CN106520699A - Recombinant HEK-293T cell and Zika virus resistant fusion protein secreted by recombinant HEK-293T cell - Google Patents
Recombinant HEK-293T cell and Zika virus resistant fusion protein secreted by recombinant HEK-293T cell Download PDFInfo
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Abstract
The invention relates to a recombinant HEK-293T cell. The recombinant HEK-293T cell is characterized in that the recombinant HEK-293T cell is obtained by transfecting an exogenous fusion gene by taking the recombinant HEK-293T cell as a host, wherein a nucleotide sequence of the exogenous fusion gene is shown as SEQ ID NO.1. The recombinant HEK-293T cell can secrete Zika virus resistant fusion protein.
Description
Technical field
The present invention relates to the cell for introducing foreign heredity substance and modifying, and in particular to the HEK-293T cells of restructuring.
Background technology
Zika virus (Zika virus) is flaviviridae (Flaviviridae) Flavivirus (Flavivirus) virus,
Mainly by bite by mosquitos propagation, transmission through sex, virus can pass through placenta in addition.Nineteen forty-seven virologist is sent out in Uganda
Existing ZIKA is viral, and patient is mainly shown as heating, maculopapule and joint pain, is showed only as fragmentary morbidity and small-scale stream afterwards
OK.Until the Yap islands eruption and prevalence in the South Pacific Ocean in 2007, subsequently card was occurred in succession in Africa, Southeast Asia and South America
Viral cases of infection.China national health State Family Planning Commission circulates a notice of on 2 9th, 2016, makes a definite diagnosis an Introduced cases Zika virus infection disease
Example, has 10 Introduced cases Zika virus cases of infection so far.Clinical research confirmation Zika viruses can be led by placenta
Fetus is caused microcephaly occur.During Guillaume Carteaux report 1 adult, Zika viruses infect the meninx brain for causing
It is scorching.Zika virus infection becomes serious worldwide public health problem, while causing sternly to the countries and regions of Outbreak
Weight economic loss., there is state in Zika virus infection, or epidemic situation be expected to large area in the data display of the newest announcement of the World Bank
The country of outburst, local international visitors quantity material will be die-offed, and therefore its tourist industry will lose 63,900,000,000 dollars.China
Geographical vast, summer mosquito grows, and is that the transmission of disease creates advantage.
Zika viral nucleic acids are single-stranded positive RNA, long 10.8kb, encode a precursor protein, Jing viruses and host protein
Digestion be cut into 3 structural proteins (C, prM/M and E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and
NS5).Zika viruses belong to flaviviridae with dengue fever virus, and the nucleic acid and albumen homology of the two is higher, current state
Inside and outside more to dengue fever virus research report, Beth-Ann G.Coller etc. adopt insect cell expression dengue fever E protein,
Mice Inoculated and non-human primate show preferable immunogenicity, and challenge test shows that the albumen has preferably protection
Power, with the potentiality for becoming dengue fever virus subunit vaccine.Additionally, Zika virus nonstructural proteins, such as NS1, NS3 and
NS5 etc. plays a significant role during virus is sticked, invades and replicated etc..
Zika virus infection has become the important public health factor for affecting health of people, and the disease does not have specific treatment
Method, and still do not have the vaccine of commercialization, E protein domain III is considered as the optimal candidate albumen for researching and developing Zika vaccines, Kim
MY and Poddar A et al. researchs show that III energy stimulating animal body of dengue fever virus E protein domain is produced and protect compared with high titre
Shield property antibody, with prepare dengue fever virus recombinant subunit vaccine potentiality (Kim MY, Kim BY, Oh SM, Reljic R,
Jang YS,Yang MS.Oral immunisation of mice with transgenic rice calli
expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen
induces antibodies to all four dengue serotypes.Plant Mol Biol.2016Oct；92(3):
347-56.Poddar A,Ramasamy V,Shukla R,Rajpoot RK,Arora U,Jain SK,Swaminathan S,
Khanna N.Virus-like particles derived from Pichia pastoris-expressed dengue
virus type 1glycoprotein elicit homotypic virus-neutralizing envelope domain
III-directed antibodies.BMC Biotechnol.2016Jun 14；16(1):50.).Document report shows, band
The fusion protein for having IgG1Fc labels can strengthen the immunogenicity of fusion protein, and its principle is probably extend fusion protein half
Decline the phase or increased expression FcR antigen presenting cells intake.In addition, human IgG1's Fc fragments are human body self components, as
The risk that immunity rejection occurs during vaccine composition is relatively low.
The content of the invention
The technical problem to be solved is to provide a kind of restructuring HEK-293T cells, restructuring HEK-293T cells
Can stably excreting express the fusion protein of anti-zika virus.
Technical proposal that the invention solves the above-mentioned problems is as follows：
A kind of restructuring HEK-293T cells, it is characterised in that restructuring HEK-293T cells are to be with HEK-293T cells
Host's transfection exogenous fusion gene is obtained, wherein the nucleotide sequence of described exogenous fusion gene is as shown in SEQ.ID.NO.1.
Above-mentioned restructuring HEK-293T cells are comprised the following steps：
(1) ZIKA virus E protein Domain IIIs (typing number be KU955594.1) and people are obtained respectively from GenBank
The nucleotide sequence of IgG1Fc (typing number is JN222933.1), and by the two using flexible peptide (GGGS)3Connection, is then carried out
Codon optimization, obtains the fusion as shown in SEQ.ID.NO.1；By the initiating terminal and clearing end of resulting fusion
Codon carries out digestion respectively, is then cloned in carrier pMD19-T, obtains recombinant vector life pMD19-ZDIII-hFc；
(2) filling-in after first taking I digestions of FUGW plasmids Pac reclaims carrier large fragment with I digestions of BamH again, then takes
Then pCDNA3.0 plasmids use Roche first with filling-in after II digestions of Bgl again with CMV promoter is reclaimed after I digestions of BamH
Ligation kit will obtain slow virus expression plasmid pLV-CMV- after the carrier large fragment for being reclaimed and CMV promoter connection
EGFP；
(3) slow virus expression plasmid pLV-CMV-EGFP double digestions are reclaimed into large fragment, then pMD19-ZDIII-hFc is double
Digestion reclaim fusion ZD III-hFc of the nucleotide sequence as shown in SEQ.ID.NO.1, then by the large fragment for being reclaimed with melt
Close III-hFc of gene ZD to connect under T4DNA connection enzyme effects, after converting stab3 competent cells, LB of the coating containing ammonia benzyl puts down
Plate, after incubated overnight picking single bacterium colony amplification cultivation and extraction obtain III-hFc of slow virus recombinant vector pLV-CMV-EGFP-ZD；
(4) III-hFc of slow virus recombinant vector pLV-CMV-EGFP-ZD are packed into helper plasmid corotation with slow virus
Dye HEK-293T cells, obtain final product restructuring HEK-293T cells.
Described restructuring HEK-293T cells can stably excreting expression zika virus and humanized IgG 1Fc fusion proteins, this melts
The amino acid sequence of hop protein is as shown in SEQ ID NO.2.Described fusion protein has anti-zika virus biologically active.
The invention has the advantages that：
1st, prior art adopts prokaryotic expression or Yeast expression albumen mostly, albumen and native protein prepared by said method
There is larger difference in higher structure method, the expression system selected by the present invention is HEK-293T cells, the restructuring for obtaining
HEK-293T cells are conducive to the large-scale production of antigen protein with the biological nature similar to parental cell；Expressing protein exists
Expression cell interior energy obtains native conformation and the modification processing for being close to virus protein, and antigenicity is good；Recombinant cell can use rolling bottle
High density fermentation culture, it is easy to a large amount of to produce.
2nd, it is of the invention on the basis of to the inclined preferendum of Zika viral gene mammalian cell codons, first using slow virus
Expression system expression E protein domain III and humanized IgG 1Fc fusion proteins, the Zika virus E protein structures in the fusion protein
By flexible peptide (GGGS) between domain III and humanized IgG 1Fc albumen3Connection, is conducive to later-period purification.
3rd, recombinant expressed clone of the invention can utilize serum free medium or low blood serum medium to carry out culture table
Reach, production of vaccine cost can be reduced；
4th, antigen presentation amount of the present invention is high, and using ordinary cells blake bottle ware culture, yield is up to 110mg/L.
Description of the drawings
Fig. 1 is that slow virus recombinant vector pLV-CMV-EGFP-ZDIII-hFc builds schematic diagram.
Fig. 2 is the electrophoretogram of slow virus recombinant vector pLV-CMV-EGFP-ZDIII-hFc digestions identification, and in figure, M is
Marker, 1 is selected clone.
Fig. 3 is that Western blot identify Zika virus E proteins domain III and people source in different positive colony cells
The electrophoretogram of IgG1Fc expressing fusion protein situations；In figure, band 1,2,3,4 is thin for four recombinant clones selected at random
Born of the same parents.
Fig. 4 is III-hFc of recombinant cell lines HEK-293T-ZD difference generation cell destination protein expression Western Blot
Electrophoretogram；In figure, band 1 be negative control, band 2-9 be respectively the 1st, 5,10,15,20, in the cell culture of 25,30,35 generations
Clear Western Blot electrophoresis results.
Fig. 5 is the RT-PCR electrophoretograms of the fusion of different generation restructuring HEK-293T cell expression；In figure, M is
Marker DL2000, band 1 be negative control, band 2-9 be respectively the 1st, 5,10,15,20,25,30,35 generation HEK-293T
The RT-PCR results of the fusion of cell expression.
Fig. 6 is the absorbance collection of illustrative plates of fusion protein purification of the present invention.
Fig. 7 is that fusion protein purification of the present invention respectively walks the electrophoretogram for keeping sample；In figure, band 1 is sample before loading；Bar
Band 2 is to flow through peak sample；Band 3 is eluting peak sample.
Specific embodiment
Material and its source involved by embodiment is as follows：
HEK-293T cells are preserved by Guangzhou Bai Nizi bio tech ltd；
Stab3 competent cells are purchased from Guangzhou FulenGen Co., Ltd.；
DL 2000Marker、DL 3000Marker、DL 5000Marker、DL10000Marker、Premix Ex Taq
Takara companies are purchased from dephosphorylation kit；Restriction enzyme Sal I Xho I BamH I Nhe I, Mlu I,
T4DNA ligases are NEB Products；Pre-dyed albumen Marker is purchased from SunShineBio companies；Bovine serum albumin(BSA) and fine jade
Lipolysaccharide is purchased from Amresco companies；Diaminobenzidine is purchased from Shanghai Mai Ruier chemical technologies Co., Ltd；DNA Ago-Gels
QIAquick Gel Extraction Kit is Axygen Products；Small amount plasmid extraction kit is purchased from OMEGA companies；Dimethyl sulfoxide (DMSO), polybrene
Purchased from Sigma companies；Tryptone and yeast extract are Oxiod Products；0.25% trypsase, DMEM culture mediums and
South America hyclone is Hyclon Products, and Zika polyclonal antibodies are purchased from Kerafast companies of the U.S..
Embodiment 1 (structure of recombinant cell lines and detection)
1st, encode Zika viral domains III to design with humanized IgG 1Fc gene orders and prepare
1.1 coding Zika viral domains III are designed with the gene order of humanized IgG 1Fc
In Zika virus Bahia03 strain (GenBank:ANA85188.1 sequence optimisation and expression are carried out on the basis of gene)
Design, added with Kozak sequences and Nhe I restriction enzyme sites and protectiveness base before initiation codon, in III base of E protein domain
Cause is with humanized IgG 1Fc coding ends added with terminator and MluI restriction enzyme sites；E protein domain III and the people source for obtaining
As shown in SEQ ID NO.1, SEQ ID NO.2 are the amino acid sequence after SEQ ID NO.1 translations to the nucleotide sequence of IgG1Fc.
1.2 coding Zika virus E proteins domains III are synthesized with humanized IgG 1Fc gene orders
Zika virus E proteins domain III is closed by Taihe county Bioisystech Co., Ltd of Sino-U.S. with humanized IgG 1Fc gene orders
Into concrete grammar is as follows, and ZIKA virus E protein Domain III (Accession are obtained from GenBank:KU955594.1) with
Human IgG1 Fc (Accession:JN222933.1) nucleotide sequence, using flexible peptide (GGGS) between two sequences3Connection, adopts
JCat softwares carry out mammalian cell expression and carry out codon optimization to sequence, obtain SEQ.ID.NO.1, and chemical synthesis process is obtained
Above-mentioned sequence, adds Nhe I restriction enzyme sites before the end of the sequence 5 ' initiation codon, adds Mlu I after 3 ' end terminator codons
Restriction enzyme site, is cloned in commercial vectors pMD19-T (Takara, lot No.D104A), and recombinant vector is named as
pMD19-ZDIII-hFc。
2nd, the structure of slow virus recombinant vector and detection
The acquisition of slow virus expression plasmid pLV-CMV-EGFP：FUGW plasmids are used into BamH I again with filling-in after I digestions of Pac
Carrier large fragment is reclaimed in digestion；PCDNA3.0 plasmids are first opened with recovery CMV after I digestions of BamH again with filling-in after II digestions of Bgl
Mover, then slow virus will be obtained after the carrier large fragment of above-mentioned recovery and CMV promoter connection with Roche ligation kit
Expression plasmid pLV-CMV-EGFP.
Above-mentioned slow virus expression plasmid pLV-CMV-EGFP is processed with I digestion of Nhe I and Mlu and reclaims large fragment, then
III-hFc the genes of ZD reclaimed with I digestion of Jing Nhe I and Mlu are connected in the case where T4DNA is connected enzyme effect, convert stab3 competence
LB flat board of the coating containing ammonia benzyl after cell, picking single bacterium colony amplification cultivation extracts plasmid after incubated overnight, with bacterium colony PCR and
I digestion of Nhe I and Mlu identifies that to which positive plasmid of identification is slow virus recombinant vector needed for the present embodiment, by which
It is named as III-hFc of pLV-CMV-EGFP-ZD.
Above-mentioned FUGW plasmids, pCDNA3.0 plasmids and Roche ligation kit are commercially available.
The above-mentioned LB flat boards containing ammonia benzyl, its culture medium prescription are 100mL：Tryptone 1g, yeast extract 0.5g, chlorine
Change sodium 1g, agar 1.5g, plus distillation water dissolves, autoclaving 20min, treat that liquid cooling, to 55 DEG C of addition ammonia benzyls, is stood after shaking up
Carve a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices；The ammonia benzyl concentration is 100mg/mL, and addition is that every 100mL culture mediums add 100ul ammonia benzyls.
The present embodiment slow virus recombinant vector build schematic diagram as shown in figure 1, extract recombinant plasmid Jing Nhe I and
After I digestions of Mlu, enter row agarose gel electrophoresis analysis, discovery there are two bands, and digestion products electrophoresis banding pattern is consistent with expected results
(see Fig. 2).
3rd, slow virus packaging and titre detection
3.1st, plating cells
Routine operation of the plating cells using those skilled in the art, comprises the following steps that shown：
The culture supernatant in HEK-293T Tissue Culture Flasks is outwelled, is washed with 1-3ml PBS one time, exhausted；Add 1ml
0.25% pancreatin, room temperature effect 1-2min；Treat that cell is all rounded, add 2-3ml DMEM complete mediums that cell is blown and beaten down
Come, collect in centrifuge tube, 1000rpm centrifugation 5min abandon supernatant；It is cell precipitation is fully resuspended with DMEM complete mediums,
Cell is made to form single cell suspension；
10ml DMEM complete mediums are added to 100mm Tissue Culture Dish, cell count are carried out with cell counting count board, often
Individual ware about adds 6 × 106Cell, 37 DEG C, 5%CO2Overnight incubation in incubator.
The formula of DMEM complete mediums is：Penicillin containing 10%FBS, 100U/ml and 0.1mg/ml streptomysins
DMEM complete mediums (Corning cellgro, USA, lot number 10-013-CVR-500ml).
Cell state is most important for virus packaging, and the good state of cell growth is conducive to viral packaging, therefore needs
Ensure good cell state.
3.2 slow virus are packed and infect
Slow virus packaging be exactly the III-hFc of slow virus recombinant vector pLV-CMV-EGFP-ZD that will prepare before with it is slow
Virus packaging helper plasmid cotransfection human embryonic kidney cell HEK-293T, transfection method adopt PEI (polyethyleneimine) method, tool
Body step is as follows.
3.2.1 rotaring redyeing system
Routine operation of the PEI infection protocols for those skilled in the art, its rotaring redyeing system are divided into A liquid and B liquid, specifically such as
Shown in lower：
A liquid
24 μ l of PEI storing liquids；
1 × HBS is mended to 1ml；
B liquid
The formula of above-mentioned PEI storing liquids is：Weigh 1.25mg PEI powder to be dissolved in 50ml × HBS (pH7.4), 0.2 μ
M membrane filtrations, be stored in 4 DEG C it is standby.
The formula of above-mentioned 1 × HBS is：8.76g NaCl are dissolved in into 900ml ultra-pure waters, the HEPES of 20ml 1M is added,
Adjust pH value to 7.4, be settled to 1L, be stored in after 0.2 μm of membrane filtration 4 DEG C it is standby.Above-mentioned slow virus packaging system plasmid gag/
Pol, Rev, VSVG are purchased from Invitrogen companies (article No.：K4975-00), these packaging plasmids provide viral genome
MRNA is packaged into structural proteins necessary to intact virions, polymerase and envelope protein.
3.2.2 transfection
Second day after plating cells, when cell fusion degree reaches 70%-90%, the A liquid of rotaring redyeing system is added to into B
Liquid, fully mixes, after being stored at room temperature 20min, is gently added drop-wise in the cells and supernatant of 100mm wares, and the light culture dish that shakes is mixed,
37 DEG C are put, 5%CO2Supernatant is collected after 48h is cultivated in incubator, then in Tissue Culture Dish adds 10ml DMEM complete again
Culture medium, collects supernatant after continuing culture 48h, merges supernatant twice, then removes cell after 3000rpm centrifugations 5min broken
Piece, then obtain required virus liquid.
The virus liquid is taken into 100 μ l for detecting virus titer, -80 DEG C of Refrigerator stores is put in after remaining packing or is used for
Follow-up cell infection.
3.3 virus titers are detected
Virus titer detection determines virus titer using RT-QPCR, and its method of operating is normal using those skilled in the art's
Rule operation and used kit illustrate step, and testing result shows that virus titer is 1 × 108Copies/ml, illustrates virus
Pack successfully, energy infection cell is simultaneously inserted into gene in cellular genome.
3.4th, cell infection
Virus liquid obtained above is infected into HEK293-T cells, is comprised the following steps that shown：
1 × 10 is diluted to DMEM complete mediums after growth conditions good HEK293-T cell dissociations are counted5/
ML, adds 24 orifice plates, and 500 μ L/ holes prepare 2 multiple holes, are put into 37 DEG C, 5%CO2Cultivate 24 hours in incubator；
Polybrene (polybrene) is added in DMEM complete mediums, (the culture of the culture medium containing polybrene is prepared
The final concentration of 6-8 μ g/mL of Polybrene in base)；
10 μ l of virus liquid prepared by above-mentioned 3.2.2 are added in the above-mentioned culture medium containing polybrene, make the final volume be
300 μ l featheriness mixing, prepare base containing Virus culture；
The old culture medium that step 1 is cultivated the HEK293-T cells after 24h is outwelled, and adds 300 μ l containing virus in each hole
Culture medium, is put into 37 DEG C, 5%CO2Cultivate in incubator.
4th, the foundation of stable gene secreting, expressing clone
The is cultivated after 3-5 days, after the cell of culture hole will be covered with pancreatin digestion, passed in 96 holes with limiting dilution assay
Continue culture in plate, the colony counts of every hole cell after 15 days, are observed under inverted microscope；
The hole containing 1 cell colony (i.e. 1 cell mass) is selected, clone cell is cultivated after 3d in 24 orifice plates,
Supernatant is collected, and supernatant is drawn after the centrifugation of electrophoresis sample-loading buffer system is added after 10 times of concentrations carries out SDS-PAGE electrophoresis, transferring film
2h is closed using 5% defatted milk (BioRad companies) afterwards, using the anti-Zika virus polyclonal antibody overnight incubations (U.S.
Kerafast companies, 1：10000 dilutions), TBST is washed 3 times, each 10min, is incubated the anti-(Tiangeng of rabbit-anti people two of HRP marks
Biochemical technology (Beijing) Co., Ltd, 1：10000 dilution), 37 DEG C of 1h, post-exposure.
Western blotting results are as shown in Figure 3.
As a result show that we screen 4 clones for obtaining and can express Zika virus E proteins domain III and people source
IgG1Fc fusion proteins, recombinant protein size are about 40kD, but expression is variant, and it is thin that we choose expression highest 4
Born of the same parents clone is preserved after continuing Amplification Culture, and is named as III-hFc of HEK-293T-ZD, and carries out stability inspection to which
Survey.
5th, the Detection of Stability of recombinant cell lines of the present invention
The Western blotting identifications of 5.1 clone cells difference generation cell
III-the hFc of recombinant cell lines HEK-293T-ZD that above-mentioned screening is obtained normally is passed on and is collected different generations (
1st, 5,10,15,20,25,30,35 generations) cells and supernatant carries out Western blotting identifications, qualification result such as Fig. 4 institutes
Show, as a result show that the cell of different generations can stable express express target protein.
The RT-PCR identifications of 5.2 clone cells difference generation cell
According to this area routine techniques and related kit specification, using RT-PCR to recombinant cell lines HEK-293T-
After the 1st, 5,10,15,20,25,30,35 generation cells of III-hFc of ZD carry out the amplification of genes of interest, as a result as shown in figure 5, restructuring
The cell of the different generations of III-hFc of clone HEK-293T-ZD can be amplified and theoretical purpose band of the same size, table
Bright genes of interest is stably blended in cellular genome, and inheritance stability.
The present embodiment be can be seen that from above-mentioned testing result obtain really and can stably express Zika virus E protein structures
III-the hFc of recombinant cell lines HEK-293T-ZD in domain III.
Embodiment 2 (purifying of fusion protein)
1st, Zika virus E proteins domain III and humanized IgG 1Fc fusion protein purifications
0.45 μm of membrane filtration of cells and supernatant Jing, rProtein A SepharoseTMFast Flow chromatograph glue post
After being correctly connected with BioLogic LP protein purifications instrument, with the sample-loading buffer (50mM pH7.0Tris) of 3 times of bed volumes,
1ml/min balances pillar, by the sample of pretreatment with the speed loading of 1ml/min, carries out stream with sample-loading buffer and wash after terminating,
1ml/min, 5 times of bed volumes subsequently use 1M pH3.0 glycine elution antibody, while with BioLogic LP protein purification instrument
It is monitored, when baseline rising is observed, that is, occurs starting to collect during eluting peak, and use the Tris-HCl of 1M, pH9.0 to adjust immediately
Whole eluent pH to 7.0.Collect eluent to eluting peak to return to after baseline, continuation balances 5-10 times of post bed body with sample-loading buffer
Product, flow velocity are adjusted to 1ml/min.5 times of bed volumes are balanced with 20% ethanol again.
2nd, protein purification analysis
Collection protein purification respectively is walked to keep sample and carries out Western blotting analyses, and method is with method in embodiment 1.By scheming
6 understand that monitored first peak is separated well with second peak (destination protein peak) for non-binding peak during loading.Western
Blotting results show Zika virus E proteins domain III and humanized IgG 1Fc fusion protein purification condition maturities, purifying effect
Rate higher (see Fig. 7).
Claims (3)
1. a kind of restructuring HEK-293T cells, it is characterised in that restructuring HEK-293T cells are with HEK-293T cells as place
Main transfection exogenous fusion gene is obtained, wherein the nucleotide sequence of described exogenous fusion gene is as shown in SEQ.ID.NO.1.
2. a kind of restructuring HEK-293T cells according to claim 1, it is characterised in that described by following methods system
：
(1) obtain the nucleotide sequence of ZIKA virus E proteins Domain III and human IgG1 Fc respectively from GenBank, and by two
Person is using flexible peptide (GGGS)3Connection, then carries out codon optimization, obtains the fusion as shown in SEQ.ID.NO.1；Will
The initiating terminal and clearing end codon of resulting fusion carries out digestion respectively, is then cloned in carrier pMD19-T, obtains
PMD19-ZDIII-hFc is ordered to recombinant vector；
(2) filling-in after first taking I digestions of FUGW plasmids Pac reclaims carrier large fragment with I digestions of BamH again, then takes pCDNA3.0 matter
Grain, then will with Roche ligation kit first with filling-in after II digestions of Bgl again with CMV promoter is reclaimed after I digestions of BamH
The carrier large fragment for being reclaimed and CMV promoter obtain slow virus expression plasmid pLV-CMV-EGFP after connecting；
(3) slow virus expression plasmid pLV-CMV-EGFP double digestions are reclaimed into large fragment, then by pMD19-ZDIII-hFc double digestions
Fusion ZD III-hFc of the nucleotide sequence as shown in SEQ.ID.NO.1 is reclaimed, then by the large fragment for being reclaimed and fusion base
Because III-hFc of ZD connect under T4DNA connection enzyme effects, LB flat board of the coating containing ammonia benzyl after stab3 competent cells, mistake are converted
Night culture after picking single bacterium colony amplification cultivation and extract obtain III-hFc of slow virus recombinant vector pLV-CMV-EGFP-ZD；
(4) III-hFc of slow virus recombinant vector pLV-CMV-EGFP-ZD are packed into helper plasmid cotransfection with slow virus
HEK-293T cells, obtain final product restructuring HEK-293T cells.
3. a kind of fusion protein of the anti-zika virus by HEK-293T cells secretion of recombinating described in claim 1, the fusion egg
White amino acid sequence is as shown in SEQ.ID.NO.2.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988239A (en) * | 2017-11-29 | 2018-05-04 | 南方医科大学 | A kind of recombination of zika virus and its preparation method and application |
| CN110981966A (en) * | 2019-11-19 | 2020-04-10 | 广州迪澳生物科技有限公司 | Fusion protein for detecting Zika virus and Zika virus specific T cell detection kit |
-
2016
- 2016-10-11 CN CN201610887189.XA patent/CN106520699A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| EUN KIM ET AL.: "Preventative vaccines for Zika virus outbreak: preliminary evaluation", 《EBIOMEDICINE》 * |
| JULIANA HELENA CHARVEZ ET AL.: "Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization", 《BIOLOGICALS》 * |
| LIANPAN DAI ET AL.: "Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody", 《CELL HOST & MICROBE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988239A (en) * | 2017-11-29 | 2018-05-04 | 南方医科大学 | A kind of recombination of zika virus and its preparation method and application |
| CN110981966A (en) * | 2019-11-19 | 2020-04-10 | 广州迪澳生物科技有限公司 | Fusion protein for detecting Zika virus and Zika virus specific T cell detection kit |
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