CN109705222A - The fusion protein and the preparation method and application thereof of flaviviridae envelope protein - Google Patents

Abstract

The invention discloses a kind of fusion proteins and the preparation method and application thereof of flaviviridae envelope protein.The present invention with antibody Fc fragment by merging flaviviridae envelope protein to obtain fusion protein, compared with flaviviridae envelope protein, has the structure closer to virus surface native conformation, can be applied to the antibody that in-vitro screening is directed to space conformation；It can also be applied to the preparation of vaccine simultaneously, induction body generates efficiently and the predicting space epitope antibody of wide spectrum, Fc segment can also enhance the response of cellular immunity.

Description

The fusion protein and the preparation method and application thereof of flaviviridae envelope protein

Technical field

The present invention relates to field of biotechnology, a kind of fusion protein in particular to flaviviridae envelope protein and its Preparation method and application.

Background technique

Zika virus (ZIKV) is a kind of flaviviridae, can lead to neonatal microcephalus simultaneously after infecting the virus The neurodevelopment of infant is influenced, Guillain Barre syndrome (GBS), which may occur, after Adult infections leads to neurotrosis.Stockaded village's card Virus mainly enters host cell by the envelope protein on its surface.In natural virus surface, zika virus envelope protein (ZE) be in the form of antiparallel dimer existing for, and some researches show that, the antibody for dimer comformational epitope is high Effect and can be in wide spectrum and zika virus and dengue fever virus.Therefore external to obtain close to flaviviridae surface cyst membrane egg The recombinant protein of white native conformation is vital for the discovery of antiviral drugs and the development of vaccine.

Summary of the invention

It is an object of the invention to be obtained in vitro closer to flaviviridae envelope protein native dimeric body conformation Recombinant protein provides the fusion protein and its system of a kind of flaviviridae envelope protein for being easily achieved external dimerization conformation Preparation Method and application.

To achieve the above object, the present invention provides a kind of fusion protein of flaviviridae envelope protein, it be by The fusion protein of the antibody Fc fragment of flaviviridae envelope protein and humanized IgG composition.

In above scheme, it is preferable that the coding gene sequence of the fusion protein is by the method for bridging PCR by jaundice The Gene Fusion of the antibody Fc fragment of the gene and humanized IgG of malicious coe virus envelope protein obtains.

In above scheme, the gene of the antibody Fc fragment of the humanized IgG passes through artificial mutation, and the mutational site is Fc The cysteine base of the hinge portion of segment.Disulfide bond is easily formed between cysteine base, is made by way of mutation The space structure for obtaining fusion protein has more freedom.

Preferably, the cysteine base mutation is the base of encoding serine.Serine is similar with cysteine, no Change the property of albumen.

The method of the present invention is passed through for the flaviviridae envelope protein for being not readily available dimerization conformation in vitro It is merged with the Fc segment of human antibody IgG, such as ZE virus and Fc segment composition, human antibody IgG-Fc segment can be by CH3 Non-covalent strong interaction between structural domain, which mediates, generates dimerization, we can form the spy of dimer by Fc segment herein Property, zika virus envelope protein is merged with Fc segment, building obtain ZE-Fc fusion protein, and thus obtain close to The recombinant protein of native dimeric body conformation obtains under the action of Fc segment native dimeric closer to the natural structure of virus surface The dimer recombinant protein of elephant.Flaviviridae is well-conserved in structure, therefore dimerization strategy provided herein It can be applied to the external reproduction of entire flaviviridae envelope protein native conformation.

The present invention also provides the fusion protein of a kind of zika virus envelope protein and Fc segment, amino acid full length sequences For Seq ID No.2, it is named as ZE-Fc.

Optionally, the coding gene sequence of the ZE and Fc fusion protein (ZE-Fc) are Seq ID No.1.

The present invention also provides the preparation method of above-mentioned zika virus envelope protein and the fusion protein of Fc segment, steps It is as follows:

(1) genetic fragment of Fc and the genetic fragment of zika virus envelope protein are prepared；The zika virus envelope protein Gene be ZE extracellular domain 1-408 amino acid coded sequence；

(2) genetic fragment of the genetic fragment of Fc and zika virus envelope protein is merged to obtain complete ZE-Fc gene；

(3) it with ZE-Fc gene order construction of expression vector, transfects, expression, purifying obtains destination protein.

In above scheme, the genetic fragment of the Fc is mutated gene, described to sport wild type Fc hinge portion Cysteine base mutation be encoding serine base, amplification obtain Fc (S.Hinge).

In above scheme, the step (3) are as follows: insect expression vector is constructed with ZE-Fc gene order, and is transfected into drosophila In S2 cell, building surely turns expression cell system, carries out eukaryotic expression, and purifying obtains destination protein.

Present invention further teaches the fusion proteins of above-mentioned flaviviridae envelope protein, are preparing antiviral antibody and epidemic disease Application in seedling.

Beneficial effects of the present invention:

(1) it is merged by the Fc segment with human antibody IgG1, under the action of Fc segment native dimeric, is obtained Closer to the dimer recombinant protein of virus surface native conformation, realizes and be not easy external Dimerized flaviviridae The native conformation of envelope protein reappears.

(2) relative to the flaviviridae envelope protein directly expressed, fusion protein can pass through Fc fragment mediates Cellullar immunologic response causes stronger vivo immunization reaction, this lays a good foundation for it as vaccine dose.

(3) retaining for the fusion protein of zika virus envelope protein and Fc segment relative to the ZE directly expressed In the case where ZE monomer conformation, the structure of antiparallel dimer has also been reappeared.

Detailed description of the invention

Fig. 1 is protein immunoblot detection figure after the expression and purification of ZE and ZE-Fc albumen.

Fig. 2 is SDS electrophoretogram after the expression and purification of ZE and ZE-Fc albumen.

Fig. 3 is SDS electrophoretogram after the expression and purification of D2E-Fc and JE-Fc albumen.

Fig. 4 is the secondary structure figure of ZE and ZE-Fc albumen.

Fig. 5 is the thermal denaturation analysis chart of ZE and ZE-Fc albumen.

Fig. 6 is ZE and ZE-Fc albumen and the elisa assay figure for identifying non-space epitope-positive antibody.

Fig. 7 is ZE and ZE-Fc albumen and the elisa assay figure for identifying predicting space epitope positive antibody.

Fig. 8 is after mouse is immunized respectively in ZE and ZE-Fc albumen, and antibody generates the elisa assay figure of titre in mice serum Wherein it is directed to the antibody titer analysis chart of predicting space epitope.

Specific embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.Following embodiment is with this Implemented under premised on inventive technique scheme, the detailed implementation method and specific operation process are given, but of the invention Protection scope is not limited to the following embodiments.

Embodiment 1: amplification ZE and ZE-Fc fragment gene

According to it has been reported that ZE gene order (GenBank:KJ776791) and humanized IgG 1 Fc fragment gene sequence (GenBank:KJ905798.1), gene chemical synthesis is carried out according to Drosophila S 2 cells codon bias, will when synthesis The sequence of TwinStrep-tag is added in the c-terminus of ZE and Fc base, and designs forward and reverse primer according to the base sequence of synthesis Expand target fragment:

Forward primer

5-AGATCTCCATGG(horizontal line is labeled as NcoI digestion to ATCCGTTGCATCGGCGTGTCCAACCGTGAC-3 (1) Site)

5-GAACCAAAAAGCAGCGATAAG-3(2)；

Reverse primer

5-CTAGA CTCGAG(horizontal line is labeled as Xho I digestion position to TTA CTTCTCAAACTGCGGGTGGCT-3 (1) Point)

PCR is carried out with forward primer (1)/(2) and reverse primer (1) by template of the base sequence of synthesis respectively, respectively ZE and Fc genetic fragment is obtained, and carries out bridging PCR using forward primer (1) and reverse primer (1), obtains ZE-Fc gene.

Agarose gel electrophoresis recycles ZE-Fc genetic fragment, with Nco I and Xho I digestion ZE-Fc gene and carrier pMT- ZE-Fc gene is connect, Transformed E .coliTop10 competence by Bip-V5-His after glue recycling with carrier pMT-Bip-V5-His, Subsequent picking monoclonal send sequencing.According to sequencing result, the correct monoclonal of picking sequence is used for subsequent experimental.

The gene order of ZE-Fc are as follows: Seq ID No.1, the amino acid sequence of coding are Seq ID No.2.

Embodiment 2: the S2 cell line of building stable transfection expression ZE-Fc/ZE albumen

The S2 that ZE-Fc/ZE plasmid and screening plasmid pCoBlast are gone to adhere-wall culture using Effectene kit is thin Born of the same parents, by the screening of about 20 days Blasticidin resistances, obtain adherent growth surely turns S2 cell line, through serum free medium After adaptation, freezes a batch and surely turn S2 cell line, and remaining cell is gone into 40ml shaking flask further expansion culture, be turning finally to In the Tissue Culture Flask of 1L, and final concentration of 500 μM of CuSO is added₄The expression of solution inducible protein is shaken in 27 DEG C, 100rpm Bed culture 8-10 days.

Then, centrifugation (6000g, 4 DEG C, 15min) collects S2 cell conditioned medium (supernatant is with the StrepTactin that HRP is marked It detects antibody and carries out protein immunoblot detection, as a result as shown in Figure 1, being filled out after the supernatant of collection is filtered with StrepTactin Expect that (IBA company) purifies ZE, ZE-Fc albumen.It is then carried out with molecular sieve secondarily purified, and is 10kD using molecular cut off Target protein is concentrated by ultrafiltration in ultra-filtration centrifuge tube (Merck Millipore company), its purity is verified through SDS-PAGE, such as Fig. 2 institute Show, the swimming lane of right figure from left to right is respectively as follows: Marker, ZE-Fc (boiling sample) and ZE-Fc (not boiling sample).It can be found that ZE-Fc melts Hop protein can form dimer by the noncovalent interaction of Fc segment.

The dimerization situation of embodiment 3:D2E-Fc and JE-Fc

In order to which other are not easy with obtain the flaviviridae envelope protein of dimerization conformation in vitro, we are also utilized with real The method of example 1 and 2 is applied to obtain fusion protein, and is verified.

Two type dengue virus envelope proteins (D2E), gene order (GenBank:KM087965.1), fusion protein are D2E-Fc, amino acid sequence be Seq ID No.3 japanese encephalitis virus envelope protein (JE), gene order (GenBank: FJ938228.1), fusion protein JE-Fc, amino acid sequence are Seq ID No.4

After expression and purification, the formational situation of dimer is demonstrated to the further SDS-PAGE of D2E-Fc and JE-Fc, such as Shown in Fig. 3, swimming lane from left to right is successively are as follows: Marker, D2E-Fc (boiling sample), D2E-Fc (not boiling sample), JE-Fc (boiling sample) and JE-Fc (does not boil sample).

The secondary structure and thermal stability analysis of embodiment 4:ZE, ZE-Fc albumen

The secondary structure of albumen is measured using circular dichroism spectrometer (CD) and carries out thermal stability analysis.Protein concentration is adjusted It is added in 0.1cm cuvette to 0.3-0.4mg/ml, and after being filtered with 0.22 μm of filter membrane, under the conditions of 25 DEG C, near ultraviolet end (190-260nm) respectively to air, PBS, protein sample is scanned measurement, every data of 0.5 second record.Experiment setting It is repeated twice, the reading that PBS is reduced after being averaged is the protein data of measurement.As a result as shown in figure 4, according to analysis, this The secondary structure of two albumen is typical beta sheet, it was demonstrated that second level knot of the introducing of Fc segment without change ZE albumen itself Structure.

The temperature range that thermal stability analysis selects is 25~95 DEG C, and experiment initial temperature is 25 DEG C, by 0.5 DEG C/min's Rate increases temperature, every 0.5s detection protein to the absorption of circularly polarized light at 216nm wavelength.According to alternating temperature temperature and egg White mean residue ellipticity MRE mapping, can analyze the thermal stability for comparing albumen and solution temperature Tm (melting temperature).As a result as shown in figure 5, apparent three-stage unfolding process is presented in ZE-Fc albumen, wherein ZE section and ZE egg Influence in terms of white unfolding process is similar, and Tm value is also roughly the same, therefore Fc fusion protein will not have stability to ZE.

Embodiment 5:ELISA analyzes identification of the positive antibody to ZE, ZE-Fc albumen

According to reported antigen antibody complex structure, it is anti-that positive antibody can be divided into identification non-space structure epi-position Body and identification space structure epitope antibodies.Non-space structure epi-position: 2A10G6 (PDB 5JHL), ZV-67 (PDB 5KVG)；Space Structure epi-position: EDE2 A11 (PDB 5LCV), EDE2 B7 (PDB 4UT6), EDE1 C10 (PDB 4UT9).It is coated with ZE and ZE- Fc albumen uses 2A10G6 and ZV-67 as primary antibody on elisa plate, and the Anti-His for using HRP to mark develops the color as secondary antibody As a result as shown in fig. 6, non-space structure epi-position can be identified effectively, and the EC50 of ZE-Fc albumen is higher.In space structure Epitope identification aspect is coated with StrepTactin albumen on elisa plate as capture antibody, ZE and ZE-Fc albumen is then added As antigen, the positive antibody of gradient dilution is as primary antibody, and the anti-Fab for using HRP to mark is as secondary antibody, and develop the color result such as Fig. 7 Shown, only ZE-Fc can preferably be identified that ZE does not show ELISA signal.Therefore ZE-Fc recombinant protein being capable of body The outer structure reappeared close to virus surface native dimeric body conformation.

The mouse immune of embodiment 6:ZE-Fc albumen

Fusion protein ZE-Fc and ZE are uniformly mixed with QuickAntibody adjuvant (Biodragon) respectively, albumen is whole Concentration is 0.2mg/ml.The female BABL/c mouse of 4-6 week old is divided into 3 groups, every group 5, respectively with 100 μ l fusion proteins ZE-Fc and recombinant protein ZE are injected at the hind leg muscle of mouse, and PBS is as negative control.Exempted from for the second time after three weeks Epidemic disease took certain serum from mouse orbit veniplex after 2 weeks, measured antibody titer therein with ELISA method；Meanwhile benefit Whether ELISA means (capture ELISA and indirect ELISA) verifying differently wherein produces for the anti-of predicting space epitope Body.As a result as shown in figure 8, mouse, which is immunized, with fusion protein ZE-Fc and ZE can generate antibody, but fusion protein ZE-Fc is induced The antibody titer of generation is apparently higher than ZE, and since fusion protein ZE-Fc remains predicting space epitope structure, only immune The mouse of fusion protein ZE-Fc produces the antibody for predicting space epitope.According to existing report, for the antibody of predicting space epitope Can very effective neutralization virus, therefore, fusion protein ZE-Fc is expected to become effective subunit vaccine.

Sequence table

<110>Wuhan Virology Institute,Chinan academy of Sciences

<120>fusion protein and the preparation method and application thereof of flaviviridae envelope protein

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caggagggag ctgtgcatac cgctctggcc ggagctctgg aggccgaaat ggatggcgcc 840

aagggccgcc tgtccagcgg acatctgaag tgccgcctga agatggacaa gctgcgcctg 900

aagggcgtgt cctacagcct gtgcaccgct gccttcacct tcacgaagat ccccgctgag 960

accctgcacg gaaccgtgac cgtggaggtg cagtacgctg gcacggatgg cccgtgtaag 1020

gtgcccgccc agatggccgt ggatatgcaa acgctgaccc ccgtgggacg cctgattacc 1080

gccaacccgg tcatcaccga gtccacggag aacagcaaga tgatgttgga gttggatccc 1140

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Thr Glu Asn Ser Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp

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Val Thr Asp Ile Ser Thr Val Ala Arg Cys Pro Thr Thr Gly Glu Ala

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His Asn Glu Lys Arg Ala Asp Ser Ser Tyr Val Cys Lys Gln Gly Phe

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Trp Thr Ser Pro Ser Ser Thr Ala Trp Arg Asn Arg Glu Leu Leu Met

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Glu Phe Glu Glu Ala His Ala Thr Lys Gln Ser Val Val Ala Leu Gly

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Ser Gln Glu Gly Gly Leu His Gln Ala Leu Ala Gly Ala Ile Val Val

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Glu Tyr Ser Ser Ser Val Lys Leu Thr Ser Gly His Leu Lys Cys Arg

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Leu Lys Met Asp Lys Leu Ala Leu Lys Gly Thr Thr Tyr Gly Met Cys

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Thr Glu Lys Phe Ser Phe Ala Lys Asn Pro Ala Asp Thr Gly His Gly

305 310 315 320

Thr Val Val Ile Glu Leu Ser Tyr Ser Gly Ser Asp Gly Pro Cys Lys

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Ile Pro Ile Val Ser Val Ala Ser Leu Asn Asp Met Thr Pro Val Gly

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Arg Leu Val Thr Val Asn Pro Phe Val Ala Thr Ser Ser Ala Asn Ser

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Lys Val Leu Val Glu Met Glu Pro Pro Phe Gly Asp Ser Tyr Ile Val

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Val Gly Arg Gly Asp Lys Gln Ile Asn His His Trp His Lys Ala Gly

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Ser Thr Leu Gly Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro

405 410 415

Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

420 425 430

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

435 440 445

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

450 455 460

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

465 470 475 480

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

485 490 495

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

500 505 510

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

515 520 525

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

530 535 540

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

545 550 555 560

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

565 570 575

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

580 585 590

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

595 600 605

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

610 615 620

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Asp Asp Asp

625 630 635 640

Lys Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly

645 650 655

Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

660 665 670

Claims (10)

1. a kind of fusion protein of flaviviridae envelope protein, it is by flaviviridae envelope protein and humanized IgG Antibody Fc fragment composition fusion protein.

2. the fusion protein of flaviviridae envelope protein according to claim 1, it is characterised in that: the fusion egg White coding gene sequence is by the method for bridging PCR by the anti-of the gene of flaviviridae envelope protein and humanized IgG The Gene Fusion of body Fc segment obtains.

3. the fusion protein of flaviviridae envelope protein according to claim 1, it is characterised in that: the source of people The gene of the antibody Fc fragment of IgG passes through artificial mutation, and the mutational site is the cysteine of the hinge portion of Fc segment Base.

4. the fusion protein of flaviviridae envelope protein according to claim 3, it is characterised in that: the half Guang ammonia Soda acid base sports the base of encoding serine.

5. a kind of fusion protein of zika virus envelope protein and Fc segment, amino acid full length sequence is Seq ID No.2.

6. the fusion protein of zika virus envelope protein and Fc segment according to claim 5, it is characterised in that: the fusion The coding gene sequence of albumen is Seq ID No.1.

7. the preparation method of zika virus envelope protein described in a kind of claim 5 and the fusion protein of Fc segment, step is such as Under:

(1) genetic fragment of Fc and the genetic fragment of zika virus envelope protein are prepared；The base of the zika virus envelope protein Because of the coded sequence of the 1-408 amino acid of ZE extracellular domain；

(2) genetic fragment of the genetic fragment of Fc and zika virus envelope protein is merged to obtain complete ZE-Fc gene；

(3) it with ZE-Fc gene order construction of expression vector, transfects, expression, purifying obtains destination protein.

8. the preparation method of zika virus envelope protein described in a kind of claim 5 and the fusion protein of Fc segment, feature exist In: the genetic fragment of the Fc is mutated gene, and described sport dashes forward the cysteine base of wild type Fc hinge portion Become the base of encoding serine.

9. the preparation method of zika virus envelope protein and the fusion protein of Fc segment, feature exist according to claim 7 In: the step (3) are as follows: insect expression vector is constructed with ZE-Fc gene order, and is transfected into Drosophila S 2 cells, building is steady Turn expression cell system, carry out eukaryotic expression, purifying obtains destination protein.

10. the fusion protein of flaviviridae envelope protein described in claim 1, in preparing antiviral antibody and vaccine Using.