CN108130341B - Recombinant hansenula polymorpha for molecular chaperone-assisted expression of Zika virus E protein and construction method thereof - Google Patents
Recombinant hansenula polymorpha for molecular chaperone-assisted expression of Zika virus E protein and construction method thereof Download PDFInfo
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Abstract
The invention provides a recombinant hansenula polymorpha for expressing a Zika virus E protein under the assistance of a molecular chaperone and a construction method thereof.A co-expression vector of a recombinant Zika virus structural protein E and a molecular chaperone hansenula polymorpha calnexin is constructed firstly, wherein the sequence of the recombinant Zika virus structural protein E is shown in SEQ ID No.1, and the gene segment of the hansenula polymorpha calnexin is shown in SEQ ID No. 2. secondly, the linearized co-expression vector is transformed into hansenula polymorpha cells through an electroporation transformation method, and after induction culture, the recombinant hansenula polymorpha fermentation product is purified, the content of a recombinant protein is detected to be 12.6 mg/L.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to establishment of a hansenula polymorpha expression system of a Zika virus glycoprotein gene.
Background
Zika virus (Zika virus) belongs to Flaviviridae (Flaviviridae) flaviviruses (Flavivivirus), has similar characteristics with dengue virus, yellow fever virus, Japanese encephalitis virus and West Nile virus, is a single-stranded positive-strand RNA virus, has an increased infection rate in recent years, and has an epidemic which not only seriously threatens the life safety of people, but also causes serious psychological influence and heavy economic burden.
The Zika virus genome is about 10.8kb in length and contains a single open reading frame, the virus protein is prepared by enzyme digestion of a single polyprotein precursor through host protease and virus proteins, and the virus proteins comprise 3 structural proteins (C, prM/M, E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS 5). Like other flaviviruses, the structural protein E (ZKE) of Zika virus is highly conserved and contains multiple neutralizing epitopes. In addition, ZKE is the major protein that binds to cell membrane receptors and fuses with cell membranes to allow the virus to enter cells, and is closely related to the effects of virus adsorption, penetration, pathogenicity, tissue tropism, and induction of host immune responses. Therefore, the structural protein E of Zika virus is the main antigen protein which causes host body immunity and generates neutralizing antibodies, and can stimulate the body to generate neutralizing antibodies and protect the body from being attacked by viruses.
Hansenula polymorpha (h. polymorpha) belongs to methanol-type yeast, and as a unicellular lower eukaryote, it has not only the advantages of easy culture, fast propagation, convenient genetic manipulation, ability to perform post-translational processing and modification on exogenous products, and non-toxic metabolites, but also its specific high temperature resistance. These advantages have led to the rapid development of Hansenula polymorpha expression systems in recent years as one of the major cell factories of great interest in the field of biotechnology. The Hansenula polymorpha expression system has been successfully used to express many pharmaceutical, industrial proteins and enzyme preparations, such as eukaryotic proteins with diagnostic and therapeutic value, some of which have been commercially promoted, such as hirudin, hepatitis B virus surface antigen B (HBsAg), human interferon (IFNa-2a), insulin, penicillin, gelatin, urate oxidase, phytase.
At present, reports of expressing structural protein E of Zika virus by using Hansenula polymorpha hosts are not seen at home and abroad. The main difficulties may be: the secretory expression of the protein depends on an extra sequence at the N end of the protein and whether the protein can be folded correctly, however, the expression efficiency of the ZKE protein from the host cell is low or even the protein is not expressed under the influence of ZKE protein structure and folding.
Disclosure of Invention
The invention aims to provide recombinant hansenula polymorpha for expressing Zika virus E protein under the assistance of molecular chaperones and a construction method thereof, and lays a foundation for further developing Zika vaccine genetic engineering bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides a secretory high-throughput expression vector of Zika virus structural protein E, a target gene expressed by the expression vector is a recombinant Zika virus structural protein E sequence shown in SEQ ID No.1 or a sequence with homology of more than 90 percent with the recombinant Zika virus structural protein E sequence (SEQ ID No.1), and the expression vector also co-expresses a molecular chaperone sequence of a target gene expression product.
The expression vector comprises two expression frames, wherein one expression frame is a recombinant Zika virus structural protein E gene expression frame, the other expression frame is a molecular chaperone expression frame, the recombinant Zika virus structural protein E gene expression frame comprises a first eukaryotic promoter (such as a MOX promoter), a secretion signal peptide sequence (such as α chaperone factor) coexpressed with the target gene and the target gene, and the molecular chaperone expression frame comprises a second eukaryotic promoter (such as an FMD promoter) and a calcium binding protein (such as Hansenula polymorpha calcium binding protein) open reading frame.
The recombinant Zika virus structural protein E sequence is designed from the Zika virus structural protein E gene, and the design key points are as follows: 1) the part of the secretory signal peptide sequence of the structural protein E gene of Zika virus itself is removed, and 2) the coding sequence of the hydrophobic region is partially removed.
The recombinant Hansenula polymorpha capable of expressing structural protein E of Zika virus in high flux is obtained by transforming a recombinant expression vector (e.g., the high-flux expression vector) into a Hansenula polymorpha host, and after induction expression, the recombinant structural protein E of Zika virus is secreted to the outside of the Hansenula polymorpha host.
Hansenula polymorpha is food grade yeast approved by FDA, during secretory expression of Hansenula polymorpha, exogenous proteins can complete glycosylation modification and post-translational processing modification such as disulfide bond formation during secretion process, so that the expressed proteins are closer to natural protein forms with biological activity, and the Hansenula polymorpha is a widely used eukaryotic expression system.
The method for constructing the recombinant hansenula polymorpha comprises the following steps:
1) artificially synthesizing the recombinant Zika virus structural protein E sequence (SEQ. ID. NO. 1);
2) amplifying the open reading frame of the molecular chaperone;
3) constructing a recombinant expression vector containing the recombinant Zika virus structural protein E sequence and an open reading frame of a molecular chaperone;
4) transforming the recombinant expression vector into a Hansenula polymorpha host to obtain the Hansenula polymorpha for expressing the recombinant Zika virus structural protein E.
The step 3) specifically comprises the following steps:
3.1) respectively constructing an expression frame capable of co-expressing the structural protein E sequence and the secretion signal peptide sequence (α chaperone factor) of the recombinant Zika virus and an expression frame capable of expressing calnexin of the host (comprising SEQ. ID. NO. 2);
3.2) constructing a linearized vector comprising the two expression frames to obtain the recombinant expression vector.
Preferably, the recombinant expression vector can be constructed by using plasmid vectors comprising pHMOXG- α -A and pHFMDG-A plasmid vectors, wherein the pHMOXG- α -A and pHFMDG-A plasmid vectors both belong to G418 resistant plasmids, pHMOXG- α -A contains a MOX promoter derived from H.polymorpha and α chaperone factors derived from Saccharomyces cerevisiae (S.cerevisiae), and pHFMDG-A contains an FMD promoter derived from H.polymorpha, pHMOXG- α -ZKE containing a target gene fragment (SEQ. ID. NO.1) can be constructed by using pHMOXG- α -A, pHFMXG- α -ZKE containing a target gene fragment (SEQ. ID. NO.1) can be constructed by using pHFMDG-A, pHFMDG-1 (Hansenula calcium binding protein gene) fragment (SEQ. ID. NO.2) can be constructed by using pHFMDG-A, and then the plasmid vectors containing HpC gene fragment HpC-8945 (pHFMXG-11. ID. NO.2) can be constructed by inserting the recombinant gene into pHFMXG-3626-7 plasmid vectors for linear expression and then carrying out recombination by using the recombinant BBXG-34 plasmid vectors, and inserting the pHFMXG-3626 plasmid vectors for expression.
The recombinant Hansenula polymorpha of the present invention can be used for producing recombinant Zika virus structural protein E. The first stage of the production process is the fermentation culture of recombinant Hansenula polymorpha (increasing cell biomass), the second stage is the induction expression (2% glycerol), and the expressed product (retaining the core functional region structure of structural protein E of Zika virus, namely structural parts related to immunogenicity and antigen presentation, such as antigenic determinants) can be enriched, separated and purified by simple steps of separating fermentation supernatant, affinity chromatography and the like.
The invention has the beneficial effects that:
the invention secretes and expresses the core structure of the structural protein E of the Zika virus in a Hansenula polymorpha expression system for the first time, the content of foreign proteins is extremely low because the expressed protein exists extracellularly, the purification step is simple, the cost is low, the high-throughput expression can be realized, and the industrial production potential is large. In addition, the obtained recombinant Hansenula polymorpha has the advantages of easy high-density fermentation and simple culture of Hansenula polymorpha, and the expression product has good immunogenicity and is suitable for the field of medicine.
Drawings
FIG. 1 is a Western blot analysis of recombinant Hansenula polymorpha fermentation products, wherein samples 1 and 2 are pHMOXG- α -ZKE (1m L supernatant concentrate) for inducing fermentation products for 72 hours after transforming Hansenula polymorpha cells, samples 3 and 4 are pHMOXG- α -ZKE-HpCne1 (1m L supernatant concentrate) for inducing fermentation products for 72 hours after transforming Hansenula polymorpha cells, and samples 5 and 6 are pHMOXG- α -A (1m L supernatant concentrate) for inducing fermentation products for 72 hours after transforming Hansenula polymorpha cells.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
The invention respectively clones the structural protein E gene fragment of Zika virus without signal peptide and the gene (HpCne1) fragment of calnexin (CNE1) derived from Hansenula polymorpha (H. polymorpha) into plasmid pHMOXG-alpha-A (or pHMOXG- α -A), constructs a recombinant co-expression vector pHMOXG- α -ZKE-HpCne1, transfects the recombinant Hansenula polymorpha D L-1 (host cell), and realizes the high-flux secretory expression of the recombinant Zika virus structural protein E by the host cell through culturing and inducing.
(I) transformation of construction vectors pHMOXG- α -ZKE-HpCne1 and H.polymorpha D L-1
Restriction endonucleases were purchased from Takara and the primer sequences used are shown in Table 1:
TABLE 1 primer sequences used in the Experimental procedures
1.1 artificially synthesized sequence (Biotechnology engineering (Shanghai) GmbH): according to the reported structural protein E gene information of Zika virus (GenBank: KU820899.2), a structural protein E gene fragment of Zika virus to be expressed is artificially designed (designed in 5 months in 2017), and EcoRI cleavage sites (carbon ends) and NotI cleavage sites (nitrogen ends) can be added at both ends of the fragment by using amplification primers (ZKE-F and ZKE-R, designed in 7 months in 2017). The sequence of the artificially designed Zika virus structural protein E gene fragment is shown in SEQ ID No. 1.
1.2 cloning and replication of fragment (Biotechnology engineering (Shanghai) Co., Ltd.) the cloning vector was pMD18-T (Takara) and the inserted target gene was the above fragment, and a plasmid (pMD18-ZKE) containing the designed fragment of the structural protein E gene of Zika virus was transferred to E.coli Top10 and cultured in L B liquid medium containing 0.1% ampicillin.
L B liquid medium containing 0.1% ampicillin comprises 1.0g/100m L ampicillin, 5g/1000m L yeast extract, 10g/1000m L tryptone, 10g/1000m L sodium chloride, NaOH adjusted to pH 7.0, and autoclaving at 121 deg.C for 20 min.
1.3 Positive clone identification, namely amplifying by PCR, wherein the reaction systems are ZKE-F (1 mu L), ZKE-R (1 mu L), 2 × TaqMaster Mix (10 mu L, Nanjing Nodezan Biotech Co., Ltd.), Escherichia coli culture solution (1 mu L) and ddH2O (7 mu L) and 20 mu L in total, wherein the amplification conditions are 94 ℃ for 5min, 94 ℃ for 35s, 52 ℃ for 35s, 72 ℃ for 1.5min and 30 cycles, 72 ℃ for 10min and 4 ℃ for storage, the amplification product is detected by 1% agarose gel electrophoresis, and the detection result of the agarose gel electrophoresis shows that the amplification product with a band of about 1515bp is detected at 1500bp of the standard molecular weight of the DNA and accords with the base number of the artificial design of the Zika virus structural protein E gene fragment.
1.4 construction of recombinant plasmid pHMOXG- α -ZKE, recovery of PCR amplification product with gel recovery kit from Beijing Quanyujin company, and after EcoRI and NotI double digestion, ligation with plasmid pHMOXG- α -A digested with the same restriction enzymes under T4 ligase at 4 ℃ for 16h, identification with EcoRI and NotI double digestion, screening of correctly ligated recombinant plasmids, pHMOXG- α -ZKE, pHMOXG- α -A (Song hohui et al, biotechnology letters, 25(23): 1999) belonging to G418 resistant plasmid, containing a MOX promoter from H.polymorpha and a transcription factor α from yeast (S.cerevisia), pHMOXG- α -8295 inserted into the insertion site of gene (SEQ. ZKE, insert site of the gene of Saccharomyces cerevisiae), and secretion promoter sequence of upstream of the gene of Saccharomyces cerevisiae (SEQ. 1.5. His), and further containing the sequence of promoter for expression of upstream transcription promoter and transcription partner of destination gene of promoter sequence of destination gene (SEQ. 5. downstream of Saccharomyces cerevisiae, SEQ. cold-5. secretory protein sequence of destination gene, and sequence of promoter of destination gene of destination.
Amplification of HpCne1 fragment HpCne1 fragment (open reading frame, SEQ. ID. NO.2) was PCR-amplified from H.polymorpha D L-1 (AT26012, obtained in ATCC AT 3 months 2010) genomic DNA (genomic DNA extraction kit, Tiangen Biotechnology (Beijing) Ltd.) using the above-mentioned HpCne1-F and HpCne1-R (designed in 7 months 2017), the amplified product was examined by 1% agarose gel electrophoresis, the band of interest was recovered, the recovered product was double-digested with EcoRI and NotI and then ligated with plasmid pHFMA (Song houhui, biotechnology letters, 25(23):1999 2006) using T4 ligase, the correct FMI and pHI plasmid ligated by double-digestion, the inserted site was identified by PCR-cloning, the promoter was derived from a pCne 67418, and the plasmid was cloned AT 1. mu.26. PCR-cloned site.
1.6 construction of ZKE Gene fragment and HpCNE1 fragment coexpression plasmid, recombinant plasmid pHFMDG-HpCne1 is digested with BglII and BamHI, then ligated with pHMOXG- α -ZKE digested with BamHI, and the correct ligated recombinant plasmid is selected, i.e., the recombinant HpCne1 gene expression cassette is inserted into the BamHI site of recombinant plasmid pHMOXG- α -ZKE, to construct plasmid vector pHMOXG- α -ZKE-HpCne 1. pHMOXG- α -ZKE-HpCne1 containing recombinant ZKE gene expression cassette composed of MOX promoter, α chaperone factor, Zika virus structural protein E gene fragment (SEQ. ID. NO.1) and His tag sequence, downstream of which is FMD promoter and HpCne1 open recombinant HpCne1 gene expression cassette composed of open reading frame, etc., the HpCne1 gene expression cassette is achieved by expressing HpCne1 in host cells, and simultaneously secreting calcium linked polymorphic gene fragment of the yeast and the recombinant HpCNE α recombinant gene.
The recombinant plasmid pHMOXG- α -ZKE-HpCne1 was transformed into E.coli TOP10 competent cells (purchased from Beijing Quanji Biotechnology Co., Ltd.) by heat shock method, incubated at 37 ℃ for 1h at 160rpm in L B medium, centrifuged to collect the cells, spread on L B solid medium containing 0.2% kanamycin, cultured at 37 ℃ for 16h to screen, 6 transformants were randomly selected and inoculated into 3m L liquid medium L B containing 0.1% kanamycin, cultured at 37 ℃ for 8h, and then colony PCR was performed using primer FMD-F (designed at 7 months in 2017) and HpCne1-R and MOX-F (designed at 7 months in 2017) and ZKE-R, respectively, wherein the reaction system was 1 μ L, the primer was 1 μ L, 2 × μ Taq Master Mix 10 μ L (Mix L (Kinao Tokyo Biotech)2O is 7 mu L, and the total volume is20 mu L, carrying out agarose gel electrophoresis detection on the PCR amplification product, and if the detected band meets the base number of the target fragment, successfully obtaining the positive escherichia coli containing the recombinant plasmid pHMOXG- α -ZKE-HpCne 1.
L B solid medium containing 0.2% kanamycin comprises 2.0g/100m L kanamycin, 5g/1000m L yeast extract, 10g/1000m L tryptone, 10g/1000m L sodium chloride, 15g/1000m L agar powder, NaOH adjusted to pH 7.0, 121 deg.C, and autoclaving for 20 min.
L B liquid medium containing 0.1% kanamycin comprises 1.0g/100m L kanamycin, 5g/1000m L yeast extract, 10g/1000m L tryptone, 10g/1000m L sodium chloride, NaOH adjusted to pH 7.0, and autoclaving at 121 deg.C for 20 min.
1.7 recombinant co-expression plasmid transformed cells:
1.7.1 preparation of competent cells of Hansenula polymorpha D L-1
1. Selecting a single colony of Hansenula polymorpha D L-1 from a fresh YPD plate, and culturing the single colony in a 5m L YPD liquid culture medium at 37 ℃ for 16h until the colony grows to saturation;
2. inoculating 1m L into 50m L YPD liquid culture medium, and culturing at 37 deg.C to OD600Centrifuging at 5000rpm for 8min after the speed is 0.8-1.2, and collecting cells;
3. adding 50m L TED (100mm Tris HCl and 50mm EDTA) with pH8.0, placing at 37 deg.C, shaking at 100rpm/min for 30min, centrifuging at 4 deg.C and 5000rpm/min for 8min, and collecting cells;
4. adding 50m L ice-cooled 270mM sucrose to gently suspend the cells, and centrifuging at 4 ℃ and 5000rpm/min for 5min to collect the cells;
5. adding 25m L ice-precooled 270mM sucrose to gently suspend the cells, and centrifuging at 4 ℃ and 5000rpm/min for 5min to collect the cells;
6. the cells were gently suspended with 1m L ice-chilled 270mM sucrose and dispensed 80. mu. L/tube to prepare competent cells of Hansenula polymorpha D L-1.
H.polymorpha D L-1 competent cells were transformed by electroporation (Faber et al, 1994)
1m L containing recombinant plasmid pHMOXG- α -ZKE-HpCne1 Escherichia coli liquid was added into 50m L L B liquid medium, cultured at 37 ℃ and 200rpm for 24h, and the recombinant plasmid pHMOXG- α -ZKE-HpCne1 was extracted using a plasmid extraction kit manufactured by Beijing Quanji corporation.
H.polymorpha D L-1 competent cells were transformed with DraI type linearized plasmid pHMOXG- α -ZKE (positive control), pHMOXG- α -A (negative control) and KpnI type linearized plasmid pHMOXG- α -ZKE-HpCne1, respectively;
and (3) linearization treatment, wherein the enzyme digestion reaction condition is 37 ℃, enzyme digestion is carried out for 8h, agarose gel electrophoresis is carried out on the linearized product, and an electrophoresis product is recovered by using a gel recovery kit produced by Beijing Quanyu gold and dissolved in a TE solution of 20-30 mu L.
Gently mixing the linearized product of 20-30 μ L with competent cells of Hansenula polymorpha D L-1 of 80 μ L, immediately placing into a groove of an ice-bath electric shock cup, ice-cooling for 5min, performing pulse (pulse setting: 50 μ F, 200 Ω, 1.5KV) electric shock on the sample with a Bio-Rad electroporator, and immediately adding ice-precooled 1m L YPD (containing 1mM MgCl)2) The liquid medium was transferred into a 2m L sterile centrifuge tube and incubated at 37 ℃ for 1-2h at 100 rpm.
1.7.3 Positive clone (transformant) screening
After the above culture for 1-2h, 100-200. mu. L were plated on YPD (containing G418 at a final concentration of 300. mu.g/m L) plates, and the presence of transformants was observed after culturing at 37 ℃ for 48-96 h.
Randomly selecting 20-30 transformants, respectively inoculating the transformants into YPD liquid culture medium containing 300 mug/m L G418 of 5m L, culturing at 37 ℃ for 24h, centrifuging at 5000rpm/min for 5min, collecting cells, extracting a genome, performing PCR amplification screening, performing agarose gel electrophoresis detection on PCR amplification products, and obtaining a positive hansenula polymorpha recombinant strain containing a recombinant plasmid pHMOXG- α -ZKE-HpCne1 if the detection result meets the base number of the artificially designed Zika virus structural protein E gene fragment and the base number of the HpCne1 fragment.
(II) Positive clone culture and recombinant Zika virus structural protein E expression
The recombinant strain of Hansenula polymorpha was inoculated into 2m L YPD liquid medium supplemented with 300. mu.g/m L G418, cultured at 37 ℃ and 180rpm for 12 hours, centrifuged at 2000G for 15min to collect cells, and 30m L YPD liquid medium (glucose)Glucose 2%, yeast extract 1%, peptone 2%), suspended thallus, shaking the thallus in 500m L flask, culturing at 37 deg.C and 180rpm for 24 hr, collecting cell, 300m L YPD liquid culture medium suspended thallus, shaking the thallus in 2L flask to make initial OD of cell600Culturing at 1.0 ℃ and 37 ℃ at 180rpm for 12h, adding glycerol for induction, wherein the final concentration of the glycerol is 2% (v/v), inducing once every 12h, continuously inducing for 72h, sampling fermentation liquor during induction and at the end of induction, centrifuging for 15min at 4000g, collecting supernatant, concentrating 1m L supernatant by using a tangential flow ultrafiltration membrane 5000MWCO PES, directly performing western blotting analysis by using a His tag antibody after concentration, detecting the expression level of the structural protein E of the recombinant Zika virus, and detecting a protein band at a protein standard molecular weight of 55kDa as shown in figure 1, wherein the target protein (the structural protein E of the recombinant Zika virus) is 55.6kDa, and a molecular chaperone (HpCne1) obviously assists (enhances) the secretory expression of the structural protein E of the recombinant Zika virus.
(III) purification of recombinant Zika Virus structural protein E
Purifying by NI-NTN agarose affinity chromatography, namely concentrating the supernatant with tangential flow ultrafiltration membrane 5000MWCO PES, passing through Ni-NTA chromatography column (BIO-RAD company), and balancing buffer solution of 50mM Na2HPO4·12H2O and 300mM NaCl, pH 7.5. Eluting protein with eluent, wherein the first elution buffer is 20mM Na2HPO4·12H2O、20mMNaH2PO4·H2O, 300mM NaCl and 20mM imidazole, pH 7.5, and the imidazole concentration in the second elution buffer is 150 mM. Protein content was measured using the Bradford method (bovine serum albumin as control).
Western blotting identification of the purified protein: the purified sample was electrophoresed and transferred to PVDF membrane, and after transfer, PBST (80mM Na) containing 2.5% casein was used2HPO4，20mM NaH2PO4100mM NaCl, and 0.1% Tween 20) was blocked for 2h at room temperature and PBST was eluted three times. The membrane was transferred to anti-His-tag murine monoclonal antibody (King-rui Biotech) diluted in PBST (1:10000) containing 1% BSA and incubated at room temperature for 1h, and eluted with PBS (containing 0.1% Tween) three times. HRP-goat anti-mouse IgG (PBST dilution 1:4000 with 1% BSA) was added,and after three times of elution, an EC L Western blotting color developing agent is used for developing color, and the color is analyzed by Bandscan 5.0 software, so that the molecular weight of the protein is about 55.6kDa, namely the purified product is the target protein.
The content of the structural protein E of the purified recombinant Zika virus increased from 5.7 mg/L to 12.6 mg/L as the time for inducible expression continued from 48h to 72 h.
Concentration and preservation of (IV) recombinant Zika Virus structural protein E
The eluate containing the target protein (recombinant Zika virus structural protein E) purified by affinity chromatography was concentrated by centrifugation at 4000rpm at 4 ℃ using an ultrafiltration tube having a pore size of more than 3.5 kD.
The concentrated protein was dialyzed with a dialysis bag for 12-16h (desalting) in PBS buffer containing 5% (volume fraction) glycerol.
The dialysate was passed through a 0.22 μm filter, injected into a sterile EP tube, and stored at-80 ℃ for further use.
(V) immunogenicity analysis of recombinant Zika Virus structural protein E
(1) Animal immunization
Selecting 24 female Balb/c mice with the age of 6-8 weeks, randomly dividing the mice into 3 groups, wherein an immunogen in an experimental group 1 is an expressed target protein (recombinant Zika virus structural protein E), an immunogen in an experimental group 2 is an inactivated Zika virus, an experimental group 3 is a negative control group (adjuvant), and subcutaneous multipoint immune injection is carried out, wherein the initial immunity of the experimental group 1 is carried out by emulsifying the recombinant Zika virus structural protein E with 30 mu g of the expressed recombinant Zika virus structural protein E and the same amount of Freund's complete adjuvant and then immunizing, the second immunity is carried out after 2 weeks of the initial immunity, the immunization is carried out by emulsifying the recombinant Zika virus structural protein E with 15 mu g of the expressed recombinant Zika virus structural protein E and the same amount of Freund's incomplete adjuvant and then carrying out the boosting immunity once a week for 2 times, the last boosting immunity is carried out for 1 week and then blood is taken after tail breaking and placed at 4500r/min5PFU (Plaque-Forming Unit) inactivated Zika virus was emulsified with the same amount of Freund's complete adjuvant and then immunized, and 2 weeks after the primary immunization, and the inactivated Zika virus was immunized with 1 × 104The PFU inactivated Zika virus is emulsified with the same amount of Freund incomplete adjuvant and then immunizedThe immunization is strengthened once a week for 2 times, the blood is taken after the tail is broken after the last immunization is strengthened for 1 week, the centrifugation is carried out for 10min at 4500r/min, and the serum is separated for standby. Experimental group 3 the primary immunization was performed with 30. mu.g Freund's complete adjuvant, the secondary immunization was performed 2 weeks after the primary immunization, the immunization was performed with 15. mu.g Freund's incomplete adjuvant, the booster immunization was performed 2 times per week, the blood was taken after 1 week of the final booster immunization, the blood was centrifuged at 4500r/min for 10min, and the serum was separated for use.
(2) Antibody titer determination
By using PRNT (Plaque reduction neutralization test), the serum of a mouse in an experimental group 2 (an inactivated virus immune mouse group) is used as positive serum, the serum of a mouse in an experimental group 3 (an immune adjuvant mouse group) is used as negative control serum, Hanks buffer solution is used as blank control, the antibody titer of an immune mouse (an experimental group 1) with expressed recombinant Zika virus structural protein E is analyzed, and the result shows that the antibody titer of the anti-Zika virus antibody of the immune mouse group with the experimental group 1, namely the expressed recombinant Zika virus structural protein E, reaches 1:1280, and the expressed recombinant Zika virus structural protein E is proved to have immunogenicity.
In a word, α chaperone factors contained in the coexpression plasmid can guide foreign proteins expressed in host bacteria to be secreted to the outside, and intracellular high expression of HpCNE1 fragments promotes the expression of the foreign proteins (by exerting the action of molecular chaperones), so that not only is high-flux secretory expression realized, but also the subsequent purification and application are facilitated, and the protein extraction process is simplified.
Sequence listing
<110> centers for disease prevention and control in Ningbo City; shanxi university of science and technology
Recombinant hansenula polymorpha for auxiliary expression of Zika virus E protein by molecular chaperone and construction method thereof
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>1515
<212>DNA
<213>Zika virus
<400>1
atgatcaggt gcataggagt cagcaatagg gactttgtgg aaggtatgtc aggtgggact 60
tgggttgatg ttgtcttgga acatggaggt tgtgtcaccg taatggcaca ggacaaaccg 120
actgtcgaca tagagctggt tacaacaaca gtcagcaaca tggcggaggt aagatcctac 180
tgctatgagg catcaatatc ggacatggct tcggacagcc gctgcccaac acaaggtgaa 240
gcctaccttg acaagcaatc agacactcaa tatgtctgca aaagaacgtt agtggacaga 300
ggctggggaa atggatgtgg actttttggc aaagggagcc tggtgacatg cgctaagttt 360
gcatgctcca agaaaatgac cgggaagagc atccagccag agaatctgga gtaccggata 420
atgctgtcag ttcatggctc ccagcacagt gggatgatcg ttaatgacac aggacatgaa 480
actgatgaga atagagcgaa ggttgagata acgcccaatt caccaagagc cgaagccacc 540
ctggggggtt ttggaagcct aggacttgat tgtgaaccga ggacaggcct tgacttttca 600
gatttgtatt acttgactat gaataacaag cactggttgg ttcacaagga gtggttccac 660
gacattccat taccttggca cgctggggca gacaccggaa ctccacactg gaacaacaaa 720
gaagcactgg tagagttcaa ggacgcacat gccaaaaggc aaactgtcgt ggttctaggg 780
agtcaagaag gagcagttca cacggccctt gctggagctc tggaggctga gatggatggt 840
gcaaagggaa ggctgtcctc tggccacttg aaatgtcgcc tgaaaatgga taaacttaga 900
ttgaagggcg tgtcatactc cttgtgtacc gcagcgttca cattcaccaa gatcccggct 960
gaaacactgc acgggacagt cacagtggag gtacagtacg cagggacaga tggaccttgc 1020
aaggttccag ctcagatggc ggtggacatg caaactctga ccccagttgg gaggctgata 1080
accgctaacc ccgtaatcac tgaaagcact gagaactcca agatgatgct ggaacttgat 1140
ccaccatttg gggactctta cattgtcata ggagtcgggg agaagaagat cacccaccac 1200
tggcacagga gtggcagcac cattggaaaa gcatttgaag ccactgtgag aggtgccagg 1260
agaatggcag tcttgggaga cacagcctgg gactttggat cagttggagg cgctctcaac 1320
tcattgggca agggcatcca tcaaattttt ggagcagctt tcaaatcatt gtttggagga 1380
atgtcctggt tctcacaaat tctcattgga acgttgctga tgtggttggg tctgaacaca 1440
aagaatggat ctatttccct tatgtgcttg gccttagggg gagtgttgat cttcttatcc 1500
acagccgtct ctgct 1515
<210>2
<211>1674
<212>DNA
<213>H.polymorpha DL-1
<400>2
atgaaagtca gccgtccaat cattcttctg tttctttcac tggtctcgta tgcggcggat 60
gttgagcgga aagacttctt gccattgagt cttgagtccg acgctttttt cgagcaattt 120
aacgagacat gggcttcaag gtggaaacct tctcacagca agcgcgatga aaattttgtg 180
tatcgaggcg aatgggatgt tgaagagcca actgtgaatc ctggattgaa aggcgacaag 240
ggtcttgttg cgaagacaga ggccgcccat catgctatca gtgcaagatt gcctactcct 300
ttcgacaaca ccaacaacac cttggtgttg caatacgagg tcaagttgca aaaaggactt 360
gaatgcggtg gtgcatatat caagttgctg agccaggaag gtggagttag tgactcggtt 420
gagtttagca acgacactcc ataccaggtc atgtttggtc ctgacaaatg tggaatgtcc 480
aacaaggtcc actttatact tcgcagaaaa aaccctaaaa caggtgaata cgaggaaaaa 540
catctcaaag tgcctccaat ggcaaggatc gtgaaaacat ctacacttta cacgttgata 600
attaagccta accaagactt tgagatcaga atcaacggcg aagttgcaaa agcaggctca 660
ctgttggatt ccagatactt cgatctgact cctccaaagg aaatcgacga tcctaacgac 720
gagaagccag ctgattgggt tgacgacgag ctgatcccag accctaatgc tgtcaagccg 780
gaggattggg atgaagacgc tccatatttg atcccagatc caaacgcagt taagccggag 840
gactgggatg aggatgctga ggcatacatt cctgatccag atgcagtcaa acctgagtac 900
tgggacgagg aagaggacgg agaatggatt gctccgaaaa tcccaaaccc agcttgtgaa 960
cagcacggct gtggaccatg gtctgcgcca aaaatcagga acccagatta caaaggcaaa 1020
tggaccccag aattgattga gaacccagat tacaaggggc catggtctcc aagaaagatt 1080
cctaacccgg aatactacga ggacccaact ccctcgaatc tcgagcctat cggtgcgttg 1140
ggatttgagc tttggactat gactgactcc attatgtttg acaacatata tcttggccat 1200
tccatcgagg aagcagagta cattggtaac aacacattcc ttccaaaggt cgagattgaa 1260
gaacagatag ctgctgccaa tgctcctcaa gccaagtatg atgctgaaga gcctgaaaaa 1320
cagattgaag agcaatcttt ggtctcggac ttgttggata agtcggtgga tagagttttg 1380
aaagtttatg cttctggaaa agcatattgg gccgacctct tggacgaccc aaccaaaact 1440
cttctgaacc gccctggtga ggcatttttc tttgcttccg tggttgttgg cactattacc 1500
acgacttttg ctatttctac taccattgtc agtatatttg ctactttgtt tgctccatcc 1560
actcctcagc cagtcaaaag aagcgaggct aaagagcgga tcgagtctgt gaaagagaag 1620
gctaccgcgt ccgattcgaa ggtcaatgag acagaagctg tcaagagagg atag 1674
<210>3
<211>30
<212>DNA
<213>ZKE-F
<400>3
ggaattcatg atcaggtgca taggagtcag 30
<210>4
<211>28
<212>DNA
<213>ZKE-R
<400>4
gcggccgcag cagagacggc tgtggata 28
<210>5
<211>28
<212>DNA
<213>HpCne1-F
<400>5
cggaattcat gaaagtcagc cgtccaat 28
<210>6
<211>37
<212>DNA
<213>HpCne1-R
<400>6
ataagaatgc ggccgcctat cctctcttga cagcttc 37
<210>7
<211>19
<212>DNA
<213>FMD-F
<400>7
tctctcagag gggggaatg 19
<210>8
<211>19
<212>DNA
<213>MOX-F
<400>8
atcacagatg gggtcagcg 19
Claims (7)
1. A secreted high-throughput expression vector for structural protein E of Zika virus, which is characterized in that: the target gene expressed by the expression vector is a recombinant Zika virus structural protein E sequence shown in SEQ ID No.1, and the expression vector also co-expresses a molecular chaperone sequence of a target gene expression product;
the expression vector comprises two expression frames, wherein one expression frame is a recombinant Zika virus structural protein E gene expression frame, the other expression frame is a molecular chaperone expression frame, the recombinant Zika virus structural protein E gene expression frame comprises a first eukaryotic promoter, a secretion signal peptide sequence co-expressed with the target gene and the target gene, and the molecular chaperone expression frame comprises a second eukaryotic promoter and a calcium-linked protein open reading frame;
the secretion signal peptide sequence is selected from α chaperone factors.
2. A recombinant Hansenula polymorpha capable of expressing structural protein E of Zika virus in a high-throughput manner, comprising: the recombinant Hansenula polymorpha is obtained by transforming a recombinant expression vector into a Hansenula polymorpha host, wherein the recombinant expression vector comprises a recombinant Zika virus structural protein E sequence shown in SEQ ID No.1 and a molecular chaperone sequence of a target gene expression product co-expressed with a target gene, and the recombinant Zika virus structural protein E sequence is secreted to the outside of the Hansenula polymorpha host after being expressed;
the recombinant expression vector comprises two expression frames, wherein one expression frame is a recombinant Zika virus structural protein E gene expression frame, the other expression frame is a molecular chaperone expression frame, the recombinant Zika virus structural protein E gene expression frame comprises a first eukaryotic promoter, a secretion signal peptide sequence co-expressed with the target gene and the target gene, and the molecular chaperone expression frame comprises a second eukaryotic promoter and a calcium-linked protein open reading frame;
the secretion signal peptide sequence is selected from α chaperone factors.
3. The recombinant Hansenula polymorpha capable of high-throughput expression of structural protein E of Zika virus according to claim 2, wherein the Hansenula polymorpha host is Hansenula polymorpha strain D L-1.
4. A method for constructing the recombinant Hansenula polymorpha capable of high-throughput expression of structural protein E of Zika virus according to claim 2, wherein: the method comprises the following steps:
1) artificially synthesizing the recombinant Zika virus structural protein E sequence;
2) amplifying the open reading frame of the molecular chaperone;
3) constructing a recombinant expression vector containing the recombinant Zika virus structural protein E sequence and an open reading frame of a molecular chaperone;
the step 3) specifically comprises the following steps:
3.1) respectively constructing an expression frame capable of co-expressing the structural protein E sequence and the secretion signal peptide sequence of the recombinant Zika virus and an expression frame capable of expressing calnexin of the host;
3.2) constructing a linearized vector comprising the two expression frames to obtain the recombinant expression vector;
4) transforming the recombinant expression vector into a Hansenula polymorpha host to obtain the Hansenula polymorpha for expressing the recombinant Zika virus structural protein E.
5. The method of claim 4, wherein the Hansenula polymorpha host is Hansenula polymorpha strain D L-1.
6. Use of the secreted high-throughput expression vector of claim 1 for the production of recombinant Zika virus structural protein E.
7. Use of the recombinant Hansenula polymorpha according to any one of claims 2 to 3 for producing recombinant structural protein E of Zika virus.
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