CN104558108A - Linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein - Google Patents
Linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein Download PDFInfo
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Abstract
The invention relates to linear epitope minimum motif peptides of PPRV (peste des petits ruminants virus) N (nucleocapsid) protein and an application of the linear epitope minimum motif peptides. Aiming at a Tibetan isolate PPRV N protein with high immunogenicity, an improved short peptide biosynthesis method and an anti-recombinant protein polyclonal antibody are adopted, a complete-sequence fine linear B cell epitope map is provided and comprises 20 minimum epitope motif contained epitope peptides which can be used for detecting the infection serum of the PPRV independently or in a combined manner. And further, an epitope map with broad spectrum applicable to various PPRVs is provided.
Description
Technical field
The invention belongs to immunology and biomedicine technical field, more specifically, the present invention relates to a kind of linear epitope minimum motif peptide of PPR virus N protein.
Background technology
PPR (peste des petits ruminants, PPR) be by PPR virus (peste despetits ruminants virus, what PPRV) cause a kind ofly infects the little Acute exposure communicable disease (Munir ruminating beasts, M., Role of Wild Small Ruminants in the Epidemiology of Peste DesPetits Ruminants.Transbound Emerg Dis, 2013).As a kind of transnational category-A zoonosis seriously, China is also defined as a class animal epidemic, is the highest concern rank.Sickness rate is up to 100% in susceptible flock of sheep for PPR, and during serious outburst, lethality rate is 100%.PPR nineteen forty-two Late Cambrian in Cote d'lvoire, after diffuse to the large portion in Africa and Asia; PPR epidemic situation was broken out twice respectively at 2007,2008 in Tibet, China area, caused huge financial loss to local peasants and herdsmen.Although Chinese scholars have developed the prevention and control for this epidemic disease of the detection of PPR virus and attenuated vaccine, because it is expensive, in the developing country that PPR frequently breaks out, being included in China's western region still can not popularization and application.Setting up novel PPRV detection kit or being domesticized by these test kits is one of extensive PPR investigation of epidemic situation problem demanding prompt solution.
PPRV belongs to Paramyxoviridae (paramyxoviridae), Morbillivirus (morbillivirus) minus-stranded rna virus, full-length genome about 16,000 Nucleotide, it only has a serotype, its genome encoding nucleocapsid protein (N), phosphorprotein (P), polysaccharase or large protein (L), stromatin (M), fusion rotein (F) and hemagglutinin (H) six structural protein and P albumen and C protein two nonstructural proteins (Munir, M. etc., Genome Organization of Peste des Petits Ruminants Virus, in Molecular Biologyand Pathogenesis of Peste des Petits Ruminants Virus.2013, Springer BerlinHeidelberg.p.1-22).Wherein N protein is the protein that in PPRV, content is the abundantest and immunogenicity is the strongest.Epitope mapping location (epitope mapping) and epitope motifs qualification (epitope motifidentifying) are the emphasis that animal virus field is paid close attention to always.Also relevant report is had in the epi-position research of PPRV structural protein N, as Kang-Seuk Choi etc. is the highest and the nucleocapsid N protein that immunogenicity is the strongest identifies 4 antigen sites (A-1 with regard to PPRV virus abundance, A-2, C-1, C-2) (Choi, K.S., et al., Antigenic and immunogenic investigation of B-cell epitopes in the nucleocapsidprotein of peste des petits ruminants virus.Clin Diagn Lab Immunol, 2005.12 (1): p.114-21), the chemical synthesising peptide method such as Dechamma identifies special B cell epitope peptide section (amino-acid residue 454-472) (Dechamma that can distinguish PPRV and RRV and infect at the C end of the N protein of PPRV, H.J., et al., Identification of T-helper and linear B epitopein the hypervariable region of nucleocapsid protein of PPRV and its use in thedevelopment of specific antibodies to detect viral antigen.VeterinaryMicrobiology, 2006.118 (3-4): p.201-211).But the research of the epitope mapping of PPR virus N protein and epitope motifs qualification is not reported so far.
Summary of the invention
The object of the present invention is to provide a kind of linear epitope minimum motif peptide of PPR virus N protein.
In a first aspect of the present invention, provide the linear epitope collection of illustrative plates of PPR virus (Tibet strain) N protein, this epitope collection of illustrative plates comprises:
PPR virus N protein 62-68 position and the linear epitope peptide shown in SEQ ID NO:1;
PPR virus N protein 96-100 position and the linear epitope peptide shown in SEQ ID NO:2;
PPR virus N protein 119-126 position and the linear epitope peptide shown in SEQ ID NO:3;
PPR virus N protein 128-133 position and the linear epitope peptide shown in SEQ ID NO:4;
PPR virus N protein 147-153 position and the linear epitope peptide shown in SEQ ID NO:5;
PPR virus N protein 152-158 position and the linear epitope peptide shown in SEQ ID NO:6;
PPR virus N protein 297-305 position and the linear epitope peptide shown in SEQ ID NO:7;
PPR virus N protein 304-312 position and the linear epitope peptide shown in SEQ ID NO:8;
PPR virus N protein 377-385 position and the linear epitope peptide shown in SEQ ID NO:9;
PPR virus N protein 384-392 position and the linear epitope peptide shown in SEQ ID NO:10;
PPR virus N protein 413-420 position and the linear epitope peptide shown in SEQ ID NO:11;
PPR virus N protein 417-424 position and the linear epitope peptide shown in SEQ ID NO:12;
PPR virus N protein 438-442 position and the linear epitope peptide shown in SEQ ID NO:13;
PPR virus N protein 452-456 position and the linear epitope peptide shown in SEQ ID NO:14;
PPR virus N protein 462-464 position and the linear epitope peptide shown in SEQ ID NO:15;
PPR virus N protein 474-479 position and the linear epitope peptide shown in SEQ ID NO:16;
PPR virus N protein 486-493 position and the linear epitope peptide shown in SEQ ID NO:17;
PPR virus N protein 492-496 position and the linear epitope peptide shown in SEQ ID NO:18;
PPR virus N protein 504-509 position and the linear epitope peptide shown in SEQ ID NO:19;
PPR virus N protein 523-525 position and the linear epitope peptide shown in SEQ ID NO:20.
In another aspect of this invention, provide the conservative property linear epitope collection of illustrative plates of PPR virus N protein, this epitope collection of illustrative plates comprises:
PPR virus N protein 62-68 position and the linear epitope peptide shown in SEQ ID NO:1;
PPR virus N protein 96-100 position and the linear epitope peptide shown in SEQ ID NO:2;
PPR virus N protein 119-126 position and the linear epitope peptide shown in SEQ ID NO:3;
PPR virus N protein 128-133 position and the linear epitope peptide shown in SEQ ID NO:4;
PPR virus N protein 147-153 position and the linear epitope peptide shown in SEQ ID NO:5;
PPR virus N protein 152-158 position and the linear epitope peptide shown in SEQ ID NO:6;
PPR virus N protein 304-312 position and the linear epitope peptide shown in SEQ ID NO:8;
PPR virus N protein 377-385 position and the linear epitope peptide shown in SEQ ID NO:9;
PPR virus N protein 384-392 position and the linear epitope peptide shown in SEQ ID NO:10;
PPR virus N protein 413-420 position and the linear epitope peptide shown in SEQ ID NO:11;
PPR virus N protein 492-496 position and the linear epitope peptide shown in SEQ ID NO:18.
In a preference, described linear epitope or conservative property linear epitope peptide are separated.
In another aspect of this invention, there is provided a kind of test kit of the linear epitope for detecting PPR virus N protein, described test kit comprises: SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ ID NO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence; Or
The encoding gene of SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ ID NO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence.
In a preference, also comprise in described test kit: SEQ ID NO:7, SEQ ID NO:12 ~ SEQ ID NO:17, the linear epitope peptide shown in SEQ ID NO:19 ~ SEQ ID NO:20 sequence; Or
The encoding gene of SEQ ID NO:7, SEQ ID NO:12 ~ SEQ ID NO:17, the linear epitope peptide shown in SEQ ID NO:19 ~ SEQ ID NO:20 sequence.
In another aspect of this invention, provide a kind of fusion rotein, described fusion rotein comprises SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ ID NO:11 and the aminoacid sequence described in SEQ ID NO:18; Above-mentioned sequence is connected mutually.
In a preference, the aminoacid sequence of described fusion rotein is as shown in SEQ ID NO:21.
In another aspect of this invention, provide a kind of fusion gene, the fusion rotein described in this fusion gene coding.
In a preference, as described in fusion gene, the nucleotide sequence of this fusion gene is as shown in SEQ ID NO:21.
In another aspect of this invention, provide the linear epitope collection of illustrative plates of described PPR virus N protein or the purposes of described test kit, ruminate the multi-epitope chimeric peptide vaccine of epizootic disease virus for the preparation of control; Or
For the preparation of detecting the virus detection reagent ruminating epizootic disease virus; Or
For the preparation of the specific antibody of specific linear epitope in anti-PPR virus N protein.
In another aspect of this invention, provide the purposes of described fusion rotein, for the preparation of detecting the virus detection reagent ruminating epizootic disease virus.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, Western blot screens positive reaction restructuring fusion 16 peptide.Wherein, M: low molecular weight protein Marker; 1-64: the series restructuring fusion 16 peptide N1-N64 of overlapped 8 amino-acid residues of covered structure protein N complete sequence.
The qualification of Fig. 2, a minimum epitope motifs of positive reaction 16 peptide (N12).
A: the SDS-PAGE that the series for positive reaction 16 peptide merges 8 peptides analyzes; Wherein, M: low molecular weight protein Marker; 1-9: serial 8 peptides;
In b:a, the western blot of series fusion 8 peptides analyzes;
The aminoacid sequence of serial 8 peptides in c:a; The common amino acid motif of positive 8 peptides is shown as in dotted line frame.
Embodiment
The present inventor adopts the biosynthesizing small peptide method of improvement and anti-recombinant protein to resist more, for the nucleocapsid protein matter (N) that Tibet strain PPRV immunogenicity is strong, provides complete sequence meticulous linear B cell antigen epitope maps; Further, the epitope maps that wide spectrum is applicable to multiple PPRV virus is additionally provided.The present invention is that development of new PPRV multi-epitope chimeric peptide vaccine and high-sensitivity detecting method provide effective way, and provides basis for the linear B cell epitopes group research of the whole coded protein of the follow-up PPRV of development.
Term
As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.
As used herein, " N protein " refers to the nucleocapsid protein being present in PPRV virus, and it is that conservative property exists in PPRV virus Different plant type.
As used herein, described " containing ", " having " or " comprising " include " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
As used herein, " specificity " of antibody refers to that antibody capable is incorporated into PPRV viral N proteins defined epitope, and is not incorporated into other epi-position.
Epitope collection of illustrative plates and uses thereof
PPR is the little acute contact category-A transmissible disease of ruminating beasts such as a kind of goat, sheep caused by PPR virus.This disease, as a kind of great transnational animal epidemic, seriously endangers animal productiong and the hygienic safety in Tibet, China and even whole west area.At present, still lack effective treatment means for PPR, main epidemiology survey and the vaccine of relying on carries out prevention and control.Epitope mapping and epitope minimum motif qualification detect virus disease and novel multi-epitope peptide vaccine development tool is of great significance.
The present inventor adopts the biosynthesizing small peptide method of improvement and anti-recombinant protein to resist more, to Tibet strain PPRV nucleocapsid protein matter (N), carries out the meticulous linear B cell antigen epitope mapping research of complete sequence.First, codon optimized synonym transformation is carried out to Tibet strain PPR virus N gene in GenBank, by 2 step PCR full chemosynthesis PPRV N gene, be cloned into prokaryotic expression carrier and in intestinal bacteria solubility expression.By the restructuring PPRV N protein immunize New Zealand White Rabbit of purifying, obtain antiserum(antisera).Utilize the anti-restructuring PPRV N protein antiserum(antisera) of preparation, cover restructuring 16 peptide fragment of the full length sequence of N protein to screen from 64 and obtain 19 immuno positive and to recombinate 16 peptide fragment; And the further minimum antigen motif determining reactive 16 peptides; Delineate the complete and meticulous linear B cell epitopes collection of illustrative plates of PPR virus N protein.Comparison different modification virus strain homologous protein sequence, finds that wherein 9 epitope motifs are complete epitopes conserved amongst's motif.
Based on the new discovery of the present inventor, provide the linear epitope collection of illustrative plates of PPR virus (Tibet strain) N protein, comprise the linear epitope peptide (small peptide) shown in SEQ ID NO:1 ~ SEQ ID NO:20.
Based on the new discovery of the present inventor, additionally provide the fusion rotein comprising all conservative property linear epitope peptides, described fusion rotein comprises SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQID NO:11 and the aminoacid sequence described in SEQ ID NO:18; Above-mentioned sequence is connected mutually, preferably, can comprise connection peptides (1-10aa, preferably 1-5aa, as 2aa, 3aa) between each epi-position.As optimal way of the present invention, the aminoacid sequence of described fusion rotein is as shown in SEQ ID NO:21.
Once obtain the sequence of described polypeptide, just this polypeptide amalgamation protein can be obtained in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, from the host cell after propagation, be then separated the fusion rotein obtaining relevant sequence by ordinary method.In addition, contrasting small peptide of the present invention, preferably adopting the method for synthetic (as synthesized by Peptide synthesizer) to synthesize relevant sequence, the method for synthetic can be easy and obtain required polypeptide rapidly.
The present invention provide simultaneously identified epitope peptide preparation " general " preventative/therapeutic PPRV recombinant multi-epitope peptide vaccine in application.Described " general " epitope peptide is: SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ ID NO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence.
Epitope peptide of the present invention can be used as the effective antigenic fragment of one of PPRV N protein, for the preparation of the universal antibody of specific binding PPRV N protein, comprises monoclonal antibody or polyclonal antibody.Epitope peptide of the present invention also can be connected with acceptable carrier (preferably itself does not have immunogenicity) in other peptide or immunology, for as immunogen Dispersal risk.
Antibody can be prepared by the various technology that those skilled in that art are known.Such as, the polypeptide of the present invention (or with in other peptide or immunology can after thorny carrier is connected) of purifying can be applied to animal to induce the generation of polyclonal antibody.Similarly, the cell of expressing polypeptide of the present invention can be used to immune animal to produce antibody.Described antibody also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare (see people such as Kohler, Nature256; 495,1975; The people such as Kohler, Eur.J.Immunol.6:511,1976; The people such as Kohler, Eur.J.Immunol.6:292,1976; The people such as Hammerling, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).The available polypeptide immune animal of the present invention of production of polyclonal antibody, as rabbit, mouse, rat etc.Multiple adjuvant can be used for strengthening immune response, includes but not limited to freund's adjuvant etc.
Present invention also offers the purposes of described epitope peptide, whether there is PPRV virus for the identification of in sample.After obtaining epitope peptide of the present invention, whether those skilled in the art detect the existence of minimum motif peptide sequence in sample delicately by number of ways, and the technology of employing can be that technology conventional in field of immunology is as PCR method.
The present invention also provides described epitope peptide separately or by combination (building recombinant multi-epitope peptide), for the preparation of the detectable antigens of diagnosis high-risk-type PPRV virus infection antibody (as being present in serum).
The present invention depicts the complete and meticulous Linear B Cell Epitopes collection of illustrative plates of animal virus structural protein first; Technical scheme of the present invention is development of new PPRV multi-epitope chimeric peptide vaccine and high-sensitivity detecting method, and solid basis has been established in the linear B cell epitopes group research of the whole coded protein of the follow-up PPRV of development.
Test kit
SEQ ID NO:1 ~ SEQ ID NO:8 of the present invention, SEQ ID NO:10 ~ SEQ ID NO:11, the linear epitope peptide (more preferably also comprising: SEQ ID NO:1 ~ SEQ IDNO:8, SEQ ID NO:10 ~ SEQ ID NO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence) shown in SEQ ID NO:18 sequence or its encoding gene or can be placed in a test kit containing the expression vector of described encoding gene or host cell, for preparing the multi-epitope chimeric peptide vaccine that epizootic disease virus is ruminated in control for those skilled in the art; Or for preparing the virus detection reagent detecting and ruminate epizootic disease virus for those skilled in the art; Or for preparing the specific antibody of specific linear epitope in anti-PPR virus N protein for those skilled in the art.In order to prepare the needs of detection reagent, vaccine or antibody, the linear epitope peptide shown in SEQ ID NO:1 ~ SEQ ID NO:20
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, immunological experiment is with reference to the step described in " antibody technique experiment "; Or according to the condition that manufacturer advises.
I. materials and methods
Biological material source
Thermal induction expression plasmid (number of patent application: 200710173305.2); The goat-anti rabbit two of e. coli bl21 (DE3) and Top10 bacterial strain, protein molecular weight standard, pre-dyed protein molecular weight standard and horseradish peroxidase mark is anti-, ECL chemoluminescence colouring reagents box is purchased from Tian Gen bio tech ltd, Beijing; Restriction enzyme, T4DNA ligase enzyme are purchased from Chao Rui bio tech ltd, Shanghai; Nitrocellulose filter is purchased from Ding Guo bio tech ltd, Shanghai; Plasmid extraction kit, PCR primer purification kit, glue reclaim test kit, DNA extraction kit matches bio tech ltd purchased from Shanghai hundred; Conventional chemical reagent is purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
PPRV N protein matter recombinant strains builds
The full chemosynthesis of PPR virus N gene and the expression reference Zhang in E.coli, G.R., et al., Development of an indirect ELISA with artificially synthesizedN protein of PPR virus.Intervirology, 2012.55 (1): p.12-20 carry out.Simply say, according to the sequence (ACQ44667.1) of PPR viral N proteins matter in Genbank, the optimization of intestinal bacteria preference codon is carried out to it, by 2 step PCR full chemosynthesis Tibet strain PPRV N gene, be inserted in the EcoRV site of prokaryotic expression carrier pET-32a after sequence verification is correct, obtain recombinant expression vector pET-32a-N, its transformation of E. coli BL21 (DE3) is obtained PPRV N protein matter recombinant strains.
The sequence of ACQ44667.1 basis being carried out after the optimization of intestinal bacteria preference codon is as follows:
atggctactttattaaaatctttagctttatttaaacgtaataaagataaagctcctactgcttctggttctggtggtgctattcgtggtattaaaaatgttattattgttcctattcctggtgattcttctattactactcgttctcgtttattagatcgtttagttcgtttagctggtgatcctgatattaatggttctaaattaactggtgttatgatttctatgttatctttatttgttgaatctcctggtcaattaattcaacgtattactgatgatcctgatgtttctattcgtttagttgaagttgttcaatctactcgttctcaatctggtttaacttttgcttctcgtggtgctgatttagataatgaagctgatatgtatttttctactgaaggtccttcttctggtggtaaaaaacgtattaattggtttgaaaatcgtgaaattattgatattgaagttcaagatcctgaagaatttaatatgttattagcttctattttagctcaagtttggattttattagctaaagctgttactgctcctgatactgctgctgattctgaattacgtcgttgggttaaatatactcaacaacgtcgtgttattggtgaatttcgtttagataaaggttggttagatgctgttcgtaatcgtattgctgaagatttatctttacgtcgttttatggtttctttaattttagatattaaacgtactcctggtaataaacctcgtattgctgaaatgatttgtgatattgataattatattgttgaagctggtttagcttcttttattttaactattaaatttggtattgaaactatgtatcctgctttaggtttacatgaatttgctggtgaattatctactattgaatctttaatgaatttatatcaacaattaggtgaagttgctccttatatggttattttagaaaattctattcaaaataaattttctgctggtgcttatcctttattatggtcttatgctatgggtgttggtgttgaattagaaaattctatgggtggtttaaattttggtcgttcttattttgatcctgcttattttcgtttaggtcaagaaatggttcgtcgttctgctggtaaagtttcttctgttattgctgctgaattaggtattactgctgaagaagctaaattagtttctgaaattgcttctcaagctggtgatgaacgtactgctcgtggtactggtcctcgtcaagctcaagtttcttttttacaacatcaaactggtggtggtgaatcttctgctcctgctactcgtgaaggtgttaaagctgctattcctaatggttctgaagaacgtgatcgtaaacaaactcgtcctggtcgtcctcgtggtgaaactcctggtcaattattattagaaattatgcctgaagatgaagtttctcgtgaatctggtcaaaatcctcgtgaagctcaacgttctgctgaagctttatttcgtttacaagctatggctaaaattttagaagatcaagaagaaggtgaagataataatcaagtttataatgataaagatttattaggttaa(SEQ ID NO:23)
The preparation of anti-PPRV N protein rabbit anti-serum
By recombinant expressed PPRV N protein after Ni column purification and SDS-PAGE tap rubber purifying, active immunity new zealand white rabbit, it is for subsequent use that booster immunization extracts serum 2 times afterwards.
PPR virus N protein linear epitope scanning mapping
(1) according to codon optimized rear improved PPRV N gene order, (normal chain 5 ＇ end adds 5 ＇-gatcc to the positive minus strand fragment of 16 peptide coding DNA of overlapped 8 amino-acid residues of design covering N gene complete sequence, and 3 ＇ ends add taag-3 ＇; Minus strand 5 ＇ end adds 5 ＇-tcgactta, and 3 ＇ ends add g-3 ＇), send DNA synthesis outside.
(2) positive for the complementation of 1 or 2 OD minus strand fragment ddH2O is dissolved into 100mmol/ml storage liquid (synthesizing the data of report according to DNA); Respectively get 10 μ l storage liquid and 20 μ l ddH
2in O and 1.5mlEppendorf pipe, 94 DEG C of heating in water bath 15min, after Temperature fall to room temperature, in 10 μ l reaction systems, add anneal fragments, 1 μ l of 2 μ l be about 200ng/ μ l through the pXXGST-1 plasmid of BamH I and Sal I double digestion, 1 μ l T4DNA ligase enzyme and 1 μ l ligase enzyme damping fluid, 22 DEG C connect 1h; Connect product conversion competence Top10 Host Strains, coating is dull and stereotyped containing the LB of penbritin (Amp), 37 DEG C of overnight incubation; Picking mono-clonal is forwarded to fresh LB substratum, extracts plasmid and cuts and sequence verification through enzyme, verify that correct plasmid (is called pXXGST-N
xplasmid) Transformed E .coli BL21 (DE3, plysS) competent cell.
(3) picking mono-clonal is seeded to fresh in the LB substratum of Amp, 37 DEG C of concussion overnight incubation, next day is forwarded to fresh in the LB substratum of Amp with the ratio of 1:50,30 DEG C of concussion cultivation 2-3 are little after cell concentration OD reaches 0.6-0.8, be warming up to 42 DEG C of thermal induction 4h, collected by centrifugation thalline, adds loading lysate and boils 5min, and standby SDS-PAGE screens high expression level bacterial strain.
(4) high expression level sample total protein SDS-PAGE electrophoresis, after electrophoresis terminates, a clotting glue coomassie brilliant blue staining, another clotting glue carries out nitrocellulose filter electrotransfer 30min;
(5) marking film TBST damping fluid repetitive scrubbing 4 times, closes with 5% skim-milk and spends the night, TBST buffer solution 4 times; Add primary antibodie (the rabbit anti-PPRV N protein matter serum of above-mentioned preparation), incubated at room 1h, TBST buffer solution 5 times; Add two anti-(goat anti-rabbit igg/HRP), room temperature reaction 1h, TBST buffer solution 6 times; ECL chemoluminescence colour developing post-exposure is in X-ray film.
(6) according to Western blot result, positive reaction linear epitope 16 peptide is determined.
The qualification of PPR virus N protein epitope peptide minimum motif
(1) according to the sequence of positive reaction linear epitope 16 peptide determined above, (normal chain 5 ＇ end adds 5 ＇-gatcc to the positive minus strand fragment of series 8 peptide coding DNA of overlapped 7 amino-acid residues of each epitope peptide full length sequence of design covering, and 3 ＇ ends add taag-3 ＇; Minus strand 5 ＇ end adds 5 ＇-tcgactta, and 3 ＇ ends add g-3 ＇), send DNA synthesis outside.
(2) adopt and recombinate serial 8 peptides with method amalgamation and expression identical above, and pass through Western blot screening positive reaction 8 peptide sequence, thus determine the minimum motif of each Linear B Cell Epitopes.
The qualification of N38 and N48 epitope peptide minimum motif
(1) respectively design and synthesis is held from N38 (and N48) epitope peptide N and C end reduces the positive minus strand fragment of small peptide (9-16 peptide) coding DNA of 1 amino-acid residue one by one (normal chain 5 ＇ end adds 5 ＇-gatcc, and 3 ＇ hold and add taag-3 ＇; Minus strand 5 ＇ end adds 5 ＇-tcgactta, and 3 ＇ ends add g-3 ＇), send DNA synthesis outside.
(2) adopt and to recombinate serial 9-16 peptide with previous methods amalgamation and expression, and screen positive reaction peptide sequence by Western blot, thus determine the minimum motif of Linear B Cell Epitopes N38 and N48.
The qualification of N64 epitope peptide minimum motif
(1) respectively the design and synthesis unique positive 8 peptide (last 8 peptide aa:518-525) N from series 8 peptide of N64 epitope peptide holds and substitutes 1-8 the positive minus strand fragment of amino acid whose 8 peptide coding DNA with glycine successively (normal chain 5 ＇ end adds 5 ＇-gatcc, and 3 ＇ ends add taag-3 ＇; Minus strand 5 ＇ end adds 5 ＇-tcgactta, and 3 ＇ ends add g-3 ＇), send DNA synthesis outside.
(2) adopt and recombinate serial 8 peptides with method amalgamation and expression identical above, and pass through Western blot screening positive reaction 8 peptide sequence, thus determine the minimum motif of Linear B Cell Epitopes N64.
II, result and discussion
1, PPR virus N protein linear epitope scanning mapping
Through the pXXGST-N checked order and digestion verification is correct
xplastid transformation E.coli BL21 (DE3, plysS) competent cell, each picking 3 clone, carries out thermal induction expression; SDS-PAGE screens the bacterial strain of high expression level.Result shows, and all positive colonies all can induce the recombinant protein band (Fig. 1) of 22kD.
Utilize the anti-PPRV N protein rabbit polyvalent antibody of preparation as primary antibodie, Western blot screens positive reaction and merges 16 peptides.From restructuring 16 peptide of 64 amalgamation and expressions, screen 19 positive reactions and merge 16 peptides (N8, N12, N15, N16, N19, N38, N48, N52, N53, N55-N64) (Fig. 1).Wherein, 7 strong positive reaction 16 peptides (N12, N52, N53, N56, N57, N58 and N64) are comprised.From the distribution of 19 positive reaction 16 peptides on N protein matter, their major parts are positioned at the two ends of PPRV N protein matter, wherein 6 positive reaction 16 peptides are positioned at the N end regions (1-312aa) of N protein matter, and 13 positive reaction 16 peptides are positioned at the C section region (377-525aa) of N protein matter.
2, positive 16 peptide minimum motif qualifications
Through the pXXGST-N checked order and digestion verification is correct
xplastid transformation E.coli BL21 (DE3, plysS) competent cell, each picking 3 clone, carries out thermal induction expression; SDS-PAGE screening obtains the recombinant bacterial strain of restructuring 8 peptide high expression level.Result shows, and all positive colonies all can induce the recombinant protein band of 21kD, and (Fig. 2 a).
The anti-PPRV N protein rabbit polyvalent antibody of preparation is utilized to verify that 8 peptides are merged in positive reaction as primary antibodie Western blot, according to aminoacid sequence common between each marking reacting positive 8 peptide, determine the minimum motif (e.g., the minimum motif qualification of Fig. 2 b, 2c N12 epitope peptide) of each positive reaction 16 peptide.The minimum motif of each epitope peptide is as table 1.
Table 1
Analysis shows, positive reaction 16 peptide N19, N38, N48 respectively comprise two epitope motifs; N52 comprises an epitope motifs and an epitope motifs shared with N53; N56 and N57 shares an epitope motifs; N57 also comprises an epitope motifs shared with N58; N59 and N60 shares an epitope motifs; N61 comprises an epitope motifs and an epitope motifs shared with N62.All the other each positive reaction 16 peptides all identify an epitope motifs.The size of each epitope motifs is 3-9 amino-acid residue.According to 20 the minimum epitope motifs obtained, delineate the Linear B Cell Epitopes collection of illustrative plates that PPRV N protein is complete and meticulous.
Utilize MEGA4.0 to known at present 45 strain PPRV N protein matter (GenBank sequence respectively: 2113440A, ABX75299.1, ABX75307.1, ABY61984.1, ABZ81035.1, ACN62115.1, ACN62116.1, ACQ44667.1, ADJ05523.1, ADM32485.1, ADN03211.1, ADN03214.1, CAD91555.1, ADX95989.1, AEH25639.1, AEX61010.1, AFC87739.1, AFC87740.1, AFC87741.1, AFC87747.1, AFC87748.1, AFC87749.1, AFC87750.1, AFC87751.1, AFC87752.1, AFC87753.1, AFC87754.1, AFC87755.1, AFC87756.1, AFC87757.1, AFC87758.1, AFC87759.1, AFC87760.1, AFC87761.1, AFC87762.1, AFC87763.1, AFC87764.1, CAA52454.1, AGG09141.1, AAS68026.1, AGJ84027.1, CAH61252.1, YP_133821.1, Q08823.1, AAA62258.1) sequence compares, and shows in 20 epitope motifs identified, have 11 epitope motifs be complete conserved epitope motif in all PPRV N protein matter (
62pDINGSK
68,
96dVSIR
100,
119rGADLDNE
126,
128dMYFST
133,
147fENREII
153,
152iIDIEVQ
158,
304qQLGEVAPY
312,
377sSVIAAELG
385,
384lGITAEEAK
392,
413rQAQVSFL
420,
492aEALF
496), there is the amino-acid residue of the variation of or more in the N protein matter gene comparision of remaining 9 epitope motifs and other strain PPRV.The size of the epi-position identified is all at 3-9 amino-acid residue; The N protein matter sequence of same rinderpest virus (Rinderpest virus, RPV) is compared, and 22 the minimum epitope motifs screened can not find on all four motif in rinderpest virus (Rinderpest virus, RPV) N protein matter; There is the difference of more than one amino-acid residue in the amino acid motif in the corresponding site of RPV N protein matter.
3, the minimum motif of above-mentioned acquisition is utilized to prepare PPR virus detectable antigens peptide
Select 11 conservative property positive epitope (
62pDINGSK
68,
96dVSIR
100,
119rGADLDNE
126,
128dMYFST
133,
147fENREII
153,
152iIDIEVQ
158,
304qQLGEVAPY
312,
377sSVIAAELG
385,
384lGITAEEAK
392,
413rQAQVSFL
420,
492aEALF
496), in order to reduce influencing each other between epitope, between adjacent antibody epitope, inserting 2 glycine build PPR virus detection multi-epitope antigen peptide P1 (PDINGSKGGDVSIRGGRGADLDNEGGDMYFSTGGFENREIIGGIIDIEVQGGQQLG EVAPYGGSSVIAAELGGGLGITAEEAKGGRQAQVSFLGGAEALF (SEQ ID NO:21)); The optimization of intestinal bacteria preference codon is carried out to it, optimizes postorder and be classified as:
CCGGATATTAACGGCAGCAAAGGGGGCGACGTGAGCATTCGTGGAGGCCGTGGCGCGGATCTGGATAATGAAGGCGGCGATATGTATTTTAGCACCGGCGGTTTTGAAAATCGTGAAATTATCGGCGGCATTATTGATATTGAAGTGCAGGGCGGTCAGCAGCTGGGCGAAGTGGCGCCGTATGGCGGAAGCAGCGTGATTGCGGCGGAACTGGGTGGTGGCCTGGGCATTACCGCGGAAGAGGCGAAAGGCGGACGTCAGGCGCAGGTGAGCTTTTTGGGTGGCGCTGAAGCGCTGTTT(SEQ ID NO:22)
P1 albumen coded sequence gene after codon optimized by the full chemosynthesis of PCR, be inserted in the BamH I/Sal I site of prokaryotic expression carrier pET32-a after sequence verification is correct, obtain recombinant expression vector pET32-a-P1, its transformation of E. coli BL21 (DE3) is obtained PPRV and detect with multi-epitope peptide P1 recombinant strains.By recombinant expressed PPRV detection multi-epitope peptide P1 after Ni column purification and SDS-PAGE tap rubber purifying, obtain PPRV detection multi-epitope peptide P1, be coated in elisa plate, carry out the detection of PPRV virus serology.When there is anti-RPV N protein matter antibody in serum sample, there is specific binding with it in this multi-epitope peptide, sensitivity and accuracy good.
To sum up, the present inventor has been separated the minimum epitope motifs of 20 B cell linear epitope peptides of PPRV Tibet strain N protein matter first time, and they can chemosynthesis or GST carrier amalgamation and expression containing each epitope motifs 8 peptide or be longer than 8 peptide antigens.The former can be used as significant antigen peptide in the detection kit of development and design PPRV multi-epitope peptide vaccine or PPRV (or chemosynthesis or amalgamation and expression) alone or in combination.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the linear epitope collection of illustrative plates of PPR virus N protein, is characterized in that, this epitope collection of illustrative plates comprises:
PPR virus N protein 62-68 position and the linear epitope peptide shown in SEQ ID NO:1;
PPR virus N protein 96-100 position and the linear epitope peptide shown in SEQ ID NO:2;
PPR virus N protein 119-126 position and the linear epitope peptide shown in SEQ ID NO:3;
PPR virus N protein 128-133 position and the linear epitope peptide shown in SEQ ID NO:4;
PPR virus N protein 147-153 position and the linear epitope peptide shown in SEQ ID NO:5;
PPR virus N protein 152-158 position and the linear epitope peptide shown in SEQ ID NO:6;
PPR virus N protein 297-305 position and the linear epitope peptide shown in SEQ ID NO:7;
PPR virus N protein 304-312 position and the linear epitope peptide shown in SEQ ID NO:8;
PPR virus N protein 377-385 position and the linear epitope peptide shown in SEQ ID NO:9;
PPR virus N protein 384-392 position and the linear epitope peptide shown in SEQ ID NO:10;
PPR virus N protein 413-420 position and the linear epitope peptide shown in SEQ ID NO:11;
PPR virus N protein 417-424 position and the linear epitope peptide shown in SEQ ID NO:12;
PPR virus N protein 438-442 position and the linear epitope peptide shown in SEQ ID NO:13;
PPR virus N protein 452-456 position and the linear epitope peptide shown in SEQ ID NO:14;
PPR virus N protein 462-464 position and the linear epitope peptide shown in SEQ ID NO:15;
PPR virus N protein 474-479 position and the linear epitope peptide shown in SEQ ID NO:16;
PPR virus N protein 486-493 position and the linear epitope peptide shown in SEQ ID NO:17;
PPR virus N protein 492-496 position and the linear epitope peptide shown in SEQ ID NO:18;
PPR virus N protein 504-509 position and the linear epitope peptide shown in SEQ ID NO:19;
PPR virus N protein 523-525 position and the linear epitope peptide shown in SEQ ID NO:20.
2. the conservative property linear epitope collection of illustrative plates of PPR virus N protein, is characterized in that, this epitope collection of illustrative plates comprises:
PPR virus N protein 62-68 position and the linear epitope peptide shown in SEQ ID NO:1;
PPR virus N protein 96-100 position and the linear epitope peptide shown in SEQ ID NO:2;
PPR virus N protein 119-126 position and the linear epitope peptide shown in SEQ ID NO:3;
PPR virus N protein 128-133 position and the linear epitope peptide shown in SEQ ID NO:4;
PPR virus N protein 147-153 position and the linear epitope peptide shown in SEQ ID NO:5;
PPR virus N protein 152-158 position and the linear epitope peptide shown in SEQ ID NO:6;
PPR virus N protein 304-312 position and the linear epitope peptide shown in SEQ ID NO:8;
PPR virus N protein 377-385 position and the linear epitope peptide shown in SEQ ID NO:9;
PPR virus N protein 384-392 position and the linear epitope peptide shown in SEQ ID NO:10;
PPR virus N protein 413-420 position and the linear epitope peptide shown in SEQ ID NO:11;
PPR virus N protein 492-496 position and the linear epitope peptide shown in SEQ ID NO:18.
3. one kind for detecting the test kit of the linear epitope of PPR virus N protein, it is characterized in that, described test kit comprises: SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ IDNO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence; Or
The encoding gene of SEQ ID NO:1 ~ SEQ ID NO:6, SEQ ID NO:8 ~ SEQ ID NO:11, the linear epitope peptide shown in SEQ ID NO:18 sequence.
4. test kit as claimed in claim 3, is characterized in that, also comprise in described test kit: SEQ IDNO:7, SEQ ID NO:12 ~ SEQ ID NO:17, the linear epitope peptide shown in SEQ ID NO:19 ~ SEQ ID NO:20 sequence; Or
The encoding gene of SEQ ID NO:7, SEQ ID NO:12 ~ SEQ ID NO:17, the linear epitope peptide shown in SEQ ID NO:19 ~ SEQ ID NO:20 sequence.
5. a fusion rotein, is characterized in that, described fusion rotein comprises SEQ ID NO:1 ~ SEQ IDNO:6, SEQ ID NO:8 ~ SEQ ID NO:11 and the aminoacid sequence described in SEQ ID NO:18; Above-mentioned sequence is connected mutually.
6. fusion rotein as claimed in claim 5, it is characterized in that, the aminoacid sequence of described fusion rotein is as shown in SEQ ID NO:21.
7. a fusion gene, is characterized in that, this fusion gene is encoded fusion rotein according to claim 5.
8. fusion gene as claimed in claim 7, it is characterized in that, the nucleotide sequence of this fusion gene is as shown in SEQ ID NO:21.
9. the purposes of the linear epitope collection of illustrative plates of the PPR virus N protein described in claim 1 or 2 or the test kit described in claim 3 or 4, is characterized in that, ruminates the multi-epitope chimeric peptide vaccine of epizootic disease virus for the preparation of control; Or
For the preparation of detecting the virus detection reagent ruminating epizootic disease virus; Or
For the preparation of the specific antibody of specific linear epitope in anti-PPR virus N protein.
10. the purposes of the fusion rotein described in claim 5 or 6, is characterized in that, for the preparation of detecting the virus detection reagent ruminating epizootic disease virus.
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| CN112213493A (en) * | 2019-07-11 | 2021-01-12 | 中国兽医药品监察所 | Peste des petits ruminants detection kit capable of distinguishing vaccine immunity from natural infection |
| CN112213493B (en) * | 2019-07-11 | 2023-07-07 | 中国兽医药品监察所 | Peste des petits ruminants detection kit capable of being used for distinguishing vaccine immunity from natural infection |
| CN112255401A (en) * | 2020-10-21 | 2021-01-22 | 中国农业科学院兰州兽医研究所 | Peste des petits ruminants virus antibody chemiluminescence detection method based on H protein epitope synthetic peptide and application |
| CN115926002A (en) * | 2022-12-29 | 2023-04-07 | 北京亿森宝生物科技有限公司 | Kit for detecting peste des petits ruminants virus and application thereof |
| CN115926002B (en) * | 2022-12-29 | 2023-12-12 | 北京亿森宝生物科技有限公司 | Kit for detecting peste des petits ruminants virus and application thereof |
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