
| 2 | 1.5×10 11 | 6×10 4 | 4×10 －7 |
| 3 | 1.5×10 11 | 2×10 5 | 1.3×10 －6 |
In table 5, data show, enrichment phenomenon has appearred in the process pnagus medius eluriating phage peptide library, and third round is higher 200 times than the output after first round elutriation.
2. the qualification of positive colony
(plaque number is lower than 100) random picking 10 independently blue plaque from the flat board of the mensuration titre of the eluate of third round screening, preparation phage original seed storage liquid after amplification.With mAb8F12 coated elisa plate, add the phage clone storage liquid of amplification, simultaneously using wild phage M13 as negative control.For avoiding screening the false positive phage clone be combined with liquid of blockading (main component is BSA) composition, the system of BSA bag quilt has separately been established to contrast.With OD
492value is higher than negative control OD
492be worth more than 2.1 times as positive criterion, 10 clones are all positive phage clones.Result following (Fig. 4).
3. the base sequence mensuration of positive colony and the analysis of encoding amino acid sequence
After extracting the single stranded DNA of 10 positive phage clones, record DNA sequence dna.The result of positive colony sequencing is the sequence (see table 6) of complementary strand, is translated as aminoacid sequence (see table 7) again and carries out sequence alignment analysis again after must being changed into coding strand sequence.
Table 6: the DNA sequence dna of the positive phage clones dodecapeptide be combined with 8F12
Table 7: phage display peptide sequence
Derived in the encoding amino acid sequence that obtains by above-mentioned 10 positive colony DNA sequence dnas, have 4 to be tumor-necrosis factor glycoproteins (RNTO 8JJ), therefore obtain 7 seed amino acid Epitope peptide sequences altogether.For further clear and definite epi-position phage is whether by monoclonal antibody 8F12 is identified, carry out Westernblot experiment (Goatanti-MouseIRDye800CW resists as biotin labeling two).Experimental result display (Fig. 5): the positive phage clones presenting above-mentioned 7 kinds of Epitope peptide sequences, in the position of about 60kDa, has all occurred the band of position consistency.And the wild phage of M13 has no band in this position.Because displayed polypeptide is connected on the secondary capsid protein pIII of phage, when carrying out SDS-PAGE electrophoresis, pIII runs usually near molecular weight 60-65kDa.Therefore can determine in the displayed polypeptide of positive bacteriophage Clone1-10 containing the epitope that mAb8F12 identifies.
4. the identification of improvement on synthesis and mAb8F12
Epitope peptide sequences contained by above-mentioned 7 kinds of positive phage clones is delivered company's synthesis epitope polypeptide, called after 8JJ (Epitope peptide sequences contained by 8A6,8A7,8A11,8A12 is separately named), 8A1,8A9,8F1,8F6,8F7,8F10 respectively.For increasing polypeptide antigen, by polypeptide and carrier proteins BSA or KLH coupling (Beijing NIVEA Corp difficult to understand carries out technology synthesis), the display of ELISA result 8JJ-BSA, 8A1-BSA, 8A9-BSA, 8F1-BSA, 8F6-BSA, 8F7-BSA, 8F10-BSA all can identify with 8F12, and the OD value that ELISA detects reaches the detected value (Fig. 6) of albumen rTs-Pmy-N1-322aa and 8F12.
embodiment 6: epi-position improvement on synthesis immune mouse also evaluates its immune protective
1. laboratory sample:
8JJ-KLH, 8A1-KLH, 8A9-KLH, 8F1-KLH, 8F6-KLH, 8F7-KLH, the 8F10-KLH of synthesis in step 4 in embodiment 5.
2. laboratory animal and grouping:
6-8 BALB/c female mice in age in week is divided into 10 groups at random, often organizes 6.Be respectively epi-position synthetic peptide group (7 groups), rTs-Pmy-N1-322aa group, and KLH group and PBS control group.
3. experimental technique:
The epitope polypeptide of coupling KLH and rTs-Pmy-N1-322aa get 50 μ g respectively and are dissolved in 75 μ lPBS and fully mix with isopyknic ISA50V2 adjuvant, the subcutaneous multi-point injection of mouse back.KLH and PBS with same dosage and mode immune animal in contrast.Every immunity in two weeks once, immunity three times are total to.After final immunization 10 days, every mouse challenge infected cultivation of larvae of Trichinella spiralis from muscle 400, cuts open and kill mouse after 45 days, checked muscle larvae worm lotus, evaluated the immune protective of each group.
4. experimental result:
Compared with KLH control group, 8A1-KLH group, 8F1-KLH group, 8F7-KLH group muscle larvae number (LPG) have significant differences (p<0.01); Obtain the muscle larvae worm reduction rate (table 12) of 15.9%, 22.2% and 26.3% respectively.Compared with KLH control group, 8F6-KLH group muscle larvae number has significant difference (p<0.05), obtains the muscle larvae worm reduction rate (table 8, Fig. 7) of 18.7%.
Table 8: the muscle larvae check result of each group immune mouse
Note: * *, through statistical test, has significant differences (p<0.01) with KLH control group;
* through statistical test, significant differences (p<0.05) is had with KLH control group
In a word, by above experiment, the present inventor obtains 4 protective epitopes of Ts-Pmy, all can induce and produce effective immanoprotection action after these epitope polypeptide immune mouses.Above result prompting: these epi-positions can be used as candidate vaccine component, thus lay a good foundation for preparing anti-trichinous polyepitope vaccines.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various amendment and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (11)
1. an epitope, its aminoacid sequence is as shown in SEQIDNO:1.
2. the nucleotide sequence of epitope described in coding claim 1.
3. nucleotide sequence according to claim 2, it is as shown in SEQIDNO:8.
4. a recombinant vectors, it contains the nucleotide sequence described in Claims 2 or 3.
5. a recombinant host cell, it contains recombinant vectors according to claim 4.
6. an epitope conjugate, comprise epitope and coupling moiety, wherein, described epitope is epitope according to claim 1, described coupling moiety be selected from radionuclide, medicine, toxin, enzyme, fluorescein, carrier proteins and vitamin H one or more.
7. epitope conjugate according to claim 6, wherein, described coupling moiety is cytokine.
8. epitope conjugate according to claim 6, wherein, described coupling moiety is carrier proteins BSA or KLH.
9. a composition, it comprises one or more epitopes according to claim 1, and/or the epitope conjugate according to any one of one or more claims 6 to 8.
10. composition according to claim 9, it is vaccine composition.
11. epitopes according to claim 1 or the epitope conjugate according to any one of claim 6 to 8 are preparing the purposes treated and/or prevented in the medicine of trichinzation.
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| CN201410005780.9A CN103724413B (en) | 2011-12-29 | 2011-12-29 | Trichina paramyosin B cell antigen epi-position 8A1 and application thereof |
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| CN201110448420.2A CN102558306B (en) | 2011-12-29 | 2011-12-29 | Antigen epitope for preventing and treating trichinosis, composition thereof and application thereof |
| CN201410005780.9A CN103724413B (en) | 2011-12-29 | 2011-12-29 | Trichina paramyosin B cell antigen epi-position 8A1 and application thereof |
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| CN201110448420.2A Expired - Fee Related CN102558306B (en) | 2011-12-29 | 2011-12-29 | Antigen epitope for preventing and treating trichinosis, composition thereof and application thereof |
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| CN103724402B (en) * | 2014-01-20 | 2016-06-15 | 首都医科大学 | The complement function associated antigen epitopes of trichina paramyosin and antibody |
| WO2017184873A2 (en) * | 2016-04-20 | 2017-10-26 | Aelan Cell Technologies, Inc. | Compositions and methods related to the methylation of histone h1.0 protein |
| CN107118267B (en) * | 2017-04-18 | 2020-07-24 | 浙江工商大学 | Modified protein mMet e1 for relieving sensitization reaction of shrimp tropomyosin, and preparation method and application thereof |
| CN110734495B (en) * | 2019-09-30 | 2022-05-31 | 吉林大学 | Hybridoma cell strain, monoclonal antibody of trichina-resistant serine protease in new larva stage and application |
| CN111303276B (en) * | 2019-12-20 | 2021-08-06 | 吉林大学 | B cell epitope polypeptide of trichina intestinal cysteine protease inhibitor, hybridoma cell strain, monoclonal antibody and application |
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Non-Patent Citations (2)
| Title |
|---|
| Identification and characterization of protective epitope of Trichinella spiralis paramyosin;Wei J.等;《Vaccine》;20110411;第29卷(第17期);第3162-3168页 * |
| 抗旋毛虫副肌球蛋白单克隆抗体的制备与鉴定;魏骏飞等;《中国科技论文在线》;20090402;第1-6页 * |
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| Publication number | Publication date |
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| CN102558306A (en) | 2012-07-11 |
| CN103724413A (en) | 2014-04-16 |
| CN102558306B (en) | 2014-11-05 |
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