CN108130332A - Recombinant poliovirus type III virus-like particle - Google Patents
Recombinant poliovirus type III virus-like particle Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C12N2800/00—Nucleic acids vectors
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Abstract
The invention discloses the gene of coding type III wild poliovirus housing precursor albumen of a kind of codon optimization and the gene of encoding Type I attenuated polio viruses 3CD albumen, forementioned gene efficiently intracellular can be spontaneously assemble into VLP particles after yeast cells is transferred into using coexpression.The invention also discloses a kind of macromoleculars with immunogenicity, mainly express generation in yeast cells by forementioned gene.Application and composition the invention also discloses the aforementioned macromolecular with immunogenicity.
Description
Technical field
The present invention relates to a kind of new pichia yeast expression system, can be used for production poliovirus type III virus
Sample particle.
Background technology
Polio is a kind of acute infectious disease, is caused by poliovirus, mainly passes through transmission.Disease
It is malicious often to invade central nervous system, ventricornu motor nerve cells are damaged, leads to limbs flaccid paralysis, is more common in children,
Also known as infantile paralysis.Part child obtains can voluntarily fully recover after being ill, but will appear after most child's illness lower limb muscles atrophy,
As a result deformity causes life-long disabilities, mostly walk lamely even cannot stand, walk at all.It there is no effectively currently for this disease
Therapy, vaccine prevention can only be passed through.
By the end of 2005, still with the presence of the prevalence of 6 country's Wild poliovirus in global range, wherein bordering on China
Have 3, Introduced cases Wild poliovirus biography has occurred in some countries for having eliminated ridge ash such as Indonesia, Yemen in succession
It broadcasts, causes some areas ridge ash outbreak of epidemic again.Such incoming event is at global 18 without Ji Hui states since two thousand four
Family recurs.Once condition is suitable, Wild poliovirus may still stage a comeback.In October, 2000, Western Pacific of the World Health Organization
Area is announced as Maintaining Polio-free Status region, indicates that China has reached Maintaining Polio-free Status target.But these three exist wild
The neighbouring country of strain forms Introduced cases to China and threatens, and the maintenance in China is made to face a severe challenge without the work of ridge ash.
Poliomyelitis Vaccine,Live(Oral) (oral polio vaccine, OPV) was succeeded in developing in 1958 in U.S.
State lists, and extends to the whole world later.The inoculation of OPV simple (oral), expense is low, can generate stable Intestinal Mucosal Immunization, have
Effect blocks Polio virus propagation etc..The popularization of OPV makes Incidence of Poliomyelitis obtain effective control, global polio
Disease incidence declines year by year.But often take 2,500,000 dose of -1,000 ten thousand doses of OPV, it may occur however that 1 vaccine- associated diseases, including vaccine
Grey (Vaccine associated paralytic poliomyelitis, the VAPP) case of related paralysis ridge, vaccine derive
The cycle of Polio virus (Vaccine-derived poliovirus, VDPV) and its caused Vaccine-derived poliovirus
(Circulating vaccine-derived polioviruses, cVDPVs) and immunodeficiency vaccines derive Polio virus
(Immunodeficient related vaccine-derived poliovirus, iVDPV) case.
IPV is traditional inactivated vaccine (Conventional by using the ridge ash strain production of formalin-inactivated
Inactivated poliovirus vaccine, cIPV).Foreign countries research and development IPV inactivated using street strain, immunogenicity and
Safety is all highly desirable, can effectively prevent the outburst of ridge ash, and IPV, there is no VAPP, cVDPVs equivalent risk is caused, group exempts from
Epidemic disease is better than OPV.But it is higher to technology and production requirement, it is expensive.The IPV of the independent research of China's listing in 2015 is used
Attenuated strain inactivates, similarly with good immunogenicity and safety.As the propulsion of ridge ash target is eliminated in the whole world, it will
More strict control ridge ash strain, including low virulent strain and street strain.So the exploitation of polio vaccine of new generation is compeled in eyebrow
Eyelash.
Poliovirus belongs to Picornaviridae (Picornaviridae) enterovirus genus
(Enteroviruse), 20~30nm of diameter, 12 face body of cubic symmetry.Virion center be single-stranded positive RNA, nucleocapsid body
Exposed no cyst membrane.Virogene of poliomyelitis group RNA is about 7.5kb.Genome is divided into 5 ' end noncoding regions, polyprotein is compiled
Code area, 3 ' end noncoding regions and 3 ' end four parts of Poly (A) tail.Wherein polyprotein code area coding generates a polyprotein
Precursor is divided into P1, P2 and P3 area；P1 areas can generate capsid protein VP1, VP2, VP3 and VP4, P2 and P3 areas through protease hydrolytic
Then hydrolyzable generates protease, rna replicon enzyme and other albumen for identifying cell, adjusting gene.Nucleocapsid contains 4 kinds of structures
Albumen VP1, VP3 and the VP2 and VP4 formed by VP0 divisions.VP1 at least closes 2 epitopes for main exposed albumen
(epitope), the generation of neutralizing antibody is can induce, VP1 has special affinity to receptor in human cell membrane, with causing a disease for virus
Property is related with toxicity.VP0 is split into VP2 and VP4, is intimately associated for integrated protein with RNA.Half exposure tool antigens of VP2 and VP3
Property.VP1, VP2, VP3 and VP4 of 5 copies constitute pentamer, and 12 pentamers form icosahedron nucleocapsid.
Virus-like particle (VLP) can be spontaneously assemble into after the virus structural protein recombinant expression of many viruses, it is most of
The antigenicity of VLP is not different, but lack viral nucleic acid because without infectivity with euvirus.VLP has become open peace
One available strategy of complete effective viral disease vaccine, therefore, the VLP for developing poliovirus is to utterly destroy ridge
The effective way of grey virus.
Poliovirus is there are three serotype, i.e. I types Mahoney, II type MEF-1, type III Saukett, it is various it
Between without cross-immune reaction.2015, WHO announced that II types strain is utterly destroyed in the world.Therefore, the application
Research be concentrated mainly on the exploitation of type III ridge ash VLP.
Invention content
An object of the present invention is：A kind of coding type III wild type polio of codon optimization is provided
The gene and encoding Type I attenuated polio viruses of viral (Polio 3) virus coat precursor protein (P1 albumen)
The gene of (Sabin 1) 3CD albumen.
First purpose of the present invention is realized by the following method：First, to natural Polio3 P1 albumen and Sabin 1
3CD protein gene is transformed, and to its all amino acid all using the highest codon of frequency of use, is designed completely new
DNA sequence dna；Then, excessively high in order to avoid translating the GC ratios of the mRNA come, the secondary structure of mRNA is to the shadow of translation efficiency
It rings, avoids some common restriction enzyme sites, certain amendment is carried out to optimal codon frequency, specifically there is following repair
Just：Asparagine (Asn) highest frequency codon AAC is modified to AAT；Lysine (Lys) highest frequency codon AAG is modified to
AAA；Aspartic acid (Asp) highest frequency codon GAT is modified to GAC；Phenylalanine (Phe) highest frequency codon TTT is repaiied
Just it is being TTC；Tyrosine (Tyr) highest frequency codon TAT is modified to TAT.But these amendments enumerated above are not limited to,
To further improve the expression quantity of target protein, meanwhile, special tune has also been carried out to P1 and two kinds of recombinant protein expression quantity of 3CD
Control so that the expression quantity of P1 albumen is higher than 3CD albumen in coexpression system, works to form VLP convenient for 3CD protease, designs
Go out two completely new DNA sequence dnas, and synthesize by modified completely new DNA sequence dna.
The gene of the P1 albumen of Polio 3 after codon optimization provided by the present invention, sequence is most preferably such as SEQ
ID NO：Shown in 1.
The gene of the 3CD albumen of Sabin 1 after codon optimization provided by the present invention, sequence is most preferably such as SEQ
ID NO：Shown in 2.
By the DNA after above-mentioned codon optimization, restructured Pichia pastoris in expression carrier and expression bacterial strain, Jin Erbiao can be built
Up to P1 albumen and 3CD albumen.P1 albumen can be spontaneously assemble into VLP particles after 3CD cleavages in intracellular.
Another object of the present invention is：A kind of Pichia anomala expression for expressing gene after above-mentioned codon optimization is provided to carry
Body.
Concrete mode is：Respectively by the gene cloning of the P1 albumen of Polio after codon optimization 3 to a Pichia pastoris table
It, will be in the gene cloning to another yeast expression vector that poliovirus 3CD albumen be encoded up in carrier.
More specific mode is：By the gene of the P1 albumen of Polio after codon optimization 3 and the 3CD eggs of Sabin 1
White gene, is cloned into yeast expression vector respectively, expression vector used include pPICZ, pPIC6, pGAPZ,
pAO815、pPIC9k.For example, the gene progress by the P1 albumen after optimization is artificial synthesized, it is connected to yeast expression vector
In pPICZa B, 3 P1 of expression vector pPICZ-Polio of 3 P1 albumen of Polio are obtained, by the base of the 3CD albumen after optimization
It is artificial synthesized because carrying out, it is connected in yeast expression vector pPIC 9K, obtains the expression vector pPIC 9K- of 3CD albumen
Sabin 1 3CD。
A further object of the present invention is：The Pichia anomala expression bacterium that a kind of table amounts to gene after above-mentioned codon optimization is provided
Strain.
Concrete mode is：By homologous recombination and the restructured Pichia pastoris in expression bacterial strain of screening structure expression 3CD genes, then
Pass through homologous recombination and the restructured Pichia pastoris in expression bacterium of screening P1 genes and 3CD gene co-expressings using this recombinant bacterium as host
Strain.
More specific mode is：Expression vector (such as 1 3CD of the pPIC 9K-Sabin) conversion of the gene containing 3CD is finished red
Yeast host bacterial strain obtains 3CD recombinant yeast pichia pastoris engineering bacterias by screening, and method for transformation can electricity consumption conversion or liposome turn
Change, host yeast used includes the bacterial strains such as Pichia pastoris GS115, KM71, SMD1168, X33, and screening mode can be group ammonia
The screening of sour deficiency.By expression vector (such as 3 P1 of the pPICZ-Polio) conversion of the gene containing P1 to above-mentioned recombinant bacterium, lead to
The recombinant yeast pichia pastoris engineering bacteria that screening obtains 3CD genes and P1 gene co-expressings is crossed, method for transformation can electricity consumption conversion or lipid
Body converts, and screening mode can be the screening of high concentration antibiotic.In this way, induced expression P1 albumen and 3CD albumen, and can
Virus-like particle is formed in intracellular.
A further object of the present invention is：A kind of Pichiapastoris expression strain for expressing gene after above-mentioned codon optimization is provided
Construction method, include the following steps：
A, the 3CD genes of codon optimization and P1 genes are designed and synthesized；
B, the yeast expression vector of the yeast expression vector of structure 3CD genes and P1 genes；
C, structure P1 albumen and the Pichiapastoris expression strain of 3CD albumen coexpression.
More specifically, above-mentioned steps A includes：Natural 3 P1 albumen of Polio and 1 3CD protein gene of Sabin are carried out
Transformation, to its all amino acid all using the highest codon of frequency of use, designs completely new DNA sequence dna；Then, it is
Avoid the GC ratios for translating the mRNA come excessively high, it is common to avoid some for the influence of the secondary structure of mRNA to translation efficiency
Restriction enzyme site, certain amendment is carried out to optimal codon frequency, specifically there is following amendment：Asparagine (Asn)
Highest frequency codon AAC is modified to AAT；Lysine (Lys) highest frequency codon AAG is modified to AAA；Aspartic acid
(Asp) highest frequency codon GAT is modified to GAC；Phenylalanine (Phe) highest frequency codon TTT is modified to TTC；Junket ammonia
Acid (Tyr) highest frequency codon TAT is modified to TAT.But these amendments enumerated above are not limited to, to further improve
The expression quantity of target protein designs two completely new DNA sequence dnas, and synthesizes by modified completely new DNA sequence dna.The present invention
The gene of the P1 albumen of Polio 3 after the codon optimization provided, sequence is most preferably such as SEQ ID NO：Shown in 1.This
The gene of the 3CD albumen of Sabin 1 after the provided codon optimization of invention, sequence is most preferably such as SEQ ID NO：2 institutes
Show.Pass through the optimization to sequence, on the one hand, P1 is suitable for pichia yeast expression system with 3CD genes, reaches the mesh of high efficient expression
, on the other hand, P1 has also carried out special regulation and control with two kinds of recombinant protein expression quantity of 3CD so that the P1 eggs in coexpression system
White expression quantity is higher than 3CD albumen, works to form VLP convenient for 3CD protease.
More specifically, above-mentioned steps B includes：By the gene of the P1 albumen of Polio after codon optimization 3 and Sabin 1
3CD albumen gene, be cloned into yeast expression vector respectively, expression vector used include pPICZ, pPIC6,
pGAPZ、pAO815、pPIC9k.For example, the gene progress by the P1 albumen after optimization is artificial synthesized, it is connected to Pichia pastoris table
Up in carrier pPICZa B, 3 P1 of expression vector pPICZ-Polio of 3 P1 albumen of Polio are obtained, by the 3CD eggs after optimization
White gene progress is artificial synthesized, is connected in yeast expression vector pPIC 9K, obtains the expression vector of 3CD albumen
pPIC 9K-Sabin 1 3CD。
More specifically, above-mentioned steps C includes：By the expression vector (such as 1 3CD of pPIC 9K-Sabin) of the gene containing 3CD
Pichia pastoris host strain is converted, 3CD recombinant yeast pichia pastoris engineering bacterias are obtained by screening, method for transformation can electricity consumption conversion or fat
Plastid transformation, host yeast used includes bacterial strains, the screening mode such as Pichia pastoris GS115, KM71, SMD1168, X33 can
Screening for histidine deficient.By expression vector (such as 3 P1 of the pPICZ-Polio) conversion of the gene containing P1 to above-mentioned recombination
Bacterium, obtains the recombinant yeast pichia pastoris engineering bacteria of 3CD genes and P1 gene co-expressings by screening, method for transformation can electricity consumption conversion or
Lipofection, screening mode can be the screening of high concentration antibiotic.Induced expression P1 albumen and 3CD albumen, and can be in intracellular
Form virus-like particle.
The present invention has the following advantages：
1. the gene by optimization is more suitable for the high-efficient expression target protein in yeast host, and disclosure satisfy that industry
The requirement of metaplasia production, it should be strongly noted that the method disclosed in the method and general literature of the application codon optimization is deposited
In difference, the application employs suboptimum codon on some critical sites.Codon replacement on these critical sites is kept away
The binding site of several introne identification sequences and possible transcription factor has been opened, and has been conducive to the stabilization of mRNA structures.It is this
There can be following two advantages after codon optimization：A, expression harvest yield is high, has apparent industrial advantages；B, codon are excellent
During change other than expression quantity, the structural stability of mRNA and the integrality of translation have also been taken into account, has been more easy to correctly after making protein expression
It folds, is embodied in that the VLP particles to be formed are more uniform, and the basic structural unit of VLP particles is high-visible, be more suitable for making epidemic disease
Seedling finished product.
2. simultaneously as using yeast expression system, thus it is at low cost, and yield is high, and product property more stable homogeneous.
Description of the drawings
The ED50 figures that 3 VLP of Fig. 1 Polio are reacted with anti-Polio virus type III immune serum National reference；
3 VLP electromicroscopic photographs of Fig. 2 Polio.
Sequence explanation
SEQ ID NO：1：The gene of the P1 albumen of Polio 3 after codon optimization；
SEQ ID NO：2：The gene of the 3CD albumen of Sabin 1 after codon optimization.
Specific embodiment
The present invention is will be more fully described by the following example, so that those of ordinary skill in the art better understood when this hair
It is bright.Following embodiments solely for illustrative purposes, are not intended to limit the scope of the present invention.It makes efforts to ensure related number
The accuracy of (such as quantity, temperature), but be specifically contemplated that can there are some errors and deviations.Unless otherwise indicated, temperature with
DEG C it is unit or is environment temperature, pressure is close or equal to atmospheric pressure.Unless otherwise indicated, it uses in each of the embodiments described below
Restriction enzyme be purchased from New England Biolab companies.It should be understood that unless otherwise indicated, following each realities
Apply the conventional equipment that instrument and equipment used in example is this field.Unless otherwise indicated, used culture medium is city
Sell available conventional medium, the known ingredient therein of those skilled in the art and content.For simplicity herein
There is a possibility that with various general abbreviations, those skilled in the art are fully able to understand its meaning.
Embodiment
Embodiment 1：Codon optimization designs
Genetic code has 64 kinds, but most biological tendencies are in utilizing the part in these codons.Finish red ferment
The utilization of female and people gene pairs codon has respective preference.Since genetic code is degeneracy, each amino acid is by one
A above codon coding, frequency of use of the codon in the gene of wild type with monoamino-acid is different.Bi Chi
The codon preference of yeast may lead to the low translation efficiency and expression of recombinant protein, and the present inventor is according to wild type
3CD albumen and P1 albumen carry out gene order transformation：To its all amino acid gene all using frequency of use highest and compared with
High codon.Pichia yeast codons frequency of use is shown in Table 1 (referring to http：//www.kazusa.or.jp/codon/).
Then on this basis, and excessively high in order to avoid translating the GC ratios of the mRNA come, the secondary structure of mRNA influences translation
Efficiency and some common restriction enzyme sites, the present inventor carry out the codon of highest frequency certain amendment, for example, by some
Asparagine (Asn) highest frequency codon AAC is modified to AAT, and lysine (Lys) highest frequency codon AAG is modified to
AAA, aspartic acid (Asp) highest frequency codon GAT are modified to GAC, and phenylalanine (Phe) highest frequency codon TTT is repaiied
Just it is TTC, tyrosine (Tyr) highest frequency codon TAC is modified to TAT, and glycine (Gly) highest frequency codon GGT is repaiied
Just it is being GGA.Improved gene order does not contain following introne identification sequence and Binding site for transcription factor：ATGACTCAT
With TGACTA (transcription factor GCN4 binding sites)；ATATAA (binding site of GAL4)；TATTTAA (TBP binding sites)；
TTAGTAA and TTACTAA (YAP1 binding sites)；ATGACTAAT；ACTAATTAGG.
The present inventor's optimization design goes out the several nucleotide sequence suitable for Pichia anomala expression as a result, according to described
Sequence fully synthetic 3CD genes and P1 genes, are cloned into existing yeast expression vector, pass through homologous recombination and sieve
Choosing structure restructured Pichia pastoris in expression bacterial strain；Fermented and cultured and methanol induction intracellular expression P1 eggs are carried out using recombinant yeast pichia pastoris
Bletilla 3CD albumen.P1 albumen can be spontaneously assemble into virus-like particle (VLP) after 3CD cleavages in intracellular；Bacteria break supernatant is passed through
Chromatography method after purification, obtains being self-assembled into virus-like particle.The nucleotide sequence of the gene of these optimization designs such as SEQ ID
NO：1、SEQ ID NO：Shown in 2.Pass through the optimization to sequence, on the one hand, P1 is suitable for Pichia anomala expression system with 3CD genes
System, achievees the purpose that high efficient expression, and on the other hand, P1 has also carried out special regulation and control with two kinds of recombinant protein expression quantity of 3CD so that
The expression quantity of P1 albumen is higher than 3CD albumen in coexpression system, works to form VLP convenient for 3CD protease.
1 Pichia yeast codons tables of table
Embodiment 2：The synthesis of 1 3CD genes of Sabin and the structure of 3CD expression vectors
The P1 albumen of poliovirus can be self-assembled into VLP structures, in view of ridge after the 3CD cleavages of specificity
The 3CD enzymes of marrow poliovirus have very high homology.The present invention selects the 3CD enzymes of polio attenuated strain sabin 1
Nickase as specificity.
In order to improve expression of the 1 3CD albumen of sabin in Pichia pastoris, closed carrying out 1 3CD genes of sabin
Cheng Qian, we have carried out the gene of 1 3CD of sabin codon preference, mRNA secondary structures, G/C content and common limitation
The optimization of property restriction enzyme site etc..We are taken under the premise of amino acid sequence is not changed, its all amino acid is preferentially adopted
With the highest codon of frequency of use in Pichia pastoris, while the GC ratios of mRNA and the secondary structure of mRNA etc. are taken into account, and
Some common restriction enzyme sites are avoided, design a completely new DNA sequence dna.
DNA sequence dna after optimization is synthesized by Shanghai Jierui Biology Engineering Co., Ltd, is connected in carrier pPIC9K, is obtained
Sabin 1 3CD pPIC9K.1 3CD gene orders of the sabin such as SEQ ID NO synthesized after codon optimization：Shown in 2.
Embodiment three：The synthesis of 3 P1 genes of Polio and the structure of expression vector
Polio type III P1 genes select the P1 genes of 3 bacterial strain Saukett of wild type polio, in order to improve
Expression of the 3 P1 albumen of Polio in Pichia pastoris, before 3 P1 gene chemical synthesis of Polio is carried out, we are to Polio 3
The gene of P1 has carried out the optimization of codon preference, mRNA secondary structures, G/C content and common restriction enzyme site etc..I
Take under the premise of amino acid sequence is not changed, to its all amino acid preferentially using frequency of use in Pichia pastoris most
High codon, while the GC ratios of mRNA and the secondary structure of mRNA etc. are taken into account, and avoid some common restriction enzymes
Enzyme site designs a completely new DNA sequence dna.
DNA sequence dna after optimization is synthesized by Shanghai Jierui Biology Engineering Co., Ltd, passes through BstBI/KpnI restriction enzyme sites
It is connected in carrier pPICZa B, obtains 3 P1 of expression vector pPICZ-Polio of 3 P1 of Polio.It is closed after codon optimization
Into 3 P1 gene orders of Polio as such as SEQ ID NO：Shown in 1.
Example IV：The construction and screening of 1 3CD Pichiapastoris expression strains of Sabin
Polio P1 albumen needs that VLP particles could be formed after 3CD digestions, it is therefore desirable to the P1 of polio
Albumen is co-expressed with 3CD albumen in same host.Present invention use first passes through carrier pPIC 9K carriers and leads 3CD genes
Enter Pichia pastoris host strain postgenome, then on its basis by pPICZa B carriers by the P1 gene integrations of polio
Onto Pichia pastoris host strain genome, the coexpression of polio P1 albumen and 3CD albumen is realized.
1 3CD expression vector pPIC 9k-sabin of Sabin, 1 3CD are used after restriction enzyme SalI linearisations
QIAGEN MinElute Gel Extraction Kit are purified, the plasmid fragments linearized with the dissolving of 10ul distilled waters,
The step of by Molecular Cloning:A Laboratory guide (third edition) electroporated Pichia yeast SMD1168 (Invitrogen).Electricity conversion
Condition is：5 microgram of DNA fragmentation, 1500 volts of voltage, 25 ohm of resistance, the electric shock time is about 5 milliseconds.Electric converted product is plated on
On MD agar, the single bacterium colony of transformed cells, the recombinant bacterial strain of screening expression 1 3CD enzymes of sabin are detached.
Embodiment five：The construction and screening of 3 P1 3CD Pichiapastoris expression strains of Polio
In order to improve integration efficiency of the recombinant plasmid on yeast chromosomal, with Sac I restriction enzyme single endonuclease digestions
3 P1 plasmids of pPICZ-Polio make its linearisation.It linearizes product and uses QIAGEN MinElute Gel Extraction
Kit is purified, with the plasmid fragments of 10ul distilled waters dissolving linearisation, the step of by Molecular Cloning:A Laboratory guide (third edition)
Electroporated Pichia yeast Sabin 13CD-SMD1168.
Electric conversion condition is：5 microgram of DNA fragmentation, 1500 volts of voltage, 25 ohm of resistance, electric shock time are 5 milliseconds.Electricity turns
Change product to be plated on containing 1000ug/ml, on the YPDS agar of zero mycin (Zeocin) of 1500ug/ml, be put down in high concentration resistance
Monoclonal is selected on plate, respectively with 4ml YPD fluid nutrient medium cultures, changes BMMY culture medium inducible gene expressions after r into for 24 hours,
Thalline is harvested by centrifugation after expression 48hr.Glass bead method breaks bacterium, and ELISA detects the expression quantity of each bacterial strain, Polio is obtained after screening
The higher 3 P1 3CD SMD1168 of bacterial strain Polio of 3 P1 expressing quantities.
Case study on implementation six：The preparation of a small amount of VLP samples
3 recombinant bacterial strains of Polio that screening obtains is taken to be inoculated in 500mlYPD culture mediums, 30 DEG C, 250rpm cultures,
It changes 500ml BMMY culture medium inducible gene expressions after r into for 24 hours, after expressing 48hr, zymotic fluid is carried out using refrigerated centrifuge
Separation of solid and liquid, centrifugal rotational speed 8000rpm are centrifuged 10 minutes.It abandons supernatant after centrifugation, collects bacterium mud, the bacterium mud of harvest is placed on-
It is frozen in 20 DEG C of refrigerators.
10g fermentation thallis are taken, add in the broken bacterium buffer solution (50mM citrate, 0.1M NaCl, pH5.2) of 4 DEG C of precoolings
20ml, using homogenizer, 70Hz shakes 6min, breaks bacterium solution 10000rpm, 30min, 8 DEG C, centrifuges, after collecting centrifugation
Supernatant.
Supernatant carries out preliminary purification using poros 50Hs chromatographic columns.Purification condition is as follows：
A liquid：0.1M NaCl, 50mM citrate, pH 5.2.
B liquid：1M NaCl, 50mM citrate, pH 5.2.
Balance：Using B liquid by pillar wash it is flat after, using A liquid by 1ml/min velocity balance pillars, 2CV.
Loading：Broken supernatant of bacteria solution, by 1ml/min speed loadings, collection flows through.
It washes miscellaneous：Impurity is rinsed using A liquid.
Elution：It is eluted using B liquid, collects virus-like particle protein sample of the eluting peak to get purifying.
Regeneration：Pillar is regenerated using 1M NaOH.
It should be noted that above-mentioned fermentation and purification process is merely illustrative rather than special limitation to the application.
Other suitable fermentation process can be：Obtained after screening bacterial strain access activation medium (YPD or BMGY or
SOC), 25~30 DEG C of overnight incubations.Remove culture medium supernatant after the activating solution of microscopy qualification is centrifuged, by 0.8~1.5OD/mL
Add in fermentation medium BMMY.Fermentation temperature is 25~30 DEG C of 180~250rpm shake flask fermentations, and 0.5% was added every 24 hours
Methanol.It is centrifuged after fermentation 48H, the bacterium mud of harvest is frozen at -20 DEG C.
Other suitable purification process can be：The thalline of the express express target protein of -20 DEG C of freezings is taken, passes through neutral buffered salt
Solution purifies water thawing and cleans thalline, removes medium component therein (salt, pigment etc.), reduces the shadow to later-period purification
It rings.Thalline after over cleaning is mixed using the appropriate broken bacterium buffer solution containing certain salinity and surfactant component：
Workable salt component has NaCl, KCl etc., and concentration range is in 0.1-0.8mol/l or so；Workable surfactant has
Tween-80, Tween-20, Triton 100 etc., concentration range is in 0.005-0.05% (w/v) left and right, workable buffer system
System has phosphate-buffered, Tris buffer solutions, MOPS buffer solutions, HEPES buffer solution etc., using concentration range in 0.02-0.2mol/
L or so.The mixed mixed thalline of thalline adds in suitable bead, and using homogenizer, 70Hz shakes broken bacterium, homogeneous
Machine selects 0.2-0.4 μm of bead diameter, shaking broken bacterium 3min can be to broken bacterium rate>More than 80%.Broken broken bacterium solution is led to
Ultracentrifugal method precipitation and separation and supernatant are crossed, the rotating speed of centrifuge is controlled in 6000-1000rpm (SORVALL, HITACHI
Deng), centrifugation time 20-60 minutes collects supernatant and is used for later-period purification.
Preliminary purification selects cation seperation column poros 50HS, and first pillar is balanced using equilibrium liquid before bacteria break supernatant loading.It is flat
The NaCl of the debita spissitudo and buffer with specific pH buffering ranges is typically contained in weighing apparatus liquid.Pillar will be above-mentioned broken after balancing
Bacterium supernatant presses the speed loading of 1ml/min, is washed down partial impurities albumen using balance buffer.Use washing containing high NaCl
De- liquid washes down albumen, obtains the 3 VLP samples of polio by preliminary purification.Used pillar is regenerated using 1M NaOH.
Case study on implementation seven：ELISA identifies virus-like particle
After poliovirus sample granule protein to debita spissitudo with PBS dilution purifying, it is dilute to carry out gradient according to 1: 3
It releases, the protein solution that 0.1ml has diluted is taken to add in ELISA Plate, 4 DEG C of coatings are overnight.Remove coating buffer, board-washing.It is added in per hole
0.3ml confining liquids (5% skimmed milk power+PBST) keep the temperature 2 hours in 37 DEG C.Coating buffer is removed, per hole addition dilution buffer
(2% skimmed milk power+PBST) keeps the temperature 1 hour with 1: 1000 diluted each 0.1ml of serum standard panel, in 37 DEG C, removes serum solution,
Board-washing.Then the goat-anti marked to every hole addition with dilution buffer (2% skimmed milk power+PBST) with 1: 5000 diluted HRP
Rabbit igg (BIO-RAD, article No. 170-8241-MSDS, Goat Anti-Rabbit IgG (H+L) HRP Conjugate) is each
0.1ml, 37 DEG C heat preservation 0.5 hour after remove enzyme standard liquid, board-washing；Then 0.1ml DAB developing solutions are added in into every hole, room temperature is kept away
Light action adds 2M H after ten minutes25O40.05ml terminate liquids terminate reaction, and measure OD450 values with enzyme mark colour comparatour,
The virus-like particle protein sample of the Polio 3 of purifying is detected by ELISA, can be exempted from anti-Polio virus type III
Epidemic disease serum National reference reacts, and ED50 curves are shown in Fig. 1, and ED50 values are about 0.00272g/L.
Case study on implementation eight：Electronic Speculum detects virus-like particle
The protein solution of polio 3 is placed on the copper mesh of 200 mesh carbon covering, adds the phosphotungstic acid of the pH7.0 of a drop 2%
(PTA), it is observed after drying with transmission electron microscope (2011 types of Japan Electronics JEM), the amplification factor of picture is 20
000 ×, virus-like particle is presented in sample as shown in Fig. 2, observing, particle diameter is in 30~40nm.
In conclusion 3CD genes provided by the invention and P1 genes are a kind of genes optimized, has the following advantages：
Gene by optimization is more suitable for the high-efficient expression target protein in yeast host, and disclosure satisfy that wanting for industrialized production
It asks；Meanwhile virus-like particle (VLPs) structure, the virus-like particle protein sample of the Polio 3 of purifying can be self-assembly of
Product are detected by ELISA, can be reacted with anti-Polio virus type III immune serum National reference, and due to using complete
Red yeast expression system, therefore this method has the following advantages：At low cost, yield is high, product property more stable homogeneous.
SEQUENCE LISTING
<110>Shanghai Runze Biotechnology Co., Ltd
<120>Recombinant poliovirus type III virus-like particle
<130> ZR1610003
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 2640
<212> DNA
<213> Artificial
<220>
<223>The gene of the P1 albumen of Polio 3 after codon optimization
<400> 1
atgggtgcac aagtctcttc tcaaaaggtc ggtgcacatg aaaactctaa cagagcttac 60
ggtggatcta ctattaatta cactaccatt aactattata aggattctgc ttctaacgct 120
gcttctaagc aagattactc tcaagatcca tctaagttca ccgaaccatt gaaggatgtc 180
ttgattaaga ccgctcctac tttgaactct ccaaacgtcg aggcttgtgg atactctgat 240
agagtcttgc aattgacctt gggaaactct actattacca ctcaagaagc tgctaactct 300
gtcgttgctt acggaagatg gccagaattt attagagatg atgaagctaa cccagtcgat 360
caaccaaccg aaccagatgt cgctacctgt agattctaca ccttggatac cgtcatgtgg 420
ggaaaggaat ctaagggatg gtggtggaag ttgccagatg ctttgagaga tatgggattg 480
ttcggacaaa acatgtatta tcattacttg ggaagatcag gatacaccgt ccatgtccaa 540
tgtaacgctt ctaaatttca tcaaggtgct cttggagtct ttgctattcc tgagtactgt 600
ttggctggag attctgataa gcaaagatac acctcttacg ctaacgctaa cccaggagag 660
aaaggaggaa agttctactc tcaattcaac agagatactg ctgtcacctc tccaaagaga 720
gagttctgtc cagtcgatta cttgttggga tgtggagtct tgttgggaaa cgctttcgtc 780
tacccacatc aaattattaa cttgagaacc aataactctg ctaccattgt cttgccatac 840
gtcaacgctt tggctattga ttctatggtc aagcataata actggggaat tgctatcttg 900
ccattgtctc cattggattt cgctcaagat tcttctgtcg aaattccaat taccgtcacc 960
attgctccaa tgtgttctga atttaacgga ttgagaaacg tcaccgctcc aaagttccaa 1020
ggattgccag tcttgaacac cccaggatct aaccaatact tgacctctga taaccatcaa 1080
tctccatgtg caattccaga atttgatgtc accccaccaa ttgatattcc aggagaagtc 1140
aagaacatga tggaattggc tgaaattgat accatgatcc cattgaactt ggagaatacc 1200
aagagaaaca ctatggatat gtatagagtt accttgtctg attctgctga tttgtctcaa 1260
ccaattcttt gtctttcttt gtctccagct tctgatccaa gattgtctca taccatgttg 1320
ggagaagtct tgaactacta tactcattgg gctggatctt tgaagtttac cttcttgttc 1380
tgtggatcta tgatggctac cggaaagatt cttgtcgctt acgctccacc tggagcacaa 1440
cctccaacct caagaaagga agctatgctt ggaacccatg tcatttggga tttgggattg 1500
caatcttctt gtactatggt cgttccttgg atttctaacg tcacctacag acaaaccacc 1560
caagattctt tcaccgaagg aggatacatt tctatgttct accaaaccag aattgtcgtt 1620
ccattgtcta ccccaaagtc tatgtcaatg ttgggattcg tctctgcttg taacgatttc 1680
tctgtcagat tgcttagaga caccactcat atttctcaat ctgctttgcc acaaggaatt 1740
gaagatttga ttactgaagt cgctcaaggt gcattgactt tgtctttgcc aaagcaacaa 1800
gattctcttc ctgataccaa ggcttctgga ccagctcatt ctaaggaagt cccagctttg 1860
accgctgtcg aaactggtgc aactaaccca ttggttccat ctgatactgt tcaaactaga 1920
catgttattc aaagaagatc aagatcagaa tctaccattg aatcattctt cgctagagga 1980
gcttgtgtcg ctattattga agtcgataac gaggaaccaa ctactagagc acaaaagttg 2040
ttcgctacat ggagaattac ctacaaagat actgtccagt tgagaagaaa gttggagttc 2100
ttcacctact caagatttga tatggaattt accttcgttg tcaccgctaa ctttaccaac 2160
actaacaacg gacatgcttt gaaccaagtc taccaaatta tgtacattcc accaggtgct 2220
cctactccta agtcttggga tgattacacc tggcaaacct catctaaccc atctattttc 2280
tacacctacg gagctgctcc agctagaatt tctgtcccat acgtcggatt ggctaacgct 2340
tactctcatt tctacgatgg attcgctaag gtcccattga agaccgatgc taacgatcaa 2400
attggagatt ctttgtactc tgctatgacc gtcgatgact tcggagtctt ggctattaga 2460
gttgtcaacg atcataaccc aaccaaggtt acctctaagg ttagaattta catgaagcca 2520
aagcatgtca gagtttggtg tccaagacca cctagagctg tcccatatta cggaccagga 2580
gtcgattaca aggataactt aaatccattg tctgaaaagg gattgaccac ttactaatag 2640
<210> 2
<211> 1941
<212> DNA
<213> Artificial
<220>
<223>The gene of the 3CD albumen of Sabin 1 after codon optimization
<400> 2
atgggtcctg gtttcgatta cgctgtggct atggctaaaa gaaacatcgt cactgctact 60
acctctaaag gtgagttcac tatgttgggt gtccatgata atgtcgctat cttgcctact 120
catgcttctc ctggtgaatc tatcgtcatt gatggtaagg aagttgaaat cttggatgct 180
aaagcattgg aggatcaagc tggtactaac ttggaaatca ctattatcac tttgaagaga 240
aatgaaaaat tcagagatat cagacctcac attcctactc aaatcactga aactaatgat 300
ggtgtcttga tcgtcaatac ttctaagtac cctaatatgt acgttcctgt tggtgctgtc 360
actgagcaag gttacttgaa cttgggtggt agacaaactg ctagaacttt gatgtacaac 420
ttccctacta gagctggtca atgcggtgga gttatcactt gcactggtaa agtcatcggt 480
atgcacgttg gtggtaacgg ttctcatggt ttcgctgccg ctcttaagag atcatacttc 540
actcagtctc aaggtgaaat ccaatggatg agaccttcta aagaagttgg ttatccaatc 600
attaacgctc cttctaaaac taagttggaa ccttcagcct tccactacgt cttcgagggt 660
gtcaaagaac ctgctgtctt gaccaaaaac gatcctagat tgaaaactaa cttcgaggaa 720
gctatcttct ctaagtacgt cggtaacaag atcactgagg ttgatgaaca catgaaagaa 780
gctgttgatc actacgctgg tcagttgatg tctttggata tcaacactga acaaatgtgc 840
ttggaagatg ctatgtacgg tactgatgga ttggaggctt tagacttgtc tacttctgct 900
ggttaccctt acgttgctat gggtaagaag aaaagagata tcttgaacaa acaaactaga 960
gatactaaag agatgcaaaa gttgttggat acttacggta tcaacttgcc tttggtcact 1020
tacgtcaaag atgaattgag atcaaaaact aaagtcgaac aaggtaagtc aagattgatc 1080
gaggcttctt ctttgaacga ttctgtcgct atgagaatgg ctttcggtaa cttgtacgct 1140
gccttccaca aaaaccctgg tgtcatcact ggttctgctg ttggttgcga tcctgacttg 1200
ttctggtcta agatccctgt cttgatggaa gagaagttgt tcgccttcga ttacactggt 1260
tacgatgctt cattgtctcc tgcttggttc gaggctcttg aaatggtctt ggagaagatc 1320
ggtttcggtg atagagttga ttacattgat tatttgaacc actctcatca cttgtacaag 1380
aacaaaactt actgcgtcaa aggtggtatg ccttctggtt gctctggtac ttctatcttc 1440
aactctatga tcaacaactt gatcattaga accttgttgt tgaaaactta caaaggtatt 1500
gacttggatc acttgaaaat gatcgcttac ggtgatgatg tcatcgcttc ttaccctcat 1560
gaagttgatg cttctttgtt ggctcaatct ggtaaagatt acggtttgac aatgactcct 1620
gctgataaat ctgctatctt cgagactgtc acttgggaaa acgtcacctt cttgaagaga 1680
ttcttcagag ctgatgaaaa gtaccctttc ttgatccatc ctgtcatgcc tatgaaagaa 1740
atccatgagt ctatcagatg gactaaagat cctagaaaca ctcaagatca tgtcagatca 1800
ttgtgcttgt tggcttggca caacggtgaa gaagagtaca acaagttctt ggctaagatc 1860
agatcagttc ctatcggtag agctttgttg ttgcctgaat actctacttt gtacagaaga 1920
tggttggatt ctttctaata g 1941
Claims (10)
1. a kind of method using pichia yeast expression system Prepare restructuring polio type III P1 albumen, including：
A, the 3CD genes of codon optimization and P1 genes are designed and synthesized；
B, the yeast expression vector of the yeast expression vector of structure 3CD genes and P1 genes；
C, structure P1 albumen and the Pichiapastoris expression strain of 3CD albumen coexpression；
Wherein, the nucleotide sequence of the P1 genes of the codon optimization such as SEQ ID NO：Shown in 1, the core of the 3CD genes
Nucleotide sequence such as SEQ ID NO：Shown in 2.
2. according to the method described in claim 1, it is characterized in that, in the step B, the yeast expression vector includes
pPICZ、pPIC6、pGAPZ、pAO815、pPIC9k。
3. according to the method described in claim 1, it is characterized in that, in the step C, the Pichi strain includes
GS115、KM71、SMD1168、X33。
4. the method as described in claim 1, which is characterized in that the step C includes that the Pichia anomala expression completed will be built
Carrier is transformed into the step of Pichiapastoris expression strain.
5. according to the method described in claim 1, it is characterized in that, the P1 albumen can be self-assembled into Pichia pastoris body
Virus-like particle.
It is 6. a kind of such as SEQ ID NO：The gene of coding type III polio P1 albumen shown in 1.
It is 7. a kind of such as SEQ ID NO：The gene of encoding Type I attenuated polio viruses 3CD albumen shown in 2.
8. a kind of yeast expression vector, which is characterized in that the expression vector has the sequence of gene described in claim 6
Row.
9. a kind of yeast expression vector, which is characterized in that the expression vector has the sequence of gene described in claim 7
Row.
10. a kind of expression claim 6 and the P1 albumen of gene code and the Pichiapastoris expression strain of 3CD albumen described in 7.
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