CN102276725B - Cryptosporidium CTL and Th mixed multi-epitope gene and fusion protein and application - Google Patents

Abstract

The invention discloses a cryptosporidium CTL and Th mixed multi-epitope fusion protein, which comprises any one or two amino acid sequences selected from SEQ ID NO. 1 and SEQ ID NO. 3. The invention also discloses a cryptosporidium mixed multi-epitope gene, which comprises a nucleotide sequence for coding the above fusion protein. The cryptosporidium mixed multi-epitope gene and fusion protein provided by the invention can be prepared into a vaccine which has a good immunity and protective effect for the murine cryptosporidium infection, and is applicable to serve as multiepitope vaccine against cryptosporidium.

Description

Cryptosporidium CTL and Th mixing multi-epitope gene and fusion rotein and application

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of Cryptosporidium CTL and Th mixing multi-epitope gene and fusion rotein and application thereof.

Background technology

Cryptosporidiosis (cryptosporidiosis) is pandemic Zoonosis parasitosis in a kind of world wide that is caused by Cryptosporidium (Cryptosporidiumspp.), can infect 170 many animals such as the mammals that comprises the mankind, birds, reptiles, batrachians, fish.Can cause self limiting diarrhoea to the normal host of immunologic function, the host of immune function depression is chronic diarrhoea and threat to life usually, bring serious threat for human health and life security, also cause huge loss to livestock industry production.Cryptosporidium is found in the section of mouse gastric gland mucosal tissue in 1907 by Tyzzer at first.Wherein, cryptosporidium parvum (Cryptosporidium parvum) is topmost Zoonosis disease pathogen.Up to now, the researchist in laboratory screening thousands of kinds of chemicalses and microbiotic, do not find yet desirable anti-Cryptosporidium medicine.Therefore the immunoprophylaxis of cryptosporidiosis and treatment just seem very important, but still lack at present effective cryptosporidiosis vaccine.

In Cryptosporidium vaccine research in the past, people are the Cryptosporidium antigen protein of a total length of coding in recombiant vaccine usually.But; the immune protective effect of the vaccine candidate molecule that this type of is single is unsatisfactory; its reason may be that the Cryptosporidium genome is huge; the life history is complicated; antigen type is various; single antigen can't thoroughly be blocked the life cycle of cause of disease in host, causes the immune protective effect of single antigen, single epi-position not ideal enough.When consider using many antigen vaccines, due to the carrier finite capacity, and high molecular weight protein may cause the animal immune pathologic reaction, so add a plurality of antigens to have significant limitation in single carrier.In recent years, world parasite educational circles begins to attempt utilizing the Epitope prediction instrument to carry out Epitope prediction, build polyepitope vaccines (multi-epitope vaccine), polyepitope vaccines also claims the cocktail type vaccine, is to carry simultaneously a plurality of epi-positions relevant to target antigen and the vaccine of complementary epi-position.Compare with traditional vaccine, polyepitope vaccines has unique advantage, can be by the major histocompatibility complex of multiple genetic background (major histocompatibility complex, MHC) molecule identify, combination, thereby obtain efficient submission, especially has significant advantage aspect cellular immunization, with many unfavorable factors in the reply variation of pathogenic micro-organism and immune response.In anti-malarial polyepitope vaccines development field, designed and synthesized a plurality of recombinant vaccines and synthetic peptide vaccine, obtained immune protective effect preferably, wherein much entered II phase or III clinical trial phase.There is no at present the report of this respect in the Cryptosporidium field.

For making body obtain best immunoprotection, need consider the immunne response feature of body and the singularity of disease when building polyepitope vaccines, select pointedly the purpose epitope.Humoral immunization mainly produces antibody by the B cell and plays a role, therefore can select the specific b cells epi-position to come directional induction host's humoral immunoresponse(HI).Cellullar immunologic response comprises CD4 ⁺Helper T cell (helper T cell, Th cell are divided into two subgroups of Th1 and Th2) is replied and CD8 ⁺Cytotoxic T cell (cytotoxicT lymphocyte, CTL) is replied.Therefore, can according to the immunne response characteristics of body to pathogenic agent, select specific Th1, Th2 or CTL epi-position to induce immune response to Th1, Th2 or the directed development of CTL acknowledgement type.

Cellular immunization plays vital effect in the anti-Cryptosporidium spp process of host.Studies show that the first and reinfect of cryptosporidium parvum all causes strong cell immune response, wherein CD4 ⁺Helper T cell participates in infection control, CD8 ⁺Cytotoxic T cell has the parasitic effect of very strong removing.

Summary of the invention

The present invention will solve the technical problem that lacks effective Cryptosporidium polyepitope vaccines, a kind of Cryptosporidium CTL and Th mixing multi-epitope gene and fusion rotein are provided, and this mixing multi-epitope gene and fusion rotein can be used for preparing the polyepitope vaccines of anti-cryptosporidiosis.

In addition, also need to provide a kind of Cryptosporidium CTL and Th mixing multi-epitope gene and the fusion rotein application in the vaccine of preparation prevention or treatment cryptosporidiosis.

In order to solve the problems of the technologies described above, the present invention is achieved through the following technical solutions:

In one aspect of the invention, provide a kind of Cryptosporidium to mix Multi-Epitope Fusion Protein, comprised: any one or two aminoacid sequences in SEQ ID NO.1, SEQ ID NO.3.

Described SEQ ID NO.1 and SEQ ID NO.3 are rich in CTL and Th epi-position, contain 4 CTL epi-positions and 2 Th epi-positions in SEQ ID NO.1, contain 3 CTL epi-positions and 1 Th epi-position in SEQ ID NO.3.

Preferably, described Cryptosporidium mixes Multi-Epitope Fusion Protein and also comprises aminoacid sequence shown in SEQ ID NO.2, contains 4 Th epi-positions in this sequence.Increase SEQ ID NO.2 sequence and can suitably improve the immune peptide molecular weight, so that immune mouse produces higher serum antibody titer, has better immunogenicity.

Preferably, be provided with the joint sequence that flexible amino acid forms between described SEQ ID NO.1, SEQ ID NO.2, three sections sequences of SEQ ID NO.3, the aminoacid sequence that this joint sequence can make each section be rich in CTL and Th epi-position is spatially independent mutually, prevent that each epitope polypeptide molecule from hindering the combination of itself and MHC (major histocompatibility complex has the antigen presentation effect) molecule because space structure changes; Joint sequence can suitably improve the immune peptide molecular weight simultaneously, to strengthen the immunogenicity of Multi-Epitope Fusion Protein.

Preferred, described fusion rotein has the aminoacid sequence shown in SEQ ID NO.7.

In another aspect of this invention, provide a kind of Cryptosporidium to mix multi-epitope gene, it comprises the nucleotide sequence of the above-mentioned fusion rotein of encoding.

Preferably, described Cryptosporidium mixes the nucleotide sequence that multi-epitope gene has aminoacid sequence shown in coding SEQ ID NO.7.Preferred, described Cryptosporidium mixes multi-epitope gene and has the nucleotide sequence shown in SEQ ID NO.8.

In another aspect of this invention, also provide a kind of recombinant vectors that above-mentioned Cryptosporidium mixes multi-epitope gene that comprises.

Described recombinant vectors comprises recombinant cloning vector or recombinant expression vector, and recombinant expression vector comprises recombinant prokaryotic expression vector, recombinant eukaryon expression vector.

In another aspect of this invention, also provide a kind of vaccine, comprised above-mentioned Cryptosporidium and mix multi-epitope gene and expression vector.

In another aspect of this invention, also provide a kind of Cryptosporidium to mix the application of Multi-Epitope Fusion Protein in the vaccine of preparation prevention or treatment cryptosporidiosis.

In the present invention, with fusion rotein and Fu Shi not exclusively and the abundant mixing such as Freund's complete adjuvant, Montanide ISA 206 prepare subunit vaccine.

In another aspect of this invention, also provide a kind of Cryptosporidium to mix the application of multi-epitope gene in the vaccine of preparation prevention or treatment cryptosporidiosis.

In the present invention, Cryptosporidium is mixed multi-epitope gene be cloned into carrier for expression of eukaryon such as pVAX1, pcDNA3.1 etc., or jointly insert cytokine gene, be built into nucleic acid vaccine.

Cryptosporidium CTL of the present invention and Th mixing multi-epitope gene; be cloned into the nucleic acid vaccine that forms after carrier for expression of eukaryon; show through animal immune protection test result; compare with control group; nucleic acid vaccine group of the present invention is discharged egg capsule quantity and is obviously reduced; beginning to discharge the egg capsule time delays, and egg capsule is discharged the time length and obviously shortened, and pointing out this nucleic acid vaccine that Cryptosporidium muris musculus cdna type is infected has good immune protective effect.

Description of drawings

The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.

Fig. 1 is the pcr amplification product electrophoresis result figure of the embodiment of the present invention 2 Cryptosporidium P1, P2 and P3 gene fragment;

Fig. 2 is the pcr amplification product electrophoresis result figure that the embodiment of the present invention 2 Cryptosporidiums mix multi-epitope fusion gene P1-2 and CpT;

Fig. 3 is the embodiment of the present invention 2 recombinant plasmid pMD-CpT double digestion product electrophoresis result figure;

Fig. 4 is that the double digestion of the embodiment of the present invention 3 recombined pronucleus expression plasmid pGEX-CpT is identified electrophorogram;

Fig. 5 is the SDS-PAGE electrophorogram of the embodiment of the present invention 3 expression of recombinant proteins products;

Fig. 6 is the SDS-PAGE electrophoresis result figures of the embodiment of the present invention 3 expression of recombinant proteins products after purified;

Fig. 7 is that the embodiment of the present invention 4 Cryptosporidiums mix multi-epitope gene pcr amplification product electrophoresis evaluation figure;

Fig. 8 is that the double digestion of the embodiment of the present invention 4 recombinant cloning vector pMD18T-CpT (e) is identified electrophoresis result figure;

Fig. 9 is the double digestion evaluation figure of the embodiment of the present invention 4 eukaryotic expression recombinant plasmid pVAX-1-CpT;

Figure 10 be the embodiment of the present invention 5 eukaryotic expression recombinant plasmid pVAX-1-CpT as nucleic acid vaccine immunity after Cryptosporidium muris egg capsule discharge curve figure.

Embodiment

In the following example, the experimental technique of unreceipted actual conditions, condition routinely usually is as " molecular cloning experiment guide " (J. Pehanorm Brooker, D.W. the Russell is outstanding, Huang Peitang, Wang Jiaxi, Zhu Houchu, Deng translating. the 3rd edition, Beijing: Science Press, 2002) described in method carry out.

The present invention utilizes bioinformatics technique, and the epi-position that cryptosporidium parvum vaccine candidate antigen and host's mhc class i molecule H2-Kd, H2-Ld and H2-Dd and MHC class Ⅱmolecule H2-Ad, H2-Ed are combined is predicted respectively, filters out desirable epitope; Choose the different antigen fragments of a plurality of CTL of being rich in and Th epi-position, structure comprises one or more antigen fragments and is rich in CTL and the multivalence multi-epitope gene of Th epi-position, links with flexible amino acid between each antigen fragment.This multi-epitope gene is cloned into carries out prokaryotic expression in prokaryotic expression carrier; This multi-epitope gene is cloned into carrier for expression of eukaryon, builds nucleic acid vaccine, observe the immune protective effect that it infects Cryptosporidium muris.Animal immune protection test result shows; compare with control group, the nucleic acid vaccine group is discharged egg capsule quantity and is obviously reduced, and begins to discharge the egg capsule time to delay; the egg capsule discharge time length obviously shortens, and pointing out this nucleic acid vaccine that Cryptosporidium muris musculus cdna type is infected has good immune protective effect.

Prediction and the screening of embodiment 1 Cryptosporidium T cell antigen epitope

Download amino acid (the Amino Acid of cryptosporidium parvum antigens c P15 (accession number L34568), CP15/60 (accession number L08612) from GenBank, AA) and gene order, obtain Cryptosporidium musculus cdna type (Cryptosporidiummousegenotype) P23 antigen amino acid and gene order from the laboratory.Use SYFPEITHI (http://www.syfpeithi.de/home.htm), ProPred-I (http://www.imtech.res.in/raghava/propred1/) and 3 Antigen Epitope Prediction servers of NetMHC 3.0 (http://www.cbs.dtu.dk/services/NetMHC/), respectively above-mentioned Cryptosporidium antigen is predicted with the restricted CTL epitope of H2-d (comprising H2-Kd, H2-Ld and H2-Dd) the type MHC class Ⅰmolecule combination of 9 amino-acid residues in mouse source.Concrete grammar is: when prediction H2-Kd, H2-Ld restricted epitope, the antigen aminoacid sequence is inputted respectively server, preserve SYFPEITH I prediction score value greater than 20 9 peptides; Preserve simultaneously front 15 that ProPred-I returns results; Seek tumor-necrosis factor glycoproteins both.According to document [Panagiotopoulos C, Qin H, Tan R, et al.Identification of a β-cell-specific HLA class I restricted epitope in type 1 diabetes[J] .Diabetes, 2003,52 (11): 2647-2651] method of report is calculated both contained MHC class Ⅰmolecules in conjunction with the score of 9 peptides and sequence, chooses score obviously greater than the sequence of other peptide sections.Predict the outcome in conjunction with NetMHC 3.0, carry out analysis-by-synthesis.During prediction H2-Dd restricted epitope, preserve front 15 that ProPred-I returns results, predict the outcome in conjunction with NetMHC 3.0 and carry out analysis-by-synthesis.

use RANKPEP (http://bio.dfci.harvard.edu/RANKPEP/), MHCPred (http://www.jenner.ac.uk/MHCPred), MHC2Pred (http://www.imtech.res.in/raghava/mhc2pred/) and MHC BindingPrediction (http://epitope.liai.org:8080/tools/matrix/iedb_input? matrixClass=I, II) 3 Cryptosporidium antigens of 4 Antigen Epitope Prediction server prediction are with the Th epi-position of mouse source H2-d (comprising H2-Ad and H2-Ed) type MHC class Ⅱmolecule combination.Concrete grammar is, respectively with the above server of FASTA form input, the epi-position data of returning are deposited the document to word with the aminoacid sequence of Cryptosporidium antigen.If length amino acid sequence during greater than 1000 AA residues, is preserved high front 20 epi-positions of score, otherwise is got front 15 epi-positions.Preserve shown in red the returning results of RANKPEP.Find out 4 server prediction as a result repetition rate get its union greater than 60% fragment (namely have more than 5 and repeat amino-acid residue), sequence is respectively extended 1-6 sequence to two ends as candidate's epi-position of mouse source H2-d type molecule combination.

Result: through prediction, find that cryptosporidium parvum CP15 antigen 5-59AA section (SEQ ID NO.1), the 1-113AA section (SEQ ID NO.2) of Cryptosporidium musculus cdna type P23 antigen, the 11-95AA section (SEQ ID NO.3) of cryptosporidium parvum CP15/60 antigen contain respectively 4,0,3 CTL epi-positions, comprise 2 of H2-Kd type epi-positions, 3, H2-Ld type, 2, H2-Dd type; Contain respectively 2,4,1 Th epi-positions.Compare with other sections, the contained t cell epitope of above-mentioned section is more, CTL and Th epi-position rich region, therefore select these 3 sections sequence construct CTL and Th mixing multi-epitope gene, and with nucleotide sequence SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, called after P1, P2 and the P3 successively of the 11-95AA section coding of the 1-113AA section of CP15 antigen 5-59AA section, P23 antigen, CP15/60 antigen.

Amplification and the evaluation of embodiment 2 Cryptosporidium CTL and Th mixing multi-epitope gene

(1) primer is synthetic

According to Cryptosporidium antigens c P15, the P23 of screening and the nucleotide sequence of CP15/60 epi-position rich region coding, according to gene splicing by overlap extension (gene splicing by overlap extension, gene SOEing) round pcr method requirement, design 3 pairs of primers, respectively amplification gene fragment P1, P2 and P3.Primer sequence sees Table 1, holds at 5 ' of P1 upstream primer (P1F) to add suitable protectiveness base, restriction enzyme EcoR I sequence; Add the DNA sequence dna of a complementary fifteen amino acid peptide linker at 5 ' end of the downstream primer (P1R) of the upstream primer (P2F) of P2 and P1; Add the DNA sequence dna of flexible amino acid joint GS at 5 ' end of the downstream primer (P2R) of P2 and P3 upstream primer (P3F); 5 ' end at P3 downstream primer (P3R) adds restriction enzyme Sal I sequence, terminator codon TAA and protectiveness base.It is synthetic that primer is transferred to the English Weihe River, Shanghai Jie Ji Bioisystech Co., Ltd.

Table 1PCR primer sequence

In table 1, the boldface type that adds of primer sequence is restriction enzyme site or Linker sequence.

(2) amplification of goal gene be connected

The Cryptosporidium recombinant plasmid pMD-18T-CP15 that preserves take the laboratory, the DH5 α transformed bacteria of pMD-18T-P23, pMD-18T-CP15/60 be as template, with the primer of table 1 by PCR increase respectively purpose fragment P1, P2, P3.Result as shown in Figure 1, amplified fragments size with expect that size conforms to.In Fig. 1, M:100bp DNA molecular amount standard; The 1:P1 amplified production; The 2:P2 amplified production; The 3:P3 amplified production.The demonstration of sequencing result, the P1 size is 225bp, and the P2 size is 393bp, and the P3 size is 276bp, and sequence variations does not occur.

The PCR product reclaims test kit with glue and reclaims, and connects P1, P2 fragment, called after P1-2 by gene SOEing round pcr.Reclaim test kit with glue and reclaim P1-2, again connect P1-2 and P3, called after CpT by gene SOEing round pcr.Electrophoresis result shows, the size of P1-2 and CpT conform to expection (seeing Fig. 2).In Fig. 2, M:100bp DNA molecular amount standard; The 1:P1-2 amplified production; The 2:CpT amplified production.

Reclaim test kit with glue and reclaim CpT, adopt the TA cloning process that goal gene is connected with the pMD18-T carrier, be converted into bacillus coli DH 5 alpha, the picking positive colony is cut with sequencing with PCR, enzyme and is verified.Through EcoR I and Sal I enzyme cut identify correct (seeing Fig. 3) after, carry out sequencing, result shows that its size is 837bp (SEQ ID NO.8), wherein 16-828 position Nucleotide is coding region sequence, in this coding region sequence, 16-18 position Nucleotide ATG is initiator codon, and 826-828 position Nucleotide TAA is terminator codon, and sequence variations does not occur whole sequence.Identify correct recombinant plasmid called after pMD-CpT.In Fig. 3, M1:100bp DNA molecular amount standard; M2:Marker IV DNA molecular amount standard; 1: the EcoR I of recombinant plasmid pMD-CpT and Sal I double digestion product.

The concrete sequence of CpT is:

CCGGAATTCCCGACCATGAAATTGGATGAGGTTGTTGAGCTTTTACCAGCACGTAAAAGACGTAAGATAGCCAGAGGTTGTCTTAACAGAAGAACTGCAGCTTTTATCGCAAAGCTCCGCAAATCTAAGGCTGAATGTCCAATGGGAGAGAAACCTGTTGCTGTTCGTACCCATTTACGTGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGATGGGTTGTTCATCATCAAAGCCAGAAACTAAAGTTGCTGAAAATAAATCTGCAGCAGATGCTAACAAACAAAGAGAATTAGCTGAAAAGAAGGCTCAATTAGCCAAGGCTGTAAAGAATCCAGCTCCAATCAGCAACCAAGCTCAACAAAAGCCAGAAGAACCAAAGAAGTCCGAGCCTGCTCCAAATAACCCTCCAGCTGCTGATGCGCCAGCAGCCCAAGCTCCTGCTGCCCCTGCCCCTGCTGCCCCTGCTTCACAGGATAAGCCAGCTGAGGCTCCAGCTGCTGAAGCTCCAGCTGCTGAACCTGCTGCTCAACAAGACAAGCCAGCTGATGCCGGATCCATGGGTAACTTGAAATCCTGTTGTTCTTTTGCCGATGAACACTCCCTAACCTCTACTCAACTAGTAGTTGGAAATGGTTCAGGAGCTTCAGAAACTGCTTCCAACCACCCCCAAGAAGAAGTTAATGATATTAATACTTTTAATGTAAAGTTAATAATGCAAGATAGAAGTAAGCTTGACTGTGAGGTAGTATTTGATAGCACAAGTATTTCGCTTTCTGGAGATGGAAAATGCAGAAATATTGCTTTGGATGAATAAGTCGACGGG(SEQ?ID?NO.8)

270 AA that encode altogether, sequence is:

MKLDEVVELLPARKRRKIARGCLNRRTAAFIAKLRKSKAECPMGEKPVAVRTHLRGGGGSGGGGSGGGGSMGCSSSKPETKVAENKSAADANKQRELAEKKAQLAKAVKNPAP?I?SNQAQQKPEEPKKSEPAPNNPPAADAPAAQAPAAPAPAAPASQDKPAEAPAAEAPAAEPAAQQDKPADAGSMGNLKSCCSFADEHSLTSTQLVVGNGSGASETASNHPQEEVNDINTFNVKLIMQDRSKLDCEVVFDSTSISLSGDGKCRNIALDE(SEQ?ID?NO.7)

Embodiment 3 Cryptosporidiums mix the prokaryotic expression of multi-epitope gene

(1) Construction and identification of prokaryotic expression plasmid

Get pGEX-4T-1 plasmid and pMD-CpT plasmid, cut with EcoR I and Sal I enzyme simultaneously respectively.Enzyme is cut and is used T after product reclaims the test kit recovery with glue ₄DNA ligase connects.Connect product and change the bacillus coli DH 5 alpha competent cell over to, screen positive bacterium colony.The alkaline lysis method of extracting recombinant plasmid is identified and checks order and identify positive colony through PCR, EcoR I and Sal I double digestion, will identify correct recombined pronucleus expression plasmid called after pGEX-CpT.Recombined pronucleus expression plasmid pGEX-CpT through EcoR I and Sal I double digestion qualification result as shown in Figure 4, in Fig. 4, the EcoR I of 1:pGEX-CpT and Sal I double digestion product; M:Marker IV DNA molecular quality standard, Fig. 4 shows that recombined pronucleus expression plasmid pGEX-CpT identifies correct through EcoR I and Sal I double digestion.

(2) abduction delivering of prokaryotic expression plasmid

PGEX-CpT is converted into e. coli bl21 (DE3) competent cell, and the positive single bacterium colony of picking is cultured to D _600nmWhen reaching 0.5 left and right, the IPTG take final concentration as 1.0mmol/L induces 7h, during every 1h sampling, carry out concentration and be 12% SDS-PAGE electrophoresis, observe the protein expression situation.Expression amount reaches the highest (seeing Fig. 5) after IPTG induces 5h, and the 57ku that the molecular weight ratio of SDS-PAGE analyzing proteins is estimated is bigger.In Fig. 5,1-3: recombinant plasmid transformed bacterium IPTG induces the 5h product; 4: the recombinant plasmid transformed bacterium is the abduction delivering product not; M: protein standard molecular weight.

Get IPTG and induce the expression bacterium of 5h, through frozen-thawed 3 times and carry out ultrasonic treatment, collect supernatant liquor, precipitation adds the dissolving of 8mol/L urea, centrifugal rear supernatant liquor and the precipitation of collecting respectively analyzed the existence form (seeing Fig. 6) of recombinant protein with SDS-PAGE after the GST column purification.In Fig. 6, M: protein standard molecular weight; The 1:pGEX-CpT transformed bacteria is induced 5h bacterium liquid; The 2:pGEX-CpT transformed bacteria is induced the supernatant after 5h bacterium liquid ultrasonic degradation; The 3:pGEX-CpT transformed bacteria induces the precipitation after 5h bacterium liquid ultrasonic degradation to be dissolved in 8mol/L urea; The 4:pGEX-CpT transformed bacteria induces the precipitation after 5h bacterium liquid ultrasonic degradation to be dissolved in PBS.The demonstration of BandScan software analysis, the recombinant protein of expression accounts for 12.3% of tropina total amount, mainly with the inclusion body formal representation.

Embodiment 4 Cryptosporidiums mix the structure of multi-epitope gene eukaryotic expression recombination plasmid

(1) design of primer and synthetic

For making Cryptosporidium multi-epitope gene CpT correctly be cloned in eukaryon expression plasmid and normal expression, require redesign primer amplification CpT (e) according to restriction enzyme site and codeword triplet on pVAX1 carrier multiple clone site, primer is introduced restriction enzyme site EcoR I and Xba I and Kozak sequence, replenishes base and guarantee that triplet codon does not misplace after EcoR I.The upstream primer CpT F (e) of this primer sequence is: CCGGAATTCCCGACCATGGCTATGAAATTGGATGAGGTTGTTG (SEQ IDNO.15); Downstream primer CpT R (e) is: CCCTCTAGATTATTCATCCAAAGCAATATTTCT (SEQ ID NO.16).

(2) amplification of goal gene, cloning and identification

Take pMD-CpT recombinant plasmid transformed bacterium as template, utilize primer CpT F (e)/CpT R (e), amplification purpose fragment CpT (e).Through the pcr amplification of 30 circulations, obtained CpT (e) gene, size fulfill the expectation (seeing Fig. 7).In Fig. 7, M:100bp DNA molecular quality standard; 1:CpT (e) pcr amplification product.

Reclaim test kit with glue and reclaim CpT (e), adopt the TA cloning process that goal gene is connected with the pMD18-T carrier, be converted into bacillus coli DH 5 alpha, the picking positive colony, verify with PCR, EcoR I/Xba I double digestion and sequencing, identify correct recombinant plasmid called after pMD-CpT (e).Recombinant plasmid pMD-CpT (e) obtains 2 bands after EcoR I/Xba I double digestion, with expectation clip size identical (seeing Fig. 8).In Fig. 8, M1:100bp DNA molecular amount standard; M2:Marker IV DNA molecular amount standard; 1: the EcoR I of recombinant plasmid pMD-CpT (e) and Xba I double digestion product.Identify that through order-checking base mutation does not occur sequence.

(3) structure of eukaryotic expression recombination plasmid

A large amount of recombinant plasmid pMD-CpT (e) and eukaryon expression plasmid pVAX-1 of extracting carry out respectively enzyme with EcoR I and Xba I and cut, and enzyme is cut product and use T after glue is reclaimed the test kit recovery ₄DNA ligase connects, connect product and change the bacillus coli DH 5 alpha competent cell over to, screen positive bacterium colony, the alkaline lysis method of extracting recombinant plasmid, through PCR, the enzyme evaluation positive colony of cutting and check order, will identify correct eukaryotic expression recombination plasmid called after pVAX-1-CpT.PVAX-1-CpT identifies through EcoR I and Xba I double digestion, obtains and estimate the fragment that clip size conforms to (seeing Fig. 9).In Fig. 9, M1:Marker IV DNA molecular amount standard; M2:100bp DNA molecular amount standard; 1: the EcoR I of eukaryotic expression recombination plasmid pVAX-1-CpT and Xba I double digestion product.Base mutation does not occur through the order-checking evaluation in pVAX-1-CpT, and the sequence of insertion meets the triplet codon order of open reading frame.

Extract in a large number the pVAX-1-CpT plasmid with alkaline lysis, polyoxyethylene glycol (PEG8000) precipitator method plasmid DNA purification, GEHeal thcare NanoVue ultra-violet and visible spectrophotometer is measured plasmid concentration.

Embodiment 5 animal immune protection tests

With 30 4 the week age BALB/c mouse be divided into 3 groups, 10 every group, comprise pVAX-1-CpT test group and pVAX-1 empty carrier, physiological saline control group.Test group is carried out the subcutaneous multiple spot immunity in back, every injected in mice plasmid 0.05mg after normal saline dilution with eukaryotic expression recombinant plasmid pVAX-1-CpT; 2 control groups are injected respectively pVAX-1 empty carrier and physiological saline with identical method, and the pVAX-1 immunizing dose is also 0.05mg/ mouse, and physiological saline control group dosage of inoculation is 0.05mL/ mouse.Every 2 week immunity 1 time, immunity is 3 times altogether.After the 3rd 2 weeks of immunity, every its mouse oral inoculation 1 * 10 ⁶Individual Cryptosporidium musculus cdna type egg capsule.Take ight soil 10g every 2d after infecting, add the 40mL water dissolution, the copper gauze filters, the centrifugal 10min of filtrate 3000r/min, collecting precipitation.Add the saturated sucrose mixing of 10mL, the centrifugal 10min of 1500r/min.Dip the liquid level top layer to slide glass with iron wire loop, covered with 10 * 40 power microscope microscopies, is counted the Cryptosporidium parvum oocysts suspended in 50 visuals field.

Result:

Compare with control group, test group begins to discharge the egg capsule time and delays.To infecting rear 31d, nucleic acid vaccine pVAX-1-CpT only detects egg capsule in infecting rear 10d; PVAX-1 empty carrier group 10d after infecting detects egg capsule, and 16d peaks, and 31d after namely inoculating to off-test still has egg capsule to discharge; And physiological saline control group 7d after infecting begins to detect egg capsule, and 13-19d peaks, and still has egg capsule to discharge (Fig. 7) to infecting rear 31d.

Compare with control group, test group is discharged egg capsule quantity and is obviously reduced.Each is organized in stool in mice Cryptosporidium parvum oocysts suspended and discharges situation as shown in figure 10, and the relative egg sac number that 31 days each groups detect altogether is followed successively by: 2 of pVAX-1-CpT immune group; PVAX-1 empty carrier contrast: 16; The physiological saline blank: 20, the difference highly significant.

Compare with control group, the test group egg capsule discharge time length obviously reduces.The pVAX-1-CpT test group only detects egg capsule in infecting rear 10d; The pVAX-1 empty carrier group ovulation capsule time length surpasses 21d; The physiological saline control group ovulation capsule time surpasses 24d (seeing Figure 10).

Figure 10 result shows that the nucleic acid vaccine that eukaryotic expression recombinant plasmid pVAX-1-CpT of the present invention makes has good immune protective effect.

The above embodiment has only expressed embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.Should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Sequence table

＜110〉China Agriculture Academe Shanghai Veterinary Institute

＜120〉Cryptosporidium CTL and Th mixing multi-epitope gene and fusion rotein and application thereof

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Met?Lys?Leu?Asp?Glu?Val?Val?Glu?Leu?Leu?Pro?Ala?Arg?Lys?Arg?Arg

1 5 10 15

Lys?Ile?Ala?Arg?Gly?Cys?Leu?Asn?Arg?Arg?Thr?Ala?Ala?Phe?Ile?Ala

20 25 30

Lys?Leu?Arg?Lys?Ser?Lys?Ala?Glu?Cys?Pro?Met?Gly?Glu?Lys?Pro?Val

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Ala?Val?Arg?Thr?His?Leu?Arg

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20 25 30

Gln?Leu?Ala?Lys?Ala?Val?Lys?Asn?Pro?Ala?Pro?Ile?Ser?Asn?Gln?Ala

35 40 45

Gln?Gln?Lys?Pro?Glu?Glu?Pro?Lys?Lys?Ser?Glu?Pro?Ala?Pro?Asn?Asn

50 55 60

Pro?Pro?Ala?Ala?Asp?Ala?Pro?Ala?Ala?Gln?Ala?Pro?Ala? Ala?Pro?Ala

65 70 75 80

Pro?Ala?Ala?Pro?Ala?Ser?Gln?Asp?Lys?Pro?Ala?Glu?Ala?Pro?Ala?Ala

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Glu?Ala?Pro?Ala?Ala?Glu?Pro?Ala?Ala?Gln?Gln?Asp?Lys?Pro?Ala?Asp

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<211>85

<212>PRT

<213>Cryptosporidium?parvum

<220>

<221>MISC_FEATURE

<222>(1)..(85)

＜223〉CP15/60 antigen fragment

<400>3

Met?Gly?Asn?Leu?Lys?Ser?Cys?Cys?Ser?Phe?Ala?Asp?Glu?His?Ser?Leu

1 5 10 15

Thr?Ser?Thr?Gln?Leu?Val?Val?Gly?Asn?Gly?Ser?Gly?Ala?Ser?Glu?Thr

20 25 30

Ala?Ser?Asn?His?Pro?Gln?Glu?Glu?Val?Asn?Asp?Ile?Asn?Thr?Phe?Asn

35 40 45

Val?Lys?Leu?Ile?Met?Gln?Asp?Arg?Ser?Lys?Leu?Asp?Cys?Glu?Val?Val

50 55 60

Phe?Asp?Ser?Thr?Ser?Ile?Ser?Leu?Ser?Gly?Asp?Gly?Lys?Cys?Arg?Asn

65 70 75 80

Ile?Ala?Leu?Asp?Glu

85

<210>4

<211>165

<212>DNA

<213>Cryptosporidium?parvum

<400>4

atgaaattgg?atgaggttgt?tgagctttta?ccagcacgta?aaagacgtaa?gatagccaga 60

ggttgtctta?acagaagaac?tgcagctttt?atcgcaaagc?tccgcaaatc?taaggctgaa 120

tgtccaatgg?gagagaaacc?tgttgctgtt?cgtacccatt?tacgt 165

<210>5

<211>339

<212>DNA

<213>Cryptosporidium?mouse?genotype

<400>5

atgggttgtt?catcatcaaa?gccagaaact?aaagttgctg?aaaataaatc?tgcagcagat 60

gctaacaaac?aaagagaatt?agctgaaaag?aaggctcaat?tagccaaggc?tgtaaagaat 120

ccagctccaa?tcagcaacca?agctcaacaa?aagccagaag?aaccaaagaa?gtccgagcct 180

gctccaaata?accctccagc?tgctgatgcg?ccagcagccc?aagctcctgc?tgcccctgcc 240

cctgctgccc?ctgcttcaca?ggataagcca?gctgaggctc?cagctgctga?agctccagct 300

gctgaacctg?ctgctcaaca?agacaagcca?gctgatgcc 339

<210>6

<211>255

<212>DNA

<213>Cryptosporidium?parvum

<400>6

atgggtaact?tgaaatcctg?ttgttctttt?gccgatgaac?actccctaac?ctctactcaa 60

ctagtagttg?gaaatggttc?aggagcttca?gaaactgctt?ccaaccaccc?ccaagaagaa 120

gttaatgata?tcaatacttt?taatgtaaag?ttaataatgc?aagatagaag?taagcttgac 180

tgcgaggtag?tatttgatag?cacaagtatt?tcgctttctg?gagatggaaa?atgcagaaat 240

attgctttgg?atgaa 255

<210>7

<211>270

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(55)

＜223〉CP15 antigen fragment

<220>

<221>MISC_FEATURE

<222>(56)..(70)

＜223〉joint sequence

<220>

<221>MISC_FEATURE

<222>(71)..(183)

＜223〉P23 antigen sequence

<220>

<221>MISC_FEATURE

<222>(184)..(185)

＜223〉joint sequence

<220>

<221>MISC_FEATURE

<222>(186)..(270)

＜223〉CP15/60 antigen fragment

<400>7

Met?Lys?Leu?Asp?Glu?Val?Val?Glu?Leu?Leu?Pro?Ala?Arg?Lys?Arg?Arg

1 5 10 15

Lys?Ile?Ala?Arg?Gly?Cys?Leu?Asn?Arg?Arg?Thr?Ala?Ala?Phe?Ile?Ala

20 25 30

Lys?Leu?Arg?Lys?Ser?Lys?Ala?Glu?Cys?Pro?Met?Gly?Glu?Lys?Pro?Val

35 40 45

Ala?Val?Arg?Thr?His?Leu?Arg?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly

50 55 60

Ser?Gly?Gly?Gly?Gly?Ser?Met?Gly?Cys?Ser?Ser?Ser?Lys?Pro?Glu?Thr

65 70 75 80

Lys?Val?Ala?Glu?Asn?Lys?Ser?Ala?Ala?Asp?Ala?Asn?Lys?Gln?Arg?Glu

85 90 95

Leu?Ala?Glu?Lys?Lys?Ala?Gln?Leu?Ala?Lys?Ala?Val?Lys?Asn?Pro?Ala

100 105 110

Pro?Ile?Ser?Asn?Gln?Ala?Gln?Gln?Lys?Pro?Glu?Glu?Pro?Lys?Lys?Ser

115 120 125

Glu?Pro?Ala?Pro?Asn?Asn?Pro?Pro?Ala?Ala?Asp?Ala?Pro?Ala?Ala?Gln

130 135 140

Ala?Pro?Ala?Ala?Pro?Ala?Pro?Ala?Ala?Pro?Ala?Ser?Gln?Asp?Lys?Pro

145 150 155 160

Ala?Glu?Ala?Pro?Ala?Ala?Glu?Ala?Pro?Ala?Ala?Glu?Pro?Ala?Ala?Gln

165 170 175

Gln?Asp?Lys?Pro?Ala?Asp?Ala?Gly?Ser?Met?Gly?Asn?Leu?Lys?Ser?Cys

180 185 190

Cys?Ser?Phe?Ala?Asp?Glu?His?Ser?Leu?Thr?Ser?Thr?Gln?Leu?Val?Val

195 200 205

Gly?Asn?Gly?Ser?Gly?Ala?Ser?Glu?Thr?Ala?Ser?Asn?His?Pro?Gln?Glu

210 215 220

Glu?Val?Asn?Asp?Ile?Asn?Thr?Phe?Asn?Val?Lys?Leu?Ile?Met?Gln?Asp

225 230 235 240

Arg?Ser?Lys?Leu?Asp?Cys?Glu?Val?Val?Phe?Asp?Ser?Thr?Ser?Ile?Ser

245 250 255

Leu?Ser?Gly?Asp?Gly?Lys?Cys?Arg?Asn?Ile?Ala?Leu?Asp?Glu

260 265 270

<210>8

<211>837

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(16)..(828)

<400>8

ccggaattcc?cgacc?atg?aaa?ttg?gat?gag?gtt?gtt?gag?ctt?tta?cca?gca 51

Met?Lys?Leu?Asp?Glu?Val?Val?Glu?Leu?Leu?Pro?Ala

1 5 10

cgt?aaa?aga?cgt?aag?ata?gcc?aga?ggt?tgt?ctt?aac?aga?aga?act?gca 99

Arg?Lys?Arg?Arg?Lys?Ile?Ala?Arg?Gly?Cys?Leu?Asn?Arg?Arg?Thr?Ala

15 20 25

gct?ttt?atc?gca?aag?ctc?cgc?aaa?tct?aag?gct?gaa?tgt?cca?atg?gga 147

Ala?Phe?Ile?Ala?Lys?Leu?Arg?Lys?Ser?Lys?Ala?Glu?Cys?Pro?Met?Gly

30 35 40

gag?aaa?cct?gtt?gct?gtt?cgt?acc?cat?tta?cgt?ggt?gga?ggc?ggt?tca 195

Glu?Lys?Pro?Val?Ala?Val?Arg?Thr?His?Leu?Arg?Gly?Gly?Gly?Gly?Ser

45 50 55 60

ggc?gga?ggt?ggc?agc?ggc?ggt?ggc?ggg?tcg?atg?ggt?tgt?tca?tca?tca 243

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Met?Gly?Cys?Ser?Ser?Ser

65 70 75

aag?cca?gaa?act?aaa?gtt?gct?gaa?aat?aaa?tct?gca?gca?gat?gct?aac 291

Lys?Pro?Glu?Thr?Lys?Val?Ala?Glu?Asn?Lys?Ser?Ala?Ala?Asp?Ala?Asn

80 85 90

aaa?caa?aga?gaa?tta?gct?gaa?aag?aag?gct?caa?tta?gcc?aag?gct?gta 339

Lys?Gln?Arg?Glu?Leu?Ala?Glu?Lys?Lys?Ala?Gln?Leu?Ala?Lys?Ala?Val

95 100 105

aag?aat?cca?gct?cca?atc?agc?aac?caa?gct?caa?caa?aag?cca?gaa?gaa 387

Lys?Asn?Pro?Ala?Pro?Ile?Ser?Asn?Gln?Ala?Gln?Gln?Lys?Pro?Glu?Glu

110 115 120

cca?aag?aag?tcc?gag?cct?gct?cca?aat?aac?cct?cca?gct?gct?gat?gcg 435

Pro?Lys?Lys?Ser?Glu?Pro?Ala?Pro?Asn?Asn?Pro?Pro?Ala?Ala?Asp?Ala

125 130 135 140

cca?gca?gcc?caa?gct?cct?gct?gcc?cct?gcc?cct?gct?gcc?cct?gct?tca 483

Pro?Ala?Ala?Gln?Ala?Pro?Ala?Ala?Pro?Ala?Pro?Ala?Ala?Pro?Ala?Ser

145 150 155

cag?gat?aag?cca?gct?gag?gct?cca?gct?gct?gaa?gct?cca?gct?gct?gaa 531

Gln?Asp?Lys?Pro?Ala?Glu?Ala?Pro?Ala?Ala?Glu?Ala?Pro?Ala?Ala?Glu

160 165 170

cct?gct?gct?caa?caa?gac?aag?cca?gct?gat?gcc?gga?tcc?atg?ggt?aac 579

Pro?Ala?Ala?Gln?Gln?Asp?Lys?Pro?Ala?Asp?Ala?Gly?Ser?Met?Gly?Asn

175 180 185

ttg?aaa?tcc?tgt?tgt?tct?ttt?gcc?gat?gaa?cac?tcc?cta?acc?tct?act 627

Leu?Lys?Ser?Cys?Cys?Ser?Phe?Ala?Asp?Glu?His?Ser?Leu?Thr?Ser?Thr

190 195 200

caa?cta?gta?gtt?gga?aat?ggt?tca?gga?gct?tca?gaa?act?gct?tcc?aac 675

Gln?Leu?Val?Val?Gly?Asn?Gly?Ser?Gly?Ala?Ser?Glu?Thr?Ala?Ser?Asn

205 210 215 220

cac?ccc?caa?gaa?gaa?gtt?aat?gat?att?aat?act?ttt?aat?gta?aag?tta 723

His?Pro?Gln?Glu?Glu?Val?Asn?Asp?Ile?Asn?Thr?Phe?Asn?Val?Lys?Leu

225 230 235

ata?atg?caa?gat?aga?agt?aag?ctt?gac?tgt?gag?gta?gta?ttt?gat?agc 771

Ile?Met?Gln?Asp?Arg?Ser?Lys?Leu?Asp?Cys?Glu?Val?Val?Phe?Asp?Ser

240 245 250

aca?agt?att?tcg?ctt?tct?gga?gat?gga?aaa?tgc?aga?aat?att?gct?ttg 819

Thr?Ser?Ile?Ser?Leu?Ser?Gly?Asp?Gly?Lys?Cys?Arg?Asn?Ile?Ala?Leu

255 260 265

gat?gaa?taa?gtcgacggg 837

Asp?Glu

270

<210>9

<211>37

<212>DNA

＜213〉artificial sequence

<220>

<22l>misc_feature

<222>(1)..(37)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉EcoR I restriction enzyme site

<400>9

ccggaattcc?cgaccatgaa?attggatgag?gttgttg 37

<210>10

<211>65

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(65)

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(45)

＜223〉joint sequence

<400>10

cgacccgcca?ccgccgctgc?cacctccgcc?tgaaccgcct?ccaccacgta?aatgggtacg 60

aacag 65

<210>11

<211>68

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(68)

＜223〉primer

<220>

<221>misc_feature

<222>(1)..(45)

＜223〉joint sequence

<400>11

ggtggaggcg?gttcaggcgg?aggtggcagc?ggcggtggcg?ggtcgatggg?ttgttcatca 60

tcaaagcc 68

<210>12

<211>29

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(29)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉BamH I restriction enzyme site

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉joint sequence

<400>12

catggatccg?gcatcagctg?gcttgtctt 29

<210>13

<211>35

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(35)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉BamH I restriction enzyme site

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉joint sequence

<400>13

gccggatcca?tgggtaactt?gaaatcctgt?tgttc 35

<210>14

<211>34

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(34)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉Sal I restriction enzyme site

<400>14

cccgtcgact?tattcatcca?aagcaatatt?tctg 34

<210>15

<211>43

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(43)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉EcoR I restriction enzyme site

<400>15

ccggaattcc?cgaccatggc?tatgaaattg?gatgaggttg?ttg 43

<210>16

<211>33

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(33)

＜223〉primer

<220>

<221>misc_feature

<222>(4)..(9)

＜223〉Xba I restriction enzyme site

<400>16

ccctctagat?tattcatcca?aagcaatatt?tct 33

Claims (7)

1. a Cryptosporidium mixes Multi-Epitope Fusion Protein, it is characterized in that, the aminoacid sequence of described fusion rotein is the aminoacid sequence shown in SEQ IDNO.7.

2. a Cryptosporidium mixes multi-epitope gene, it is characterized in that, the nucleotides sequence of described mixing multi-epitope gene is classified the nucleotide sequence of aminoacid sequence shown in coding SEQ ID NO.7 as.

3. Cryptosporidium according to claim 2 mixes multi-epitope gene, it is characterized in that, the nucleotides sequence of described mixing multi-epitope gene is classified the nucleotide sequence shown in SEQ ID NO.8 as.

4. a recombinant vectors, is characterized in that, comprises Cryptosporidium claimed in claim 2 and mix multi-epitope gene.

5. a vaccine, is characterized in that, comprises Cryptosporidium claimed in claim 2 and mix multi-epitope gene and expression vector.

6. Cryptosporidium claimed in claim 1 mixes the application of Multi-Epitope Fusion Protein in the vaccine of preparation prevention or treatment cryptosporidiosis.

7. Cryptosporidium claimed in claim 2 mixes the application of multi-epitope gene in the vaccine of preparation prevention or treatment cryptosporidiosis.

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