CN103724413A - B cell epitope 8A1 of trichina paramyosin and application thereof - Google Patents
B cell epitope 8A1 of trichina paramyosin and application thereof Download PDFInfo
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- 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
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- 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
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- 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
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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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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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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Abstract
The invention belongs to the field of immunobiology, relates to epitope for preventing and controlling trichinellosis, a composition and an application thereof, and in particular relates to epitope of which the amino acid sequence is shown as an arbitrary item of SEQ ID NO:1-4. The epitope has an effective trichina reduction rate, thus having potential for preventing and controlling trichinization or medicines, such as vaccines, for treating diseases caused by trichinization.
Description
The present invention is that application number is dividing an application of female case of 201110448420.2, and the applying date of this mother's 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 immunobiology field, relate to for preventing and treating trichinous epitope, its composition and purposes.
Background technology
Trichonematosis is a kind of Zoonosis parasitosis that is global distribution.People's infection be mainly derived from highly pathogenic trichina(Trichinella spiralis) (Pozio E.World distribution of Trichinella spp.infections in animals and humans.[J] .Vet Parasitol, 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, caused certain difficulty and pharmacological agent can not solve repeated infection problem (the Gottstein B of this worm to pharmacological agent timely, Pozio E, Nockler K.Epidemiology, diagnosis, treatment, and control of trichinellosis.[J] .Clin Microbiol Rev, 2009,22 (1): 127-145.).Trichinous popular, not only directly threatening the healthy of the mankind, and livestock industry, meat product industry and foreign export etc. are being caused to heavy economic losses, be one of China's food-borne parasitic disease that the Eleventh Five-Year Plan period emphasis is captured.
Paramyosin (paramyosin, Pmy) be the main structural protein of multiple invertebrates, different developmental phases at parasitic worm is persistent expression, main component (the Epstein H F that participates in the thick filament of Muscle contraction, Miller D R, Ortiz I, et al.Myosin and paramyosin are organized about a newly identified core structure[J] .J Cell Biol, 1985,100 (3): 904-915.).Non-muscle position at some parasitic worm, as body surface, digestive tube and reproductive tract also have distribution (the Zhao Q P of paramyosin, Moon S U, Na B K, et al.Paragonimus westermani:biochemical and immunological characterizations of paramyosin.[J] .Exp Parasitol, 2007,115 (1): 9-18.Matsumoto Y, Perry G, Levine R J, et al.Paramyosin and actin in schistosomal teguments[J] .Nature, 1988,333 (6168): 76-78.).Paramyosin is also immune modulatory molecules simultaneously, in parasitic worm and host's interaction, bring into play critical function (Loukas A, Jones M K, King L T, et al.Receptor for Fc on the surfaces of schistosomes.[J] .Infect Immun, 2001, 69 (6): 3646-3651.Deng J, Gold D, Loverde P T, et al.Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin.[J] .Infect Immun, 2003, 71 (11): 6402-6410.).The paramyosin gene of Trichinella spiralis (Ts-Pmy GenBank accession No.:EF429310) total length is 2996bp, its open reading frame (ORF) 2655bp, and 885 amino acid of encoding, theoretical molecular is 102kDa.
But at present to the research of the epitope of trichina paramyosin still not deeply.Use infection by Onchocerca volvulus patient's serum to studies show that the epitope mapping of heart worm paramyosin, infect serum and mainly identify aminoterminal (the Steel C of paramyosin molecule, Limberger R J, Mcreynolds L A, et al.B cell responses to paramyosin.Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.[J] .J Immunol, 1990, 145 (11): 3917-3923), and show in the correlative study of Schistosoma japonicum and taeniasis suis, infect serum and mainly identify carboxyl terminal (the Vazquez-Talavera J of albumen, Solis C F, Medina-Escutia E, et al.Human T and B cell epitope mapping of Taenia solium paramyosin.[J] .Parasite Immunol, 2001, 23 (11): 575-579.Nara T, Tanabe K, Mahakunkijcharoen Y, et al.The B cell epitope of paramyosin recognized by a protective monoclonal IgE antibody to Schistosoma japonicum[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 trichinzation and associated diseases thereof are prevented and treated effectively.
Summary of the invention
The inventor is through deep research and performing creative labour, discovery divides three segment tables to reach partly overlapping protein fragments (rTs-Pmy-N1-322aa, rTs-Pmy-M286-600aa and rTs-Pmy-C571-885aa) Ts-Pmy, identify with the mouse pooled serum that infects latter 27-45 days, that paramyosin is wherein mainly identified in discovery is N end fragment (rTs-Pmy-N1-322aa).The inventor carries out the research of paramyosin antigen molecule epi-position based on this N end fragment, has obtained epitope.The inventor is surprised to find, and the epitope obtaining has effective Trichinella spiralis worm reduction rate, thereby has for example, potentiality as the medicine (vaccine) of control trichinzation or its associated diseases.Following invention is provided thus:
One aspect of the present invention relates to a kind of epitope, and it has the aminoacid sequence described in any one in SEQ ID NO:1-4:
ALSTPTFSTLPA （SEQ ID NO：1）
LPWHFKSRHRYQ （SEQ ID NO：2）
SHWNSHSTPARA （SEQ ID NO：3）
LSTPYSKSQAST （SEQ ID NO：4）。
In one embodiment of the invention, the aminoacid sequence of described epitope is as shown in any one in SEQ ID NO:1-4.
Described epitope can obtain by the synthetic mode of artificial chemistry, and other biological chemistry or the molecular biological method also can those skilled in the art known obtain.
The peptide species that also relates to of the present invention, it has the aminoacid sequence described in any one in SEQ ID NO:1-4.
Of the present inventionly relate in one aspect to again a kind of nucleotide sequence, the epitope described in its code book invention any one; Particularly, described nucleotide sequence is respectively as shown in any one in SEQ ID NO:8-11:
GCGCTGAGTACTCCGACTTTTTCGACTCTGCCTGCG（SEQ ID NO：8）
CTTCCGTGGCATTTTAAGTCGCGTCATACGTATCAG（SEQ ID NO：9）
TCTCATTGGAATAGTCATTCGACTCCTGCGCGTGCG（SEQ ID NO：10）
CTGTCGACGCCTTATTCGAAGTCTCAGGCGTCGACT（SEQ ID NO：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 SEQ ID NO:16.
MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETERELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESEDSLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAAEKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQLEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKSKFDAEVSLHQEE（SEQ ID NO：16）
The nucleotide sequence that also relates in one aspect to coding antigen of the present invention of the present invention; Particularly, the coding nucleotide sequence of described antigen is as shown in SEQ ID NO:15.
ATGTCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCATGCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCAACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGATTTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCAACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACGCCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTGGAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCAGCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGCAGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCGGAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCTGGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATTTGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAGACTCAACTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCAAATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAGAGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCCCAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAA（SEQ ID NO：15）
Of the present inventionly relate in one aspect to a kind of recombinant vectors, it contains the nucleotide sequence described in any one of the present invention again.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 to prepare recombinant expression vector, this carrier can comprise 1 or a plurality of restriction site easily, to insert or replace the nucleotide sequence of coded polypeptide in these sites.Or, can express nucleotide sequence of the present invention by nucleotide sequence or the nucleic acid construct that comprises this sequence are inserted to suitable expression vector.While preparing expression vector, can make encoding sequence 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, regulating and controlling sequence will comprise promotor and the termination signal of transcribing and translating.In order to import specific restriction site, to regulating and controlling sequence is connected with the coding region of the nucleotide sequence of coded polypeptide, can provide the regulating and controlling sequence of belt lacing.Term " is operatively connected " and is defined as in the text a kind of like this conformation, and wherein regulating and controlling sequence is positioned at the appropriate location of the encoding sequence of relative DNA sequence dna, so that regulating and controlling sequence instructs the expression of polypeptide.
Recombinant expression vector can be any carrier (for example plasmid or virus) of being convenient to carry out recombinant DNA operation express nucleic acid sequence.The consistency of the host cell that carrier and it will import is depended in the selection of carrier conventionally.Carrier can be linearity or closed loop plasmid.
Of the present inventionly relate in one aspect to a kind of recombinant host cell, it contains the recombinant vectors described in any one of the present invention again.
The recombinant vectors of the nucleotide sequence that comprises the present invention can be imported to host cell, thereby this carrier is maintained with the outer carrier format of karyomit(e) of above-mentioned chromosomal integration body or self-replacation.Any offspring different from parental cell due to the sudden change occurring between replicative phase contained in term " host cell ".Peptide coding gene 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, for example bacterium or yeast cell.Can carrier be imported to host cell by technology well known to those skilled in the art.
Of the present inventionly relate in one aspect to again a kind of epitope conjugate, comprise epitope and coupling part, wherein, described epitope is the epitope described in any one of the present invention, and described coupling is partly for being selected from one or more in radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H; Particularly, described coupling is partly carrier proteins BSA or KLH.
Of the present inventionly relate in one aspect to a kind of composition, 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 again; Particularly, described composition is vaccine composition.
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 of relating in one aspect to again of the present invention treats and/or prevents the purposes in the medicine of trichinzation in preparation.
The data presentation of embodiment 6, epitope of the present invention has effective Trichinella spiralis worm reduction rate, can effectively prevent and treat trichinzation or trichinzation associated diseases, there are for example, potentiality as the medicine (vaccine) of control trichinzation or its associated diseases.
Of the present inventionly relate in one aspect to a kind of antibody, it can be 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 again.
In the present invention, term " specific binding " has immunologic general sense, for example the combination between antigen-antibody.
Of the present inventionly relate in one aspect to again a kind of antibody coupling matter, comprise antibody moiety and coupling part, wherein, described antibody moiety is the antibody described in any one of the present invention, and described coupling is partly for being selected from one or more in radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H.
Of the present inventionly relate in one aspect to a kind of pharmaceutical composition, it comprises antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention again; Alternatively, described pharmaceutical composition also comprises pharmaceutically acceptable carrier or auxiliary material.
Of the present inventionly relate in one aspect to a kind of test kit, it comprises antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention again.In one embodiment of the invention, described test kit is detection or the diagnostic kit of trichinzation or trichinzation associated diseases.
Antibody described in any one of the present invention or the antibody coupling matter described in any one of the present invention of relating in one aspect to again of the present invention treats and/or prevents the purposes in the medicine of trichinzation in preparation.
The beneficial effect of the invention
The effective Trichinella spiralis worm reduction rate of epitope tool of the present invention, thus for example, potentiality as the medicine (vaccine) of control trichinzation or its associated diseases there are.
Accompanying drawing explanation
Fig. 1: the ascites 8F12mAb of SDS-PAGE purification Identification.1, low molecular weight protein (LMWP) marker; 2,8F12 strain is purifying ascites not; Antibody after 3,8F12 strain column purification.Note: the assorted band at 65kDa place should be the position of Human Serum Albumin, and 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.
The passive immunization protectiveness of Fig. 3: mAb8F12 and the anti-trichinzation of natural infection serum.The muscle larvae number that muscle larvae worm lotus (LPG) is every gram of muscle.With mean ± standard deviation (n=6), represent.Note:
*be and the comparison of PBS control group, p<0.01.
The specific recognition of Fig. 4: mAb8F12 and positive phage clones (evaluation of ELISA method).Using wild phage M13 as negative control, and the coated hole of BSA is in order to get rid of cross reaction.
The specific recognition of Fig. 5: mAb8F12 and positive phage clones (Western blot)
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 synthetic polypeptide and mAb8F12 specific binding detects (polypeptide coupling carrier BSA is coated).Note: mAb5A3 contrasts as irrelevant ascites.
Fig. 7: each organizes the muscle larvae number of immune mouse.Note: with the comparison of 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 are only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition (such as with reference to works such as J. Pehanorm Brookers, the < < molecular cloning experiment guide > > that Huang Peitang etc. translate, the third edition, Science Press) or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be 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-Pmy GenBank accession No.:EF429310) reading frame sequence, can obtain the nucleotide sequence of Ts-Pmy-N1-322aa, as follows:
ATGTCTCTGTATCGCAGTCCCAGTGCGTCAGTGATGAGATCAGCAAGCATGCTCAGCCGAAGTGGCGGATTCGATGCTTACGGATTTGGAGGTTACGGTGCGCCAAGCCTCAACGTTGCCGACTTGGGTTCTTTGACCAGACTCGAGGATAAAATTCGCCTGCTTCAAGATGATTTGGAAACGGAAAGAGAATTGCGAAACCGAATTGAACGCGAACGTGCCGATTTGTCCTGCCAACTGATCAGCTTAACCGATCGATTGGAAGAGGCTGAAGGAACCACCGATGCCCAGATCGACGCCAATCGAAAGCGTGAATCCGAATTGCAAAAGTTGAGAAAAATATTGGAAGATTCGCAATTGGAAAGCGAAGATTCGCTGAACCAGCTGCGCAAGAAGCACCAAGAATCCCTTTTAGATTATCAGCAGCAAATTGAACAACTTCAAAAGAAAAATAGCAAAATCGACAGAGAACGACAACGTTTGCAGCATGAAGTCATTGAACTTACTGCCGGAATTGATCAGATGCAAAAAGACAAGCATGCCGCGGAAAAAGCTGCCGAAAAGCACGAAGCGCATGCCAGAGAGCTTCAGAACAGAGTTGACGATCTGGCAAAAAATTTGAACGACCTGGCCTCGCAGCGTCAACGTCTGCAACAGGAAAACAACGATTTGATGAAAGAGTTGCACGATGTCAAAGTGCAAATGGAAAATATTCAACACGTCAAGACTCAACTTGCTCAACAGCTCGAAGAAGCACGTCGTCGACTCGAAGATGCGGAACGTGAACGTTCGCAAATGCAAACCCAGTTGCATCAGATGCAGCTGGAATTGGATTCAATTCAAGGTGCGTTGGAAGAGGAATCGTCCGCACGTGCCGAAGCAGAGCACAAATTGTCGTTGGCAAATACGGAAATTTCCCAGTGGAAGAGCAAATTCGACGCCGAAGTTTCACTCCACCAAGAAGAA（SEQ ID NO：15）
The aminoacid sequence of its coding is as follows:
MSLYRSPSASVMRSASMLSRSGGFDAYGFGGYGAPSLNVADLGSLTRLEDKIRLLQDDLETERELRNRIERERADLSCQLISLTDRLEEAEGTTDAQIDANRKRESELQKLRKILEDSQLESEDSLNQLRKKHQESLLDYQQQIEQLQKKNSKIDRERQRLQHEVIELTAGIDQMQKDKHAAEKAAEKHEAHARELQNRVDDLAKNLNDLASQRQRLQQENNDLMKELHDVKVQMENIQHVKTQLAQQLEEARRRLEDAERERSQMQTQLHQMQLELDSIQGALEEESSARAEAEHKLSLANTEISQWKSKFDAEVSLHQEE（SEQ ID NO：16）
Ts-Pmy-N1-322aa gene fragment can obtain by synthetic according to SEQ ID NO:15, also can adopt the method for pcr amplification below to obtain.
Design of primers: as shown in Table 1 below.Primer is synthetic to be completed by Shanghai Ying Jun company with sequencing.
Table 1: primer data
Amplification condition: 95 ℃ of 5min; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min; PCR product carries out 1% agarose gel electrophoresis, observes amplification.Reaction system:
Note: Ts-Pmy plasmid bacterium liquid has for transforming the intestinal bacteria that contain paramyosin gene (Ts-Pmy GenBank accession No.:EF429310) reading frame total length plasmid, being equal to publication number is that CN100999737A(application number is 200710000018.1) patent application in the conversion of embodiment 2 preparation have the e. coli bl21 of paramyosin gene (Ts86cDNA), its preparation method can also be 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) enzyme of the PCR product of Ts-Pmy-N1-322aa gene fragment and pET-28a (+) plasmid is cut
Enzyme tangent condition: 37 ℃, water-bath 2h.Enzyme is cut product and is carried out 1% agarose gel electrophoresis detection, and endonuclease bamhi reclaims with test kit.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: ratio solution B) adds solution B;
2) 50 ℃ of water-bath 10min, until glue dissolves completely, during vortex vibration 3 times;
3) solution is added in centrifugal column to high speed centrifugation 1min after standing 2min;
4) Xiang Zhuzhong adds 500 μ l solution C (with dehydrated alcohol dilution in 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 to a new centrifuge tube, adds 20 μ l solution D of 50 ℃ of preheatings, standing 2min, centrifugal collection DNA;
7) agarose gel electrophoresis is observed recovering effect, and-20 ℃ save backup.
(3) the ligation condition that Ts-Pmy-N1-322aa gene fragment and pET-28a (+) carrier enzyme are cut product: 4 ℃ 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 ℃ of heat-shocked 90sec;
(3) centrifuge tube is placed on ice fast, makes the cooling 2min of cell;
(4) add 900 μ l LB substratum, 37 ℃, 50rpm, 45min, makes bacteria resuscitation;
(5) get the above-mentioned bacterium of 100 μ l and coat on the LB solid medium containing kantlex and IPTG, X-gal, cultivate 12-16h for 37 ℃;
After conversion, carry the evaluation of the plasmid bacterial strain of Ts-Pmy-N1-322aa gene fragment:
In flat board after transforming, picking list bacterium colony is in containing in the LB liquid nutrient medium of kantlex, extracts that plasmid carries out PCR and enzyme is cut evaluation after incubated overnight.Or the evaluation of checking order.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 to contain in the LB nutrient solution of kantlex (30 μ g/ml).37 ℃, 150rpm shaking culture is spent the night;
(2) next day, 5ml culture is added to containing in the LB substratum of kantlex of 500ml.37 ℃, 300rpm, continues shaking culture until OD
600≈ 0.6;
(3) adding IPTG is 1mM abduction delivering to final concentration, and 37 ℃, 150rpm, continues to cultivate 4h;
(4) centrifugal collection thalline, 200ml20mM Tris-HCl(pH7.9) bacterial precipitation fully suspends;
(5) fully ultrasonic on ice, ultrasonic output intensity 40%, each 8sec, interval 5sec, totally 10 times;
(6) the centrifugal supernatant of abandoning, 100ml20mMTris-HCl(pH7.9) suspends and precipitates;
(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, the centrifugal supernatant of abandoning, throw out is inclusion body and cell debris;
(9) add 50ml to contain 1 * binding buffer of 6M Guanidinium hydrochloride, ultrasonicly make to precipitate abundant suspension, dissolving, 4 ℃ 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-Bind Resin(binding resin), concussion suspends.In affinity column, add 2.5ml His-Bind Resin, flick cylinder to remove bubble;
(2) add successively 15ml DDW, 12ml1 * Charge Buffer, 9ml1 * Binding Buffer(is containing 6M Guanidinium hydrochloride) balance chromatography column;
(3) the inclusion body solution centrifugal being dissolved in containing in 1 * Binding Buffer of 6M Guanidinium hydrochloride is got to supernatant, 45 μ m filters slowly add in post after filtering;
(4) add successively again 12ml1 * Binding Buffer(containing 6M Guanidinium hydrochloride), 15ml20mM imidazole buffer (11ml1 * Binding Buffer, 4ml1 * Wash Buffer, all containing 6M Guanidinium hydrochloride) clean chromatography column, 12ml1 * Elute Buffer(is containing 6M Guanidinium hydrochloride) wash-out target protein;
(5) dialysis: by the collection liquid impouring dialysis tubing of 1 * Elute Buffer wash-out (molecular weight cut-off is 12-14kDa), put into the 20mM Tris-HCl(pH7.9 of 50 times of volumes), more than 4 ℃ of dialysis 3h.
(6) proceed in the dialyzate of same volume 4 ℃ of dialysed overnight;
(7) in dialysis tubing, deactivated protein precipitation is separated out, and by the solution impouring centrifuge tube that contains 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 appropriate 1 * IB Solubilization Buffer(prepares from 10 * storage liquid.10 * IB Solubilization Buffer comprises 200mM Tis-HCl, pH7.5,100mM EDTA, 10%Triton X-100), and add 0.3%N-lauroylsarcosine and 1mM DTT;
(2) with the concentration suspension albumen of 1-2mg/ml, the standing 10min of room temperature, fully dissolves albumen;
(3) after centrifugal, supernatant is proceeded in dialysis tubing;
(4) put into 1 * Dialysis Buffer(50 times of volumes, that contain 0.1mM DTT by 50 * Dialysis Buffer dilution and obtain, wherein composition is 1M Tris-HCl, pH8.5), in dialyzate, more than 4 ℃ of dialysis 3h, then change same dialyzate and continue dialysis 3h;
(5) put into dialyzate 50 times of volumes, that do not contain 1 * Dialysis Buffe of DTT, 4 ℃ of dialysis 3h, change same dialyzate and continue dialysed overnight;
Next day, collect recombinant protein liquid, obtain rTs-Pmy-N1-322aa albumen (solution).
By BCA method, survey protein concentration ,-20 ℃ of preservations.
embodiment 2: the foundation of hybridoma cell strain
By the immune BALB/c mouse of rTs-Pmy-N1-322aa albumen (solution) of embodiment 1 preparation, through 4 immunity, after last immunity, mouse antibodies is tired and is reached 1: 128000.The SP2/0 myeloma cell of immune mouse spleen cell and logarithmic phase is merged, through HAT nutrient solution, select to cultivate.In 96 well culture plates, visible fused cell growth, when cell colony grows to 1/3 hole, by indirect ELISA and Western blot, identify, pick out in culture supernatant and have anti-rTs-Pmy-N1-322aa antibody to exist, and the positive hybridoma cell strain that can identify with rTs-Pmy-N1-322aa and larva polypide albumen.Through 3 subclones, will screen and obtain a strain of hybridoma strain called after 8F12, the antibody of its secretion belongs to the IgG1 Subclass Antibodies of secretion Th2 type, through continuous passage, cultivated for 20 generations, F5, the F10, F15 and the F20 that get respectively wherein preserve the recovery cells and supernatant after half a year for cells and supernatant and liquid nitrogen, with indirect ELISA method, measure antibody titer, the results are shown in Table 2.Hybridoma antibody-secreting after continuous passage and cryopreservation resuscitation is stable, selects use for further study.
Table 2: the ELISA of hybridoma cell strain 8F12 stability identifies
| Passage number | 8F12 (A value) |
| F5 | 2.096 |
| F10 | 1.886 |
| F15 | 1.973 |
| F20 | 1.896 |
| Cryopreservation resuscitation cell | 1.796 |
the evaluation of the monoclonal antibody (mAb) of embodiment 3:8F12 cell strain secretion
In the cell culture supernatant of the 8F12 cell strain in ELISA detection embodiment 2 and ascites, the titre of mAb, is respectively 1: 3200 and 1: 64000.The Subclass of antibody of secretion is IgG1 subclass κ type (table 3).Monoclonal antibody ascites is carried out SDS-PAGE electrophoresis after HiTrap rProtein A column purification, respectively at 50kDa and 25kDa place, has band to manifest, and meets the position (Fig. 1) of IgG heavy chain of antibody and light chain.
The evaluation of table 3: monoclonal antibody 8F12
For clear and definite mAb under certain antigen concentration when antigen is combined the size of relative affinity, adopt indirect non-competing ELISA method.Logarithmic value with extension rate is lg(C
0/ C) be X-coordinate (C
0for mAb starting point concentration, C is concentration after mAb dilution), take A value as ordinate zou, draw the relative affinity curve (Fig. 2) of two strain mAb.The antibody affinity costant Ka value of calculating 8F12mAb by formula is 9.1 * 10
7m
-(table 3).
embodiment 4: monoclonal antibody (mAb) passive immunization protective effect
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, 6 every group.
3. experimental technique:
Use the method passive immunization mouse of tail vein injection antibody, the antibody purification of every injected in mice of monoclonal antibody group 500 μ g (being dissolved in 100 μ l PBS); The BALB/c mouse of every injected in mice of natural infection serologic group 100 μ l infects serum (serum titer is 1: 3000); The PBS of every injected in mice of PBS control group 100 μ l.After passive immunization, 2 hours every mouse are attacked 400 of worms.Within the 4th day after attacking worm, with same dosage, distinguish booster immunization once.Attack after worm and within 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 has obtained the immune protective efficiency of anti-trichinzation in BALB/c mouse body.The experimental group of injection mAb8F12 is compared with the control group of injection PBS with natural infection serologic group mouse, obtains respectively 25.6% and 24.6% muscle larvae worm reduction rate (p<0.01).But muscle larvae number (LPG) difference of monoclonal antibody group and natural infection serologic group is without significance (Fig. 3).Confirm mAb8F12 tool passive immunization protectiveness.The B cell antigen epi-position that can be used for follow-up screening tool immune protective.
Table 4: each organizes the muscle larvae check result of immune mouse
Note:
*learn by statistics check, have 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, calculated every rate of recovery of taking turns phage.Every phage virus of taking turns input is 2 * 10
11, measure every titre of taking turns the phage under wash-out, calculate recovery rate, result following (table 5):
Table 5: the variation of the three-wheel phage display peptide library pnagus medius rate of recovery
| Screening number of times | The phage adding | The phage of wash-out | The phage rate of |
| 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 of eluriating phage peptide library, the output height after third round was eluriated than the first round 200 times.
2. the evaluation of positive colony
10 of (plaque number is lower than 100) random pickings blue plaque independently 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, using wild phage M13 as negative control simultaneously.For avoiding screening the false positive phage clone of being combined with the liquid of blockading (main component is BSA) composition, separately established the coated system contrast of BSA.With OD
492value is higher than negative control OD
492more than being worth 2.1 times, as positive criterion, clone whole positive phage clones for 10.Result following (Fig. 4).
3. the analysis of the base sequence of positive colony mensuration and encoding amino acid sequence
Extract after the single stranded DNA of 10 positive phage clones, record DNA sequence dna.The result of positive colony sequencing is the sequence (in Table 6) of complementary strand, must be changed into be translated as aminoacid sequence (in Table 7) after coding strand sequence again and to carry out sequence alignment analysis again.
Table 6: the DNA sequence dna of the positive phage clones dodecapeptide of being combined with 8F12
Table 7: phage display peptide sequence
By above-mentioned 10 positive colony DNA sequence dnas, derived in the encoding amino acid sequence obtaining, have 4 to be tumor-necrosis factor glycoproteins (RNTO 8JJ), therefore obtain altogether 7 seed amino acid epitope peptide sequences.For whether further clear and definite epi-position phage is identified by monoclonal antibody 8F12, carried out Western blot experiment (Goat anti-MouseIRDye800CW is anti-as biotin labeling two).Experimental result shows (Fig. 5): present the positive phage clones of above-mentioned 7 kinds of epitope peptide sequences in the position of 60kDa left and right, all occurred the band of position consistency.And the wild phage of M13 has no band in this position.The less important capsid protein pIII that is connected in phage due to displayed polypeptide is upper, and while carrying out SDS-PAGE electrophoresis, pIII runs conventionally near molecular weight 60-65kDa.Therefore can determine the epitope that contains mAb8F12 identification in the displayed polypeptide of positive bacteriophage Clone1-10.
4. the identification of synthetic polypeptide and mAb8F12
The contained epitope peptide sequence of above-mentioned 7 kinds of positive phage clones is delivered to the synthetic epitope polypeptide of company, respectively called after 8JJ (8A6,8A7, the contained epitope peptide sequence of 8A11,8A12 are separately named), 8A1,8A9,8F1,8F6,8F7,8F10.For increasing polypeptide antigen, by polypeptide and carrier proteins BSA or KLH coupling (it is synthetic that Beijing NIVEA Corp difficult to understand carries out technology), ELISA result shows that 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 has reached the detected value (Fig. 6) of albumen rTs-Pmy-N1-322aa and 8F12.
embodiment 6: the synthetic polypeptide immune mouse of epi-position is also evaluated its immune protective
1. laboratory sample:
Synthetic 8JJ-KLH, 8A1-KLH, 8A9-KLH, 8F1-KLH, 8F6-KLH, 8F7-KLH, 8F10-KLH 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, 6 every group at random.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 respectively 50 μ g 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 are with same dosage and mode immune animal in contrast.Every immunity in two weeks once, be total to immunity three times.Latter 10 days of last immunity, 400 of every mouse challenge infection cultivation of larvae of Trichinella spiralis from muscles, cutd open and kill mouse after 45 days, checked muscle larvae worm lotus, evaluated immune protective of each group.
4. experimental result:
Compare with KLH control group, 8A1-KLH group, 8F1-KLH group, 8F7-KLH group muscle larvae number (LPG) have significant differences (p<0.01); Obtain respectively 15.9%, 22.2% and 26.3% muscle larvae worm reduction rate (table 12).Compare with KLH control group, 8F6-KLH group muscle larvae number has significant difference (p<0.05), and the muscle larvae worm reduction rate of acquisition 18.7% (table 8, Fig. 7).
Table 8: each organizes the muscle larvae check result of immune mouse
Note: * * learns check by statistics, has significant differences (p<0.01) with KLH control group;
* learn by statistics check, have significant differences (p<0.05) with KLH control group
In a word, by above experiment, the inventor has obtained 4 protective epitopes of Ts-Pmy, all can induce and produce effective immanoprotection action after these epitope polypeptide immune mouses.Above results suggest: these epi-positions can be used as candidate vaccine component, thus for resisting trichinous polyepitope vaccines, preparation lays a good foundation.
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 modifications 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 (10)
1. an epitope, its aminoacid sequence is as shown in SEQ ID NO:1.
2. the nucleotide sequence of epitope described in the claim 1 of encoding; Particularly, described nucleotide sequence is as shown in SEQ ID NO:8.
3. a recombinant vectors, it contains nucleotide sequence claimed in claim 2.
4. a recombinant host cell, it contains recombinant vectors claimed in claim 3.
5. an epitope conjugate, comprise epitope and coupling part, wherein, described epitope is epitope claimed in claim 1, and described coupling is partly for being selected from one or more in radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H; Particularly, described coupling is partly carrier proteins BSA or KLH.
6. a composition, it comprises one or more epitopes claimed in claim 1, and/or one or more epitope conjugates claimed in claim 5; Particularly, described composition is vaccine composition.
7. epitope claimed in claim 1 or epitope conjugate claimed in claim 5 treat and/or prevent the purposes in the medicine of trichinzation in preparation.
8. an antibody, it can be specifically in conjunction with epitope claimed in claim 1 or epitope conjugate claimed in claim 7.
9. an antibody coupling matter, comprise antibody moiety and coupling part, wherein, described antibody moiety is antibody claimed in claim 8, and described coupling is partly for being selected from one or more in radionuclide, medicine, toxin, cytokine, enzyme, fluorescein, carrier proteins and vitamin H.
10. a pharmaceutical composition, it comprises antibody claimed in claim 8 or antibody coupling matter claimed in claim 9; Alternatively, described pharmaceutical composition also comprises pharmaceutically acceptable carrier or auxiliary material.
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| WO2017184895A2 (en) * | 2016-04-20 | 2017-10-26 | Aelan Cell Technologies, Inc. | Compositions and methods related to κ180 dimethylated h1.0 protein |
| 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 |
|---|
| WEI J.等: "Identification and characterization of protective epitope of Trichinella spiralis paramyosin", 《VACCINE》, vol. 29, no. 17, 11 April 2011 (2011-04-11), pages 3162 - 3168 * |
| 魏骏飞等: "抗旋毛虫副肌球蛋白单克隆抗体的制备与鉴定", 《中国科技论文在线》, 2 April 2009 (2009-04-02), pages 1 - 6 * |
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| CN107118267A (en) * | 2017-04-18 | 2017-09-01 | 浙江工商大学 | It is a kind of to be used to alleviate improvement albumen mMete1 of prawn tropomyosin sensitivity response and its preparation method and application |
| 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 |
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| CN103724413B (en) | 2016-02-24 |
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