CN104711289A - Recombinant vector, recombinant baculovirus prepared with the same and application of virus in preparation of malaria vaccines - Google Patents
Recombinant vector, recombinant baculovirus prepared with the same and application of virus in preparation of malaria vaccines Download PDFInfo
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Abstract
The invention provides a recombinant vector, a recombinant baculovirus prepared with the vector and application of the virus in preparation of malaria vaccines. The recombinant vector is constructed by inserting a section of recombinant sequence into the pFastBacDual vector. The recombinant sequence is formed by: according to CMV, Ph, SP and TM sequences, adding EcoR I enzyme site and Xho I enzyme site between SP and TM, adding KpnI enzyme site in front of CMV-F, and adding HindIII enzyme site after TM-R. The recombinant baculovirus is obtained by: inserting Plasmodium yoelii Pys25 antigen gene into the recombinant vector, conducting homologous recombination with the genome of a shuttle vector Bacmid through transposition, then transfecting a bombyx mori cell, and performing packaging in the bombyx mori cell. Through simple separation and purification of the recombinant baculovirus, an antibody with good titer can be prepared, thus providing a reference for study of P25 malaria vaccines.
Description
Technical field
The invention belongs to biomedicine technical field, be specifically related to a kind of baculovirus vector through transformation and recombinant vectors, the recombinant baculovirus containing goal gene (Plasmodium yoelii surface protein Pys25 gene) utilizing this carrier to prepare, the preparation method of this recombinant baculovirus, the fusion rotein of this recombinant baculovirus expression, and the application in malaria vaccine preparation of this recombinant baculovirus and this fusion rotein.
Background technology
The malaria propagated by Mosquito Vectors is a kind of infectious diseases of serious threat human life health.Point out, 2010 according to WHO in 2011 " world's malaria report in 2011 ", in the whole world, 106 malaria prevalences countries and area find about 2.16 hundred million case histories altogether, and wherein the malaria case history of 86% is less than 5 years old children.According to estimates, in 2010, the whole world is total more than 650,000 routine malaria Records of Deaths.The Study and Development of malaria vaccine is one of Critical policies of propagating of malaria control, and is found along with increasing plasmodium persister, and the development of malaria vaccine seems particularly important.
Plasmodium yoelii surface protein Pys25 is the surface protein in vermicule period in its in syngenesis stage, its structure height is guarded, and have higher homology with other plasmodial P25 albumen, be often used as the research that laboratory Modling model is other malaria and reference is provided.
At present conventional intestinal bacteria and yeast expression albumen prepare vaccine in the world, and by the most of non-immunogenicity of the albumen of escherichia coli expression and protectiveness, vaccine effect prepared by yeast expression neither be very desirable.Mammalian cell expression system (CHO) although etc. expression system effective, because expression amount is low, need mass propgation base and bovine serum albumin, cost is high simultaneously, is not suitable for need of production.
Summary of the invention
Technical problem to be solved by this invention is for above the deficiencies in the prior art, a kind of recombination bacillary viral vector is provided, utilize the recombinant baculovirus of this vector construction surface display Plasmodium yoelii Pys25 antigen, by separation and purification virus particle simply, Dispersal risk has good antibody titer, can provide reference for the research of P25 malaria vaccine.
The technical solution adopted in the present invention is:
A kind of recombinant vectors pFastBacDual-CMV-Ph-SP-TM, this recombinant vectors is inserted in pFastBacDual carrier to build by one section of recombination sequence and forms, described recombination sequence is according to CMV, Ph, SP, TM known array, EcoR I restriction enzyme site and Xho I restriction enzyme site is added between SP and TM nucleotide sequence, KpnI restriction enzyme site is added before CMV-F, add HindIII restriction enzyme site after TM-R to be formed, described recombination sequence total length is 923bp, as shown in SEQ ID NO:1.
Particularly, described recombination sequence is inserted between the KpnI restriction enzyme site of pFastBacDual carrier and HindIII restriction enzyme site.
The present invention further provides the recombinant baculovirus BmPys25 utilizing above-mentioned recombinant vectors pFastBacDual-CMV-Ph-SP-TM to build, this recombinant baculovirus is inserted into recombinant vectors pFastBacDual-CMV-Ph-SP-TM by Plasmodium yoelii Pys25 antigen gene and carries out homologous recombination by the genome of swivel base and shuttle vectors Bacmid, obtain recombinant baculovirus genomic dna, then by recombinant baculovirus genomic dna transfection bombyx mori cell, obtain in bombyx mori cell internal packing the recombinant baculovirus that surface display has Plasmodium yoelii Pys25 antigen.
Particularly, described Plasmodium yoelii Pys25 antigen gene is inserted between the EcoR I restriction enzyme site of recombinant vectors pFastBacDual-CMV-Ph-SP-TM and Xho I restriction enzyme site.
Particularly, described bombyx mori cell is Bombyx noriN cell.
Shuttle vectors Bacmid(and Baculovirusplasmid) be plasmid with Baculovirus Gene group, can shuttle back and forth between bacterium and insect cell, one of member of Bac-to-bac baculovirus expression system, this system also comprises donor plasmid and helper plasmid and intestinal bacteria, is prior art.
Carrier pFastBacDual is the donor plasmid of Bac-to-bac expression system, can insert foreign gene and under the transposase effect of helper plasmid coding, is inserted in Bacmid by foreign gene.Carrier pFastBacDual commercialization; CMV promoter is mammalian promoter, after being inserted into baculovirus, baculovirus can in Mammals expression alien gene.
The preparation method of above-mentioned recombinant baculovirus BmPys25, comprises the following steps:
(1) Plasmodium yoelii Pys25 antigen gene is obtained by the method for pcr amplification, then Plasmodium yoelii Pys25 antigen gene is connected between the EcoR I restriction enzyme site of recombinant vectors pFastBacDual-CMV-Ph-SP-TM and Xho I restriction enzyme site, obtains restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM;
(2) restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM is transformed the intestinal bacteria DH10Bac competent cell containing baculovirus shuttle vector Bacmid, carry out homologous recombination, LB culture plate containing kantlex, gentamicin, tsiklomitsin, X-gal and IPTG carries out blue hickie screening, picking hickie after lucifuge cultivation 40-48h, after hickie continues to cultivate 24-48h, extracting recombinant baculovirus genomic dna carries out PCR qualification;
(3) get step (2) and identify that correct recombinant baculovirus genomic dna is by liposome mediated-method transfection Bombyx noriN cell, generation viral suspension is obtained after morbidity, extract viral genome and again carry out PCR qualification, identify the correct recombinant baculovirus BmPys25 being surface display Plasmodium yoelii Pys25 antigen.
Fusion protein S P-Pys25-TM expressed by above-mentioned recombinant baculovirus BmPys25, by Plasmodium yoelii Pys25 albumen N termination gp64 secretes property signal peptide (SP), C termination gp64 membrane spaning domain (TM) forms, its aminoacid sequence is as shown in SEQ ID NO:10.
The present invention further provides the application of above-mentioned recombinant baculovirus BmPys25 in malaria vaccine preparation.
The application of the fusion protein S P-Pys25-TM that the present invention also provides above-mentioned recombinant baculovirus BmPys25 to express further in malaria vaccine preparation.
Compared with prior art, the present invention has following remarkable advantage and beneficial effect:
(1) the present invention is by analyzing Plasmodium yoelii Pys25 gene, find its coding 217 amino acid, its two ends are hydrophobic region, interlude is hydrophilic area, and its N-holds the signal peptide containing 20 amino-acid residues, Pys25 albumen the 177 to 192 amino-acid residue is for EGF-like structural domain (CdCkdGFklsveeekC) is as shown in SEQ ID NO:9, show that it has the correlation functions such as Urogastron, BLAST analyzes and shows simultaneously, the P25 albumen of itself and other malaria has very high homology, therefore can be used for the research of other malaria as a kind of model.
(2) the present invention is by Plasmodium yoelii Pys25 protein gene and silkworm baculovirus envelope protein gp64 gene fusion, silkworm baculovirus envelope protein gp64 contains Liang Ge very hydrophobic district: the secretion signal peptide (SP) of N-end and the membrane spaning domain (TM) of C-end, what be connected with membrane spaning domain is hydrophilic domain, glycoprotein in virus envelope and host cell membrane can be linked together, thus make to present invention achieves the surface display of object Pys25 albumen on viral capsid.
(3) recombinant baculovirus of the surface display Plasmodium yoelii Pys25 albumen of the present invention's structure, Bombyx noriN cell can be utilized as bio-reactor, by baculovirus expression vector system high expression Plasmodium yoelii Pys25 albumen, it can prepare vaccine, for the research of other malaria provides reference.Silkworm biological reactor, as a kind of eukaryotic expression system, is applicable to scale operation, and cost is low, and output is high, and the vaccine using value of producing is large.
(4) current Pys25 vaccine there is no and utilizes baculovirus surface display technologies to produce, baculovirus expression vector system is eukaryotic expression, there is posttranslational modification function, its structure of albumen of expression can be made closer to its native conformation, by separation and purification virus particle simply, immunity Balb/c mouse, the antibody of acquisition has good antibody titer.
Accompanying drawing explanation
Shown in Fig. 1 is the structural representation of carrier pFastBacDual.
Shown in Fig. 2 is the design of graphics of restructuring swivel base plasmid pFstBacDual-CMV-Ph-SP-Pys25-TM, wherein CMV: mammalian promoter; Ph: polyhedron promoter; The signal peptide sequence of SP:gp64 gene; Pys25 albumen: Plasmodium yoelii vermicule surface protein in period; The transmembrane domain of TM:gp64 gene; Kpn I, EcoR I, Xho I, Hind III: restriction enzyme site.
Shown in Fig. 3 is recombinant vectors pFastBacDual-CMV-Ph-SP-TM electroresis appraisal figure, wherein M:DNA molecular weight standard; 1:CMV-Ph-SP-TM gene PCR amplified production, sequence for the purpose of arrow locations, 923bp; 2: recombinant vectors double digestion product.
Shown in Fig. 4 is that the PCR of restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM identifies electrophorogram, wherein M:DNA molecular weight standard, 1:Pys25 goal gene pcr amplification product, 2:Pys25 goal gene double digestion product.
Shown in Fig. 5 is the PCR primer electroresis appraisal result of recombinant baculovirus genome BmPys25, wherein, M:DNA molecular weight standard, 1:Pys25-F and Pys25-R is the fragment of primer amplification, 2:Pys25-F and M13-R is the fragment of primer amplification, 3:Pys25-R and M13-F is the fragment of primer amplification, 4:M13-F and M13-R is the fragment of primer amplification.
Shown in Fig. 6 is the Pys25 albumen WesternBlot detected result (detection of Pys25 albumen mouse-anti) of recombinant baculovirus BmPys25, M: Protein Marker, 1: cell expressing Pys25 albumen, 2: negative control.
Shown in Fig. 7 is the result of tiring that indirect ELISA measures antibody, and the line is wherein the serum containing Pys25 antibody, and line is below negative control, and negative control is the serum that injection equivalent PBS gets.
Sequence table information:
SEQ ID NO:1:CMV-Ph-SP-TM sequence;
SEQ ID NO:2: upstream primer CMV-F;
SEQ ID NO:3: downstream primer TM-R;
SEQ ID NO:4:Pys25 gene;
SEQ ID NO:5: upstream primer Pys25-F;
SEQ ID NO:6: downstream primer Pys25-R;
SEQ ID NO:7: upstream primer M13-F;
SEQ ID NO:8: downstream primer M13-R;
SEQ ID NO:9:EGF-like structural domain;
SEQ ID NO:10: fusion protein S P-Pys25-TM.
Embodiment
It is all exemplary for below illustrating, and is intended to the invention provides further instruction.Except as otherwise noted, all Science and Technology terms used in the present invention have the identical meanings usually understood with the technical field of the invention personnel.
The present invention obtains the coding gene sequence of Plasmodium yoelii Pys25 albumen by pcr amplification, obtain Pys25 gene, increase CMV-Ph-SP-TM gene simultaneously, by CMV-Ph-SP-TM gene and pFastBacDual carrier by connecting together after KpnI and HindIII double digestion, be built into recombinant vectors pFastBacDual-CMV-Ph-SP-TM, this recombinant vectors and Pys25 gene are passed through EcoR I, Xho I double digestion, link together, obtain restructuring swivel base plasmid (called after pFastBacDual-CMV-Ph-SP-Pys25-TM), restructuring swivel base Plastid transformation is containing the intestinal bacteria DH10Bac competent cell of baculovirus shuttle vector Bacmid, carry out homologous recombination, obtain recombinant baculovirus genomic dna (recombinant plasmid called after Bacmid-Pys25), passed through liposome transfection Bombyx noriN cell, in cell, assembling forms the recombinant baculovirus (called after BmPys25) of surface display Plasmodium yoelii Pys25 albumen, and copy amplification, Bombyx noriN cell is inoculated with third generation BmPys25, virus liquid is collected after 3 ~ 5 days, centrifugal, separation and purification, make malaria vaccine, its antibody produced has good antibody titer, to the research of other P25 albumen, there is good reference value.
Particular content of the present invention is elaborated below in conjunction with embodiment.
1, the composition sequence CMV-Ph-SP-TM that relates to of the present embodiment, Pys25 gene and each primer, synthesized by Beijing Hua Da gene company limited.
2, material:
Carrier pFastBacDual, containing the intestinal bacteria DH10Bac competent cell of baculovirus shuttle vector Bacmid, Bombyx noriN cell purchased from Invitrogen company, LB culture plate, nutrient solution are purchased from Shanghai Sheng Gong biotech firm, Balb/c mouse purchased from Test Animal Centre, Academy of Military Medical Sciences, P.L.A's (animal credit number: SCXK(capital) 2012-0001, conformity certification number: 11400700020779).
3, main agents:
Taq archaeal dna polymerase, all kinds of restriction endonuclease are purchased from Fermentas company, and lipofectamine Cellfectin II Reagent is purchased from Invitrogen company, and other reagent used all belongs to common commercially available Bioexperiment reagent.
Embodiment 1: the structure of recombinant vectors pFastBacDual-CMV-Ph-SP-TM
According to CMV, Ph, SP, TM known array, EcoRI restriction enzyme site (GAATTC) and Xho I restriction enzyme site (CTCGAG) is added between SP and TM nucleotide sequence, KpnI restriction enzyme site is added before CMV-F, HindIII restriction enzyme site is added after TM-R, synthesis CMV-Ph-SP-TM sequence (as shown in SEQ ID NO:1), and design primer CMV-F(as shown in SEQ ID NO:2), TM-R(is as shown in SEQ ID NO:3).Primer is as follows:
CMV-F5'-CCC
GGTACCTAGTTATTAATAG-3'
TM-R5'-CCC
AAGCTTTTAATATTGTCTAC-3'
Wherein underscore place is its restriction enzyme site.
With the CMV-Ph-SP-TM sequence of synthesis for template, with CMV-F, TM-R for upstream and downstream primer PCR amplification object fragment, the reaction system of PCR is 50 μ L, concrete composition is: 10 × PCRBuffer5 μ L, the each 1 μ L of CMV-F and TM-R of the dNTPs5 μ L of 2.5mmol/mL, 0.01nmol/ μ L, template 2 μ L, Taq DNA polymerase 2 μ L, ddH
2o34 μ L.After each component mixing, put into PCR instrument, PCR reaction parameter: 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations, and 72 DEG C extend 10min.After question response terminates, electroresis appraisal amplified production segment, object segment size is 923bp, cuts glue simultaneously and reclaims object segment.
The PCR object fragment and the pFastBacDual carrier (as shown in Figure 1) that reclaim are used KpnI and HindIII double digestion simultaneously, and connect, transform, coated plate choose spot and identify, be just built into recombinant vectors pFastBacDual-CMV-Ph-SP-TM.The PCR of recombinant vectors pFastBacDual-CMV-Ph-SP-TM and double digestion qualification electrophorogram are as shown in Figure 3.
Like this, after we insert foreign gene between SP and TM, foreign gene N-end has SP signal peptide sequence, C-end has TM cross-film district, the albumen of expressing is the fusion rotein with signal peptide and cross-film district, so just fusion rotein can be illustrated in cell surface, when it is at silkworm cells, silkworm polyhedrin promoter Ph works, start and express, when being imported after in Mammals, mammalian promoter CMV works, expressed fusion protein, the virus that we build in the later stage like this can at silkworm cells, can express in Mammals again, there is dual function.
Embodiment 2: the structure of restructuring swivel base plasmid pFstBacDual-CMV-Ph-SP-Pys25-TM
With Pys25 goal gene (as shown in SEQ ID NO:4) for template, with Pys25-F(as shown in SEQ ID NO:5), Pys25-R(is as shown in SEQ ID NO:6) be upstream and downstream primer PCR amplifying target genes Pys25.
Pys25-F5'-CG
GAATTCATGAACACATACTAC-3'
Pys25-R5'-G
GAATTCATGTTGAGCTTCTTTGGC-3'
Wherein underscore place is its restriction enzyme site.
The reaction system of PCR is 50 μ L, and concrete composition is: each 1 μ L of Pys25-F and Pys25-R of the dNTPs5 μ L of 10 × PCRBuffer5 μ L, 2.5mmol/mL, 0.01nmol/ μ L, template 2 μ L, Taq DNA polymerase 2 μ L, ddH
2o34 μ L.After each component mixing, put into PCR instrument, PCR reaction parameter: 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 53 DEG C of annealing 30s, 72 DEG C extend 45s, 30 circulations, and 72 DEG C extend 10min.After question response terminates, electroresis appraisal amplified production segment, object segment size is 651bp, cuts glue simultaneously and reclaims object segment.
The recombinant vectors that the PCR object fragment of recovery and embodiment 1 build is used simultaneously EcoR I and Xho I double digestion, and connect, transform, coated plate choose spot qualification, be just built into recombinant plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM.The structure schematic diagram of restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM as shown in Figure 2.After the restructuring swivel base plasmid built is correct by restriction analysis and two-way order-checking identified gene sequence, the success of restructuring swivel base plasmid construction.Fig. 4 is PCR and the double digestion qualification electrophorogram of restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM.
Embodiment 3: the acquisition of silkworm with recombinant baculovirus BmPys25
The swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM that qualification restructuring successfully recombinated transforms the intestinal bacteria DH10Bac competent cell containing baculovirus shuttle vector Bacmid, containing kantlex, gentamicin, tsiklomitsin, the LB culture plate (operating to specifications) of X-gal and IPTG is upper to be cultivated, blue hickie screening is carried out after carrying out homologous recombination by swivel base, picking hickie after lucifuge cultivation 48h, hickie continues containing tsiklomitsin, kantlex, gentamicin, shake in the LB nutrient solution of X-gal and IPTG after bacterium cultivates 48h and use Virahol extracting recombinant baculovirus genomic dna, with M13 universal primer, (M13-F is as shown in SEQ ID NO:7, M13-R is as shown in SEQ ID NO:8), Pys25-F and Pys25-R inserts situation by goal gene in pcr amplification qualification restructuring Bacmid, insert successful plasmid called after Bacmid-Pys25(and recombinant baculovirus genome).
Wherein, M13 universal primer sequence:
M13-F：5'-GTTTTCCCAGTCACGAC-3'。
M13-R5'-CAGGAAACAGCTATGAC-3'。
Identify that successful plasmid Bacmid-Pys25 is by liposome mediated-method transfection Bombyx noriN cell, transfection use Invitrogen company lipofectamine Cellfectin II Reagent, transfection method with reference to this transfection reagent specification sheets, transfection concrete steps:
The plasmid Bacmid-Pys25 of 16 μ L and 8 μ L transfection reagents are added in the serum free medium of 76 μ L by evening before that day, incubated at room 20min, liposome is made fully to wrap up plasmid Bacmid-Pys25, then joined in the well-grown Bombyx noriN cell of 1mL, insert incubator overnight incubation, serum free medium siphons away by the next morning, has changed blood serum medium into and has cultivated 5 ~ 7 days, treated that cell is fallen ill.
Cell morbidity rear (microscopic examination) obtains generation viral suspension 4 DEG C preservation, extract viral genome M13-F, M13-R, Pys25-F, Pys25-R to identify, qualification result is shown in Fig. 5, result shows virus formulation success, obtain the recombinant baculovirus of surface display Plasmodium yoelii Pys25 albumen, called after BmPys25.
Embodiment 4:Pys25 albumen is in the expression of Bombyx noriN cell
By recombinant baculovirus BmPys25 with 3 × 10
-6the dosage infected silkworm BmN cell of pfu/cell carries out virus amplification, infect after 3 ~ 5 days, collect virus liquid, through separation and purification, get 10 μ L supernatant liquors and add isopyknic 2 × protein sample-loading buffer (100MmTris-HCl, 4%SDS, 0.15% tetrabromophenol sulfonphthalein, 10% glycerine), 100 DEG C of heating 10min, get the mixed solution after heating 10 μ L and carry out SDS-PAGE and Westernblotting analysis, result shows, this silkworm with recombinant baculovirus successful expression Pys25 fusion protein S P-Pys25-TM(is as shown in SEQ ID NO:10), see Fig. 6, wherein swimming lane 1 is the fusion rotein of eukaryotic expression, swimming lane 2 is blank results, and blank is normal bombyx mori cell.
Embodiment 5: separation and purification BmPys25 virus and the preparation of antibody and bioactivity from Bombyx noriN cell
(1) by the 3rd generation BmPys25 virus a large amount of inoculation BmN cell, after cell morbidity is almost all floating, virus liquid is collected;
(2) add in 50mL centrifuge tube by the virus liquid that step (1) obtains, the centrifugal 30min of 8000rpm, gets supernatant, in triplicate to remove cell residue;
(3) pour the centrifuged supernatant that step (2) obtains into 50mL centrifuge tube, the centrifugal 60min of 15000rpm, gets supernatant, in triplicate;
(4) by the centrifuged supernatant that step (3) obtains, put into Hitachi CP70MX whizzer with the centrifugal 40min of rotating speed 50000rpm, gained black group ultrafiltrated (i.e. phosphoric acid buffer PBS) is resuspended, obtains the recombinant baculovirus of purifying.
(5) recombinant baculovirus of step (4) acquisition purifying is diluted to 1mg/mL, first time and the emulsification of equal-volume Freund's complete adjuvant equal-volume, immune Balb/c mouse, abdominal injection, every only 50 μ L, immunity is once week about later, by virus particle and the emulsification of equal-volume Freund's incomplete adjuvant equal-volume, immunity Balb/c mouse, abdominal injection, every only 50 μ L, totally 3 times, after immunity completes, get blood by eyeball, obtain the serum containing antibody.
(6) serum that step (5) obtains detects antibody titer by indirect ELISA, and result shows that antibody titer can reach 1:12800, sees Fig. 7.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Sequence table:
SEQ ID NO：1：
TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTATCATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTACTGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTATTCATACCGTCCCACCATCGGGCGCGATGGTAGGCGCTATTGTTTTATACGTGCTTTTGGCGGCGGCGCATTCTGCCTTTGCGGCGGAATTCCTCGAGATGGCTGAAGGCGAATTGGCCGCCAAATTGACTTCGTTCATGTTTGGTCATGTAGCCACTTTTGTAATTGTATTTATTGTAATTTTATTTTTGTACTGTATGGTTAGAAACCGTAATAGTAGACAATATTAA
SEQ ID NO：2：
CCCGGTACCTAGTTATTAATAG
SEQ ID NO：3：
CCCAAGCTTTTAATATTGTCTAC
SEQ ID NO：4：
ATGAACACATACTACAGCGTGTTCCTGTTCATTTACGCGTTCCTGGGCATTAACTACTACAACGCGGCGATTACACCGGCGACACAATGCAAGAACGGCTTCCTGGCGCAAATGAGCAACCACCTGGAATGCCGCTGCAACAACAACTTCGTGCACGTGAGCAACGACACATGCGAAACAAAGGTGGAATGCAGCAAGGCGACAGTGAACAAGCCGTG CGGCGAGTTCAGCAAGTGCACCATGCACGAAGACGAAGGCGTGGAAACATACACATGCGACTGCCTGGACACATACATTAAGAAGAACGACGTGTGCGTGCCGGAAACCTGCCAAAACATTGACTGCGGCAACGGCAAGTGCATTATTAACGAAGACGCGGTGAACGACCCGCCGACATGCAGCTGCAACATTGGCTACGTGGTGAACGTGGACGACGGCAGCAAATGCACCAAAGAAGGCGACACACTGTGCAGCCTGGTGTGCAACAAGGAAGAACAAATTTGCAAGAAGGTGGACAGCTACTACCGCTGCGACTGCAAGGACGGCTTCAAGCTGAGCGTGGAAGAAGAAAAGTGCATTAGCCACAGCATTTACAGCATGTTCAACCTGAGCATTATTTTCGCGATTCTGCTGCTGTTCAGCAACATCATC
SEQ ID NO：5：
CGGAATTCATGAACACATACTAC
SEQ ID NO：6：
GGAATTCATGTTGAGCTTCTTTGGC
SEQ ID NO：7：
GTTTTCCCAGTCACGAC
SEQ ID NO：8：
CAGGAAACAGCTATGAC
SEQ ID NO：9：
CdCkdGFklsveeekC
SEQ ID NO：10：
MVGAIVLYVLLAAAHSAFAAEFMNTYYSVFLFIYAFLGINYYNAAITPATQCKNGFLAQMSNHLECRCNNNFVHVSNDTCETKVECSKATVNKPCGEFSKCTMHEDEGVETYTCDCLDTYIKKNDVCVPETCQNIDCGNGKCIINEDAVNDPPTCSCNIGYVVNVDDGSKCTKEGDTLCSLVCNKEEQICKKVDSYYRCDCKDGFKLSVEEEKCISHSIYSMFNLSIIFAILLLFSNIILEMAEGELAAKLTSFMFGHVATFVIVFIVILFLYCMVRNRNSRQY
Claims (9)
1. a recombinant vectors pFastBacDual-CMV-Ph-SP-TM, it is characterized in that: be inserted into structure in pFastBacDual carrier by one section of recombination sequence and form, described recombination sequence is according to CMV, Ph, SP, TM known array, EcoR I restriction enzyme site and Xho I restriction enzyme site is added between SP and TM nucleotide sequence, KpnI restriction enzyme site is added before CMV-F, add HindIII restriction enzyme site after TM-R to be formed, described recombination sequence total length is 923bp, as shown in SEQ IDNO:1.
2. recombinant vectors pFastBacDual-CMV-Ph-SP-TM according to claim 1, is characterized in that: described recombination sequence is specifically inserted between the KpnI restriction enzyme site of pFastBacDual carrier and HindIII restriction enzyme site.
3. utilize the recombinant baculovirus BmPys25 that the recombinant vectors pFastBacDual-CMV-Ph-SP-TM described in claim 1 builds, it is characterized in that: be inserted into recombinant vectors pFastBacDual-CMV-Ph-SP-TM by Plasmodium yoelii Pys25 antigen gene and carry out homologous recombination by the genome of swivel base and shuttle vectors Bacmid, obtain recombinant baculovirus genomic dna, then by recombinant baculovirus genomic dna transfection bombyx mori cell, obtain in bombyx mori cell internal packing the recombinant baculovirus that surface display has Plasmodium yoelii Pys25 antigen.
4. recombinant baculovirus BmPys25 according to claim 3, is characterized in that: described Plasmodium yoelii Pys25 antigen gene is specifically inserted between the EcoR I restriction enzyme site of recombinant vectors pFastBacDual-CMV-Ph-SP-TM and Xho I restriction enzyme site.
5. recombinant baculovirus BmPys25 according to claim 3, is characterized in that: described bombyx mori cell is Bombyx noriN cell.
6. the preparation method of the recombinant baculovirus BmPys25 described in the arbitrary claim of claim 3-5, is characterized in that comprising the following steps:
(1) Plasmodium yoelii Pys25 antigen gene is obtained by the method for pcr amplification, then Plasmodium yoelii Pys25 antigen gene is connected between the EcoRI restriction enzyme site of recombinant vectors pFastBacDual-CMV-Ph-SP-TM and XhoI restriction enzyme site, obtains restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM;
(2) restructuring swivel base plasmid pFastBacDual-CMV-Ph-SP-Pys25-TM is transformed the intestinal bacteria DH10Bac competent cell containing baculovirus shuttle vector Bacmid, carry out homologous recombination, LB culture plate containing kantlex, gentamicin, tsiklomitsin, X-gal and IPTG carries out blue hickie screening, picking hickie after lucifuge cultivation 40-48h, after hickie continues to cultivate 24-48h, extracting recombinant baculovirus genomic dna carries out PCR qualification;
(3) get step (2) and identify that correct recombinant baculovirus genomic dna is by liposome mediated-method transfection Bombyx noriN cell, generation viral suspension is obtained after morbidity, extract viral genome and again carry out PCR qualification, identify the correct recombinant baculovirus BmPys25 being surface display Plasmodium yoelii Pys25 antigen.
7. the fusion protein S P-Pys25-TM expressed by recombinant baculovirus BmPys25 described in the arbitrary claim of claim 3-5, it is characterized in that: by Plasmodium yoelii Pys25 albumen N termination gp64 secretes property signal peptide (SP), C termination gp64 membrane spaning domain (TM) forms, its aminoacid sequence is as shown in SEQ ID NO:10.
8. the application of the recombinant baculovirus BmPys25 described in the arbitrary claim of claim 3-5 in malaria vaccine preparation.
9. the application of fusion protein S P-Pys25-TM according to claim 7 in malaria vaccine preparation.
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| CN112359053A (en) * | 2020-11-10 | 2021-02-12 | 江苏科技大学 | Fusion gene and application thereof |
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| CN102533677A (en) * | 2012-01-10 | 2012-07-04 | 特菲(天津)生物医药科技有限公司 | Malaria vaccine and preparation method thereof |
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| CN102533677A (en) * | 2012-01-10 | 2012-07-04 | 特菲(天津)生物医药科技有限公司 | Malaria vaccine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112359053A (en) * | 2020-11-10 | 2021-02-12 | 江苏科技大学 | Fusion gene and application thereof |
| CN112359053B (en) * | 2020-11-10 | 2022-05-13 | 江苏科技大学 | Fusion gene and application thereof |
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