CN104479028A - Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof - Google Patents
Bovine rotavirus VP8* subunit recombinant chimeric protein and application thereof Download PDFInfo
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Abstract
The invention provides a bovine rotavirus VP8* subunit recombinant chimeric protein. According to the bovine rotavirus VP8* subunit recombinant chimeric protein, a T cell epitope polypeptide P2 in a tetanus toxin is introduced into an epitope vaccine of a bovine rotavirus VP8* subunit multi-copy chimeric gene recombinant protein, so that the immune efficacy of the epitope vaccine can be greatly enhanced, cross neutralizing immune protection can be induced, higher neutralizing antibody titer can be induced, and a high-titer anti-P[5] and P[11] genotype-specific rotavirus neutralizing antibody can be induced. The bovine rotavirus VP8* subunit recombinant chimeric protein provided by the invention can be used for preventing and controlling both G6 and G10 type rotaviruses which are mainly pandemic within a world range, and is suitable for preparing a safe and low-cost bovine rotavirus vaccine; in addition, the bovine rotavirus vaccine can be used for effectively reducing the economic loss brought by rotavirus gastroenteritis to cattle rearing by being matched with other rotavirus vaccines researched and developed at present, and has wide market prospects.
Description
Technical field
The invention belongs to molecular biology and genetically engineered field, specifically, relate to bovine rota VP8* subunit restructuring chimeric protein and application thereof.
Background technology
Rotavirus is Reoviridae, rotavirus member, is the main pathogens causing multiple new born animal and infant's gastro-enteritis.Bovine rota diarrhoea, apocleisis, diarrhoea depressed with spirit, dehydration are principal character, and the most easy infection of calf within 7 ages in days, sickness rate can up to 90% ~ 100%, and mortality ratio can reach 10% ~ 50%.If secondary e. coli septicemia, mortality ratio can be caused to rise further, the economic benefit of serious harm 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 all can induce neutralizing antibody independently.Research shows, VP4 albumen has various functions, as viral hemagglutinin, absorption and cell invade, virulence is relevant.VP4 albumen is cracked into VP8* (28kDa, aa 1 ~ 240) and VP5* (60kDa, aa 247-775) Liang Zhong subunit through pancreatin, wherein VP8* has the Main Antigenic of VP4 albumen, and determines VP4 albumen serological specificity neutralization reaction.
There is no special effect medicine therapeutic rotaviral gastroenteritis at present, vaccine inoculation is the major measure of this disease of prevention.The immune me chanism of rotavirus is mainly through immune Pregnant cows, and new-born calve obtains natural passive immunity by colostrum.Widespread use inactivated vaccine carries out the immune me chanism of rotavirus all the time, but inactivated vaccine not always safety, often there is serious side reaction.And rotavirus amplification efficiency in the MA104 clone of monkey source in ox source is lower, virus titer is lower, generally can only reach 10
4~ 10
5cfu/ml, does not reach the required virus titer of inactivated vaccine far away, must concentrated and purifying after cultivating, causes the increase of production cost.So different research group attempts DNA vaccination, virus sample particle vaccines, plant edible vaccine in world wide.But there is the shortcoming such as complicated process of preparation, production cost height in these vaccines, being not suitable for commercialized vaccine produces, and only rests on the laboratory study stage at present.Sheep rotavirus LLR-85 strain (G10P [15]) VP7 gene is replaced with ox source G6 type VP7 gene and then develops the weak malicious candidate vaccine of bovine rota reprovision in power seminar by Harbin Veterinary Medicine Inst., China Academy of Agriculture, but any rotavirus living vaccine all exists the problem of toxin expelling.The more important thing is, living vaccine can not be used for Pregnant cows immunity.Therefore urgently research and develop safety, efficient novel bovine rotavirus vaccine is used for anti-Bovine Rotavirus Diarrhea processed.
In recent years, the use of epiposition vaccine is more and more extensive, and it utilizes engineered means, the epi-position of vivoexpression or synthetic pathogenic micro-organism, it can be used as a kind of vaccine to use.Epiposition vaccine can overcome fall apart malicious risk and living vaccine of traditional vaccine exactly relative to maximum advantage traditional vaccine can not be used for the defects such as pregnant animal.On people Wa strain rotavirus vp 8*, identify 5 epitopes, wherein 3 is linear neutralizing epitope (aa 1-10, aa55-66 and aa 223-234), and aa 1-10 epi-position high conservative between multiple strain, significant to the Rotavirus Vaccine of the anti-multiple strain of development or serotype.
The combination of rotavirus gene type is numerous, and the cross immunity between different genotype is very few, and this brings difficulty to vaccine development.But research discovery P genotype may cover several G genotype, and namely certain P type can combine from different G types, and same P type can induce the heterotypic immunity of anti-different G type in theory.And determine that the VP4 albumen of P type contains multiple linearizing epitope, and this albumen is without the need to posttranslational modification, the VP8* fragment that it is formed after pancreatin cracking, contain VP4 type specificity Main Antigenic, be all linearly and comparatively conservative, prompting can utilize VP8* albumen to carry out development of new PRV subunit vaccine.
The bovine rota G genotype of world-wide prevalence is mainly G6 and G10 two kinds, and its P genotype is mainly P [5] and P [11], in addition also there is P [1], P [21] and P [29].Conventional vaccine development is mainly developed bivalent vaccine and is prevented and treated G6P [5] and G10P [11] rotavirus strain simultaneously, and this gives vaccine development and produce and brings certain difficulty, is doomed to cause production of vaccine cost to improve.
Summary of the invention
The object of this invention is to provide a kind of novel bovine rota VP8* subunit restructuring chimeric protein and application thereof.
In order to realize the object of the invention, bovine rota VP8* subunit of the present invention restructuring chimeric protein, it is the recombinant protein that the clipped form a of the bovine rota VP8* subunit of G6P [5] and clipped form b are fitted together to by tetanus toxin t cell epitope polypeptide P2 and genotype; Wherein, without additional amino acid between clipped form a and clipped form b, and be multiple copied, be connected by flexible Linker between the clipped form a of the bovine rota VP8* subunit of G6P [5] and clipped form b in the genotype of tetanus toxin t cell epitope polypeptide P2, multiple copied.
Wherein, the clipped form a of described tetanus toxin t cell epitope polypeptide P2, the genotype bovine rota VP8* subunit that is G6P [5] and the aminoacid sequence of clipped form b are respectively as shown in SEQ ID No.1-3.
The present invention also provides the gene of above-mentioned restructuring chimeric protein of encoding, and its nucleotide sequence is as shown in SEQ ID No.5.
The present invention also provides expression vector, host cell and engineering bacteria containing the above-mentioned restructuring chimeric protein gene of coding.
The present invention also provides the method preparing above-mentioned restructuring chimeric protein, expression vector containing the above-mentioned restructuring chimeric protein gene of coding is converted in competent escherichia coli cell, screening positive clone being inoculated in LB liquid nutrient medium is cultivated, and adds IPTG abduction delivering and obtains restructuring chimeric protein.
Wherein, 0.02g/mL glucose, 1 ~ 5v/v% ethanol and 2% glycerine is also added in described LB liquid nutrient medium.
Particularly, positive colony is inoculated in above-mentioned LB liquid nutrient medium and is cultured to OD
600about 0.5, adding IPTG to final concentration is 0.5mM, abduction delivering restructuring chimeric protein at 18 DEG C.
The present invention further provides described bovine rota VP8* subunit restructuring chimeric protein and prepare the application in bovine rotavirus vaccine.
T cell epitope polypeptide P2 (the 830-844 amino acids of tetanus toxin TT) in tetanus toxin introduces in bovine rota VP8* subunit multiple copied mosaic gene recombinant protein epiposition vaccine by the present invention; greatly can improve the immune efficacy of epiposition vaccine; and cross neutralization immunoprotection can be induced; and the higher NAT of induction, and can the anti-P [5] of induced high titers and the special rotavirus neutralizing antibody of P [11] genotype.Utilize bovine rota VP8* subunit of the present invention restructuring chimeric protein can prevent and treat G6 and the G10 type rotavirus of Major Epidemic in world wide simultaneously, be applicable to prepare safety, bovine rotavirus vaccine with low cost, the Rotavirus Vaccine researched and developed at present with other coordinates, effectively can reduce rotavirus gastroenteritis to the financial loss of cattle-raising, there are wide market outlook.
Accompanying drawing explanation
Fig. 1 is VP4RT-PCR electrophorogram in the embodiment of the present invention 1; Wherein, M:DL2000Marker; The PCR primer of 1:Bo-VP4 full genome; 2: negative control.
Fig. 2 is annealing amplification VP8*-ab template and VP8*-a in the embodiment of the present invention 1; Wherein, M:DL2000Marker; The PCR primer of 1:VP8*-ab gene; The PCR primer of 2:VP8*-a gene; 3: negative control.
Fig. 3 is total length VP8* gene PCR amplification in the embodiment of the present invention 1; Wherein, M:DL2000Marker; 1:VP8*PCR amplified production; 2: negative control.
Fig. 4 is P2-VP8*-(ab) in the embodiment of the present invention 1
3with P2-VP8*-ab pcr amplification product electrophoresis result; Wherein, M:DL2000Marker; 1:P2-VP8*-(ab)
3pcr amplification product; 2:P2-VP8*-ab pcr amplification product; 3: negative control.
Fig. 5 is that in the embodiment of the present invention 1, bacterium liquid PCR identifies pET28a-P2-VP8*-(ab) 3 and pET32a-P2-VP8*-ab result; Wherein, M:DL2000Marker; 1:pET28a-VP8*-(ab)
3t7PCR product; 2:pET28a-P2-VP8*-(ab)
3t7PCR product; 3:pET32a-VP8*-ab T7PCR product; 4:pET32a-P2-VP8*-ab T7PCR product; 5: negative control.
Fig. 6 is the expression of results of recombinant protein in intestinal bacteria in the embodiment of the present invention 2; Wherein, in figure A, M: protein standard; 1,2,3 pET-28a-VP8*-(ab) is respectively
3before induction, on whole cell, the rear cellular lysate of induction, after cleer and peaceful induction, cellular lysate precipitates; 4,5,6 cellular lysate precipitation after cleer and peaceful induction is respectively after whole cell before pET-32a-VP8*-ab induction, induction on cellular lysate; 7,8,9 cellular lysate precipitation after cleer and peaceful induction is respectively after whole cell before pET-32a-VP8*-a induction, induction on cellular lysate; 10,11 pET-28a-BL21 (DE3), the rear whole cell whole cell of pET-32a (+)-BL21 (DE3) empty carrier recombinant bacterium induction is respectively; In figure B, M: protein standard; 1,2,3 pET-28a-P2-VP8*-(ab) is respectively
3before induction, on whole cell, the rear cellular lysate of induction, after cleer and peaceful induction, cellular lysate precipitates; 4,5,6 cellular lysate precipitation after cleer and peaceful induction is respectively after whole cell before pET-32a-P2-VP8*-ab induction, induction on cellular lysate; 7,8 the rear whole cell of pET-28a (+) induction and the rear whole cell of pET-32a (+) induction is respectively; 9,10,11 cellular lysate precipitation after cleer and peaceful induction is respectively after whole cell before pET-28a-VP8* induction, induction on cellular lysate.
Fig. 7 is the Western blot analytical results of recombinant protein in the embodiment of the present invention 3; Wherein, M: protein standard; 1: restructuring VP8*-(ab)
3albumen; 2: restructuring VP8*-ab albumen; 3: restructuring VP8*-a albumen; 4: restructuring VP8* albumen; 5: restructuring P2-VP8*-(ab)
3albumen; 6: restructuring P2-VP8*-ab albumen; 7:pET-28a (+) contrasts; 8:pET-32a (+) contrasts.
Fig. 8 is the purification result of recombinant protein in the embodiment of the present invention 4; Wherein, M: protein standard; 1: restructuring VP8*-(ab)
3albumen; 2: restructuring VP8*-ab albumen; 3: restructuring VP8*-a albumen; 4: restructuring 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 5
3, P2-VP8*-ab, VP8*-(ab)
3, VP8*-ab, VP8* recombinant protein Analysis of Immunogenicity result.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's specification sheets advising.
The source of the biomaterial related in following examples: prokaryotic expression carrier pET28a is purchased from Novagen company; E.coli BL21 (DE3) is purchased from Novagen company; The primer is designed, designed and entrusts Beijing six directions Hua Da Gene Tech. Company Limited to synthesize.
The structure of the expression vector of embodiment 1 containing object fragment
Material therefor is the bovine rota of genotype G5P [6], cultivates with primary African Green Monkey kidney (AGMK) cell.Used medium is EMEM, and wherein adding pancreatin to final concentration is 0.5 μ g/ml, penicillin 100IU/ml, Streptomycin sulphate 100 μ g/ml, amphotericin B 2.5 μ g/ml.EMEM substratum (high glycoform) formula is in table 1.
Table 1 EMEM culture medium prescription
(1) design of primers
Bovine rota VP4 gene order (gene accession number: JF693062.1) disclosed in GenBank, utilize Oligo7.0 and DNAStar software design primer, primer sequence is in table 2.Wherein pair of primers VP8*-ab-0-F, VP8*-ab-0-R annealing is as the template of following amplification, and a, b represent aa 1-11 and aa 218-235 respectively), italic dashed part is restriction enzyme site sequence.
Table 2 primer sequence
(2) rotavirus RNA extracts
Through AGMK cell cultures obtain bovine rota supernatant liquor for extracting RNA, concrete steps are as follows: add 500 μ L TRizol in every 200 μ L virus liquids (enchylema), and fully vibrate 1min (manually vibrating with the utmost dispatch) standing 5 ~ 10min of room temperature afterwards; Add 400 μ L chloroforms, as above static 5 ~ 10min, 4 DEG C of centrifugal 15min of 12000r/min after vibration; Get upper strata aqueous phase and be placed in new EP pipe, add the Virahol of 1/2TRizol volume, room temperature leaves standstill 5 ~ 10min (or-20 DEG C are placed 2 ~ 3h or-70 DEG C of placement more than 30min) and precipitates, 4 DEG C of centrifugal 15min of 12000r/min; Abandon supernatant, RNA is deposited in natural drying at room temperature about 15min in super clean bench; Do not precipitate containing the water dissolution RNA of RNase with 10 μ L.
(3) the RT-PCR amplification of rotavirus vp 4 full-length gene
Reverse transcription system (20 μ L system) is as follows: downstream universal primer (20 μMs) 0.5 μ L; RNA template 5.5 μ L; Not containing the water 4 μ L of RNase; The dNTP 4 μ L of 2.5mM; RNase inhibitor 0.5 μ L (20u); M-MuLV ThermoScript II 1 μ L (40u); 5 × reaction buffer 4 μ L.Concrete steps are as follows: first add RNA and primer, and add DMSO (about 0.6 μ L) by 10% of their cumulative volumes, and 95 DEG C of 3min make double-stranded RNA sex change, place 2min cooling on ice; Add dNTP, RNase inhibitor after cooling and not containing the water of RNase, hatch 3 ~ 5min for 37 DEG C, place 2min cooling on ice; Add M-MuLV ThermoScript II after cooling, hatch 2h for 37 DEG C; Hatch 10min termination reaction, place 2min on ice for last 70 DEG C, be placed in-20 DEG C or-70 DEG C save backup.PCR reaction system following (25 μ L system): each 1 μ L (20 μMs) of primer; CDNA template 1 μ L; The dNTP 8 μ L of 2.5mM; Pfu archaeal dna polymerase 0.25 μ L (1.25u); 10 × pfu buffer 2.5 μ L; ddH
2o 6.25 μ L.Reaction conditions is as follows: 95 DEG C of 3min denaturations; 95 DEG C of sex change 40s, 50 DEG C of annealing 40s, 72 DEG C extend 2.5min, 30 circulations; 72 DEG C extend 10min.By increased total length goal gene with 1% agarose gel electrophoresis qualification (Fig. 1), blend compounds reclaim test kit (Promega, A9303) carry out recovery purifying.It is as follows that product reclaims purification step: after agarose gel electrophoresis terminates, cut by target DNA band, be placed in 1.5mL centrifuge tube; Every 10mg glue in add 10 μ L films in conjunction with liquid, hatch until agarose dissolves completely (if PCR primer, then directly add isopyknic film in conjunction with liquid in PCR primer in 50 ~ 60 DEG C; Adsorption column is inserted in collection tube; Solution is transferred in adsorption column, and room temperature places about 1min; The centrifugal 1min of 16000 × g, discards collected waste liquid, and is again inserted in collection tube by adsorption column; Add 700 μ L films washing lotion (adding ethanol), the centrifugal 1min of 16000 × g, discards collected waste liquid, and is again inserted in collection tube by adsorption column; Repeat once by the film washing lotion of 500 μ L, the centrifugal 5min of 16000 × g; Discard the waste liquid in collection tube, again centrifugal 1min; Adsorption column is transferred in a new 1.5mL centrifuge tube; In adsorption column, add 50 μ L not containing the water of nuclease, room temperature places the centrifugal 1min of 1min, 16000 × g; Discard adsorption column, DNA is stored in-20 DEG C.
(4) structure of pMD18-T-Bo-VP4 carrier
The VP4 full-length gene PCR primer of getting the recovery of above-mentioned glue recovery test kit is connected with pMD18-T carrier, and linked system (10 μ L system) is as follows: VP4 full-length gene PCR primer 4.5 μ L; PMD18-T carrier 0.5 μ L; Solution I connects damping fluid 5 μ L, 16 DEG C of connections of spending the night after mixing, brief centrifugation.Take out the competent cell one deposited in-80 DEG C of refrigerators to manage (100 μ L/ manage) and be placed in mixture of ice and water, when competent cell is close to and melts completely, add 10 μ L connect products, be positioned over 30min in mixture of ice and water after pressure-vaccum mixing gently; 42 DEG C of heat shock 90s are placed on 2 ~ 3min in mixture of ice and water; Xiang Guanzhong adds about 1mL substratum, 200rpm, 37 DEG C of shaking culture 45min ~ 1h; Thalline and supernatant mixing coated plate (kalamycin resistance is dull and stereotyped) will be remained after sucking-off is about 1mL supernatant after the centrifugal 3min of 5000rpm, cultivate about 12h for air-dry latter 37 DEG C.Plasmid extraction step is as follows: the single bacterium colony in super clean bench on picking flat board is connected in test tube, 37 DEG C, 200r/min spends the night and shake bacterium; Get the centrifugal 1min of connect bacterium liquid 2mL, 12000rpm and collect thalline, discard supernatant, residual solution rifle head exhausts; Add Solution I 100 μ L, by resuspended for bacterium liquid, whirlpool mixing; Add Solution II 200 μ L, gentleness puts upside down 4 ~ 5 times immediately, ice bath 5min; Add Solution III 150 μ L, leniently turn upside down 6 ~ 8 times, ice bath 5min; The centrifugal 15min of 12000rpm; Supernatant sucking-off is proceeded in a new 1.5mL centrifuge tube, in centrifuge tube, add two volumes dehydrated alcohol, mixing of turning upside down; The centrifugal 5min of 12000rpm, abandons supernatant, precipitation 1mL 75% washing with alcohol, and the centrifugal 3min of 12000rpm, abandons supernatant; Repeated washing step twice, is deposited in drying at room temperature 10min; With the TER dissolution precipitation of 20 μ L, preserve institute's upgrading grain for-20 DEG C.The plasmid utilizing the restriction enzyme site BamHI on pMT18-T to cut to extract with qualification extraction plasmid whether be successfully connected on pMT18-T, single endonuclease digestion system is as follows: recombinant plasmid 6.6 μ L; Buffer Bam H I 1.0 μ L; Bam H I 0.5 μ L; ddH
2o 1.9 μ L, 37 DEG C of water-bath 2h after mixing, brief centrifugation, 0.8% agarose gel electrophoresis qualification.Single endonuclease digestion is identified the bacterium liquid of correct recombinant plasmid is forwarded in fresh LB to spend the night and shake bacterium, after adding 25% glycerine next day, deliver to Beijing Hua Da gene sequencing.
(5) clone of single copy gene and multiple copied mosaic gene
The amplification of VP8*-ab: reaction system is as follows: primer VP8*-ab-0F, VP8*-ab-0R each 3 μ L, dNTP 2 μ L (2.5mM), pfu archaeal dna polymerase 0.2 μ L, 10 × pfu Buffer is (containing MgSO
4) 2.5 μ L, ddH
2o supplements system to 25 μ L.Reaction conditions is: 95 DEG C of denaturation 3min; 95 DEG C of 40s, 70 DEG C of 40s, 72 DEG C of 30s, 7 circulations; 72 DEG C extend 10min.
Introduce the VP8*-ab amplification of restriction enzyme site: carry out PCR to increase three sections of VP8*-ab-1, VP8*-ab-2 and VP8*-ab-3 gene fragments respectively with primer VP8*-ab-1-F and VP8*-ab-1-R, VP8*-ab-2-F and VP8*-ab-2-R, VP8*-ab-3-F and VP8*-ab-3-R, reaction system is as follows: 10 × pfu Buffer is (containing MgSO
4) 2.5 μ L; DNTP (2.5mM) 2 μ L; The each 0.5 μ L of primer (20 μMs); Template DNA 1 μ L; PfuDNA polysaccharase 0.2 μ L; ddH
2o 18.3 μ L.Reaction conditions is: 95 DEG C of denaturation 3min; 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C extend 10min.
The amplification of VP8*-a: directly by this object fragment of two primer VP8*-a-F, VP8*-a-R annealing amplification, annealing temperature is 50.5 DEG C, and all the other are the same.
The amplification of VP8* full length fragment: with the recombinant plasmid pMD18-T-Bo-VP4 preserved for template, VP8*-F, VP8*-R are that primer increases, and annealing temperature is 55 DEG C, and 72 DEG C extend 100s, all the other the same (pcr amplification result as shown in Figure 2).
(6) clone of rotavirus vp 8 full-length gene
The pMD18-T-Bo-VP4 plasmid getting above-mentioned structure is template, with primer VP8*-F and VP8*-R for amplification VP8* gene fragment, and reaction system following (25 μ L): 10 × pfu buffer2.5 μ L; DNTP (2.5mM) 2 μ L; The each 1.0 μ L of primer (20 μMs); Pfu archaeal dna polymerase 0.2 μ L; ddH
2o 18.3 μ L.PCR reaction conditions is: 95 DEG C of denaturation 3min; 95 DEG C of sex change 40s, 55 DEG C of annealing 40s, 72 DEG C extend 100s, 30 circulations; 72 DEG C extend 10min.Amplified production carries out 1.2% agarose gel electrophoresis qualification (Fig. 3).Glue reclaims test kit recovery, purified pcr product ,-20 DEG C of preservations.
(7) connection of PCR primer and carrier and qualification
PCR primer and carrier utilize corresponding restriction enzymes double zyme cutting, and glue reclaims test kit and reclaims object fragment, and T4DNA ligase enzyme 4 DEG C is converted in E.coli DH5 α competent cell after spending the night and connecting, and the LB coated containing corresponding resistant is dull and stereotyped, 37 DEG C of overnight incubation.Next day picking list colony inoculation in containing corresponding resistant LB liquid nutrient medium in, 37 DEG C shake bacterium 12h after extract plasmid.PCR (carrying out PCR with universal primer T7) identifies positive plasmid, progressively clones the 2nd section and the 3rd fragment gene.Finally restructuring positive plasmid called after pET28a-VP8*-(ab)
3, deliver to Beijing six directions Hua Da Gene Tech. Company Limited and carry out order-checking qualification.Other 3 kinds of recombinant plasmids build with method, and wherein VP8*-ab, VP8*-a used carrier is pET32a (+), total length VP8* fragment used carrier is pET28a (+).Positive recombinant plasmid called after pET32a-VP8*-ab, pET32a-VP8*-a and pET28a-VP8*.
(8) P2-VP8*-(ab)
3with P2-VP8*-ab gene clone
With constructed recombinant plasmid pET28a-VP8*-(ab)
3for template, with P2-VP8*-(ab)
3-F and VP8*-ab-3-R is primer amplification P2-VP8*-(ab)
3; With constructed recombinant plasmid pET32a-VP8*-ab for template, with P2-VP8*-(ab)
3-F and VP8*-ab-2-R is primer amplification P2-VP8*-ab.Reaction system all following (25 μ L): 10 × pfu buffer 2.5 μ L, dNTP (2.5mM) 2 μ L, each 1.0 μ L of primer (20 μMs), pfu archaeal dna polymerase 0.2 μ L, ddH
2o 18.3 μ L.Pcr amplification object fragment reaction conditions is: 95 DEG C of denaturation 3min; 95 DEG C of sex change 30s, 55 DEG C (P2-VP8* (ab) 3) or 60 DEG C (P2-VP8*-ab) anneal 30s, and 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 10min.Amplified production carries out 1.2% agarose gel electrophoresis qualification (Fig. 4).Glue reclaims test kit recovery, purified pcr product ,-20 DEG C of preservations.
(9) P2-VP8*-(ab)
3with the structure of P2-VP8*-ab recombinant plasmid
PCR primer P2-VP8*-(ab)
3, P2-VP8*-ab and carrier pET28a/pET32a utilizes Nco I and Xho I enzymes double zyme cutting, glue reclaims test kit and reclaims object fragment, T4DNA ligase enzyme 4 DEG C is converted in E.coli DH5 α competent cell after spending the night and connecting, and the LB coated containing corresponding resistant is dull and stereotyped, 37 DEG C of overnight incubation.Next day picking list colony inoculation in containing corresponding resistant LB liquid nutrient medium in, 37 DEG C shake bacterium 12h after extract plasmid.PCR (carrying out PCR with universal primer T7) identifies positive plasmid, progressively clones the 2nd section and the 3rd fragment gene (bacterium colony PCR qualification result as shown in Figure 5).Finally restructuring positive plasmid called after pET28a-P2-VP8*-(ab)
3with pET32a-P2-VP8*-(ab), recombinant plasmid entrusts Beijing six directions Hua Da Gene Tech. Company Limited to carry out order-checking qualification.
The expression of embodiment 2 bovine rota VP8* subunit restructuring chimeric protein
Adopt 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 in E.coli BL21 (DE3) competent cell.In the LB liquid nutrient medium that picking mono-clonal is inoculated in containing 50 μ g/ml kantlex (pET28a carrier) or 50 μ g/ml ammonia benzyls mycin (pET32a carrier) from agar plate (adding 2% glucose, 1% ethanol and 2% glycerine).When under 600nm, absorbance reaches 0.5, adding IPTG to its final concentration is 0.5mM, 18 DEG C of induction expression proteins that spend the night.Then the centrifugal 15min of 4 DEG C of 10000g collect restructuring E.coli cells ,-80 DEG C of storages (recombinant protein SDS-PAGE electrophoresis result as shown in Figure 6 A and 6 B, P2-VP8*-(ab) for subsequent use
3aminoacid sequence as shown in SEQ ID No.4).
The Western blot of embodiment 3 recombinant protein detects
The recombinant protein of expressing is after 15%SDS-PAGE electrophoresis, electricity goes on pvdf membrane, with the PBST 37 DEG C of closed 1h containing 5% skimming milk, PBST washs 3 times, anti-His-tag monoclonal antibody 4 DEG C of overnight incubation of doubly diluting with 1:2000, PBST washs 3 times, and the sheep anti-mouse igg 37 DEG C doubly diluting HRP mark with 1:5000 hatches 1h; PBST washs 3 times, and DAB develops the color (Fig. 7).
The purifying of embodiment 4 recombinant protein
With the cell walls destroying E.Coli cell containing the BugBuster Master Mix (Novagen) of protease inhibitor cocktail (Roche), or with ultrasonic fragmentation, collection soluble product ,-80 DEG C of storages are for subsequent use.With ProBond nickel-NTA agar affinity chromatography (Invitrogen), to step, purifying is carried out to often kind of albumen according to manufacturer.After washing away cell protein with the elution buffer containing different concns imidazoles (20-100mM), 250mM imidazoles wash-out recombinant protein.SDS-PAGE analyzes the purity of recombinant protein, then uses super filter tube (Millipore) to remove imidazoles.Illustrate according to BCA quantification kit (Thermo) manufacturer, the recombinant protein concentration after purifying with the determination of BCA quantification kit.Recombinant protein after purifying saves backup (output of often kind of recombinant protein can reach 20mg/L) in-80 DEG C.According to manufacturer give step, use Toxinsensor
tMchromogenic LAL intracellular toxin detection kit (GenScript) detects level of endotoxin in the albumen after purifying.Result shows, and level of endotoxin is all at about 1.8-2.0EU/ml, and it is well below the maximum permission level of endotoxin of recombinant subunit vaccine (< 20EU/ml) (Fig. 8).
Immunogenicity between the different immunogen of embodiment 5
1, mouse immune experiment
By the female Balb/c mouse random packet in 6 ~ 8 week age, often organize 10 and carry out intramuscular injection immunity.Control group is PBS+ aluminum hydroxide adjuvant, and other 9 groups is experimental group, with P2-VP8*-(ab)
3, P2-VP8*-ab, VP8*-(ab)
3, VP8*-ab, VP8*-a, VP8* totally 6 kinds of recombinant proteins respectively with aluminum hydroxide adjuvant emulsification mixed immunity animal, every injected in mice 20 μ g albumen+600 μ g aluminum hydroxide adjuvant, not enough volume with PBS polishing; 14d carries out tail vein blood to mouse before each immunity and after final immunization, and separation of serum preserves standby inspection in-20 DEG C.
2, the detection of antibody horizontal
First antigen coated concentration and two anti-extent of dilution are optimized, to determine best antigen coated concentration and two anti-working concentrations.Then with the antigen coated concentration optimized and two anti-extent of dilution, gathered mice serum sample is detected, detect the antibody level of serum that each immunized mice produces.The specified operational procedure of indirect ELISA is as follows: be buffered liquid with the carbonate bag of 0.05M pH 9.6 and diluted by VP8* albumen, wrap by 96 hole ELISA Sptting plates with every hole 100 μ L, 4 DEG C of bag quilts that spend the night, wrap by after wash 3 times with PBST, confining liquid (PBST containing 5% skimming milk) is added after patting dry, every hole 200 μ L, 37 DEG C of closed 2h.After washing pat dry for 3 times with PBST after closing, add in hand-hole after serum to be checked is carried out doubling dilution, establish negative control hole simultaneously, every hole 100 μ L, hatches 1h for 37 DEG C.With PBST wash pat dry for 3 times after add with containing 5% the sheep anti-mouse igg antibody that marks of HRP (horseradish peroxidase) of PBST dilution of skimming milk, hatch 1h for 37 DEG C, PBST adds tmb substrate nitrite ion after washing and patting dry for 3 times, room temperature lucifuge effect 15min.Add 2mol/L sulfuric acid termination reaction, microplate reader detects OD
450value.2.1 are more than or equal to for positive threshold value with P/N value.P2-VP8*-(ab)
3induction of splendid immunne response after 3 immunity, its antibody titers is higher than (the P < 0.05) of total length VP8*.VP8*-(ab)
3, VP8*-ab two groups of serum titers compare and show, the multiple copied of mosaic epitopes repeats the immunogenicity (P < 0.05) considerably improving antigen.P2-VP8*-ab, VP8*-ab two groups compares and shows, the introducing of T cell antigen epi-position considerably improves the immunne response level of antigen (P < 0.01) (ELISA measures serum antibody titer comparative result as shown in Figure 9).
The neutralization test of embodiment 6 recombinant protein
Adopt 500-550g/ outbreeding system Female guinea pigs only, often organize 4, random assignment.Within every two weeks, give cavy intramuscularly (IM) immunity once, P2-VP8*-(ab)
3, P2-VP8*-ab, VP8*-(ab)
3, VP8*-ab, VP8* immune 20 μ g (adding aluminum phosphate (AP) adjuvant) (every agent containing 100 μ g aluminium) at every turn, altogether immunity 3 times, final immunization was taken a blood sample after 7 days.
The NAT that every part of serum sample is determined in neutralization (PRN) test is reduced by 60% plaque.0.25ml G6P [5] type or G10P [11] type rotavirus, with after the serum mixing 1h of equal-volume doubling dilution, add in 6 orifice plates, 37 DEG C of absorption 1h, add 3ml agarose (containing MEM, 0.5 μ g/ml pancreatin), after agarose solidifies, be inverted for 37 DEG C and cultivate.After 4d, spread the 2nd layer of agarose (containing 2% toluylene red), calculate Virus plaque is suppressed to reach 60% extent of dilution by serum, calculate serum Neutralizing titer.
Table 3 P2-VP8*-(ab)
3, P2-VP8*-ab, VP8*-(ab)
3, VP8*-ab, VP8* neutralizing antibody level
Note:
a60% bites plaque reduction neutralization test serum antibody titer (inverse)
As can be seen from Table 3, VP8*-(ab)
3the NAT that restructuring mosaic epitopes vaccine is induced is significantly higher than the NAT that VP8*-ab induces, but lower than VP8* whole protein, consistent with expection.The introducing of tetanus toxin t cell epitope polypeptide P2 can improve VP8*-(ab) significantly
3the immune efficacy of restructuring mosaic epitopes vaccine, higher than the neutralizing antibody level of inducing of VP8* whole protein.Can the anti-P [5] of induced high titers and the special rotavirus neutralizing antibody of P [11] genotype, be suitable for preparing bovine rotavirus vaccine.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. bovine rota VP8* subunit restructuring chimeric protein, it is characterized in that, it is the recombinant protein that the clipped form a of the bovine rota VP8* subunit of G6P [5] and clipped form b are fitted together to by tetanus toxin t cell epitope polypeptide P2 and genotype; Wherein, without additional amino acid between clipped form a and clipped form b, and be multiple copied, be connected by flexible Linker between the clipped form a of the bovine rota VP8* subunit of G6P [5] and clipped form b in the genotype of tetanus toxin t cell epitope polypeptide P2, multiple copied;
Wherein, the clipped form a of described tetanus toxin t cell epitope polypeptide P2, the genotype bovine rota VP8* subunit that is G6P [5] and the aminoacid sequence of clipped form b are respectively as shown in SEQ ID No.1-3.
2. restructuring chimeric protein according to claim 1, is characterized in that, its aminoacid sequence is as shown in SEQ ID No.4, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.
3. to encode the gene of chimeric protein of recombinating described in claim 2.
4. gene as claimed in claim 3, it is characterized in that, its nucleotide sequence is as shown in SEQID No.5.
5. the expression vector containing gene described in claim 3 or 4.
6. the host cell containing gene described in claim 3 or 4.
7. prepare the method for chimeric protein of recombinating described in claim 1 or 2, it is characterized in that, be converted in competent escherichia coli cell by expression vector according to claim 5, screening positive clone being inoculated in LB liquid nutrient medium is cultivated, and adds IPTG abduction delivering and obtains restructuring chimeric protein.
8. method according to claim 7, is characterized in that, also adds 0.02g/mL glucose, 1 ~ 5v/v% ethanol and 2% glycerine in described LB liquid nutrient medium.
9. method according to claim 7, is characterized in that, is inoculated in by positive colony in LB liquid nutrient medium and is cultured to OD
600=0.5, adding IPTG to final concentration is 0.5mM, abduction delivering restructuring chimeric protein at 18 DEG C.
10. the restructuring chimeric protein described in claim 1 or 2 is preparing the application in bovine rotavirus vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690126A (en) * | 2018-06-07 | 2018-10-23 | 西南民族大学 | A kind of yak source rotavirus recombination VP6 proteantigens and application |
| US20200319174A1 (en) * | 2019-04-17 | 2020-10-08 | Jiangnan University | Method for Detecting Human Soluble Asialoglycoprotein Receptor |
| CN113861277A (en) * | 2021-09-30 | 2021-12-31 | 西南民族大学 | Bovine rotavirus recombinant VP8 protein and application thereof |
-
2014
- 2014-11-20 CN CN201410668656.0A patent/CN104479028B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| KOVACS-NOLAN J等: "Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice", 《BIOCHIM BIOPHYS ACTA》 * |
| LEE J 等: "A neutralizing monoclonal antibody to bovine rotavirus VP8 neutralizes rotavirus infection without inhibiting virus attachment to MA- 104 cells", 《CAN J VET RES》 * |
| LEE J 等: "Immunological response to recombinant VP8* subunit protein of bovine rotavirus in pregnant cattle", 《J GEN VIROL》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108690126A (en) * | 2018-06-07 | 2018-10-23 | 西南民族大学 | A kind of yak source rotavirus recombination VP6 proteantigens and application |
| US20200319174A1 (en) * | 2019-04-17 | 2020-10-08 | Jiangnan University | Method for Detecting Human Soluble Asialoglycoprotein Receptor |
| CN113861277A (en) * | 2021-09-30 | 2021-12-31 | 西南民族大学 | Bovine rotavirus recombinant VP8 protein and application thereof |
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| CN104479028B (en) | 2017-11-21 |
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