CN104164446B - The method for generating HPV33L1 albumen with expressed by Hansenula yeast system - Google Patents
The method for generating HPV33L1 albumen with expressed by Hansenula yeast system Download PDFInfo
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Abstract
The present invention relates to the methods for generating HPV33L1 albumen with expressed by Hansenula yeast system.Specifically, the invention discloses the method for the recombination Hansenula yeast cell for generating expression HPV33L1 albumen and the recombination Hansenula yeast cells generated by the method.The invention also discloses preparing the purposes in preventative vaccine using the method and generated HPV33L1 albumen of the recombination Hansenula yeast cell generation HPV33L1 albumen.
Description
Invention field
The invention belongs to medical bioengineering technical field, the method for being related to generating HPV33L1 albumen, more particularly to use the Chinese
The method that inferior yeast expression system generates HPV33L1 albumen.
Background technique
Human papilloma virus (human papillomavirus, HPV) is a kind of nonencapsulated closed loop double-stranded DNA virus,
Belong to papovaviridae polyomavirus subfamily, the main epithelium mucous membrane tissue for invading human body, and then induces various benign and pernicious
Preneoplastic lesions.
Having identified the different subtype HPV come at present is more than 200 kinds, and HPV infection has apparent tissue specificity, different
The HPV of type is different for the thermophilic tropism of skin with mucous membrane, can induce different papillary lesions, about more than 30 kinds of HPV types
It is not related with reproductive tract infection, wherein have more than 20 kinds it is related to tumour.The good pernicious difference of lesion is induced according to HPV, HPV can be big
Cause is divided into two classes: 1) high-risk-type (such as HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52,
HPV56, HPV58, HPV59, HPV68 etc.): high-risk HPV and mankind's Various Tissues malignant tumour are closely related, mainly cause weight
Spend atypical hyperplasia and infiltrating carcinoma；2) low risk (such as HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54,
HPV72, HPV81 etc.): low risk HPV can cause epidermal cell benign proliferative venereal disease, such as condyloma acuminatum and condyloma.
HPV is mainly made of virus coat and genomic DNA.Genome is about 7900bp, there is 8 encoding hiv protease bases
Cause.Wherein the albumen of 6 ORF coding is in the early expression of virus replication, referred to as early protein；The albumen of 2 ORF coding is in disease
The advanced stage expression of poison duplication, referred to as late protein.Late protein includes major cat protein L1 and secondary coat protein L2, and is joined
With the formation of virus coat.
HPV viruse coat protein is able to carry out self assembly, the L1 albumen of single expression or by L1 in a variety of expression systems
Albumen and L2 albumen coexpression Shi Junneng be self-assembled into virus-like particle (virus-like particle, VLP), wherein with
The VLP of the generations such as Yeast system, Baculovirus insect expression system and mammalian cell expression system is closer to natural viral
Structure.Generation neutralizing antibody can be induced in vivo after being immunized using the VLP of heterogenous expression system production, obtain good exempts from
Epidemic disease protecting effect.
Multiple-shaped nuohan inferior yeast (Hansenula Polymorpha), also referred to as Pichia augusta are currently generally acknowledged
One of ideal heterologous gene expression system.It is raw both to have protokaryon as single celled eukaryotic microorganism for multiple-shaped nuohan inferior yeast
Object fast growing is easy to the features such as genetic manipulation, and has the functions such as eukaryocyte post translational processing and modification.In addition, the inferior ferment of the Chinese
Mother is also equipped with that safety is good, be easy to cultivate, low in cost, expression quantity is high and the advantages such as inheritance stability, and can overcome such as
Saccharomyces cerevisiae (Saccharomyces cerevisiae) bacterial strain is unstable, low output and glycosylation side chain are too long and finish red ferment
The lower problem of female (Pichia Pastoris) exogenous origin gene integrator copy number.Currently, using expressed by Hansenula yeast system production
Drug (such as insulin, trade name Wosulin) and HBV vaccine (trade name Hepavax-Gene) list marketing.
It discloses in CN102586287A and CN102719453A and is disclosed using expressed by Hansenula yeast in Chinese invention patent
The method of system expression HPV16 and HPV18 type L1VLP.However whether can be in Hansenula yeast about the L1 albumen of HPV33 type
High efficient expression is realized in system and is assembled into VLP particle, and there is no open reports at present.Above-mentioned 2 published patent Shens
Please in, do not provide the exogenous origin gene integrator copy number of HPV16 and HPV18L1 result and improve copy number of foreign gene side
Method.In terms of the inducing expression of foreign protein, the induction time of HPV16 and HPV18L1 in the fermenter is required more than 20 hours,
And without providing the specifying information of protein expression level.In addition, as an important protein purification index, about purification process and
The purity of foreign protein HPV16 and HPV18L1 are also without open when purifying is completed.
It is to realize high efficient expression of the HPV33L1 albumen in Hansenula yeast and can purify to obtain high-purity
HPV33VLP albumen applies the Double Selection strategy of Zeocin resistance screening combination G418 resistance screening in the present invention, special
The recombination expressed by Hansenula yeast bacterial strain after the G418 resistant panel screening at concentrations up to 16mg/ml is passed on and stablized is not applied.
By detection, the copy number for screening the HPV33 high expression bacterial strain of acquisition can be more than 60 copies.During fermentation inducement, lead to
The expression of HPV33L1 albumen can be significantly improved to 35 DEG C by crossing raising inducing expression temperature.In addition, being purified for VLP
Common chromatography media POROS50HS is not easy to elute (when high concentration NaCl elutes target protein when purifying HPV33VLP in journey
Still have 50% target protein hanging column) the characteristics of, the present invention uses POROS XS chromatography media, so that target HPV33L1 albumen is in height
Concentration NaCl can be realized the elution of 80% or more albumen when eluting, so that the yield of HPV33L1 albumen greatly improved.
Summary of the invention
In the first aspect, the present invention provides a kind of recombination Hansenula yeast cells for generating expression HPV33L1 albumen
Method, it includes following steps:
A) by will include to encode the exogenous polynucleotide of the nucleotide sequence of HPV33L1 albumen to be inserted into carrier and construct table
Expression constructs；
B) Hansenula yeast cell is converted with the expression construct obtained in step a)；With
C) the Hansenula yeast cell obtained in step b) is screened, obtains the recombination for containing the exogenous polynucleotide
Hansenula yeast cell.
In the second aspect, the present invention also provides the recombination Hansenula yeast cells generated according to the above method.
In the third aspect, the present invention also provides a kind of methods for generating HPV33L1 albumen, comprising the following steps:
I) recombination Hansenula yeast cell of the invention is cultivated under conditions of being suitable for HPV33L1 protein expression；With
Ii it) is recycled from culture and purifies HPV33L1 albumen.
In terms of the last one, the present invention also provides the HPV33L1 albumen generated according to the method for the present invention to prepare
The purposes in vaccine for preventing HPV33 infection.
Detailed description of the invention
Fig. 1 recombinates the SDS-PAGE detection of Hansenula yeast cellular expression levels.1-8:8 plants of different recombination Hansenula yeast bacterial strains
Inducing expression；9: standard items (10 μ g/mL)；10: protein molecular weight marker.
Fig. 2 is using MOX promoter fragment as the Southern trace testing result of probe.It is with ATCC26012 genomic DNA
Control.1: control (applied sample amount 1000ng)；2: control (applied sample amount 500ng)；3: control (applied sample amount 250ng)；4: right
According to (applied sample amount 125ng)；5: height expression recombinant bacterium HP-1#/pRMHP2.1-33hp genomic DNA (applied sample amount 7.8ng,
Its brightness is between 500ng and 1000ng control)；6: the genomic DNA of height expression recombinant bacterium HP-2#/pRMHP2.1-33hp
(applied sample amount 7.8ng, brightness is between 500ng and 1000ng control).
The Western Blot of HPV33L1 protein expression detects (inducing expression under the conditions of 35 DEG C) in Fig. 3 fermentation process.1:
Induction 0 hour；2: induction 1 hour；3: induction 3 hours；4: induction 5 hours；5: induction 7 hours；6: induction 8 hours；7: induction 9
Hour；8: induction 10 hours；9: standard items (10 μ g/mL)；10: pre-dyed protein molecular weight marker.
The POROS50HS of Fig. 4 A:HPV33L1 albumen purifies electrophoretogram.1: upper prop sample；2: flowing through liquid；3-5: different dense
Spend NaCl eluent；6: cleaning solution；7: protein molecular weight marker.
The POROS XS of B:HPV33L1 albumen purifies electrophoretogram.1: upper prop sample；2: flowing through liquid；3-6: various concentration
NaCl eluent；7: cleaning solution；8: protein molecular weight marker.
The CHT of C:HPV33L1 albumen purifies electrophoretogram.1: upper prop sample；2: flowing through liquid；3,4: various concentration phosphate is washed
De- liquid；5: cleaning solution；6: protein molecular weight marker.
The transmission electron microscope observing result of the HPV33L1 albumen of Fig. 5 purifying.
Detailed description of the invention
The present inventor has been successfully set up the method for generating HPV33L1 albumen using Hansenula yeast, generated HPV33L1
Albumen can be self-assembled into virus-like particle, can be used for preparing the vaccine of prevention HPV infection.
Present invention firstly provides a kind of method of recombination Hansenula yeast cell for generating expression HPV33L1 albumen, packets
Containing following steps:
A) by will include to encode the exogenous polynucleotide of the nucleotide sequence of HPV33L1 albumen to be inserted into carrier and construct table
Expression constructs；
B) Hansenula yeast cell is converted with the expression construct obtained in step a)；With
C) the Hansenula yeast cell obtained in step b) is screened, obtains the recombination for containing the exogenous polynucleotide
Hansenula yeast cell.
The invention also includes the recombination Hansenula yeast cells generated according to the method.
Amino acid sequence from the HPV33L1 albumen of different HPV33 Strain can have differences.The present inventor passes through
HPV33L1 protein sequence all in database is compared, chooses and occurs on each amino acid position of HPV33L1 albumen
The highest amino acid residue of frequency, obtains the amino acid sequence for being shown in SEQ ID NO:1, the sequence be HPV33L1 albumen most
Representative consensus sequence.Therefore, the HPV33L1 albumen in the present invention preferably has amino acid shown in SEQ ID NO:1
Sequence.
In order to efficiently express HPV33L1 albumen, inventor's amino according to shown in SEQ ID NO:1 using Hansenula yeast
Acid sequence carries out the codon optimization of nucleotide sequence for Hansenula yeast.Optimization principles include: a) according to Hansenula yeast heredity
Password frequency of use table (http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi? species=
4905) frequency of use highest or higher codon are selected；B) avoid thering is potential impact to bear genetic transcription or protein translation
Controlling element, such as the area PolyAT, the area PolyGC, the silencer area (Sliencer) and internal splice site；C) to include 5 ' end
The code area UTR, HPV33L1 and 3 ' end UTR including mRNA secondary structure carry out comprehensive analysis, avoid complicated RNA secondary structure
Formation, reduce the free energy of mRNA secondary structure；D) upstream of coding region use as far as possible under Hansenula yeast promoter
Swim 5 ' the completely the same areas UTR of native sequences；E) common restriction enzyme enzyme recognition site is eliminated.By the nucleotide of optimization
Sequence is shown in SEQ ID NO:2.It is preferably SEQ ID that the nucleotide sequence of HPV33L1 albumen is encoded used in the present invention
Sequence shown in NO:2.
The adaptable polymorpha expression vector of the present invention is that application No. is the Chinese patent Shens of 201210021524.X
Please described in polymorpha expression vector pRMHP2.1 (sequence comprising being shown in SEQ ID NO:9).By that will include coding
The exogenous polynucleotide of the nucleotide sequence of HPV33L1 albumen is cloned into pRMHP2.1 carrier, and expression of the invention can be obtained
Construct.It will be understood by those skilled in the art that expression construct of the invention can also use other vector constructions, such as award
Carrier described in the Chinese patent CN100400665C of power.
In order to be expressed in Hansenula yeast, exogenous polynucleotide in the expression construct operably with promoter
It is connected with terminator.
As used herein, the function connects for referring at least two polynucleotides " are operably connected ".For example, operably connecting
It connects including the connection between promoter and another polynucleotides, wherein the promoter sequence originates and mediates this another is more
The transcription of nucleotide.Operably connection includes the connection between terminator and another polynucleotides, wherein the terminator
Terminate the transcription of another polynucleotides.
Being suitable for the invention promoter includes but is not limited to MOX, FMD, AOX1 and DHAS promoter.In some embodiment party
In case, promoter used in the present invention is the MOX promoter from Hansenula yeast.Be suitable for the invention terminator include but
It is not limited to the MOX terminator from Hansenula yeast.
Expression construct is converted to Hansenula yeast cell can be carried out with a variety of methods known in the art, including but not
It is limited to the conversion that electroporation and PEG are mediated.
In addition, developed in this field it is a variety of for expressing foreign protein Hansenula yeast bacterial strain, including but not limited to
CGMCC2.2498 Hansenula yeast, ATCC34438 Hansenula yeast and ATCC26012 Hansenula yeast cell.These Hansenula yeast bacterial strains
Also it can be applied to the present invention.In some embodiments, for converting the Hansenula yeast cell of expression construct of the invention
It is ATCC26012 Hansenula yeast cell.
In post-conversion, recombination Hansenula yeast cell can be selected according to the resistant gene carried on carrier.It is suitable anti-
Property gene includes but is not limited to Zeocin resistant gene and G418 resistant gene.According to used carrier, it is also possible to use battalion
Defect culture medium is supported to screen recombination Hansenula yeast cell.In one embodiment, the recombination inferior ferment of the Chinese is selected using Zeocin
Mother cell.In another embodiment, recombination Hansenula yeast cell is selected using G418.In a preferred embodiment, group
Conjunction selects recombination Hansenula yeast cell using Zeocin and G418.The screening concentration of Zeocin can be 0.25,0.5,0.75,
1.0 or 1.5mg/ml, preferably 0.5mg/ml.The screening concentration of the G418 used can be 2,4,8,10,12,14,16,18 or
20mg/ml, preferably 16mg/ml.
Expression and its copy number in Hansenula yeast of the exogenous polynucleotide in Hansenula yeast are positively correlated.Cause
This, further screening can also be carried out by the methods of Southern trace or quantitative PCR and contains multicopy external source multicore glycosides
The recombination Hansenula yeast cell of acid.It is preferred that exogenous polynucleotide copy number is greater than 15 recombination Hansenula yeast cell, it is more preferably outer
Source polynucleotide copies number is greater than 60 recombination Hansenula yeast cell.
The present inventor is it has surprisingly been found that be applied in combination Zeocin and G418 to select recombination Hansenula yeast cell, spy
It is not to carry out selection using the up to G418 of 16mg/ml to obtain exogenous polynucleotide copy number greater than 15, especially greatly
In 60 stable recombination Hansenula yeast cell.
On the other hand, the present invention also provides a kind of methods for generating HPV33L1 albumen, comprising the following steps:
I) recombination Hansenula yeast cell of the invention is cultivated under conditions of being suitable for HPV33L1 protein expression；With
Ii it) is recycled from culture and purifies HPV33L1 albumen.
The various culture mediums and basic condition of culture known in the art that can be used for cultivating Hansenula yeast, those skilled in the art
It can be selected or be modified as needed.The culture of recombination expressed by Hansenula yeast bacterial strain of the invention can according to required protein content
To carry out in flask or be carried out in the bioreactor (fermentor of such as 30L) of different scales.It, can according to selected promoter
With the expression for suitable inducer being added in culture to induce the HPV33L1 albumen.Using MOX or FMD promoter
In the case of, methanol can be added as inducer.Yeast fermented and cultured routinely carries out at 30 DEG C, and the present inventor makes us frightened
It finds oddly, the induction that recombination Hansenula yeast cell of the invention carries out protein expression at 35 DEG C can make exogenous protein expression amount
It significantly improves.
Various protein purification modes known in the art can be used in the purifying of generated HPV33L1 albumen, such as saltout,
The combination of ultrafiltration, precipitating, chromatography etc. or these modes.In the experimental program that one optimizes, chromatography media POROS is used first
XS carries out preliminary purification, further pure followed by chromatography media Macro-Prep ceramic hydroxyapatite (Type II, 40 μm)
Change.
The HPV33L1 albumen of the purifying prepared using method of the invention can be self-assembled into virus-like particle (embodiment
9, Fig. 5) good immunogenicity (embodiment 10), and in mouse is shown, therefore present invention provides the HPV33L1
Albumen is in preparation for preventing the purposes in the vaccine that HPV33 infects.
Embodiment
The present invention will be further illustrated by way of embodiment below, but is not therefore limited the present invention to described
Scope of embodiments in.
The analysis of embodiment 1:HPV33L1 consensus amino acid sequences
The HPV33L1 albumen of overall length is made of 499 amino acid, soft using Vector NTI after GenBank is retrieved
Part AlignX function carries out amino acid alignment analysis, obtains most representative HPV33L1 consensus amino acid sequences
(it is highest to be all made of the frequency of occurrences in each amino acid position of HPV33L1 by consensus amino acid sequence
The sequence of amino acid residue), sequence is as shown in SEQ ID NO:1.
The optimization design of embodiment 2:HPV33L1 encoding gene and artificial synthesized
In order to efficiently express HPV33L1 albumen, inventor's amino according to shown in SEQ ID NO:1 using Hansenula yeast
Acid sequence carries out the codon optimization of nucleotide sequence for Hansenula yeast.Optimization principles include: a) according to Hansenula yeast heredity
Password frequency of use table selects frequency of use highest or higher codon；B) it avoids having genetic transcription or protein translation potential
The negative regulatory element of influence, such as the area PolyAT, the area PolyGC, the silencer area (Sliencer) and internal splice site；C) right
Comprehensive analysis is carried out comprising the mRNA secondary structure including 5 ' the end code areas UTR, HPV33L1 and 3 ' end UTR, avoids complicated RNA
The formation of secondary structure reduces the free energy of mRNA secondary structure；D) it is used as far as possible and Hansenula yeast in upstream of coding region
5 ' the completely the same areas UTR of promoter downstream native sequences；E) common restriction enzyme enzyme recognition site is eliminated.By optimization
Nucleotides sequence be shown in SEQ ID NO:2.
According to the above nucleotide sequence, the complete sequence for entrusting Sinogenomax Co., Ltd. to carry out is artificial
Synthesis, is cloned in carrier T and (is named as T-33hp), and carry out sequence verification to it.
Embodiment 3: the expression construct for carrying HPV33L1 nucleotide sequence is generated
Polymorpha expression vector applied by the present invention is that application No. is the Chinese patent applications of 201210021524.X
Described in polymorpha expression vector pRMHP2.1 (SEQ ID NO:9).
(1) MOX promoter and the PCR amplification of MOX terminator
Using the mixed genomic DNA of Hansenula yeast strains A TCC26012 and ATCC34438 as template, with following primer pair
Amplification obtains the MOX promoter that size is 1518bp, while introducing NotI restriction enzyme site in upstream；
MOX promoter upstream primer: 5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ
ID NO:3)
MOX promoter downstream primer: 5 '-TTTGTTTTTGTACTTTAGATTGATGTC-3 ' (SEQ ID NO:4)
Using the mixed genomic DNA of Hansenula yeast strains A TCC26012 and ATCC34438 as template, with following primer pair
Amplification obtains the MOX terminator that size is 311bp, while introducing BglII restriction enzyme site in downstream；
MOX terminator upstream primer: 5 '-GGAGACGTGGAAGGACATACCGC-3 ' (SEQ ID NO:5)
MOX terminator downstream primer: 5 '-GAAGATCTCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:6)
(2) expression construct for carrying HPV33L1 nucleotide sequence is generated
To carry the recombinant plasmid T-33hp of 33hp as template, obtaining size with following primer pair amplifies is 1548bp's
HPV33L1hp gene, while the overlap that MOX promoter region 3 ' is held is introduced in upstream, MOX terminator 5 ', which is introduced, in downstream holds
Overlay region；
33 upstream primers:
5 '-CATCAATCTAAAGTACAAAAACAAAATGTCGGTGTGGAGACCATCTGAAG-3 ' (SEQ IDNO:7)
33 downstream primers: 5 '-GCGGTATGTCCTTCCACGTCTCCTTATTTCTTGACTTTCTTTCTCTTGGC-3 '
(SEQ ID NO:8)
Using three MOX promoter, 33hp gene, MOX terminator segments as hybrid template, obtained with following primer pair amplifies
Size is the 33hp expression cassette of 3.4Kb, and amplified production carries NotI restriction enzyme site in upstream, carries BglII digestion position in downstream
Point.
MOX promoter upstream primer: 5 '-AAGGAAAAAAGCGGCCGCAACGATCTCCTCGAGCTGCTCGC-3 ' (SEQ
ID NO:3)
MOX terminator downstream primer: 5 '-GAAGATCTCAATCTCCGGAATGGTGATCTG-3 ' (SEQ ID NO:6)
33hp expression cassette is cloned into pRMHP2.1 carrier by NotI+BglII double digestion, obtains expression construct
pRMHP2.1-33hp。
Embodiment 4: the generation of recombination Hansenula yeast cell
(1) extraction and digestion of recombinant expression construct constitution grain
Picking is transformed into the E. coli clones of the recombinant expression construct constitution grain obtained in embodiment 3, after expanding culture
Plasmid is extracted using E.Z.N.A Plasmid Mini Kit kit (Omega Bio-Tek company), and carries out list with BglII
Digestion is recycled using E.Z.N.A Gel Extraction Kit kit (Omega Bio-Tek company), pre- with 50 μ L
The sterile water of heat to 55 DEG C is eluted, by measuring OD260It is quantitative to carry out DNA, and the segment of linearisation is diluted to 100ng/
μ l is stored in -20 DEG C of refrigerators, spare.
(2) processing of Hansenula yeast cell
Picking Hansenula yeast strains A TCC26012 single colonie accesses in the small test tube of the YPD fluid nutrient medium containing 5ml, 30
DEG C culture 12 hours；Take bacterium solution 5ml to be forwarded in 200ml YPD culture medium, 30 DEG C culture 4-6 hours, until OD600nmAbout 1.0-
1.5,10min is centrifuged in 5000rpm；It is resuspended with 200ml0.1mol/L phosphate buffer (DTT containing 25mmol/L, pH7.5)
Thallus mixes well, and is centrifuged 10min in 30 DEG C of incubations 30min, 5000rpm, abandons supernatant, stay thallus.With the STM solution of pre-cooling
200ml washes thallus, and thallus pressure-vaccum is uniform, is centrifuged 3min in 4 DEG C of 5000rpm, abandons supernatant, stay precipitating.With ice-cold STM solution
Thallus is resuspended in 100ml, is centrifuged 3min in 4 DEG C of 5000rpm, abandons supernatant, stay precipitating.It is ice-cold with 50-200 μ l according to biomass
Thallus is resuspended in STM solution, and bacterium solution is transferred in the centrifuge tube after high pressure, and ice bath prepares conversion.
(3) electrotransformation of Hansenula yeast cell
By plasmid: thallus=1:2 amount, which is added, recombinates 15 μ l of expressed by Hansenula yeast plasmid, 30 μ l of bacterium solution, and abundant pressure-vaccum is equal
It is even, it is placed in be transformed in ice bath；It will be impregnated in advance with alcohol, and after ultraviolet irradiation, take out, add in the electric revolving cup that -20 DEG C refrigerate
Enter plasmid thallus mixed liquor；It shocks by electricity by the condition of voltage 2500V, 150 Ω of resistance, 50 μ F of capacitor；It is rapidly added after electric shock
1ml has been balanced to the YPD solution of room temperature, is transferred in EP pipe after mixing gently；Thallus after electricity is turned is put in 30 DEG C of water-baths
1h is set, is gently overturned 3 times at interval of 15min；It will be incubated for the bacterium solution of 2h, has been centrifuged 10min in 5000rpm, abandons supernatant；With 200 μ
Thallus is resuspended in l YPD solution, is coated in the YPD plate containing 0.5mg/mlZeocin with 100 μ l/ plates, cultivates in 30 DEG C of inversions
3-7 days.
(4) passage, the stabilization of expressed by Hansenula yeast bacterial strain are recombinated
The recombinant bacterial strain single colonie grown in picking Zeocin resistant panel, is inoculated in 5ml Zeocin's containing 0.5mg/ml
In YPD fluid nutrient medium, in 30 DEG C, 200rpm shaking table culture 24-48 hours, until OD value is turned up to after 50 with the ratio of 1:1000
It is connected in the YPD fluid nutrient medium of 5ml Zeocin containing 0.5mg/ml, culture to OD value turns up to after 50, then with the ratio of 1:1000
It is connected in the YPD fluid nutrient medium of 5ml Zeocin containing 0.5mg/ml, and so on, the continuous guarantor passed 10 times and carry out strain
It deposits.Preservation system is bacterium solution: 60% glycerol=1:1, and amount saves strain as needed, and usually+500 μ l60% of 500 μ l bacterium solution is sweet
Oil；
The recombination expressed by Hansenula yeast bacterial strain access 5ml for reaching 10 times is free of to the YPD fluid nutrient medium of Zeocin resistance
In, in 30 DEG C, 200rpm shaking table culture to OD value up to after 50, transferred in 5ml YPD fluid nutrient medium with the ratio of 1:1000,
And so on, it is continuously passed 5 times in the YPD fluid nutrient medium without Zeocin resistance.
By the YPD plate of bacterium solution coating G418 containing 16mg/ml after stabilization, cultivated 2-3 days in 30 DEG C of inversions.
Embodiment 5: the expression study of recombination Hansenula yeast bacterial strain
The multiple recombination Hansenula yeast single colonies of picking from the YPD plate of the G418 containing 16mg/ml access the YPG containing 5ml
In the small test tube of fluid nutrient medium, 30 DEG C are cultivated 24 hours；Bacterium solution is transferred in the 100ml tri- of the induced medium of YPM containing 30ml
In the bottle of angle, initial density OD600=1, it induces 72 hours, was added in bacterium solution every 12 hours final concentration of in 30 DEG C of shaking tables
0.5% methanol solution.
Bacterium solution after taking 10ml to induce is transferred in the centrifuge tube of 50ml, is centrifuged 10min in 10000rpm, is abandoned supernatant；With
Bacterial sediment is resuspended in the cell cracking of 50ml, after mixing well, presses in Ultrasound Instrument and " power 60%, time 20min, opens 5s, closes
The ultrasonication program of 5s " carries out bacterial cell disruption, needs to keep ice bath in shattering process；By the bacterium solution of ultrasonication, in
10000rpm is centrifuged 10min, carries out SDS-PAGE detection, induction result display (as shown in Figure 1): highly expressed after collecting supernatant
Hansenula yeast bacterial strain expression is recombinated up to 50 μ g/ml or more.
Embodiment 6: the exogenous polynucleotide copy number detection of recombination Hansenula yeast cell
(1) extraction of pastoris genomic dna and quantitative
It is inoculated with high expression yeast strain culture medium into the YPD fluid nutrient medium of 5ml that 2 plants of embodiments 5 obtain, 30 DEG C of trainings
Feeding 16~for 24 hours；2ml Yeast Cultivation liquid is taken, room temperature 4500g is centrifuged 3min and collects thallus；Using 500 μ l SCED solution (1mol/L
Sorbierite, 10mmol/L sodium citrate, 10mmol/L EDTA, 10mmol/L DTT dithiothreitol (DTT)) thallus is resuspended, and be added
5min fullys shake in 50mg bead, and 50 μ l10mg/ml lywallzymes, 37 DEG C of warm bath 1h are added；The 10%SDS of 60 μ l, 30 μ l is added
Proteinase K, the RNaseA enzyme of 10 μ l is placed at room temperature for and cultivates 2h in 55 DEG C of water-bath after ten minutes；350 μ l saturated phenols are added
And 350 μ l chloroforms is centrifuged 10 minutes in 13000rpm after being sufficiently mixed, the upper liquid after collecting layering；It is added in equal volume (about
700 μ l) chloroform, in 13000rpm, centrifugation 10 minutes, the upper liquid after collecting layering；It is added 140 μ l's into upper liquid
3mol/L sodium acetate solution adds 700 μ l isopropanols, is placed at room temperature for 5min after mixing after mixing gently, in 13000rpm, from
The heart 10 minutes.Supernatant is abandoned, 1ml70% ethyl alcohol is added and is cleaned, in 13000rpm, is centrifuged 10 minutes, supernatant, DNA precipitating are gone
It is placed in after being placed at room temperature for 30min, 100 μ l TE dissolution is added.By measuring OD260nmIt carries out genomic DNA to quantify, and by line
The segment of property is diluted to 100ng/ μ l, is stored in -20 DEG C of refrigerators, spare.
(2) Southern immunoblot method carries out quantifying for exogenous polynucleotide copy number
A: probe preparation
MOX described in Chinese patent application of the MOX probe application No. is 201210021524.X applied by the present invention
Probe, using Roche company DIG DNA Labeling and Detection kit kit (Cat No:
11093657910) probe preparation is carried out.Specific steps are as follows: the PCR product of 10 μ l MOX promoter regions is added into EP tubule
(200ng/ μ l), and sealed up with sealed membrane, boiling water boils 10 minutes.It is immediately placed in the dehydrated alcohol capsule of pre-cooling (- 20 DEG C)
(cooling down suddenly).Pipe outer wall ethyl alcohol is dried, is centrifuged (about 10s), sequentially adds 5 μ l tiny electrolytic cell water, 2 μ l10x
Hexanucleotide Mix, 2 μ l10x dTP Labeling Mixture, 1 μ l7Klenow Enzyme(labeling
Grade), mix, sealed membrane sealing.37 DEG C of water-baths are stayed overnight, -20 DEG C of preservations.
B:Southern trace
Using the digoxin hybridization check kit I(Cat No:DIGD- of Beijing Mei Laibo medical science and technology Co., Ltd
110) and the efficient hybridization solution of HyB (Cat No:Hyb-500) is according to product description progress Southern trace detection.
The display (as shown in Figure 2) of Southern trace testing result: it is obtained in the g418 resistant panel screening of 16mg/ml
Height expression recombination Hansenula yeast HP-1#/pRMHP2.1-33hp bacterial strain and HP-2#/pRMHP2.1-33hp bacterial strain external source multicore glycosides
Sour copy number is more than 60.The Double Selection strategy using zeocin antibody screening combination g418 resistance screening is shown, especially
It is to facilitate to obtain high copy integration using the g418 resistant panel screening of up to 16mg/ml concentration and can be carried out HPV33L1 egg
White highly expressed recombinant bacterial strain.
The zymotechnique of embodiment 7:HPV33L1 recombination expressed by Hansenula yeast bacterial strain
Fermentation seed liquid: taking 1 to freeze glycerol stock (HP-1#/pRMHP2.1-33hp), and 50 μ l access 5ml is drawn after thawing
In YPD culture medium, in 30 DEG C, 200rpm shaking table culture 20-24hr, A600nmAbout 2-5 respectively draws 1ml after assay approval and accesses 2 bottles
In 500ml YPD culture medium, in 30 DEG C, 200rpm shaking table culture 20-24hr to A600nmAbout 15-20, as hair after assay approval
Ferment seed liquor is stand-by.
Zymotechnique: fermentation initial culture medium contains yeast powder 300g, peptone 150g, glycerol 100g, basic salt
(K2SO4273g, MgSO4100g, 85%H3PO4400ml, KOH62g), 10L purified water sufficiently dissolves, and is added in 30L fermentor, pure
Change water and be settled to 14L, 121 DEG C, 60ml PTM1 liquid microelement (CuSO is added in 30min sterilizing after being cooled to 30 DEG C4·
5H2O6.0g, KI0.088g, MnSO4·H2O3.0g, Na2MoO4·2H2O0.2g, H3BO30.02g, CoCl2·6H2O0.5g,
ZnCl220.0g FeSO4·7H2O65.0g, Biotin0.2g, dense H2SO45.0ml, purified water are settled to 1L, 0.22 μm of filter membrane mistake
Filter out bacterium), ammonium hydroxide adjusts pH5.6, is inoculated with 1 bottle of 500ml fermentation seed liquid, and fermentation volume is 15L at this time.Originating speed of agitator is
200rpm, air mass flow 0.5Nm3/hr, tank press 0.5bar, and control oxygen dissolving value is 20-80% in fermentation.The initial growth phase maintains about
After 25hr, bacterium solution A600nmReach 20 or so, dissolved oxygen starts rapid increase, starts with 100ml/hr flow velocity flow feeding culture medium
(50% glycerol (W/V), 12ml PTM1) enters stream plus growth period at this time.After cultivating about 6-8hr, bacterium solution A600nmReach 90 or so,
Stop stream adding, ammonium hydroxide adjusts pH value to 6.0.Start stream after dissolved oxygen bottom out plus methanol (PTM1 containing 12ml/L) enters induction
Expression phase, induction period increase temperature to 35 DEG C, and the initial flow rate of methanol is 50ml/hr, is sampled per hour, and methanol electrode is surveyed
Determine methanol concentration, makes methanol concentration control in < 5g/L by adjusting methanol feeding rate.
Lower tank centrifugation and expression quantity detection: lower tank after 10hr is induced, is collected under the conditions of 4 DEG C with 5000rpm centrifugation 30min wet
Thallus, -20 DEG C freeze it is spare.In addition, 10ml fermentation liquid is taken to be transferred in the centrifuge tube of 50ml, it is centrifuged 10min in 10000rpm,
Supernatant is abandoned, bacterial sediment is resuspended with the cell pyrolysis liquid of 50ml, ultrasonication is carried out after mixing well, ultrasonication liquid carries out
Western blot detection, western blot testing result are as shown in Figure 3: entire induction duration can terminate in 10 hours,
The fermentation liquid expression quantity of HPV33 is up to 150 μ g/mL or more.
The purifying process of embodiment 8:HPV33L1 recombinant protein is studied and optimization
Bacterial cell disruption: the HPV33 expression wet thallus of -20 DEG C of preservations is taken, is added 0.9% according to the ratio of 10ml/g wet thallus
Physiological saline cleaning.After cell washing, by 20ml buffer/g wet thallus be added disruption buffer (NaCl containing 0.5mol/L,
0.02%Tween-80,0.05mol/L MOPS) sufficiently dissolution, broken using high-pressure homogenization, cracking pressure 1500bar, circulation is broken
Broken 5-8 times, microscopy percentage of damage > 90%.Bacterial cell disruption liquid is centrifuged 30min under the conditions of 4 DEG C with 10000rpm, collects supernatant,
50% ammonium sulfate is added, after precipitating 30min, 10000rpm is centrifuged 30min under the conditions of 4 DEG C, collects supernatant.
Chromatographic purifying is step 1: 1) common process: the bacterial cell disruption supernatant filtered through 1 μm is splined on chromatography media
POROS50HS, destination protein are adsorbed on chromatography media, with 0.5M~1.5M NaCl gradient elution；2) optimization technique: through 1 μm
The bacterial cell disruption supernatant of filtering is splined on chromatography media POROS XS, with 0.5M~1.5M NaCl gradient elution.Using with
Upper 2 kinds different chromatography medias adsorb HPV33L1 albumen, are cleaned again with 0.5M NaOH after various concentration NaCl gradient elution
Pillar.The purifying situation of HPV33L1 albumen during SDS-PAGE detection chromatography, as a result as shown in fig. 4 a and fig. 4b: 1) applying
Chromatography media POROS50HS still remains 50% or more HPV33L1 albumen and is not eluted down after high concentration NaCl elution
Come；2) chromatography media POROS XS is applied, after high concentration NaCl elution, the HPV33L1 albumen only less than 20% is not washed
It takes off.Show to be conducive to when carrying out the preliminary chromatographic purifying of HPV33L1 albumen using chromatography media POROS XS
The elution of HPV33L1 albumen, to greatly improve the yield of HPV33L1.
Chromatographic purifying is step 2: be splined on chromatography media Macro-Prep ceramics for the HPV33L1 albumen after preliminary purification
Hydroxyapatite (Type II, 40 μm), destination protein is incorporated on chromatography media, is washed with 20~200mM phosphate concn gradient
De-, destination protein is separated with impurity, collects the HPV33L1 albumen of elution (see Fig. 4 C).
Embodiment 9: the HPV33L1 recombinant protein of transmission electron microscope observing purifying
With the HPV33L1 protein sample of 3 times of sterile water dilutions after purification, a droplet is dripped on cured disk.Copper mesh is taken to make have branch
The surface for holding film is contacted with sample liquid surface, is stood 1min and is taken out, takes out copper mesh, extra drop is absorbed with filter paper item, is slightly dried in the air
It is dry.2% acetic acid uranium solution is taken, drips a droplet on cured disk.The copper mesh for being adsorbed with sample is placed in dye liquor surface (sample and dye liquor
Contact), stand 2min.Copper mesh is taken out, extra drop is absorbed with filter paper item, is dried under incandescent lamp.Using JEOL-1400 model
Transmission electron microscope observing VLP particle shape is simultaneously taken pictures (shown in result figure 5).
Embodiment 10: with the immunogenicity research for the HPV33L1VLP that Hansenula yeast is prepared by recombinant
Using measurement humoral immunity effect ED50The immunogenicity of the method evaluation HPV33L1VLP of (median effective dose)
(1) mouse is immune: 85 6 week old Balb/c female mices (are purchased from Chinese Academy of Medical Sciences's Animal Experimental Study
Institute), cleaning grade raising.It is divided into 6 groups, including 5 experimental groups and 1 control group, by required immunizing dose by HPV33L1 egg
White sample is diluted (table 1).Immune programme are as follows: the 0th, 3,6 week each immune primary, killed within 14 days after last time is immune mouse and point
From serum.
The grouping of 1 mouse of table
(2) serological conversion rate after mouse, specific steps are immunized in ELISA method measurement HPV33L1VLP are as follows: with coating buffering
Escherichia coli recombination HPV33L1 albumen is diluted to 0.5 μ g/ml by liquid, and every hole adds 0.1ml, and 4 DEG C overnight.Next day washing buffer
Washing 3 times, gets rid of most residual liquid.With antibody diluent closing 30 minutes, washing buffer was washed 3 times, was detected after drying, or
4 DEG C of damp proof preservations after drying.Each mice serum sample is diluted with 1:10000 with sample diluting liquid, takes 0.1ml in above-mentioned
In the reacting hole being coated with, sets 37 DEG C and be incubated for 1 hour, wash 5 times.(while doing blank, negative hole control).Add in reacting hole
Enter the sheep anti-mouse igg secondary antibody 0.1ml of the HRP label of antibody diluent 1:10000 diluted fresh, 37 DEG C are incubated for 30 minutes,
Washing 5 times, last time are washed with distilled water.It is added the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C
Colour developing 10 minutes.50 μ l2M sulfuric acid 0.05ml are added in each reacting hole to terminate reaction.In microplate reader, at 450nm
(630nm is reference wavelength) surveys each hole OD value after returning to zero with blank control wells.Cutoff value calculates and positive findings determine:
Cutoff value=negative control value × 2.1；Sample OD value > Cutoff value is then judged to the positive.
(3) humoral immunity effect ED50Calculating
According to the mouse positive rate calculated result of variant dosage level, the humoral immunity effect ED of HPV33L1VLP50Value
For 0.145 μ g, show that HPV33L1VLP has good immunogenicity.
Claims (3)
1. a kind of method for generating HPV33 L1 albumen, comprising the following steps:
A) generates the recombination Hansenula yeast cell of expression 33 type L1 of human papilloma virus (HPV33 L1) albumen；
B) cultivates the recombination Hansenula yeast cell obtained in a) under conditions of being suitable for the HPV33 L1 protein expression；With
C) is recycled from culture and is purified the HPV33 L1 albumen；
Wherein a) in by the inclusion of the method for following steps generate expression 33 type L1 of human papilloma virus (HPV33 L1) albumen
Recombinate Hansenula yeast cell:
A1) by that will include HPV33 shown in the SEQ ID NO:2 operably being connect with MOX promoter and MOX terminator
The exogenous polynucleotide insetion sequence of L1 albumen coded sequence is shown in the carrier of SEQ ID NO:9 to construct expression construct；
A2) ATCC26012 Hansenula yeast cell is converted with the expression construct obtained in step a1)；With
A3) the Hansenula yeast cell obtained in step a2) is screened with Zeocin and G418, acquisition is greater than containing copy number
The recombination Hansenula yeast cell of 60 exogenous polynucleotide；
Wherein b) in culture include the following steps b1) and b2):
B1) prepare fermentation seed liquid: taking in the 50 μ l of recombination Hansenula yeast cell access 5ml YPD culture medium, in 30 DEG C,
200rpm shaking table culture 20-24hr, A600nm2-5, each 1ml that draws is accessed in 2 bottles of 500ml YPD culture mediums, in 30 DEG C, 200rpm
Shaking table culture 20-24hr to A600nm15-20, it is stand-by as fermentation seed liquid；
B2) ferment: the fermentation initial culture medium into 30L fermentor, fermentation volume 15L is added in 500ml fermentation seed liquid；It rises
Beginning speed of agitator is 200rpm, air mass flow 0.5Nm3/ hr, tank press 0.5bar, and control oxygen dissolving value is 20-80% in fermentation；It rises
After growth period beginning maintains 25hr, bacterium solution A600nmReach 20, dissolved oxygen starts rapid increase, starts with 100ml/hr flow velocity flow feeding
Culture medium；After cultivating 6-8hr, bacterium solution A600nmReach 90, stop stream and add, ammonium hydroxide adjusts pH value to 6.0；It is opened after dissolved oxygen bottom out
Begin to flow plus the methanol of the PTM1 containing 12ml/L enters the inducing expression phase, inducing temperature is set as 35 DEG C, and the initial flow rate of methanol is
50ml/hr is sampled per hour, methanol determination of electrode methanol concentration, so that methanol concentration control is existed by adjusting methanol feeding rate
< 5g/L；Induce lower tank after 10hr, wet thallus collected with 5000rpm centrifugation 30min under the conditions of 4 DEG C, -20 DEG C freeze it is spare；
Wherein c) in recycling and purifying include the following steps c1) and c2):
C1 the HPV33-L1 expression wet thallus for) taking -20 DEG C of preservations, is added 0.9% physiology salt according to the ratio of 10ml/g wet thallus
Water cleaning；After cell washing, disruption buffer is added by 20ml buffer/g wet thallus and sufficiently dissolves, it is broken using high-pressure homogenization
Broken, cracking pressure 1500bar, circulation is 5-8 times broken, microscopy percentage of damage > 90%；Bacterial cell disruption liquid under the conditions of 4 DEG C with
10000rpm is centrifuged 30min, collects supernatant；
C2) the bacterial cell disruption supernatant filtered through 1 μm, is splined on chromatography media POROS XS, with 0.5M~1.5M NaCl gradient
Elution, collects the HPV33 L1 albumen of elution；HPV33 L1 albumen after preliminary purification is splined on chromatography media Macro-
Prep ceramic hydroxyapatite, destination protein are incorporated on chromatography media, with 20~200mM phosphate concn gradient elution, mesh
Albumen separated with impurity, collect the HPV33 L1 albumen of elution.
2. the method according to claim 1, wherein step a3) in the G418 of the Zeocin and 16mg/ml of 0.5mg/ml to step
Rapid a2) in obtain Hansenula yeast cell screened.
3. the method according to claim 1, wherein step a3) described in screening include
1) the Hansenula yeast cell obtained in step a2) is coated in the YPD plate of the Zeocin containing 0.5mg/ml, in 30 DEG C
It is inverted culture 3-7 days；
2) the recombinant bacterial strain single colonie grown in picking Zeocin resistant panel, is inoculated in 5ml Zeocin's containing 0.5mg/ml
In YPD fluid nutrient medium, in 30 DEG C, 200rpm shaking table culture 24-48 hours, until OD value is turned up to after 50 with the ratio of 1:1000
It is connected in the YPD fluid nutrient medium of 5ml Zeocin containing 0.5mg/ml, culture to OD value turns up to after 50, then with the ratio of 1:1000
It is connected in the YPD fluid nutrient medium of 5ml Zeocin containing 0.5mg/ml, it is continuous to pass 10 times；
3) the recombination expressed by Hansenula yeast bacterial strain access 5ml for reaching 10 times is free of in the YPD fluid nutrient medium of Zeocin resistance,
In 30 DEG C, 200rpm shaking table culture to OD value up to after 50, transferred in 5ml YPD fluid nutrient medium with the ratio of 1:1000,
It is continuously passed in YPD fluid nutrient medium without Zeocin resistance 5 times；With
4) product of acquisition is coated on to the YPD plate of the G418 containing 16mg/ml, is cultivated 2-3 days in 30 DEG C of inversions, picking single bacterium
Drop into the detection of row copy number.
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