CN109111507A - Viral recombinant glycoprotein and its eukaryocyte high-efficiency expression method and application - Google Patents

Viral recombinant glycoprotein and its eukaryocyte high-efficiency expression method and application Download PDF

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CN109111507A
CN109111507A CN201810963128.6A CN201810963128A CN109111507A CN 109111507 A CN109111507 A CN 109111507A CN 201810963128 A CN201810963128 A CN 201810963128A CN 109111507 A CN109111507 A CN 109111507A
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recombinant glycoprotein
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glycoprotein
leu
gly
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孙乐
张翠娟
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Zhejiang Pedichang Science And Technology Development Co ltd
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Beijing Paidichang Science And Technology Development Co Ltd
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Abstract

The present invention provides a kind of viral recombinant glycoprotein, is the recombinant glycoprotein for substituting the signal peptide of target viral glycoprotein with the signal peptide of human or animal's secreted protein and being formed.The present invention also provides the methods using recombinant glycoprotein described in eukaryocyte high efficient expression.The present invention solves the world-technology problem that viral recombinant glycoprotein expression quantity in eukaryocyte is low, secretion is poor, establishes solid foundation for vaccine development and viral protective antibody test reagent, has a good application prospect.

Description

Viral recombinant glycoprotein and its eukaryocyte high-efficiency expression method and application
Technical field
The present invention relates to gene engineering technology fields, specifically, being related to a kind of viral recombinant glycoprotein and its eukaryon is thin Born of the same parents' high-efficiency expression method and application.
Background technique
Rabies are the Category B notifiable disease of China's statutory report management, are that human death leads highest acute biography so far One of catch an illness, cause of disease is rabies viruses (Rabies virus), and people and all warm-blooded animals are susceptible to the virus.Rabies Lethality close to 100%, although human source anti-rabies virus antibody medicine has a great development in recent years, still do not have at present Very effective curative drug is developed, therefore, rabies vaccine is still current antirabic most effective means.
Rabies viruses genome is the sub-thread strand RNA of non-segmented negative, and overall length about 12kb is separately encoded zymoprotein (L), sugar Five albumen (G), stromatin (M), phosphorylated protein (P), nucleoprotein (N) major structural proteins.Wherein, G-protein contains more A protective antigens site can induce and generate rabies poison protection antibody.
Precautionary measures after the rabies exposure that the World Health Organization (WHO) is recommended, to the prevention after the exposure of rabies three-level The method that rabies poison immunoglobulin is mainly combined using rabies vaccine injection.In order to improve the safety of vaccine, weight Group hepatitis B vaccine, recombination human papillomavirus' vaccine list one after another.But rabies viruses recombinant glycoprotein eukaryotic expression amount is too Low, prokaryotic expression protein conformation is not right, and the research for recombinating rabies vaccine is never in progress.The human rabies vaccine generally used Inactivated vaccine is remained as, there is a certain security risk.
In addition, expressing viral host is different since the Strain that different producers uses is different, production technology is different, matter Amount is also irregular, directly influences rabic prevention and treatment.It generallys use at present and the experiment of virus infected cell is inhibited to carry out Vaccine efficacy evaluation, it is time-consuming and laborious.The mad dog guarantor of ELISA indirect method kit detection is developed with mad dog G-protein wrapper sheet is recombinated Shield property antibody titer, easy, accurate, repeatability height have very big social benefit to China's rabies vaccine quality is improved.
Summary of the invention
The object of the present invention is to provide viral recombinant glycoprotein and its eukaryocyte high-efficiency expression methods.
It is a further object of the present invention to provide the various uses of viral recombinant glycoprotein.
In order to achieve the object of the present invention, in a first aspect, virus recombinant glycoprotein provided by the invention, is to use human or animal The signal peptide of the signal peptide substitution target viral glycoprotein of secreted protein and the recombinant protein that is formed.
Optionally His label, flexibility Linker can be connected in the N-terminal and/or C-terminal of viral recombinant glycoprotein of the invention.
The signal peptide of human or animal's secreted protein of the present invention includes but is not limited to human antibody IgG heavy chain and/or people The signal peptide of albumin.
Target viral glycoprotein of the present invention includes but is not limited to rabies virus G protein, Ebola virus GP1 albumen.
Second aspect, the present invention provide a kind of rabies viruses recombinant glycoprotein, are the signal peptides with human IgG ferritin heavy chain The recombinant glycoprotein for substituting the signal peptide of rabies virus G protein and being formed, the amino acid sequence of the recombinant glycoprotein such as SEQ Shown in ID NO:1.
The third aspect, the present invention provide a kind of Ebola virus recombinant glycoprotein, are replaced with the signal peptide of human albumin For Ebola virus GP1 albumen signal peptide and the recombinant glycoprotein that is formed, the amino acid sequence of the recombinant glycoprotein such as SEQ Shown in ID NO:3.
Fourth aspect, the present invention provide the nucleic acid for encoding the recombinant glycoprotein.
5th aspect, the present invention provide the biomaterial for containing the nucleic acid, and the biomaterial is expression cassette, expression carries Body, cloning vector, engineering bacteria or host cell etc..
6th aspect, the present invention provide the method using recombinant glycoprotein described in eukaryocyte high efficient expression, including following Step:
1) PCR method, DNA are utilized by codon corresponding relationship and host expresses frequency according to known protein sequence Recombination method or artificial synthesized method obtain the nucleic acid of simultaneously any one of Optimized Coding Based claim 1-4 recombinant glycoprotein；
2) nucleic acid clone is entered into carrier for expression of eukaryon, then converted or transfection host cell, with the proliferation of host cell, thus It realizes expression of the viral recombinant glycoprotein in eukaryocyte, and collects target protein from cell culture.
Wherein, the carrier for expression of eukaryon includes but is not limited to pcDNA3.The host cell includes but is not limited to 293 thin Born of the same parents, CHO.
7th aspect, the present invention provide the monoclonal antibody developed based on the recombinant glycoprotein or polyclonal antibody.
Eighth aspect, the present invention provide a kind of vaccine, and effective component is the viral recombinant glycoprotein, optionally comprising medicinal Auxiliary material.
9th aspect, the present invention provide a kind of colloidal gold colloidal gold detection test paper strip, and the recombination sugar is coated in the test strips Albumen, or target viral antibody test reagent or target viral infection diagnostic reagent using recombinant glycoprotein preparation.
Tenth aspect, the present invention provide following any application of the recombinant glycoprotein:
1) application in vaccine and antiviral drugs is being prepared；
2) application in target viral antibody test reagent or target viral infection diagnostic reagent is being prepared；
3) application in colloidal gold colloidal gold detection test paper strip is being prepared；
4) application in preparation monoclonal antibody or polyclonal antibody；
5) application in the detection of target viral antibody ELISA.
In the specific embodiment of the present invention, eukaryocyte high efficient expression rabies viruses recombinant glycoprotein is utilized Method is as follows:
1) according to the amino acid sequence of rabies viruses recombinant glycoprotein, pass through codon corresponding relationship and host expresses frequency Rate obtains the nucleic acid of recombinant glycoprotein described in simultaneously Optimized Coding Based using PCR method, DNA recombination method or artificial synthesized method；
2) nucleic acid clone is entered into carrier for expression of eukaryon pcDNA3, then converted or rotaring redyeing 293 cell, with the increasing of host cell It grows, to realize expression of the viral recombinant glycoprotein in eukaryocyte, and collects target protein from cell culture.
The present invention further provides application of the rabies viruses recombinant glycoprotein in vaccine preparation.
The present invention further provides the rabies viruses recombinant glycoproteins for animal or the immune generation antibody of people.
The present invention further provides application of the rabies viruses recombinant glycoprotein in rabies virus antibodies detection.
The present invention further provides application of the rabies viruses recombinant glycoprotein in rabies virus antibodies diagnosis.
The present invention further provides the rabies viruses recombinant glycoproteins in rabies virus antibodies ELISA detection method Using.
The present invention further provides rabies viruses recombinant glycoprotein the answering in rabies virus antibodies gold label test strip With.
Rabies viruses recombinant glycoprotein provided by the invention is immunized the antibody generated after mouse and identifies different rabies viruses Strain can be used for rabies vaccine exploitation.The rabies viruses recombinant glycoprotein of the purifying animal immune from different rabies vaccines or people Serum have cross reaction, can be used for developing the exploitation of animal or human rabies viruses resisting antibody diagnostic reagent.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) present invention successfully solves that viral recombinant glycoprotein expression quantity in eukaryocyte is low, secretion poor (is not easy to wear Cell membrane) world-technology problem, establish solid foundation for vaccine development and viral protective antibody test reagent, have Good application prospect.
(2) eukaryocyte high efficient expression rabies viruses recombinant glycoprotein is utilized, turns rabies in 293 cell supernatants in wink Malicious recombinant glycoprotein expression quantity is up to 10mg/L, can carry out commercialization volume production.
(3) antibody generated after mouse being immunized with rabies viruses recombinant glycoprotein of the invention identifies different rabies Strain, can be used for rabies vaccine exploitation, and market prospects are huge；The rabies viruses recombinant glycoprotein of purifying and different rabies vaccines The serum of immune animal or people has cross reaction, can be used for developing animal or human rabies viruses resisting antibody diagnostic reagent Exploitation, has a good application prospect.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of plasmid pcDNA3 in the embodiment of the present invention 2.
Fig. 2 is the SDS-PAGE testing result that mad dog G glycoprotein is recombinated in the embodiment of the present invention 2.
Fig. 3 is the Westernblot testing result of mad dog G glycoprotein (being not added with signal peptide) in the embodiment of the present invention 2.Its In, swimming lane 1-4 is respectively as follows: cell supernatant, empty carrier expression cell supernatant, cell pyrolysis liquid, empty carrier cell pyrolysis liquid.
Fig. 4 is that rabies viruses after purification is recombinated the coating that G glycoprotein carries out various concentration in the embodiment of the present invention 2, It is detected using the antibody of anti-glycoprotein, gradient signal is presented for the glycoprotein of different package amounts in antibody.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The synthesis of 1 rabies viruses recombinant glycoprotein of embodiment and its nucleic acid and the building of expression vector
The signal peptide of rabies virus glycoprotein is substituted with the signal peptide of human IgG ferritin heavy chain, gained recombinant protein is as mad Dog disease poison recombinant glycoprotein, amino acid sequence is as shown in SEQ ID NO:1.The rabies viruses comes from Rabies virus strain CTN-1。
According to protein sequence shown in SEQ ID NO:1, by codon corresponding relationship and host expresses frequency, using artificial Synthetic method obtains the nucleic acid of rabies viruses recombinant glycoprotein described in simultaneously Optimized Coding Based.
Embodiment 2 utilizes eukaryotic cell expression rabies viruses recombinant glycoprotein
(1) building of recombinant plasmid
Rabies viruses recombinant glycoprotein nucleic acid sequence after optimization is subjected to digestion using restriction enzyme and is inserted into table Up in carrier pcDNA3 (Fig. 1), bacillus coli DH 5 alpha competent cell is converted.After checking carrier constructs successfully, recombination matter is carried out Grain preparation.The partial sequence of obtained recombinant plasmid is constructed as shown in SEQ ID NO:2.
(2) plasmid extracts and (uses Tiangeng plasmid extraction kit)
Shake bacterium: culture medium of the 4ml containing corresponding antibiotic is added in 14ml lock nut pipe, and sterilizing toothpick dips gene chemical synthesis and punctures bacterium It is put into LB culture medium, is incubated overnight.
1, column equilibration: into adsorption column CP4, the equilibrium liquid BL of 500 μ L is added in (adsorption column is put into collecting pipe), 12000rpm (about 13400g) is centrifuged 1min, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.
2, the bacterium solution for taking 5-15ml to be incubated overnight is added in centrifuge tube, and 12000rpm (about 13400g) is centrifuged 1min, as far as possible Absorb supernatant.Note: bacterial sediment can be collected into a centrifuge tube by being centrifuged several times when bacterium solution is more, biomass with It can sufficiently crack and be preferred, the insufficient extraction efficiency that can reduce plasmid of excessive cellular lysate.
3. 500 μ L solution Pl (RNaseA is added in advance) are added into the centrifuge tube there are bacterial sediment, pipettor is used Or the thorough suspended bacterial cell precipitation of turbula shaker.
4. 500 μ L solution P2 are added into centrifuge tube, leniently spinning upside down 6-8 times cracks thallus sufficiently.
5. 700 μ L solution P3 are added into centrifuge tube, leniently spin upside down 6-8 times, mix well immediately, can go out at this time Existing white flock precipitate.12000rpm (about 13400g) is centrifuged 10min, forms precipitating in centrifugation bottom of the tube at this time.
6. Filter column Cs (Filter column is put into collecting pipe) is added by several times in the supernatant that previous step is collected, 12000rpm (about 13400g) is centrifuged 2min, solution obtained in pipe will carefully be collected after centrifugation, (adsorption column in adsorption column CP4 is added by several times It is put into collecting pipe).
7.12000rpm (about 13400g) is centrifuged l min, outwells the waste liquid in collecting pipe, adsorption column CP4 is put into collection Guan Zhong.
8. 500 μ L protein liquid removal P4,12000rpm (about 13400g) centrifugation l min is added into adsorption column CP4, receipts are outwelled Adsorption column CP4 is put into collecting pipe by the waste liquid in collector.
9. 600 μ L rinsing liquid PW (check whether and dehydrated alcohol has been added) are added into adsorption column CP4,12000rpm is (about It 13400g) is centrifuged 1min, the waste liquid in collecting pipe is outwelled, adsorption column CP4 is put into collecting pipe.
10. 600 μ L rinsing liquid PW, 12000rpm (13400g) centrifugation l min is added into adsorption column CP4, collection is outwelled Waste liquid in pipe.
It is placed in 12000rpm (about 13400g) centrifugation 2min 11. adsorption column CP4 is placed back in collecting pipe, by adsorption column The rinsing liquid of middle remnants removes.To ensure that downstream experiment is not influenced by residual ethanol, adsorption column CP4 is uncapped, room temperature is placed in It places several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material.
12. adsorption column is placed in a clean centrifuge tube, 100-300 μ L is vacantly added dropwise to the intermediate position of adsorbed film Elution buffer TB, is placed at room temperature for 2min, and 12000rpm (about 13400g) is centrifuged l min and plasmid solution is collected into centrifuge tube In.
(3) plasmid DNA concentration and purity detecting
Obtained Plasmid DNA micro-spectrophotometer detectable concentration and purity (OD 260/280).
(4) ethanol precipitation
Plasmid adds the dehydrated alcohol and 0.1 times of volume 3M sodium acetate overnight precipitation of 3 times of volumes.12000rpm, 4 DEG C, centrifugation 10min is dissolved after drying precipitating in Biohazard Safety Equipment with no heat source water, micro-spectrophotometer detectable concentration, is guaranteed dense Degree is about 1 μ g/ μ l.
(5) POLOdelivererTM3000 293 cells of transient transfection determine transfection conditions
1, day before transfection inoculating cell is into 6 orifice plates, every hole inoculation 3 × 105~3.5 × 105A cell is complete in 1.5ml Full culture medium, at 37 DEG C, 5%CO2Under the conditions of be incubated overnight, cell density reaches 60%-80% before making to transfect.
2,4 μ g DNA and 8 μ l POLOdeliverer are takenTM3000 are diluted to respectively in 250mlOpti-MEM culture medium, mix 5min is placed at room temperature for after even.
3, by POLOdelivererTM3000 solution are added drop-wise in DNA solution, room temperature item after being mixed with rifle piping and druming or whirlpool 15min is placed under part.
4, said mixture is dropped evenly in culture medium, gently shakes up culture dish, do not need to change liquid before transfection.
5, by the cell of transfection at 37 DEG C, 5%CO2Under the conditions of cultivate 48-72h, liquid CD293 is changed in transfection afterwards for 24 hours.
6, supernatant ELISA detection progress transient expression assay is collected after transfecting 48-72h.
(6) POLOdelivererTM3000 a large amount of transfections
1,100ml cell is carried out using determining transfection conditions largely to transiently transfect, and collect supernatant detection.
2, supernatant purify by nickel column and ELISA detects purification result.
3, purity of protein, BCA Quantitative Western concentration are verified using SDS-PAGE.
(7) experimental result
1, the SDS-PAGE testing result for recombinating mad dog G glycoprotein is shown in Fig. 2.
Mad dog G glycoprotein (control group) the Western blot testing result for being not added with signal peptide is shown in Fig. 3.It can be with from Fig. 3 Find out, G-protein only exists in cell pyrolysis liquid, and G-protein is not detected in cell supernatant.
2, the expression quantity of mad dog G glycoprotein is recombinated in eukaryocyte supernatant and purification yield is shown in Table 1 and Fig. 4.
Using cell supernatant gradient coated elisa plate, 1:10,1:30,1:100,1:300,1:1000,1:3000,1: 10000,100 μ L, 4 DEG C of reaction overnights are added in every hole；It is incubated overnight rear PBS solution board-washing 3 times, uses the PBS liquid for containing 5% milk 1hr is closed in room temperature；It uses PBS solution board-washing 1 time, is added using the diluted 2 μ g/ of PBS liquid for containing 5% milk after the completion of closing Ml primary antibody NM57 reacts at room temperature 1hr；PBS solution board-washing 3 times after reaction, pat dry, and are added using the PBS liquid for containing 5% milk By the sheep anti-Mouse secondary antibody of the diluted HRP label of 1:2000,1hr is reacted at room temperature；PBS solution board-washing 5 times after reaction are clapped Dry, 100 μ l substrates (substrate A liquid and substrate B liquid mix in equal volume) is added in every hole, is protected from light, reacts 20min under room temperature；So 50 μ L terminate liquid (0.1M H are added in every hole afterwards2SO4), OD is read after mixing in microplate reader450Value.
Reality is 32mg/L with the albumen that Ni column purification obtains.
Table 1
3, the coating that rabies viruses recombination G glycoprotein after purification is carried out to various concentration, uses the antibody of anti-glycoprotein It is detected, gradient signal is presented for the glycoprotein of different package amounts in antibody.Mad dog glycoprotein specific monoclonal antibody identification purifying The ELISA of mad dog recombinant G protein the results are shown in Table 2.Wherein monoclonal antibody 7G3 identifies that G-protein linear epitope, monoclonal antibody NM57 identify conformation table Position.Being coated with NM57 concentration is that 2 μ g/ml, NM57 identify that the sensitivity of G-protein is 10ng/well.Monoclonal antibody NM57 is purchased from North China pharmacy New Drug Research Co., Ltd of group, 7G3 are provided by AbMax Biotechnology Co., Ltd..
Table 2
4, the titre results of human serum are shown in Table 3 after detection vaccine immunity after the mad dog glycoprotein of recombination.Sensitivity reaches 100ng/ ml。
Table 3
Using mad dog G-protein coated elisa plate after purification, 1 μ g/ml, 100 μ L, 4 DEG C of reaction overnights are added in every hole；Overnight PBS solution board-washing 3 times after incubation, using 3%BSA-PBS in 37 DEG C of closing 1hr；PBS solution board-washing 3 is used after the completion of closing It is secondary.NM57 is used into bare substrate human serum gradient dilution, reuses after 3%BSA carries out 20 times and 100 times dilutions respectively and adds 100 μ L, 37 DEG C of reaction 1hr are added in sample, every hole；PBS solution board-washing 3 times after reaction, pat dry；It is added using containing 3%BSA's PBS liquid reacts at room temperature 1hr by goat-anti people's secondary antibody of the diluted HRP label of 1:5000；PBS solution board-washing 3 times after reaction, It pats dry, 100 μ l substrates (substrate A liquid and substrate B liquid mix in equal volume) is added in every hole, is protected from light, reacts 5min under room temperature；So 50 μ L terminate liquid (0.1M H are added in every hole afterwards2SO4), OD is read after mixing in microplate reader450Value.
5, the titre results of dog serum are shown in Table 4- table 5 after detection vaccine immunity after the mad dog glycoprotein of recombination.50 times of dilutions are equal Value is 0.291, Nc+3*SD=0.482.
Table 4
Using mad dog G-protein coated elisa plate after purification, 1 μ g/ml, 100 μ L, 4 DEG C of reaction overnights are added in every hole；Overnight PBS solution board-washing 3 times after incubation are stayed overnight using 3%BSA-PBS in 4 DEG C of closings；PBS solution board-washing 3 is used after the completion of closing It is secondary.11 parts of dog bare substrate serum are carried out using 20%CS-PBS respectively 20 times, 50 times, 100 times, be loaded after 200 times of dilutions, 100 μ L, 37 DEG C of reaction 1hr are added in every hole；PBS solution board-washing 3 times after reaction, pat dry；It is added and uses the PBS containing 3%BSA Liquid reacts at room temperature 1hr by the rabbit-anti dog secondary antibody of the diluted HRP label of 1:10000；PBS solution board-washing 3 times after reaction are clapped Dry, 100 μ l substrates (substrate A liquid and substrate B liquid mix in equal volume) is added in every hole, is protected from light, reacts 6min under room temperature；Then 50 μ L terminate liquid (0.1M H are added in every hole2SO4), OD is read after mixing in microplate reader450Value.
Table 5
Three parts of dog blood background informations: 432621# > 10.26,43263#=1.5,202145#=5.92.
Using mad dog G-protein coated elisa plate after purification, 1 μ g/ml, 100 μ L, 4 DEG C of reaction overnights are added in every hole；Overnight PBS solution board-washing 3 times after incubation are stayed overnight using the PBS liquid containing 3%BSA in 4 DEG C of closings；It is washed after the completion of closing using PBS solution Plate 3 times.4 parts of dog positive serums are carried out using 20%CS-PBS respectively 0 times, 10 times, 25 times, be directly loaded after 50 times of dilutions, 100 times first reuse 50 times of dilutions of PBS liquid progress containing 20%CS using 2 times of dilutions of bare substrate dog serum progress when diluting and add 100 μ L, 37 DEG C of reaction 1hr are added in sample, every hole；PBS solution board-washing 3 times after reaction, pat dry；It is added and uses 3%BSA-PBS The rabbit-anti dog secondary antibody of the diluted HRP label of solution 1:10000, reacts at room temperature 1hr；PBS solution board-washing 3 times after reaction are clapped Dry, 100 μ l substrates (substrate A liquid and substrate B liquid mix in equal volume) is added in every hole, is protected from light, reacts 6min under room temperature；Then 50 μ L terminate liquid (0.1M H are added in every hole2SO4), OD is read after mixing in microplate reader450Value.
6, antibody purification 7G3 identifies that rabies viruses recombination G glycoprotein titre is about 10 μ g/ml (table 6).
Table 6
7, the cross reaction ELISA that the rabies viruses of mouse tail blood and different strains after mouse is immunized in mad dog glycoprotein is recombinated It the results are shown in Table 7.
Table 7
As can be seen from Table 7, it is most strong to step rich signal for PDC001 mouse tail blood identification Jilin, and at big, promise is really raw in identification Liaoning Object has signal, between 0.8-1.0, identifies that the long-range OD in Henan 0.4 or so, identifies that other vaccine signals are weak.
Mad 1 μ g/ml of dog G-protein peridium concentration, is coated with, every 100 μ L of hole is coated with enzyme respectively after each producer's vaccine 1:100 dilution Target, 4 DEG C of reaction overnights；It is incubated overnight rear PBS solution board-washing 3 times, closes 1hr in room temperature using the PBS liquid containing 5% milk； It is used PBS solution board-washing 1 time after the completion of closing, is added and presses the diluted mouse tail blood of 1:500, room using the PBS liquid containing 5% milk Temperature reaction 1hr；PBS solution board-washing 3 times after reaction, pat dry, and are added and are diluted using the PBS liquid containing 5% milk by 1:2000 HRP label sheep anti-Mouse secondary antibody, react at room temperature 1hr；PBS solution board-washing 5 times after reaction, pat dry, and every hole is added 100 μ l substrate (substrate A liquid and substrate B liquid mix in equal volume), is protected from light, reacts 20min under room temperature；Then 50 μ L are added in every hole Terminate liquid (0.1M H2SO4), OD is read after mixing in microplate reader450Value.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing Pai Dichang development in science and technology Co., Ltd
<120>viral recombinant glycoprotein and its eukaryocyte high-efficiency expression method and application
<130> KHP171115145.6
<150> 201710723877.7
<151> 2017-08-22
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 460
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly
1 5 10 15
Val Gln Thr Glu Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly
20 25 30
Pro Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu
35 40 45
Val Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met
50 55 60
Glu Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr
65 70 75 80
Cys Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly
85 90 95
Tyr Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp
100 105 110
Ala Cys Arg Ser Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr
115 120 125
Glu Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr
130 135 140
Val Lys Thr Thr Lys Glu Ser Val Val Ile Ile Ser Pro Ser Val Ala
145 150 155 160
Asp Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Arg
165 170 175
Gly Lys Cys Ser Gly Ile Thr Val Ser Ser Ala Tyr Cys Ser Thr Asn
180 185 190
His Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser
195 200 205
Cys Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Ser
210 215 220
Lys Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser Leu Lys
225 230 235 240
Gly Ala Cys Lys Leu Lys Leu Cys Gly Val Leu Gly Leu Arg Leu Met
245 250 255
Asp Gly Thr Trp Val Ala Ile Gln Thr Ser Asn Glu Thr Lys Trp Cys
260 265 270
Pro Pro Asp Gln Leu Val Asn Leu His Asp Phe His Ser Asp Glu Ile
275 280 285
Glu His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu
290 295 300
Asp Ala Leu Glu Ser Ile Met Thr Thr Lys Ser Val Ser Phe Arg Arg
305 310 315 320
Leu Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr
325 330 335
Ile Phe Asn Lys Thr Leu Met Glu Ala Asp Ala His Tyr Lys Ser Val
340 345 350
Arg Thr Trp Asn Glu Ile Ile Pro Ser Lys Gly Cys Leu Arg Val Gly
355 360 365
Gly Arg Cys His Pro His Val Asn Gly Val Phe Phe Asn Gly Ile Ile
370 375 380
Leu Gly Pro Asp Gly His Val Leu Ile Pro Glu Met Gln Ser Ser Leu
385 390 395 400
Leu Gln Gln His Met Glu Leu Leu Glu Ser Ser Val Ile Pro Leu Met
405 410 415
His Pro Leu Ala Asp Pro Ser Thr Val Phe Lys Asp Gly Asp Glu Val
420 425 430
Glu Asp Phe Val Glu Val His Leu Pro Asp Val His Lys Gln Val Ser
435 440 445
Gly Val Asp Leu Gly Leu Pro Asn Trp Gly Lys Asp
450 455 460
<210> 2
<211> 1457
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtaccgcca ccatggagtt ggggctgagc tgggttttcc ttgttgctat attagaaggt 60
gtccagtgtg agaaattccc catttacacg ataccagaca aactcggccc ctggagtccc 120
atcgatatac atcacctcag ctgtccgaac aatctggttg tggaggacga aggatgtacc 180
aatctgtcag gattctcata catggagctt aaagtaggat atatttcggc cataaaggtg 240
aacgggttca cttgtacggg tgtggtaacg gaagcagaaa cctacactaa ctttgtcggt 300
tatgtcacca ccacgtttaa gagaaagcac ttccgaccaa caccggatgc atgcagatca 360
gcatacaatt ggaagatggc aggtgacccc agatatgaag agtctctgca caatccctat 420
cctgattatc attggctccg gactgtaaaa accaccaaag agtctgttgt tatcatatct 480
ccaagtgtgg cagacttaga cccgtacgat aaatcacttc attcgagagt ttttcctaga 540
ggaaaatgct caggaataac ggtgtcttct gcctactgct ctaccaacca tgattatacc 600
atctggatgc ctgaaaatcc tagactgggg acctcttgtg atattttcac caacagcaga 660
gggaagagag catccaaagg gagcaagacc tgtggatttg tggatgagag aggcttgtac 720
aaatctctaa aaggagcatg caaactgaag ctgtgtggag ttcttggact taggcttatg 780
gacggaacct gggtcgcgat tcagacatca aacgagacca agtggtgccc tcctgatcaa 840
ctagtgaatc tacatgactt tcattcagat gagattgaac atcttgttgt ggaggagttg 900
gttaagaaga gggaggagtg tctagatgca ctggagtcca tcatgaccac caagtccgtg 960
agtttcagac gtctcagtca cttgaggaag cttgtgcctg gatttggaaa agcatacacc 1020
atattcaaca agaccttaat ggaggctgat gctcactaca aatcggtccg aacttggaat 1080
gagatcatcc cctcgaaagg gtgtttaaga gtcgggggga gatgtcatcc tcatgtgaac 1140
ggagtatttt tcaatggtat catcctaggc cctgacggcc atgtcttaat cccggaaatg 1200
cagtcatccc tcctccagca gcatatggag ttgttggaat cctcggtcat ccccttaatg 1260
catcccttgg cagatccatc aacggttttt aaagatggtg acgaggtgga ggattttgtt 1320
gaggttcacc ttccagatgt gcataagcag gtctcagggg ttgatctcgg tctcccaaac 1380
tgggggaagg atggcggggg cagcgggggc agcggcggga ccggtcatca tcatcatcat 1440
cattagtaag cggccgc 1457
<210> 3
<211> 668
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Arg Gly Val Phe Arg Arg Ile Pro Leu Gly Val Ile His Asn
20 25 30
Ser Thr Leu Gln Val Ser Asp Val Asp Lys Leu Val Cys Arg Asp Lys
35 40 45
Leu Ser Ser Thr Asn Gln Leu Arg Ser Val Gly Leu Asn Leu Glu Gly
50 55 60
Asn Gly Val Ala Thr Asp Val Pro Ser Ala Thr Lys Arg Trp Gly Phe
65 70 75 80
Arg Ser Gly Val Pro Pro Lys Val Val Asn Tyr Glu Ala Gly Glu Trp
85 90 95
Ala Glu Asn Cys Tyr Asn Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu
100 105 110
Cys Leu Pro Ala Ala Pro Asp Gly Ile Arg Gly Phe Pro Arg Cys Arg
115 120 125
Tyr Val His Lys Val Ser Gly Thr Gly Pro Cys Ala Gly Asp Phe Ala
130 135 140
Phe His Lys Glu Gly Ala Phe Phe Leu Tyr Asp Arg Leu Ala Ser Thr
145 150 155 160
Val Ile Tyr Arg Gly Thr Thr Phe Ala Glu Gly Val Val Ala Phe Leu
165 170 175
Ile Leu Pro Gln Ala Lys Lys Asp Phe Phe Ser Ser His Pro Leu Arg
180 185 190
Glu Pro Val Asn Ala Thr Glu Asp Pro Ser Ser Gly Tyr Tyr Ser Thr
195 200 205
Thr Ile Arg Tyr Gln Ala Thr Gly Phe Gly Thr Asn Glu Thr Glu Tyr
210 215 220
Leu Phe Glu Val Asp Asn Leu Thr Tyr Val Gln Leu Glu Ser Arg Phe
225 230 235 240
Thr Pro Gln Phe Leu Leu Gln Leu Asn Glu Thr Ile Tyr Thr Ser Gly
245 250 255
Lys Arg Ser Asn Thr Thr Gly Lys Leu Ile Trp Lys Val Asn Pro Glu
260 265 270
Ile Asp Thr Thr Ile Gly Glu Trp Ala Phe Trp Glu Thr Lys Lys Asn
275 280 285
Leu Thr Arg Lys Ile Arg Ser Glu Glu Leu Ser Phe Thr Val Val Ser
290 295 300
Asn Gly Ala Lys Asn Ile Ser Gly Gln Ser Pro Ala Arg Thr Ser Ser
305 310 315 320
Asp Pro Gly Thr Asn Thr Thr Thr Glu Asp His Lys Ile Met Ala Ser
325 330 335
Glu Asn Ser Ser Ala Met Val Gln Val His Ser Gln Gly Arg Glu Ala
340 345 350
Ala Val Ser His Leu Thr Thr Leu Ala Thr Ile Ser Thr Ser Pro Gln
355 360 365
Ser Leu Thr Thr Lys Pro Gly Pro Asp Asn Ser Thr His Asn Thr Pro
370 375 380
Val Tyr Lys Leu Asp Ile Ser Glu Ala Thr Gln Val Glu Gln His His
385 390 395 400
Arg Arg Thr Asp Asn Asp Ser Thr Ala Ser Asp Thr Pro Ser Ala Thr
405 410 415
Thr Ala Ala Gly Pro Pro Lys Ala Glu Asn Thr Asn Thr Ser Lys Ser
420 425 430
Thr Asp Phe Leu Asp Pro Ala Thr Thr Thr Ser Pro Gln Asn His Ser
435 440 445
Glu Thr Ala Gly Asn Asn Asn Thr His His Gln Asp Thr Gly Glu Glu
450 455 460
Ser Ala Ser Ser Gly Lys Leu Gly Leu Ile Thr Asn Thr Ile Ala Gly
465 470 475 480
Val Ala Gly Leu Ile Thr Gly Gly Arg Arg Thr Arg Arg Glu Ala Ile
485 490 495
Val Asn Ala Gln Pro Lys Cys Asn Pro Asn Leu His Tyr Trp Thr Thr
500 505 510
Gln Asp Glu Gly Ala Ala Ile Gly Leu Ala Trp Ile Pro Tyr Phe Gly
515 520 525
Pro Ala Ala Glu Gly Ile Tyr Ile Glu Gly Leu Met His Asn Gln Asp
530 535 540
Gly Leu Ile Cys Gly Leu Arg Gln Leu Ala Asn Glu Thr Thr Gln Ala
545 550 555 560
Leu Gln Leu Phe Leu Arg Ala Thr Thr Glu Leu Arg Thr Phe Ser Ile
565 570 575
Leu Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln Arg Trp Gly Gly Thr
580 585 590
Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu Pro His Asp Trp Thr
595 600 605
Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp Phe Val Asp
610 615 620
Lys Thr Leu Pro Asp Gln Gly Asp Asn Asp Asn Trp Trp Thr Gly Trp
625 630 635 640
Arg Gln Trp Ile Pro Ala Gly Ile Gly Val Thr Gly Val Ile Ile Ala
645 650 655
Val Ile Ala Leu Phe Cys Ile Cys Lys Phe Val Phe
660 665

Claims (10)

1. viral recombinant glycoprotein, which is characterized in that it is the signal peptide substitution target viral with human or animal's secreted protein The signal peptide of glycoprotein and the recombinant glycoprotein formed.
2. recombinant glycoprotein according to claim 1, which is characterized in that the signal peptide of human or animal's secreted protein Signal peptide from human antibody IgG heavy chain and/or human albumin.
3. recombinant glycoprotein according to claim 1, which is characterized in that the target viral glycoprotein includes rabies viruses G-protein, Ebola virus GP1 albumen.
4. recombinant glycoprotein according to claim 1-3, which is characterized in that it is with human IgG ferritin heavy chain The signal peptide of signal peptide substitution rabies virus G protein and the recombinant glycoprotein that is formed, the amino acid sequence of the recombinant glycoprotein As shown in SEQ ID NO:1；Or
It is the recombinant glycoprotein for substituting the signal peptide of Ebola virus GP1 albumen with the signal peptide of human albumin and being formed, institute The amino acid sequence of recombinant glycoprotein is stated as shown in SEQ ID NO:3.
5. encoding the nucleic acid of any one of the claim 1-4 recombinant glycoprotein.
6. the biomaterial containing nucleic acid described in claim 5, the biomaterial be expression cassette, expression vector, cloning vector, Engineering bacteria or host cell.
7. utilizing the method for any one of the eukaryocyte high efficient expression claim 1-4 recombinant glycoprotein, which is characterized in that packet Include following steps:
1) it is recombinated by codon corresponding relationship and host expresses frequency using PCR method, DNA according to known protein sequence Method or artificial synthesized method obtain the nucleic acid of simultaneously any one of Optimized Coding Based claim 1-4 recombinant glycoprotein；
2) nucleic acid clone is entered into carrier for expression of eukaryon, then converted or transfection host cell, with the proliferation of host cell, to realize Expression of the viral recombinant glycoprotein in eukaryocyte, and target protein is collected from cell culture.
8. according to the method described in claim 5, it is characterized in that, the carrier for expression of eukaryon includes pcDNA3, the host Cell includes 293 cells, CHO.
9. monoclonal antibody or polyclonal antibody based on any one of the claim 1-4 recombinant glycoprotein exploitation.
10. following any application of any one of the claim 1-4 recombinant glycoprotein:
1) application in vaccine and antiviral drugs is being prepared；
2) application in target viral antibody test reagent or target viral infection diagnostic reagent is being prepared；
3) application in colloidal gold colloidal gold detection test paper strip is being prepared.
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