CN107022559A - A kind of preparation method of varicellazoster virus glycoprotein E extracellular region protein - Google Patents
A kind of preparation method of varicellazoster virus glycoprotein E extracellular region protein Download PDFInfo
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- CN107022559A CN107022559A CN201611119488.5A CN201611119488A CN107022559A CN 107022559 A CN107022559 A CN 107022559A CN 201611119488 A CN201611119488 A CN 201611119488A CN 107022559 A CN107022559 A CN 107022559A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
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Abstract
The present invention discloses a kind of preparation method of varicellazoster virus glycoprotein E extracellular region protein, the recombinant vector that is related to varicellazoster virus glycoprotein E extracellular region protein, the transformant prepared with the carrier, the albumen produced using transformant, and this albumen is in the application in the field such as related vaccines research and development and corresponding antigens, antibody test such as chicken pox vaccine, herpes zoster vaccine and polyvaccine, combined vaccine containing this composition.The present invention utilizes technique for gene engineering, and VZV gE extracellular region nucleotide sequences are cloned and are connected on prokaryotic expression carrier pET 21b, recombinant vector is obtained；Recombinant vector is converted to e. coli bl21（DE3）In, obtain transformant；Transformant induced expression is obtained into destination protein.Expression system in the present invention can have good immunogenicity to VZV gE extracellular region antigen protein high efficient expressions, expressed VZV gE extracellular region protein antigens, research and development and antigen and antibody field available for corresponding vaccine.
Description
Technical field：
The present invention provides a kind of preparation method of varicella virus glycoprotein E extracellular region protein, relates in particular to contain
Varicella virus glycoprotein E Extracellular domain recombinant plasmid vector, the transformant prepared with the carrier, and utilization turn
Change body and prepare varicella virus glycoprotein E extracellular region protein, be related to the albumen in unit price, multivalence varicella or banding blister
Rash vaccine, and the research and development of the combined vaccine containing this composition, production, the application for examining detection field, belong to biotechnology
Field.
Background technology：
Varicella virus（Varicella-zoster virus, VZV）It is to cause varicella and the cause of disease of herpes zoster
Body, it is initial during children to cause varicella, it can once again be activated after hiding and cause herpes zoster.At present, control varicella and herpes zoster
Main method be vaccine inoculation.
Chicken pox vaccine and herpes zoster vaccine are now attenuated live vaccine.The preparation requirement of attenuated live vaccine is through state's familia
Prestige department detects in qualified cellular matrix and carried out, and preparation technology is complicated, and technology controlling and process is more difficult.In addition, according to water
The acne attenuated vaccine market application analysis of nearly 20 years, inoculation chicken pox vaccine can not protect inoculator to be invaded from varicella completely
Attack, have and break through case, and vaccine strain has latent risk, here it is varicella virus virus is special
Part.Herpes zoster attenuated live vaccine is applied to adult, has prevention effect to herpes zoster, according to attenuation herpes zoster
The Clinical Application Analysis of vaccine, overall protective rate is 62%, and effect is barely satisfactory.GlaxoSmithKline PLC company expressing cho cell
VZV gE glycoprotein, be aided with AS04 adjuvants preparation herpes zoster vaccine of new generation, the cellular immunity of effective provocative inoculation person and
Humoral immunity level.According to the analysis of the III phases clinical effectiveness of its offer, effective percentage is more than 90%, and effect is preferable.
VZV-gE glycoprotein is encoded by ORF68, is made up of 623 amino acid, is I type transmembrane glycoproteins, by signal peptide, born of the same parents
Outskirt, transmembrane region and intracellular region are constituted.GE glycoprotein enriches the most in virus and infection cell surface expression, and it is thin to have T, B concurrently
Born of the same parents' epitope, so the research for VZV-gE is the most extensive, expects that it turns into subunit's varicella or herpes zoster epidemic disease of a new generation
Seedling.Also the 37-161 amino acid consensus sequences for having researcher only to intercept gE develop subunit vaccine, but because its fragment is smaller, cover
Epitope is less, immunogenicity is weak and is unfavorable for application.
The expressing quantity of eukaryotic cell expression system expression is low, its protein glycosylation is inconsistent, causes albumen to have heterogeneous
Sex differernce, and the immunogenicity of some albumen has no tight association with glycosylation.Accordingly, we utilize Bacillus coli expression system
System express express target protein, evaluates its immunogenicity, is that the vaccine research in future lays the first stone.Utilize escherichia expression system table
Up to destination protein, highlight that escherichia expression system expressing quantity is high, easy to operate, process controllability is strong, it is easy to accomplish
Automation, without host cell nucleic acid and the features such as protein residue.
The content of the invention：
The present invention provides a kind of preparation method of varicella virus glycoprotein E extracellular region protein, utilizes Escherichia coli table
Up to system expression destination protein, highlight that escherichia expression system expressing quantity is high, easy to operate, process controllability strong,
It is easy to automate, without host cell nucleic acid and the features such as protein residue.
A kind of recombinant plasmid vector disclosed by the invention, by the varicella for being optimized for Escherichia coli preference codon
Simplex virus glycoprotein E Extracellular domains are connected built-up with expression vector pET-21b, its nucleotide sequence such as SEQ NO.1 institutes
Show, its corresponding amino acid sequence is as shown in SEQ NO.2.
A kind of transformant disclosed by the invention, it is characterised in that e. coli bl21 is converted by above-mentioned recombinant plasmid vector
(DE3) obtain.
A kind of method for preparing varicella virus glycoprotein E extracellular region protein of the present invention, its feature exists
In comprising the following steps：
1）Synthetic plasmid to be optimized for Escherichia coli preference codon is designed as Nhe I and EcoR I enzymes with two ends as template
The primer of enzyme site enters performing PCR amplification, purified pcr product；
Forward primer is（5'-3'）：TCCTAGCTAGCATGACCAATCC GGTTCGCG CCAGCGTGC；Reverse primer is
（5'-3'）：TTCCGGAATTCTTAATGATGATGATGATGATGACGCAGCAGC GGGCTTGTACCCGGAT；
2）By step 1）The PCR primer and pET-21b carriers of acquisition restriction endonuclease Nhe I and EcoR I double digestions, then by digestion
Product is connected, construction of recombinant plasmid vector；Recombinant plasmid vector is converted into competent escherichia coli cell, the positive is obtained through screening
Clone.
3）Positive colony plasmid is transferred to BL21（DE3）Express in bacterium, selected clone, induced expression, obtain positive expression
Bacterium.
4）Positive expression bacterium largely expands, and induces 4-6h with 0.1-10mMIPTG at 37 DEG C, can obtain VZV-gE extracellular regions
Albumen.
A kind of chicken pox vaccine or herpes zoster vaccine of the present invention, are by preparing varicella according to the present invention
Simplex virus glycoprotein E extracellular region proteins method prepare VZV-gE extracellular region proteins antigenic component and vaccine adjuvant prepare and
Into.
A kind of combined vaccine of the present invention or polyvaccine, containing preparing varicella-zoster according to the present invention
VZV-gE extracellular region proteins antigenic component prepared by viral glycoprotein E extracellular region proteins method is formulated with vaccine adjuvant.
The invention provides a kind of recombinant plasmid vector, the carrier is by varicella virus glycoprotein E extracellular region
Gene is formed by connecting with pET-21b carriers；The VZV-gE Extracellular domains contained in this recombinant vector are to be optimized for large intestine bar
The gene order of bacterium preference codon；Wherein described VZV-gE Extracellular domains can be using synthetic plasmid as template, can be with
Expanded and obtained with PCR；Described carrier can be the prokaryotic expression carriers such as pET-21b.Described recombinant plasmid vector was built
Primer synthesis, PCR amplifications for being related in journey etc. can use conventional method in the art to obtain.Described DNA polymerases, inscribe
Enzyme, ligase can use conventional biology tool enzyme, such as PrimeSTAR HS DNA polymerase, Nhe
I, EcoR I, T4DNA ligase etc..The VZV-gE Extracellular domains that wherein described template plasmid contains are to be optimized for large intestine bar
The gene order of bacterium preference codon.Wherein described connection can use conventional method in the art to obtain.Example is as mentioned
Connection can be with restricted digestion recognition site using endonuclease digestion processing containing VZV-gE Extracellular domains both sides
VZV-gE Extracellular domains obtained from cloned plasmids, connect at ambient temperature with the expression vector pET-21b through equally handling
Connection overnight under the conditions of 5-6h, or 16 DEG C is connect, recombinant expression plasmid is built.
It is the VZV-gE Extracellular domains by being optimized for Escherichia coli preference codon the invention provides a kind of transformant
It is connected what the recombinant expression plasmid conversion host cell built was obtained with recombinant expression carrier pET-21b, this host cell is
Competence e. coli bl21（DE3）.Described conversion process can use conventional method in the art.
The invention provides a kind of method for preparing VZV-gE extracellular region proteins, including with the present invention provide by being optimized for
The VZV-gE Extracellular domains of Escherichia coli preference codon are connected the recombination expression matter built with recombinant expression carrier pET-21b
Grain conversion Escherichia coli, clone's culture and induced expression VZV-gE extracellular region proteins, purified acquisition VZV-gE extracellular region proteins
Antigen.Described clone builds, converted, expression process can use conventional method in the art to obtain.
The invention provides a kind of albumen, it is encoded by VZV-gE Extracellular domains, is VZV-gE extracellular region proteins, its
Amino acid sequence is as shown in Figure 2.Its preparation method may comprise steps of：VZV-gE Extracellular domains are optimized for large intestine bar
Bacterium preference codon, synthetic plasmid pUC-gE；Using this synthetic plasmid as template, Nhe I and EcoR I digestions are designed as with two ends
The primer in site enters performing PCR amplification, purified pcr product；The PCR primer of acquisition and pET-21b carriers are subjected to double digestion, connected
Connect, construction of recombinant plasmid vector；Connection product is converted to competence escherichia coli DH5a, positive bacteria is screened；Extract positive bacteria
Plasmid transformed competence colibacillus expression bacterium BL21 (DE3), picking positive bacteria induced expression obtains VZV-gE extracellular region eggs through preliminary purification
Bai Kangyuan.
Above-mentioned VZV-gE extracellular region protein antigens be transformant provided by the present invention through amplification cultivation, induced expression,
Purify and obtain.Described incubation can use culture under the conditions of conventional 37 DEG C of shaking table.Described induction can be used
Common derivant IPTG, concentration is 0.1-10mM, and induction time is more than 4 hours.
The protein expression that the present invention is provided is inclusion body, it is possible to use conventional method extracts inclusion body, such as ultrasonic
Broken, centrifugation；Conventional method can be utilized by solubilization of inclusion bodies, such as 8M urea, 6M guanidine hydrochlorides；Can be in conventional buffering
Preliminary renaturation and purifying are carried out in system, the buffer system such as PBS, Tris.
The present invention can use conventional purification method to purify VZV-gE extracellular region proteins；Molecular sieve, ion exchange can be used
Chromatography and（Or）The method of metal ion-chelant affinity chromatography and other known or conventional methods obtain destination protein.
The albumen that the present invention is provided is VZV-gE extracellular region proteins, and its amino acid quantity can be 546,544,539,537
Deng.
The invention provides a kind of method for preparing chicken pox vaccine and herpes zoster vaccine, the above method and process will be utilized
The antigen protein of preparation, is aided with single adjuvant or composite adjuvant, prepared after rational proportion with VZV-gE extracellular region eggs
It is in vain main proteantigen, vaccine for the purpose of preventing varicella or herpes zoster.
The albumen that the present invention is provided is the recombinant protein antigen obtained using technique for gene engineering, and such a proteantigen can be with
As chicken pox vaccine of new generation, the major antigen composition of herpes zoster vaccine, combined vaccine of new generation, list can be used as（It is many）Valency
The major antigen composition of vaccine, can be used as antigen, antibody qualitative and quantitative analysis in VZV correlative studys.For example, with this
Antigen carries out antigen, the qualitative research of antibody and antibody titer research as envelope antigen；Carried out by normative reference of this antigen
Qualitative, quantitative research of related antigen etc..
The albumen that the present invention is provided has stronger immunogenicity.This antigen is aided with Freund's adjuvant subcutaneous inoculation Balb/c
Mice serum potency reaches 1 after mouse, reinforcement:105More than.Experimental result illustrates, utilizes the method and process of the invention provided
The VZV-gE extracellular region protein antigens of preparation have preferable immunogenicity, can be used for chicken pox vaccine and herpes zoster vaccine etc.
The research and development of preventative vaccine candidate vaccine and related combined vaccine.
The positive effect of the present invention is：
Recombinant vector and transformant have high efficient expression VZV-gE extracellular region proteins, it is easy to breed, the advantages of safe.This hair
The recombinant protein antigen of bright preparation has the advantages that purity is good, immunogenicity is good, security is good.The method that the present invention is provided has
Simply, efficient advantage.Preparing vaccine using the antigen that provides of the present invention can reach that purity is high, quality controllable, production process
In virus-free operating process, without worrying that advantage occurs etc. for virus mutation and atavism.
Brief description of the drawings：
Fig. 1 .VZV-gE pcr amplifications（lane1：DL5000；lane2：Purpose fragment）；
Fig. 2 .pET-21b-gE carrier digestion qualification results（lane1：DL5000；lane2：Recombinant plasmid double digestion result, purpose
Clip size meets expection）；
Fig. 3 recombinantly expresses bacterium induced expression result（lane1：pageruler plus prestained protein ladder；
lane2：Group is not induced；lane3：Induction group, it is seen that newly-increased protein expression, size meets expection）；
Fig. 4 protein expression form results（lane1：pageruler plus prestained protein ladder；lane2：
Bacterial cell disruption supernatant；lane3：Bacterial cell disruption is precipitated, it is seen that the expression-form of destination protein is inclusion body）；
The nickel glue purification result of Fig. 5 destination proteins（lane1：pageruler plus prestained protein ladder；
lane2：Prick post；lane3：50mM imidazole elutions；lane4：100mM imidazole elutions；lane5：200mM imidazole elutions；
lane6：300mM imidazole elutions；lane7：500mM imidazole elutions）；
Fig. 6 destination protein WB qualification results（lane1：pageruler plus prestained protein ladder；
lane2：Destination protein）.
Embodiment
Following specific embodiment will be described further to the present invention, specific embodiment be not intended to make the present invention into
One step is limited.But according to the synonymous replacement of the invention done or be correspondingly improved, still fall within protection scope of the present invention.
Embodiment 1
The preparation of varicella virus glycoprotein E proteantigen
1st, target gene is expanded
（1）Design of primers
According to the varicella virus glycoprotein E gene sequence announced on Genbank, amino acid sequence 25-537 is chosen
Corresponding nucleotide sequence design two ends carry Nhe I and EcoR I restriction enzyme sites（Italic is marked）Primer：
Forward primer is（5'-3'）：TCCTAGCTAGCAT GACCAATCCGGTTCGCGCCAGCGTGC；
Reverse primer is（5'-3'）：TTCCGGAATTCTTAATGATGATGATGATGATGACGCAGC
AGCGGGCTTGTACCCGGAT；
（2）PCR is expanded
Using the plasmid of synthesis as template, nucleic acid amplification is carried out using above-mentioned primer, specific reaction system is PrimeSTAR HS
DNA polymerase buffer（5×）10ul, dNTPs 4ul, Primer-F 1ul, Primer-R 1ul, template 1ul,
PrimeSTAR HS DNA polymerase 0.5ul, add deionized water to cumulative volume 50ul；Reaction tube is put into PCR instrument
In, first 94 DEG C of pre-degeneration 5min, then 94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 68 DEG C of extension 2min, this circulation carries out 35
Circulation, last 72 DEG C of extensions 10min, amplification obtains purpose fragment and meets expection, as a result sees Fig. 1；
2nd, the clone of PCR primer and sequencing
（1）PCR primer and carrier double digestion
PCR primer 30ul or pET-21b the carrier 5ul of target gene is separately added into 10x K buffer 5ul, Nhe I
Enzyme cutting, each 1ul of EcoR I restriction endonucleases, plus deionized water are to 50ul, 37 DEG C of water-bath 2h；
（2）The connection of PCR primer and cloning vector
After target gene and carrier digestion recovery product are quantified, 10ul and 2ul are taken respectively, add 10 × T4 ligases
Buffer2ul, T4 ligase 1ul, room temperature connection 6h, obtain connection product pET-21b-gE537；
（3）Conversion and screening
Connection product 10ul is transformed into 100ulE.coli In DH5 α competent cells, coated plate, 37 DEG C of overnight incubations.Select
Monoclonal bacterium colony, extracts plasmid and carries out double digestion identification, select positive colony, as a result see Fig. 2；
3rd, the expression identification of the structure of transformant and target gene
（1）Correct expression plasmid will be sequenced to be transformed intoE.coliBL21（DE3）Express in bacterial strain；
（2）Induced expression of the recombinant protein in e. coli bl21 (DE3)
In LB solid culture flat boards（Amp+）On select monoclonal, be inoculated into 4ml LB fluid nutrient mediums（Amp+）In, 37 DEG C/
220rpm shakes bacterium 4h；2ml is taken out in every group of 4ml bacterium solution, is added in another middle test tube, by 1:1000 add IPTG（Induction
Afterwards）, and and former pipe（Before induction）Continue to cultivate 4h together；200ul bacterium solutions are taken before every group of induction and after induction in test tube, plus
Enter into 1.5mlEP pipes, 12000rpm centrifugation 10min abandon net supernatant；Thalline is hanged with 80ul 1 × SDS sample-loading buffers to enter
Row SDS-PAGE is analyzed, and as a result sees Fig. 3；
（3）The identification of expression of recombinant proteins form
5ml is stayed overnight into bacterium to be seeded in 100mlLB culture mediums, 37 DEG C/220rpm shakes bacterium to OD600Reach 0.4 or so, addition
0.1mMIPTG induced expressions, 4h harvest thalline；Thalline 20ml 20mMtris (pH7.5) are hanged, washed 3 times；Use again
20ml cellular lysate liquid（20mMTris+2mMEDTA+0.1%DTT, pH7.5）Hang, ice-bath ultrasonic（60%AMP, 5 sec ON,
10sec OFF, 15min）, crush thalline.4 DEG C, 8000rpm centrifuges 30min, takes supernatant precipitation to carry out SDS_PAGE respectively,
As a result Fig. 4 is seen；
4th, the purifying of recombinant protein
（1）The purifying of destination protein
According to 3（2）Step and parameter extraction inclusion body.By inclusion body 20ml lysates（20mMtris+8MUrea +
2mMEDTA, pH8.0）Hang, more than room-temperature dissolution 2h.By solubilization of inclusion bodies liquid 10000rpm, 1h is centrifuged, supernatant is taken；By supernatant
Liquid is configured to loading sample（20mMtris+300mMNaCl+2mMEDTA+8MUrea+50mM
NaH2PO4 , pH8.0）.GE companies Ni-sepharose fast flow 6FF 1ml are taken, are added in loading sample, room
Temperature mixing 2h；Sample is added in recovery column, and successively with containing 50mM, 100mM, 200mM, 300mM, 500mM imidazoles
Elution buffer is eluted（Imidazoles+20mMtris+8Murea+300mMNaCl+50mMNaH2PO4, pH8.0）, reclaim elution sample
Product, carry out SDS-PAGE, as a result see Fig. 5；
（2）Protein renaturation
Will（2）In imidazole elution containing 300mM elute albumen successively containing 6M, 4M, 2M, 0M urea 20mMPBS buffering
4 DEG C of dialysed overnights in liquid, by dialysis sample 8000rpm centrifugation 30min, collect supernatant, as destination protein；
5th, the WB identifications of destination protein
Destination protein is subjected to SDS-PAGE, then carries out WB identifications, Fig. 6 is as a result seen.
Embodiment 2
The preparation of varicella virus glycoprotein E extracellular region protein antigen
（1）Design of primers
According to the varicella virus glycoprotein E gene sequence announced on Genbank, amino acid sequence 25-539 is chosen
Corresponding nucleotide sequence design two ends carry Nhe I and EcoR I restriction enzyme sites（Italic is marked）Primer：
Forward primer is（5'-3'）：TCCTAGCTAGCATGACCAATCCGGTTCGCGCCAGCGTGC；Reverse primer is（5'-
3'）：TTCCGGAATTCTTAATGATGATGATGATGATGCGCATAACGC AGCAGCGGGCTTGTAC；
（2）The connection of PCR primer and cloning vector
According to the primer of design expand purpose fragment, it is recovered after respectively with carrier carry out digestion, by target gene and carrier enzyme
After cutting back to close product quantitatively, 12ul and 2ul are taken respectively, 10 × T4 ligase buffer 2ul, T4 ligase 1ul are added, and are added
Deionized water connects, obtains recombinant vector pET-21b-gE to 20ul overnight under the conditions of 16 DEG C539；
（3）Induced expression of the recombinant protein in e. coli bl21 (DE3)
Recombinant vector is converted into BL21 (DE3) thalline, transformant is obtained.In LB solid culture flat boards（Amp+）On select transformant
Monoclonal, is inoculated into 4ml LB fluid nutrient mediums（Amp+）In, 37 DEG C/220rpm shakes bacterium 4h；Taken out in every group of 4ml bacterium solution
2ml, is added in another middle test tube, by 1:1000 add IPTG（After induction）, and and former pipe（Before induction）Continue to shake bacterium 4h together；
200ul bacterium solutions are taken before every group of induction and after induction in test tube, are added in 1.5mlEP pipes, 12000rpm centrifugation 10min,
Abandon net supernatant；Thalline, which has been hanged, with 80ul 1 × SDS sample-loading buffers carries out SDS-PAGE analyses；
（4）The purifying of recombinant protein
Transformant is expanded under the conditions of 37 DEG C, 6h is induced with 1mMIPTG, thalline is harvested, identified after ultrasonication, destination protein
Expressed with inclusion bodies.By inclusion body 20ml lysates（20mMPB+8MUrea+2mMEDTA, pH8.0）Hang, room temperature
Dissolving is stayed overnight.By solubilization of inclusion bodies liquid 10000rpm, 1h is centrifuged, supernatant is taken.This supernatant is configured to loading sample（20mMPB
+ 300mMNaCl+2mMEDTA+8MUrea, pH8.0）.GE companies Ni-sepharose fast flow 6FF 1ml are taken, are added
Into loading sample, mixed at room temperature 2h；Sample is added in recovery column, and successively with containing 50mM, 100mM, 200mM,
The elution buffer elution of 300mM, 500mM imidazoles（Imidazoles+8Murea+300mMNaCl+20mMPB, pH8.0）, reclaim elution
Sample, carries out SDS-PAGE.As a result most of destination protein has been eluted in visible 300mM imidazole elutions；
The albumen that imidazole elution containing 300mM is eluted is successively 4 in the 20mMPBS buffer solutions containing 6M, 4M, 2M, 0M urea
DEG C dialysed overnight, will dialysis sample 8000rpm centrifugation 30min, collect supernatant, as destination protein.
Embodiment 3
The preparation of varicella virus glycoprotein E extracellular region protein antigen
1st, design of primers
According to the varicella virus glycoprotein E gene sequence announced on Genbank, amino acid sequence 25-546 is chosen
Corresponding nucleotide sequence, design two ends carry Nhe I and EcoR I restriction enzyme sites（Italic is marked）Primer,
Forward primer is（5'-3'）：TCCTAGCTAGCATGACCAATCCGGTTCGCGCCAGCGTGC；
Reverse primer is（5'-3'）：TTCCGGAATTCTTAATGATGATGATGATGATGGCCCAGG
CCCGCGGTCCACG CCGCA；
2nd, the connection of PCR primer and cloning vector
According to the primer of design expand purpose fragment, it is recovered after respectively with carrier carry out digestion, by target gene and carrier enzyme
After cutting back to close product quantitatively, 12ul and 2ul are taken respectively, 10 × T4 ligases buffer2ul, T4 ligase 1ul is added, and are added
Deionized water connects, obtains recombinant vector pET-21b-gE to 20ul overnight under the conditions of 16 DEG C546；
3rd, induced expression of the recombinant protein in e. coli bl21 (DE3)
Recombinant vector is converted into BL21 (DE3) thalline, transformant is obtained；In LB solid culture flat boards（Amp+）On select transformant
Monoclonal, is inoculated into 4ml LB fluid nutrient mediums（Amp+）In, 37 DEG C/220rpm shakes bacterium 4h；Taken out in every group of 4ml bacterium solution
2ml, is added in another middle test tube, by 1:1000 add IPTG（After induction）, and and former pipe（Before induction）Continue to shake bacterium 4h together；
200ul bacterium solutions are taken before every group of induction and after induction in test tube, are added in 1.5mlEP pipes, 12000rpm centrifugation 10min,
Abandon net supernatant；Thalline, which has been hanged, with 80ul 1 × SDS sample-loading buffers carries out SDS-PAGE analyses；
4th, the purifying of recombinant protein
Transformant is expanded under the conditions of 37 DEG C, 6h is induced with 10mMIPTG, thalline is harvested, identified after ultrasonication, purpose egg
Expressed in vain with inclusion bodies.By inclusion body 20ml lysates（20mMPB+8MUrea+2mMEDTA, pH8.0）Hang, room
Temperature dissolving is stayed overnight.By solubilization of inclusion bodies liquid 10000rpm, 1h is centrifuged, supernatant is taken.This supernatant is configured to loading sample
（20mMPB+300mMNaCl+2mMEDTA+8MUrea, pH8.0）.Take GE companies Ni-sepharose fast flow 6FF
1ml, is added in loading sample, mixed at room temperature 2h；Sample is added in recovery column, and successively with containing 50mM, 100mM,
The elution buffer elution of 200mM, 300mM, 500mM imidazoles（Imidazoles+8Murea+300mMNaCl+20mMPB, pH8.0）, return
Elution samples are received, SDS-PAGE is carried out.As a result most of destination protein has been eluted in visible 300mM imidazole elutions；
, will by 300mM imidazole elutions successively 4 DEG C of dialysed overnights in the 20mMPBS buffer solutions containing 6M, 4M, 2M, 0M urea
Dialyse sample 8000rpm centrifugation 30min, collects supernatant, as destination protein.
SEQ no.1
<110>Changchun QiJian Biology Products Co., Ltd
<120>
<140>
<160> 1
<210> 1
<211> 37
<212> protein
<213>VZV-gE extracellular region proteins
<400> 1
ATGACCAATCCGGTTCGCGCCAGCGTGCTGCGCTACGACGATTTCCACATTGACGAGGACAAGCTGGACACCA
ATAGCGTGTACGAGCCGTACTACCATAGCGACCATGCAGAAAGCAGCTGGGTTAACCGTGGTGAAAGCAGCCGTAAA
GCCTACGACCACAATAGCCCGTATATTTGGCCGCGCAACGACTATGATGGTTTCCTGGAGAACGCCCATGAACACCA
CGGCGTGTACAATCAGGGCCGCGGCATCGATAGTGGTGAGCGTCTGATGCAGCCGACCCAGATGAGCGCCCAGGAAG
ATCTGGGCGATGATACCGGCATCCATGTGATCCCGACCCTGAATGGCGATGATCGTCATAAAATCGTTAATGTGGAT
CAACGCCAGTACGGCGACGTGTTTAAAGGTGACCTGAACCCGAAACCGCAAGGCCAACGCCTGATTGAGGTGAGCGT
GGAGGAAAATCACCCGTTCACATTACGTGCCCCGATCCAGCGTATTTACGGTGTGCGCTATACCGAAACCTGGAGCT
TTCTGCCGAGCCTGACCTGTACCGGTGATGCAGCCCCGGCCATCCAGCATATCTGCCTGAAGCATACCACCTGTTTT
CAGGATGTGGTTGTGGACGTGGACTGCGCAGAAAACACCAAAGAAGATCAGCTGGCCGAAATTAGCTATCGCTTTCA
GGGCAAAAAAGAAGCTGATCAGCCGTGGATTGTGGTGAACACCAGCACCCTGTTTGACGAGCTGGAACTGGATCCCC
CTGAAATTGAGCCGGGCGTGCTGAAAGTGCTGCGTACCGAAAAGCAGTATTTAGGCGTTTACATCTGGAACATGCGC
GGTAGCGATGGCACCAGCACCTATGCCACCTTTCTGGTGACCTGGAAGGGTGACGAGAAAACCCGTAACCCTACCCC
GGCCGTTACACCTCAACCGCGCGGTGCAGAATTTCATATGTGGAATTATCATAGTCACGTGTTCAGTGTGGGCGATA
CCTTCAGCCTGGCCATGCACCTGCAGTATAAAATCCACGAAGCCCCGTTCGATCTGCTGCTGGAGTGGCTGTATGTT
CCGATCGATCCGACCTGTCAGCCGATGCGTTTATACAGCACCTGCCTGTACCATCCGAATGCCCCGCAATGTCTGAG
TCACATGAATAGCGGCTGCACCTTTACCAGCCCGCATCTGGCACAGCGTGTGGCCAGCACCGTGTATCAGAACTGCG
AGCATGCCGATAACTATACCGCCTACTGTCTGGGCATTAGCCACATGGAGCCGAGTTTTGGTCTGATCCTGCACGAC
GGCGGCACAACCTTAAAATTTGTGGACACCCCGGAAAGTCTGAGCGGCCTGTATGTTTTTGTTGTGTATTTTAACGG
TCACGTGGAGGCCGTGGCCTATACCGTGGTGAGCACCGTGGACCACTTCGTGAACGCAATCGAAGAACGTGGCTTCC
CCCCTACAGCAGGTCAGCCGCCGGCAACCACAAAGCCGAAAGAGATTACCCCGGTGAATCCGGGTACAAGCCCGCTG
CTGCGTTAA
SEQ NO.2:
<110>Changchun QiJian Biology Products Co., Ltd
<120>
<140>
<160> 1
<210> 1
<211> 37
<212> protein
<213>VZV-gE extracellular region proteins
<400> 1
TNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHE
HHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEV
SVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYR
FQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNP
TPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQC
LSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYF
NGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLR
Claims (5)
1. a kind of recombinant plasmid vector, by the varicella virus glycoprotein E for being optimized for Escherichia coli preference codon
Extracellular domain is connected built-up with expression vector pET-21b, and its nucleotide sequence is as shown in SEQ NO.1, its corresponding amino
Acid sequence is as shown in SEQ NO.2.
2. a kind of transformant, it is characterised in that as the recombinant plasmid vector conversion e. coli bl21 (DE3) described in claim 1
Obtain.
3. a kind of method for preparing varicella virus glycoprotein E extracellular region protein, it is characterised in that including following step
Suddenly：
1）Synthetic plasmid to be optimized for Escherichia coli preference codon is designed as Nhe I and EcoR I enzymes with two ends as template
The primer of enzyme site enters performing PCR amplification, purified pcr product；
Forward primer is（5'-3'）：TCCTAGCTAGCATGACCAATCCGGTTCGCGCCAGCGTGC；
Reverse primer is（5'-3'）：TTCCGGAATTCTTAATGATGATGATGATGATGACGC AGCA
GCGGGCTTGTACCCGGAT；
2）By step 1）The PCR primer and pET-21b carriers of acquisition restriction endonuclease Nhe I and EcoR I double digestions, then by digestion
Product is connected, construction of recombinant plasmid vector；Recombinant plasmid vector is converted into competent escherichia coli cell, the positive is obtained through screening
Clone；
3）Positive colony plasmid is transferred to BL21（DE3）Express in bacterium, selected clone, induced expression, obtain positive expression bacterium；
4）Positive expression bacterium largely expands, and induces 4-6h with 0.1-10mMIPTG at 37 DEG C, can obtain VZV-gE extracellular region proteins.
4. a kind of chicken pox vaccine or herpes zoster vaccine, are by preparing the sugared egg of varicella virus according to claim 3
VZV-gE extracellular region proteins antigenic component and vaccine adjuvant prepared by white E extracellular region proteins method is formulated.
5. a kind of combined vaccine or polyvaccine, containing preparing varicella virus glycoprotein E according to claim 3
VZV-gE extracellular region protein antigenic components prepared by extracellular region protein method, are formulated with vaccine adjuvant.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108315344A (en) * | 2018-02-14 | 2018-07-24 | 武汉博沃生物科技有限公司 | VZV glycoprotein E genes expression vector and its restructuring yeast strains and application |
| CN109602901A (en) * | 2019-01-08 | 2019-04-12 | 成都迈科康生物科技有限公司 | A kind of herpes zoster virus vaccine and its preparation method and application |
| CN110237248A (en) * | 2019-07-01 | 2019-09-17 | 大连民族大学 | A kind of preparation method of shingles zoster vaccine |
| CN112142829A (en) * | 2019-06-28 | 2020-12-29 | 怡道生物科技(苏州)有限公司 | Varicella-zoster virus gE protein mutant and expression method thereof |
| CN112961221A (en) * | 2021-02-28 | 2021-06-15 | 长春祈健生物制品有限公司 | Preparation method of varicella-zoster virus glycoprotein E extracellular domain protein |
| CN113164586A (en) * | 2018-09-27 | 2021-07-23 | 武汉博沃生物科技有限公司 | Immune composition and preparation method and application thereof |
| CN113683704A (en) * | 2021-07-28 | 2021-11-23 | 安徽智飞龙科马生物制药有限公司 | Varicella-zoster virus r-gE fusion protein, recombinant varicella-zoster vaccine, and preparation method and application thereof |
| CN114703205A (en) * | 2022-03-11 | 2022-07-05 | 上海博唯生物科技有限公司 | Recombinant protein of herpesvirus glycoprotein gE, vaccine, preparation method and application |
| WO2023179588A1 (en) * | 2022-03-21 | 2023-09-28 | 厦门大学 | Truncated varicella-zoster virus envelope glycoprotein ge |
| WO2024051266A1 (en) * | 2022-09-07 | 2024-03-14 | 远大赛威信生命科学(南京)有限公司 | Mrna for expressing varicella-zoster virus antigen protein and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101189254A (en) * | 2005-03-03 | 2008-05-28 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
-
2016
- 2016-12-08 CN CN201611119488.5A patent/CN107022559A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101189254A (en) * | 2005-03-03 | 2008-05-28 | 葛兰素史密丝克莱恩生物有限公司 | Vaccine |
Non-Patent Citations (3)
| Title |
|---|
| BAO LIDAO.等: "Immunogenicity of varicella zoster virus glycoprotein E DNA vaccine", 《EXPERIMENTAL AND THERAPEUTIC MEDICINE》 * |
| 李福民 等: "水痘-带状疱疹病毒糖蛋白E原核表达载体的表达及产物纯化", 《临床皮肤科杂志》 * |
| 登录号：: "Q9J3M8", 《UNIPROT》 * |
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| CN108315344A (en) * | 2018-02-14 | 2018-07-24 | 武汉博沃生物科技有限公司 | VZV glycoprotein E genes expression vector and its restructuring yeast strains and application |
| CN113164586B (en) * | 2018-09-27 | 2024-04-16 | 武汉博沃生物科技有限公司 | Immune composition and preparation method and application thereof |
| EP3868399A4 (en) * | 2018-09-27 | 2022-07-20 | Bravovax Co., Ltd. | Immune composition, preparation method therefor, and application thereof |
| CN113164586A (en) * | 2018-09-27 | 2021-07-23 | 武汉博沃生物科技有限公司 | Immune composition and preparation method and application thereof |
| CN109602901B (en) * | 2019-01-08 | 2022-05-27 | 成都迈科康生物科技有限公司 | Herpes zoster virus vaccine and preparation method and application thereof |
| CN109602901A (en) * | 2019-01-08 | 2019-04-12 | 成都迈科康生物科技有限公司 | A kind of herpes zoster virus vaccine and its preparation method and application |
| CN112142829A (en) * | 2019-06-28 | 2020-12-29 | 怡道生物科技(苏州)有限公司 | Varicella-zoster virus gE protein mutant and expression method thereof |
| CN112142829B (en) * | 2019-06-28 | 2022-02-22 | 怡道生物科技(苏州)有限公司 | Varicella-zoster virus gE protein mutant and expression method thereof |
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| CN112961221B (en) * | 2021-02-28 | 2022-05-17 | 长春祈健生物制品有限公司 | Preparation method of varicella-zoster virus glycoprotein E extracellular domain protein |
| CN112961221A (en) * | 2021-02-28 | 2021-06-15 | 长春祈健生物制品有限公司 | Preparation method of varicella-zoster virus glycoprotein E extracellular domain protein |
| CN113683704A (en) * | 2021-07-28 | 2021-11-23 | 安徽智飞龙科马生物制药有限公司 | Varicella-zoster virus r-gE fusion protein, recombinant varicella-zoster vaccine, and preparation method and application thereof |
| CN114703205A (en) * | 2022-03-11 | 2022-07-05 | 上海博唯生物科技有限公司 | Recombinant protein of herpesvirus glycoprotein gE, vaccine, preparation method and application |
| WO2023179588A1 (en) * | 2022-03-21 | 2023-09-28 | 厦门大学 | Truncated varicella-zoster virus envelope glycoprotein ge |
| WO2024051266A1 (en) * | 2022-09-07 | 2024-03-14 | 远大赛威信生命科学(南京)有限公司 | Mrna for expressing varicella-zoster virus antigen protein and use thereof |
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Application publication date: 20170808 |