CN104479028B - Bovine rota VP8* subunits recombinate chimeric protein and its application - Google Patents
Bovine rota VP8* subunits recombinate chimeric protein and its application Download PDFInfo
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Abstract
The present invention provides a kind of bovine rota VP8* subunits restructuring chimeric protein; it is that t cell epitope polypeptide P2 in tetanus toxin is introduced into bovine rota VP8* subunits multicopy mosaic gene recombinant protein epiposition vaccine; the immune efficacy of epiposition vaccine can be greatly improved; and cross-neutralization immunoprotection can be induced; and higher neutralizing antibody titers are induced, and can be with the anti-P [5] of induced high titers and P [11] genotype special rotavirus neutralizing antibody.G6 the and G10 type rotavirus of Major Epidemic in world wide can be prevented and treated simultaneously using the bovine rota VP8* subunits restructuring chimeric protein of the present invention, the bovine rotavirus vaccine cheap suitable for preparing safety, cost, the Rotavirus Vaccine researched and developed at present with other coordinates, economic loss of the rotavirus gastroenteritis to cattle-raising can be effectively reduced, there are wide market prospects.
Description
Technical field
The invention belongs to molecular biology and genetic engineering field, specifically, is related to bovine rota VP8* subunits
Recombinate chimeric protein and its application.
Background technology
Rotavirus is Reoviridae, rotavirus member, is to cause a variety of new born animals and infant's stomach and intestine
Scorching main pathogens.Bovine rota diarrhoea is using spiritual depressed, apocleisis, diarrhoea, dehydration as principal character, calf within 7 ages in days
Ox most easy infection, the incidence of disease may be up to 90%~100%, and the death rate is up to 10%~50%.If secondary escherichia coli loses
Mass formed by blood stasis can then cause the death rate further up, seriously endanger the economic benefit of cattle-raising.
Rotavirus particle is made up of 3 layers of capsid, and outermost layer capsid is made up of VP7 and spike protein VP4, and both of which can be with
Independently induce neutralizing antibody.Research shows that VP4 albumen has various functions, as viral hemagglutinin, absorption and cell intrusion,
Virulence is related.VP4 albumen is cracked into two kinds of VP8* (28kDa, aa 1~240) and VP5* (60kDa, aa 247-775) through pancreatin
Subunit, wherein VP8* have the Main Antigenic of VP4 albumen, and determine VP4 albumen serological specificity neutralization reactions.
Special effect medicine therapeutic rotaviral gastroenteritis is there is no at present, and vaccine inoculation is to prevent the sick major measure.Wheel
For the immune me chanism of shape virus mainly by the way that Pregnant cows are immunized, newborn calf obtains natural passive immunity by colostrum.Always with
To carry out the immune me chanism of rotavirus extensively using inactivated vaccine, but inactivated vaccine is not always safe, often occurs tight
The side reaction of weight.And rotavirus amplification efficiency in the MA104 cell lines of monkey source in ox source is relatively low, virus titer is relatively low, typically
It can only achieve 104~105Cfu/ml, the required virus titer of inactivated vaccine is not reached much, it is necessary to after culture
Concentration and purifying, cause the increase of production cost.So different research groups attempt DNA vaccination, virus-like in world wide
Grain vaccine, plant edible vaccine.But these vaccines have the shortcomings of preparation technology is complicated, production cost is high, be not suitable for business
Product production of vaccine, the laboratory research stage is only rested at present.Harbin Veterinary Medicine Inst., China Academy of Agriculture is in power class
Sheep rotavirus LLR-85 strains (G10P [15]) VP7 genes are replaced with ox source G6 type VP7 genes and then developed by topic group
Bovine rota matches somebody with somebody weak malicious candidate vaccine again, but any rotavirus live vaccine all has the problem of toxin expelling.Importantly,
Live vaccine cannot be used for Pregnant cows and be immunized.Therefore urgently researching and developing safe and efficient new bovine rotavirus vaccine is used for anti-system
Bovine Rotavirus Diarrhea.
In recent years, the use of epiposition vaccine is more and more extensive, and it is the means using genetic engineering, vivoexpression or artificial
The epitope of pathogenic microorganism is synthesized, is used as a kind of vaccine.Epiposition vaccine is maximum excellent for traditional vaccine
Gesture can exactly overcome traditional vaccine to dissipate malicious risk and the defects of live vaccine cannot be used for pregnant animal.In people's Wa strain colyliforms
5 epitopes are identified on viral VP8*, wherein 3 are linear neutralizing epitope (aa 1-10, aa55-66 and aa 223-
234), and aa 1-10 epitopes are highly conserved between a variety of strains, and the Rotavirus Vaccine of a variety of strains or serotype is resisted to developing
It is significant.
The combination of rotavirus gene type is numerous, and the cross immunity between different genotype is very few, and this brings to vaccine development
Difficulty.But research finds that a P genotype may cover several G genotype, i.e. some p-type can be from different G type groups
Close, same p-type can induce the heterotypic immunity of anti-different G types in theory.And determine the VP4 albumen of p-type and contain multiple linearisations
Epitope, and the albumen its VP8* fragment formed after pancreatin cracks, it is special to contain VP4 types without posttranslational modification
Property Main Antigenic, be all linear and more conservative, prompting can develop new PRV subunits epidemic disease using VP8* albumen
Seedling.
The bovine rota G genotype of world-wide prevalence is mainly two kinds of G6 and G10, and its P genotype is mainly P
[5] and P [11], P [1], P [21] and P [29] in addition also be present.It is same that conventional vaccine development mainly develops bivalent vaccine
When prevent and treat G6P [5] and G10P [11] rotavirus strain, this to vaccine development and produce bring certain difficulty, be doomed to cause
Production of vaccine cost improves.
The content of the invention
It is an object of the invention to provide a kind of new bovine rota VP8* subunits restructuring chimeric protein and its application.
In order to realize the object of the invention, bovine rota VP8* subunits of the invention restructuring chimeric protein, it is by breaking
Toxoid T cell epitopes polypeptide P2 and the genotype of catching cold be G6P [5] bovine rota VP8* subunits clipped form a and
The chimeric recombinant proteins formed of clipped form b；Wherein, without additional amino acid between clipped form a and clipped form b, and it is more
Copy, the bovine rota VP8* subunits for being G6P [5] in the genotype of tetanus toxin t cell epitope polypeptide P2, multicopy
Clipped form a and clipped form b between connected by flexible Linker.
Wherein, the tetanus toxin t cell epitope polypeptide P2, the bovine rota VP8* that genotype is G6P [5] are sub- single
The clipped form a and clipped form b of position amino acid sequence are respectively as shown in SEQ ID No.1-3.
The present invention also provides the gene for encoding above-mentioned restructuring chimeric protein, and its nucleotide sequence is as shown in SEQ ID No.5.
The present invention is also provided containing expression vector, host cell and the engineering bacteria for encoding above-mentioned restructuring chimeric protein gene.
The present invention also provides the method for preparing above-mentioned restructuring chimeric protein, will contain and encode above-mentioned restructuring chimeric protein gene
Expression vector convert into competent escherichia coli cell, screening positive clone and being inoculated in LB fluid nutrient mediums is cultivated,
Add IPTG induced expressions and obtain restructuring chimeric protein.
Wherein, 0.02g/mL glucose, 1~5v/v% ethanol and 2% glycerine are also added in the LB fluid nutrient mediums.
Specifically, positive colony is inoculated in above-mentioned LB fluid nutrient mediums and cultivated to OD600About 0.5, IPTG is added to end
Concentration is 0.5mM, and induced expression recombinates chimeric protein at 18 DEG C.
The present invention further provides bovine rota VP8* subunits restructuring chimeric protein to prepare bovine rota
Application in vaccine.
The present invention is by t cell epitope polypeptide P2 (tetanus toxin TT 830-844 amino acids) in tetanus toxin
It is introduced into bovine rota VP8* subunits multicopy mosaic gene recombinant protein epiposition vaccine, epiposition vaccine can be greatly improved
Immune efficacy, and cross-neutralization immunoprotection can be induced, and induce higher neutralizing antibody titers, and height can be induced
The anti-P [5] and P [11] genotype of titre special rotavirus neutralizing antibody.It is sub- using the bovine rota VP8* of the present invention
Unit restructuring chimeric protein can prevent and treat G6 the and G10 type rotavirus of Major Epidemic in world wide simultaneously, suitable for preparing
The cheap bovine rotavirus vaccine of safety, cost, the Rotavirus Vaccine researched and developed at present with other coordinate, can be effectively reduced
Economic loss of the rotavirus gastroenteritis to cattle-raising, there are wide market prospects.
Brief description of the drawings
Fig. 1 is VP4RT-PCR electrophoretograms in the embodiment of the present invention 1；Wherein, M:DL2000Marker；1:The full bases of Bo-VP4
The PCR primer of cause；2:Negative control.
Fig. 2 is annealing amplification VP8*-ab templates and VP8*-a in the embodiment of the present invention 1；Wherein, M:DL2000Marker；
1:The PCR primer of VP8*-ab genes；2:The PCR primer of VP8*-a genes；3:Negative control.
Fig. 3 is total length VP8* gene PCR amplifications in the embodiment of the present invention 1；Wherein, M:DL2000Marker；1:
VP8*PCR amplified productions；2:Negative control.
Fig. 4 is P2-VP8*- (ab) in the embodiment of the present invention 13With P2-VP8*-ab pcr amplification product electrophoresis results；Its
In, M：DL2000Marker；1:P2-VP8*-(ab)3Pcr amplification product；2:P2-VP8*-ab pcr amplification products；3:It is negative
Control.
Fig. 5 is that bacterium solution PCR identifies pET28a-P2-VP8*- (ab) 3 and pET32a-P2-VP8*- in the embodiment of the present invention 1
Ab results；Wherein, M:DL2000Marker；1:pET28a-VP8*-(ab)3T7PCR products；2:pET28a-P2-VP8*-(ab)3T7PCR products；3:PET32a-VP8*-ab T7PCR products；4:PET32a-P2-VP8*-ab T7PCR products；5:It is negative right
According to.
Fig. 6 is expression of results of the recombinant protein in Escherichia coli in the embodiment of the present invention 2；Wherein, scheme in A, M：Albumen
Matter molecular mass standard；1st, 2,3 be respectively pET-28a-VP8*- (ab)3Cellular lysate supernatant after whole cell, induction before induction
Cellular lysate precipitates after induction；4th, 5,6 be respectively whole cell before pET-32a-VP8*-ab is induced, cellular lysate supernatant after induction
Precipitated with cellular lysate after induction；7th, 8,9 be respectively whole cell before pET-32a-VP8*-a is induced, cellular lysate supernatant after induction
Precipitated with cellular lysate after induction；10th, 11 be respectively pET-28a-BL21 (DE3), pET-32a (+)-BL21 (DE3) empty carrier
Whole cell whole cell after recombinant bacterium induction；Scheme in B, M：Protein standard；1st, 2,3 be respectively pET-28a-P2-
VP8*-(ab)3Cellular lysate precipitates after the induction of cellular lysate supernatant after whole cell, induction before induction；4th, 5,6 be respectively pET-
Cellular lysate precipitates after the induction of cellular lysate supernatant after whole cell, induction before 32a-P2-VP8*-ab inductions；7th, 8 are respectively
Whole cell after whole cell and pET-32a (+) are induced after pET-28a (+) induction；9th, 10,11 be respectively pET-28a-VP8* inductions
Cellular lysate precipitates after the induction of cellular lysate supernatant after preceding whole cell, induction.
Fig. 7 is the Western blot analysis results of recombinant protein in the embodiment of the present invention 3；Wherein, M：Protein molecule
Quality standard；1：Recombinate VP8*- (ab)3Albumen；2：Recombinate VP8*-ab albumen；3：Recombinate VP8*-a albumen；4：Recombinate VP8* eggs
In vain；5：Recombinate P2-VP8*- (ab)3Albumen；6：Recombinate P2-VP8*-ab albumen；7：PET-28a (+) control；8：pET-32a(+)
Control.
Fig. 8 is the purification result of recombinant protein in the embodiment of the present invention 4；Wherein, M：Protein standard；1：Weight
Group VP8*- (ab)3Albumen；2：Recombinate VP8*-ab albumen；3：Recombinate VP8*-a albumen；4：Recombinate VP8* albumen；5：P2-VP8*-
(ab)3Albumen；6：P2-VP8*-ab albumen.
Fig. 9 is P2-VP8*- (ab) in the embodiment of the present invention 53、P2-VP8*-ab、VP8*-(ab)3, VP8*-ab, VP8* weight
The Analysis of Immunogenicity result of histone.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.
The source for the biomaterial being related in following examples：Prokaryotic expression carrier pET28a is purchased from Novagen companies；
E.coli BL21 (DE3) are purchased from Novagen companies；The primer is designed, designed and entrusts Beijing six directions Hua Da Gene science
Co., Ltd synthesizes.
The structure of expression vector of the embodiment 1 containing purpose fragment
Material therefor is genotype G5P [6] bovine rota, is trained with primary African Green Monkey kidney (AGMK) cell
Support.Used medium is EMEM, wherein adding pancreatin to final concentration of 0.5 μ g/ml, penicillin 100IU/ml, the μ of streptomysin 100
G/ml, the μ g/ml of amphotericin B 2.5.EMEM culture mediums (high glycoform) formula is shown in Table 1.
The EMEM culture medium prescriptions of table 1
(1) design of primers
According to the bovine rota VP4 gene order (gene accession numbers disclosed in GenBank：JF693062.1), utilize
Oligo7.0 and DNAStar software Design primers, primer sequence are shown in Table 2.Wherein pair of primers VP8*-ab-0-F, VP8*-
The template that ab-0-R anneals as following amplification, a, b represent aa 1-11 and aa 218-235 respectively), italic dashed part is enzyme
Enzyme site sequence.
The primer sequence of table 2
(2) rotavirus RNA extracts
The bovine rota supernatant obtained through AGMK cell culture is used to extract RNA, comprises the following steps that：Every 200 μ L
500 μ L TRizol are added in virus liquid (cell liquid), 5 are stored at room temperature after fully vibrating 1min (being vibrated manually with prestissimo)
~10min；400 μ L chloroforms are added, static 5~10min after as above vibrating, 4 DEG C of 12000r/min centrifuge 15min；Take upper water
Mutually be placed in new EP pipes, add 1/2TRizol volumes isopropanol, be stored at room temperature 5~10min (or -20 DEG C place 2~3h,
Or -70 DEG C of placement more than 30min) precipitated, 4 DEG C of 12000r/min centrifuge 15min；Supernatant is abandoned, RNA precipitate is in super-clean bench
Natural drying at room temperature 15min or so；RNA precipitate is dissolved with water of the 10 μ L without RNase.
(3) the RT-PCR amplifications of the full-length gene of rotavirus vp 4
Reverse transcription system (20 μ L systems) is as follows：The μ L of downstream universal primer (20 μM) 0.5；The μ L of RNA templates 5.5；Be free of
The RNase μ L of water 4；The 2.5mM μ L of dNTP 4；The μ L (20u) of RNase inhibitor 0.5；The μ L (40u) of M-MuLV reverse transcriptase 1；5×
The μ L of reaction buffer 4.Comprise the following steps that：RNA and primer are firstly added, and DMSO is added (about by the 10% of their cumulative volumes
0.6 μ L), 95 DEG C of 3min are denatured double-stranded RNA, place 2min coolings on ice；DNTP, RNase inhibitor and not is added after cooling
Water containing RNase, 37 DEG C of 3~5min of incubation, places 2min coolings on ice；M-MuLV reverse transcriptase is added after cooling, 37 DEG C incubate
Educate 2h；Last 70 DEG C of incubations 10min terminating reactions, place 2min, are placed in -20 DEG C or -70 DEG C and save backup on ice.PCR reacts
System is following (25 μ L systems)：Each 1 μ L of primer (20 μM)；The μ L of cDNA templates 1；The 2.5mM μ L of dNTP 8；Pfu archaeal dna polymerases
0.25μL(1.25u)；10×pfu buffer 2.5μL；ddH2O 6.25μL.Reaction condition is as follows：95 DEG C of 3min pre-degenerations；
95 DEG C of denaturation 40s, 50 DEG C of annealing 40s, 72 DEG C of extension 2.5min, 30 circulate；72 DEG C of extension 10min.The total length that will be expanded
Target gene identifies (Fig. 1) with 1% agarose gel electrophoresis, and blend compounds QIAquick Gel Extraction Kit (Promega, A9303) is returned
Receive purifying.Product recovery purifying step is as follows：After agarose gel electrophoresis terminates, target DNA band is cut, is placed in 1.5mL
In centrifuge tube；Per 10mg glue in add 10 μ L film combination liquid, in 50~60 DEG C be incubated until agarose be completely dissolved (if
It is PCR primer, then is directly added into isometric film combination liquid in PCR primer；Adsorption column is inserted in collecting pipe；Solution shifts
Into adsorption column, room temperature places about 1min；16000 × g centrifuges 1min, discards collected waste liquid, and adsorption column is inserted again
Into collecting pipe；700 μ L films washing lotions (plus ethanol) are added, 16000 × g centrifugation 1min, discard collected waste liquid, and will inhale
Attached column is inserted into collecting pipe again；It is repeated once with 500 μ L film washing lotion, 16000 × g centrifugations 5min；Discard in collecting pipe
Waste liquid, 1min is centrifuged again；Adsorption column is transferred in a new 1.5mL centrifuge tube；50 μ L are added into adsorption column to be free of
The water of nuclease, room temperature place 1min, 16000 × g centrifugations 1min；Adsorption column is discarded, DNA is stored in -20 DEG C.
(4) structure of pMD18-T-Bo-VP4 carriers
Take the VP4 full-length genes PCR primer of above-mentioned glue reclaim kit recovery to be attached with pMD18-T carriers, connect
System (10 μ L systems) is as follows：The μ L of VP4 full-length genes PCR primer 4.5；The μ L of pMD18-T carriers 0.5；Solution I connections are slow
The μ L of fliud flushing 5, mix, 16 DEG C of connections overnight after brief centrifugation.Take out the competent cell one deposited in -80 DEG C of refrigerators and manage (100 μ
L/ is managed) it is placed in mixture of ice and water, 10 μ L connection products are added when competent cell almost melts completely, gently pressure-vaccum mixes
After be positioned over 30min in mixture of ice and water；2~3min in mixture of ice and water is placed in after 42 DEG C of heat shock 90s；Xiang Guanzhong is added about
1mL culture mediums, 200rpm, 37 DEG C of shaken cultivation 45min~1h；Suctioned out residue after 5000rpm centrifugations 3min after about 1mL supernatants
Thalline and supernatant mix coated plate (kalamycin resistance flat board), and 12h or so is cultivated for 37 DEG C after air-drying.Plasmid extraction step is as follows：
Single bacterium colony in super-clean bench on picking flat board is connected in test tube, 37 DEG C, 200r/min shake bacterium overnight；Connect bacterium solution 2mL is taken,
12000rpm centrifugations 1min collects thalline, discards supernatant, residual solution is exhausted with pipette tips；The μ L of Solution I 100 are added, by bacterium
Liquid is resuspended, whirlpool mixes；The μ L of Solution II 200 are added, reverse 4~5 times, ice bath 5min gentle immediately；Add
The μ L of Solution III 150, leniently turn upside down 6~8 times, ice bath 5min；12000rpm centrifuges 15min；Supernatant is suctioned out
It is transferred in a new 1.5mL centrifuge tube, two volumes absolute ethyl alcohol, mixing of turning upside down is added into centrifuge tube；12000rpm
5min is centrifuged, abandons supernatant, precipitation is washed with the ethanol of 1mL 75%, 12000rpm centrifugation 3min, abandons supernatant；Repeated washing step two
It is secondary, it is deposited in drying at room temperature 10min；Precipitation is dissolved with 20 μ L TER, -20 DEG C preserve institute's upgrading grain.Using on pMT18-T
Restriction enzyme site BamHI cuts extracted plasmid, and to identify, whether extraction plasmid is successfully connected on pMT18-T, single endonuclease digestion body
System is as follows：The μ L of recombinant plasmid 6.6；buffer Bam H I 1.0μL；Bam H I 0.5μL；ddH2The μ L of O 1.9, mix, be instantaneous
37 DEG C of water-bath 2h after centrifugation, the identification of 0.8% agarose gel electrophoresis.The bacterium solution that single endonuclease digestion identifies correct recombinant plasmid is forwarded to
Bacterium is shaken in fresh LB overnight, Beijing Hua Da gene sequencing is delivered to after 25% glycerine of next day addition.
(5) clone of single copy gene and multicopy mosaic gene
VP8*-ab amplification：Reaction system is as follows：Each μ L of 3 μ L, dNTP 2 of primer VP8*-ab-0F, VP8*-ab-0R
(2.5mM), pfu archaeal dna polymerases 0.2 μ L, 10 × pfu Buffer (contain MgSO4) 2.5 μ L, ddH2O supplements system to 25 μ L.Instead
The condition is answered to be：95 DEG C of pre-degeneration 3min；95 DEG C of 40s, 70 DEG C of 40s, 72 DEG C of 30s, 7 circulations；72 DEG C of extension 10min.
Introduce the VP8*-ab amplifications of restriction enzyme site：With primer VP8*-ab-1-F and VP8*-ab-1-R, VP8*-ab-2-F
Enter performing PCR respectively with VP8*-ab-2-R, VP8*-ab-3-F and VP8*-ab-3-R to expand three sections of VP8*-ab-1, VP8*-ab-
2 and VP8*-ab-3 genetic fragments, reaction system are as follows：10 × pfu Buffer (contain MgSO4)2.5μL；dNTP(2.5mM)2μ
L；Each 0.5 μ L of primer (20 μM)；The μ L of template DNA 1；The μ L of pfu archaeal dna polymerases 0.2；ddH2O 18.3μL.Reaction condition is：95
DEG C pre-degeneration 3min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C of extension 10min.
VP8*-a amplification：Directly this purpose fragment, annealing temperature are expanded with two primer VP8*-a-F, VP8*-a-R annealing
Spend for 50.5 DEG C, remaining is same as above.
The amplification of VP8* full length fragments：Using the recombinant plasmid pMD18-T-Bo-VP4 of preservation as template, VP8*-F, VP8*-R
Expanded for primer, annealing temperature is 55 DEG C, and 72 DEG C of extension 100s, remaining is ibid (PCR amplifications are as shown in Figure 2).
(6) clone of the full-length gene of rotavirus vp 8
The pMD18-T-Bo-VP4 plasmids for taking above-mentioned structure are template, using primer VP8*-F and VP8*-R as amplification VP8* bases
Because fragment, reaction system are following (25 μ L)：10×pfu buffer2.5μL；dNTP(2.5mM)2μL；Primer (20 μM) each 1.0 μ
L；The μ L of pfu archaeal dna polymerases 0.2；ddH2O 18.3μL.PCR reaction conditions are：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 40s, 55
DEG C annealing 40s, 72 DEG C extension 100s, 30 circulation；72 DEG C of extension 10min.Amplified production carries out 1.2% agarose gel electrophoresis
Identify (Fig. 3).The recovery of glue reclaim kit, purified pcr product, -20 DEG C of preservations.
(7) connection and identification of PCR primer and carrier
PCR primer utilizes corresponding restriction enzymes double zyme cutting to carrier, and glue reclaim kit reclaims purpose fragment,
Conversion is coated on the LB containing corresponding resistant and put down into E.coli DH5 α competent cells after the connection overnight of 4 DEG C of T4DNA ligases
Plate, 37 DEG C of overnight incubations.Next day picking single bacterium colony is inoculated in the LB fluid nutrient mediums containing corresponding resistant, after 37 DEG C are shaken bacterium 12h
Extract plasmid.PCR (entering performing PCR with universal primer T7) identifies positive plasmid, progressively clones the 2nd section and the 3rd section of gene.Finally weigh
Group positive plasmid is named as pET28a-VP8*- (ab)3, deliver to Beijing six directions Hua Da Gene Tech. Company Limited and carry out sequencing mirror
It is fixed.Other 3 kinds of recombinant plasmids are built with method, and wherein VP8*-ab, VP8*-a used carrier is pET32a (+), total length VP8* fragments
Used carrier is pET28a (+).Positive recombinant plasmid is named as pET32a-VP8*-ab, pET32a-VP8*-a and pET28a-
VP8*。
(8)P2-VP8*-(ab)3With P2-VP8*-ab gene clonings
With constructed recombinant plasmid pET28a-VP8*- (ab)3For template, with P2-VP8*- (ab)3- F and VP8*-ab-
3-R is that primer expands P2-VP8*- (ab)3；Using constructed recombinant plasmid pET32a-VP8*-ab as template, with P2-VP8*-
(ab)3- F and VP8*-ab-2-R is that primer expands P2-VP8*-ab.Reaction system is following (25 μ L)：10×pfu buffer
2.5 μ L, dNTP (2.5mM) 2 μ L, each 1.0 μ L, pfu archaeal dna polymerase 0.2 μ L, ddH of primer (20 μM)2O 18.3μL.PCR expands
Increasing purpose fragment reaction condition is：95 DEG C of pre-degeneration 3min；95 DEG C of denaturation 30s, 55 DEG C (P2-VP8* (ab) 3) or 60 DEG C of (P2-
VP8*-ab) anneal 30s, 72 DEG C of extension 30s, 30 circulations；72 DEG C of extension 10min.Amplified production carries out 1.2% agarose and coagulated
Gel electrophoresis identify (Fig. 4).The recovery of glue reclaim kit, purified pcr product, -20 DEG C of preservations.
(9)P2-VP8*-(ab)3With the structure of P2-VP8*-ab recombinant plasmids
PCR primer P2-VP8*- (ab)3, P2-VP8*-ab and carrier pET28a/pET32a be using in Nco I and Xho I
Enzyme cutting double digestion, glue reclaim kit recovery purpose fragment, converted after the connection overnight of 4 DEG C of T4DNA ligases to E.coli DH5 α
In competent cell, the LB flat boards containing corresponding resistant, 37 DEG C of overnight incubations are coated on.Next day picking single bacterium colony is inoculated in containing corresponding
In the LB fluid nutrient mediums of resistance, 37 DEG C shake bacterium 12h after extract plasmid.PCR (entering performing PCR with universal primer T7) identifies positive matter
Grain, progressively clone the 2nd section and the 3rd section of gene (bacterium colony PCR qualification results are as shown in Figure 5).Finally restructuring positive plasmid is named as
pET28a-P2-VP8*-(ab)3With pET32a-P2-VP8*- (ab), recombinant plasmid entrusts Beijing six directions Hua Da Gene science to have
Limit company carries out sequencing identification.
The bovine rota VP8* subunits of embodiment 2 recombinate the expression of chimeric protein
Using heat shock, by expression plasmid pET28a-P2-VP8*- (ab)3、pET28a-P2-VP8*-ab、pET28a-
VP8*-(ab)3, pET32a-VP8*-ab, pET32a-VP8*-a and pET28a-VP8* be transformed into E.coli BL21 (DE3) sense
In by state cell.Picking monoclonal is inoculated in containing 50 μ g/ml kanamycins (pET28a carriers) or 50 μ g/ml ammonia from agar plate
(2% glucose, 1% ethanol and 2% glycerine are added in the LB fluid nutrient mediums of benzyl mycin (pET32a carriers)).When under 600nm
When absorbance is up to 0.5, IPTG to its final concentration of 0.5mM, 18 DEG C of overnight induction expression proteins are added.Then 4 DEG C of 10000g
Centrifuge 15min and collect restructuring E.coli cells, -80 DEG C of storages standby (recombinant protein SDS-PAGE electrophoresis results such as Fig. 6 A and figure
Shown in 6B, P2-VP8*- (ab)3Amino acid sequence as shown in SEQ ID No.4).
The Western blot detections of the recombinant protein of embodiment 3
After 15%SDS-PAGE electrophoresis, electricity is gone on pvdf membrane the recombinant protein of expression, with the PBST containing 5% skimmed milk
37 DEG C of closing 1h, PBST is washed 3 times, with 1:4 DEG C of overnight incubations of anti-His-tag monoclonal antibodies of 2000 times of dilutions, PBST are washed 3 times,
With 1:37 DEG C of incubation 1h of sheep anti-mouse igg of 5000 times of dilution HRP marks；PBST is washed 3 times, DAB colour developings (Fig. 7).
The purifying of the recombinant protein of embodiment 4
Destroyed with the BugBuster Master Mix (Novagen) containing protease inhibitor cocktail (Roche)
The cell membrane of E.Coli cells, or it is standby with ultrasonic fragmentation, collection soluble product, -80 DEG C of storages.Use ProBond
Nickel-NTA agar affinity chromatography (Invitrogen), every kind of albumen is purified to step according to institute of manufacturer.With containing not
With concentration imidazoles (20-100mM) elution buffer wash away cell protein after, 250mM imidazoles wash-out recombinant proteins.SDS-PAGE
The purity of recombinant protein is analyzed, then removes imidazoles with super filter tube (Millipore).According to BCA quantification kits (Thermo)
Manufacturer illustrates, with the recombinant protein concentration of BCA quantification kits determination after purification.Recombinant protein after purification is in -80 DEG C of preservations
Standby (yield of every kind of recombinant protein can reach 20mg/L).Step is given according to manufacturer, uses ToxinsensorTM
Level of endotoxin in the albumen of chromogenic LAL endotoxins detection kits (GenScript) detection after purification.As a result show
Show, level of endotoxin is in 1.8-2.0EU/ml or so, and it is well below the maximum allowable level of endotoxin of recombinant subunit vaccine
(＜ 20EU/ml) (Fig. 8).
Immunogenicity between 5 different immunogenes of embodiment
1st, mouse immune is tested
The female Balb/c mouse of 6~8 week old are grouped at random, every group 10 carry out intramuscular injection and are immunized.Control group is
PBS+ aluminum hydroxide adjuvants, other 9 groups are experimental group, with P2-VP8*- (ab)3、P2-VP8*-ab、VP8*-(ab)3、VP8*-
Totally 6 kinds of recombinant proteins emulsify mixed immunity animal with aluminum hydroxide adjuvant respectively by ab, VP8*-a, VP8*, and every mouse injects 20 μ
The μ g aluminum hydroxide adjuvants of g albumen+600, insufficient volume with PBS polishings；14d enters to mouse after immune preceding and final immunization every time
End of line venous blood collection, separation serum preserve standby inspection in -20 DEG C.
2nd, the detection of antibody level
Antigen coat concentration and secondary antibody dilution factor are optimized first, to determine optimal antigen coat concentration and secondary antibody work
Make concentration.Then the mice serum sample gathered is detected with the antigen coat concentration and secondary antibody dilution factor that have optimized,
Detect antibody level of serum caused by each immunized mice.The specified operational procedure of indirect ELISA is as follows：With 0.05M pH
VP8* albumen is diluted by 9.6 carbonate coating buffer solution, and 96 hole ELISA reaction plates, 4 DEG C of mistakes are coated with every μ L of hole 100
Night is coated with, and is washed 3 times with PBST after coating, and confining liquid (PBST for containing 5% skimmed milk) is added after patting dry, per the μ L of hole 200,37 DEG C of envelopes
Close 2h.Washed after closing with PBST after patting dry for 3 times, serum to be checked carried out after doubling dilution in adding hole, at the same set it is negative right
According to hole, per hole 100 μ L, 37 DEG C of incubation 1h.The HRP diluted after patting dry plus with the PBST containing 5% skimmed milk is washed 3 times with PBST
The sheep anti-mouse igg antibody of (horseradish peroxidase) mark, 37 DEG C of incubation 1h, PBST is washed pat dry for 3 times after plus tmb substrate colour developing
Liquid, room temperature lucifuge effect 15min.Add 2mol/L sulfuric acid terminating reactions, ELIASA detection OD450Value.It is more than or equal to P/N values
2.1 be positive threshold value.P2-VP8*-(ab)33 times it is immune after induction of splendid immune response, its antibody titer be higher than total length
VP8* (P ＜ 0.05).VP8*-(ab)3, two groups of serum titers of VP8*-ab compared to showing, the multicopies of mosaic epitopes repeats aobvious
Improve the immunogenicity (P ＜ 0.05) of antigen with writing.Two groups of P2-VP8*-ab, VP8*-ab relatively show, T cell antigen table
The introducing of position considerably improves immune response horizontal (P ＜ 0.01) (the ELISA measure serum antibody titer comparative results of antigen
As shown in Figure 9).
The neutralization test of the recombinant protein of embodiment 6
Using the outbreeding system Female guinea pigs of 500-550g/ only, every group 4, it is randomly assigned.Cavy intramuscular injection is given every two weeks
(IM) it is immunized once, P2-VP8*- (ab)3、P2-VP8*-ab、VP8*-(ab)3, VP8*-ab, VP8* every time be immunized 20 μ g (phosphorate
Sour aluminium (AP) adjuvant) (every dose contains 100 μ g aluminium), it is immunized 3 times altogether, final immunization is taken a blood sample after 7 days.
Reduced by 60% plaque and neutralize the neutralizing antibody titers that (PRN) experiment determines every part of serum sample.0.25ml
G6P [5] types or G10P [11] type rotavirus are with after the serum mixing 1h of isometric doubling dilution, adding in 6 orifice plates, 37 DEG C
1h is adsorbed, adds 3ml agaroses (containing MEM, 0.5 μ g/ml pancreatin), after agarose solidification, 37 DEG C are inverted culture.After 4d, paving
2nd layer of agarose (containing 2% dimethyl diaminophenazine chloride), Virus plaque is calculated by serum suppression up to 60% dilution factor, calculates in serum and imitates
Valency.
The P2-VP8*- of table 3 (ab)3、P2-VP8*-ab、VP8*-(ab)3, VP8*-ab, VP8* neutralizing antibody it is horizontal
Note：a60% bites plaque reduction neutralization test serum antibody titer (inverse)
From table 3 it can be seen that VP8*- (ab)3The neutralizing antibody titers that restructuring mosaic epitopes vaccine is induced are significantly higher than
The neutralizing antibody titers that VP8*-ab is induced, but it is less than VP8* holoproteins, with being expected unanimously.Tetanus toxin t cell epitope is more
Peptide P2 introducing can significantly increase VP8*- (ab)3The immune efficacy of mosaic epitopes vaccine is recombinated, higher than VP8* holoproteins
The neutralizing antibody induced is horizontal.Can be with the anti-P [5] of induced high titers and P [11] genotype special rotavirus and anti-
Body, it is suitable for preparing bovine rotavirus vaccine.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. bovine rota VP8* subunits recombinate chimeric protein, it is characterised in that it is more by tetanus toxin t cell epitope
Peptide P2 and genotype are fitted together to what is formed for the clipped form a and clipped form b of G6P [5] bovine rota VP8* subunits
Recombinant protein；Wherein, without additional amino acid between clipped form a and clipped form b, and it is multicopy, it is thin in tetanus toxin T
The clipped form a and truncation shape for the bovine rota VP8* subunits that born of the same parents' epitope polypeptide P2, the genotype of multicopy are G6P [5]
Connected between formula b by flexible Linker；
Wherein, the tetanus toxin t cell epitope polypeptide P2, genotype are G6P [5] bovine rota VP8* subunits
Clipped form a and clipped form b amino acid sequence are respectively as shown in SEQ ID No.1-3.
2. restructuring chimeric protein according to claim 1, it is characterised in that its amino acid sequence such as SEQ ID No.4 institutes
Show.
3. encode the gene that chimeric protein is recombinated described in claim 2.
4. the expression vector containing gene described in claim 3.
5. the host cell containing gene described in claim 3.
6. prepare the method for the restructuring chimeric protein of claim 1 or 2, it is characterised in that by the expression described in claim 4
Carrier is converted into competent escherichia coli cell, and screening positive clone and being inoculated in LB fluid nutrient mediums is cultivated, and is added
IPTG induced expressions obtain restructuring chimeric protein.
7. according to the method for claim 6, it is characterised in that also add 0.02g/mL grapes in the LB fluid nutrient mediums
Sugar, 1~5v/v% ethanol and 2% glycerine.
8. according to the method for claim 6, it is characterised in that positive colony is inoculated in LB fluid nutrient mediums cultivate to
OD600=0.5, IPTG to final concentration of 0.5mM is added, induced expression recombinates chimeric protein at 18 DEG C.
9. application of the restructuring chimeric protein in bovine rotavirus vaccine is prepared described in claim 1 or 2.
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