CN102212139A - Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof - Google Patents
Fusion protein of tick-borne encephalitis virus envelop E protein and human antibody Fc fragment, and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biomedical engineering, a tick-borne encephalitis virus is a neurotropic virus with extremely strong toxicity, and the tick-borne encephalitis caused by the tick-borne encephalitis virus is an acute infectious disease of a central nervous system. At present, both the crude vaccines and purified vaccines for the tick-borne encephalitis are inactivated vaccines, and have the problems of complicated antigen components, low content of effective antigen, multiple side actions and the like. Envelop E protein is an important structural protein of the tick-borne encephalitis virus, and a neutralizing antibody induced by the envelop E protein is a main immunoprotection mechanism of an organism against the tick-borne encephalitis virus. The invention utilizes a molecular cloning method to construct a fusion protein of the tick-borne encephalitis virus envelop E protein and human IgG1 (immune gamma globulins 1) Fc fragment, and then carries out transfection on CHO (Chinese hamster ovary) cells, thus expressing a recombinant fusion protein. The invention proves that the fusion with human IgG1 Fc fragment can obviously improve the expression level of the E protein and facilitate the purification of protein, and can obviously improve the immunogenicity of the E protein and enhance the immune response of E antibody and cells induced by the E protein. The fusion protein provided by the invention has a good application prospect in being used as an antigen for the tick-borne encephalitis vaccines.
Description
Technical field
The present invention relates to bio-pharmaceutical engineer technology domain, be specifically related to a kind of fores encephalitis virus envelope E protein and people's antibody I g G1 Fc section fusion rotein, and the application in the preparation Teck-borne Encephalitis Vaccine.
Background technology
Fores encephalitis virus also claims tick-brone encephalitis virus (Tick-borne encephalitis virus, TBEV), be the extremely strong neurotropic virus of a kind of virulence, mainly invade people's central nervous system, the forest encephalitis that causes is the acute central nervous system transmissible disease, and case fatality rate and disability rate are quite high.Fores encephalitis virus is one of important virus biological agent, and the American National Center for Disease Control is C viroid class biological weapon with this virus taxis.China, forest encephalitis is the legal occupational illness that is caused by biotic factor.Teck-borne Encephalitis Vaccine is one of national emergency stock vaccine.
Fores encephalitis virus has three hypotypes at least, and promptly West Europe hypotype, Far East hypotype and siberian hypotype mainly are distributed in states such as China, Russia, Czech, Bulgaria, Poland and Austria.China mainly is distributed in Changbai Mountain, Daxing'an Mountainrange, the Xiaoxinanlin Mountains, Tianshan Mountains, six natural focuss in Altai Mountains and cross-section mountain in northeast, northwest, three big epidemic-stricken areas, southwest, and wherein the epidemic-stricken area, northeast is the most serious.Epidemic situation was spread to grassland, hills, farming region by the forest zone in recent years, in addition along with forest zone expanding economy and forest trip, exploring tour, the rise of field survivorship trip and the change of forest zone ecotope, the crowd who enters the forest zone increases year by year, cause the popular scope of forest encephalitis to rise year by year, to the also increase year by year of needs of vaccine inoculation.
Rough vaccine of present forest encephalitis and purified vaccine all belong to inactivated vaccine, the antigenic component complexity, and effective antigenic content is lower, and side effect is more, relates to the Biosafety problem in the preparation process, and there is technical difficulty in mass preparation.
The fores encephalitis virus envelope E protein is the important structure albumen of fores encephalitis virus, the biological function that mediation virus and receptors bind and film merge, this protein induced neutralizing antibody is that the main immunoprotection mechanism of the anti-fores encephalitis virus of body (is taken charge of bright silver, Zhu Qingyu, the protein E of tick borne encephalitis virus progress of research, institute of Military Medical Science Institute periodical, 1998; 22 (4): 305-308).Exploitation is that the recombiant vaccine of target antigen is feasible in theory based on envelope E protein, is the developing direction of Teck-borne Encephalitis Vaccine of new generation.
Still not having based on envelope E protein at present is the report of the recombiant vaccine of target antigen.
Summary of the invention
The purpose of this invention is to provide a kind of fores encephalitis virus envelope E protein and people's antibody I g G1 Fc section fusion rotein, and the application of described albumen in the preparation Teck-borne Encephalitis Vaccine.
The fores encephalitis virus envelope E protein is one of primary structure albumen of fores encephalitis virus; be positioned at the surface of virosome; can induce protection antibody; the envelope E protein immune animal can watch for animals and avoid the attack of virus, so the contriver thinks: this albumen can be used as the important antigen of Teck-borne Encephalitis Vaccine in theory.In order to make this albumen energy high level expression, the present invention optimizes the proteic encoding gene of E, uses the synthetic E gene of Mammals preference codon, can improve the proteic translation efficiency of E, thereby improve its expression level.
Antibody is the sphaeroprotein that, excretory one class synthetic by the B cell of differentiation and maturation can have immunologic function with corresponding antigens specificity bonded, promptly immunoglobulin (Ig) (immunoglobulin, Ig).Immunoglobulin (Ig) can be divided into five classes, i.e. IgG, IgA, IgM, IgD and IgE.Wherein IgG is topmost immunoglobulin (Ig), accounts for 70% of human plasma gamma-globulin.Immunoglobulin (Ig) has the symmetrical structure of 4 polypeptide chains, wherein 2 identical heavy chains (H chain) long, that relative molecular weight is bigger; Article 2, short, the less identical light chain (L chain) of relative molecular weight.Connect by disulfide linkage and non covalent bond between heavy chain and light chain and form a monomer molecule that constitutes by 4 polypeptide chains.Immunoglobulin molecules can be produced different fragments by many protease hydrolysiss.Papoid becomes two identical Fab sections and a Fc section with the IgG molecular degradation.The Fab section is Fab (antigen binding fragment), still has antigen-binding activity.The Fc section is FC (crystallizable fragment), can form crystallization under certain condition, the Fc section can not combine with antigen, but have many other biologicals learn active, as complement-fixing, affine cell (scavenger cell, NK cell and granulocyte etc.), mediation and combining of bacterioprotein (as staphylococcus aureus protein A and streptococcus protein G) etc.Several genes engineering medicine can improve proteic expression and stability with the carboxyl terminal of antibody Fc section fusion at activated protein, and convenient proteic purifying.
The invention provides a kind of fusion rotein, this fusion rotein is the Fc section that has merged people's IgG antibody 1 at the carboxyl terminal of fores encephalitis virus envelope E protein, wherein encode the gene order of fores encephalitis virus envelope E protein shown in SEQ ID NO:1, and the gene order of the Fc section of coding people IgG antibody 1 is shown in SEQ ID NO:2.
Fusion rotein provided by the invention, the gene order of this fusion rotein of encoding is shown in SEQ ID NO:3.
In addition, the contriver thinks, because E albumen is glycosylated protein, can be modified by host cell processing and form complicated space conformation.The proteic natural space conformation of E is for the mediation virus infection and induce neutralizing antibody that material impact is all arranged.Selecting proper host cell to express E albumen, make it to have natural glycosylation modified natural with formation functional space conformation, is as the antigenic essential condition of effective vaccine.Therefore, the contriver thinks: with mammalian cell expression E albumen is desirable selection.Chinese hamster ovary celI is a Mammals engineering cell commonly used at present, and expression product can be modified, is processed to form natural conformation and had natural biological learns function and activity, is the internationally recognized cell strain that the human genetically engineered drug is produced that can be used for.Compare with intestinal bacteria, the aggregate level of expressing cho cell foreign protein is lower, but except the antibody.The antibody that gives expression to the gram level in present every liter of Chinese hamster ovary celI culture system has not been problem technically.
The present invention's molecular cloning method, utilize Mammals preference codon to synthesize China gloomy strain fores encephalitis virus coating E gene, E gene expression plasmid and E gene and people's IgG antibody 1Fc section fusion gene expression plasmid have been made up then respectively, transfection Chinese hamster ovary cell (Chinese hamster ovary celI), successfully construct stably excreting and express the cell strain of E albumen or E-Fc fusion rotein, method purifying protein with affinity chromatography, be adjuvant with ISA720 then, immune mouse and rabbit, detect the E antibody in mouse and the rabbit anteserum, and the analysis mouse boosting cell stimulates secreted cytokine external for E albumen.
Fusion rotein provided by the invention obtains by the following method: at first make up and contain just like the expression of gene plasmid shown in the SEQ IDNO:3, transfection Chinese hamster ovary cell then, this fusion rotein of secreting, expressing, and separation and purification.
The concrete technical scheme of the present invention is as follows:
1. gloomy strain fores encephalitis virus of China coating E gene extracellular fragment is synthetic: according to the aminoacid sequence of China's gloomy strain E of tick borne encephalitis virus genes encoding, the codon that utilizes mammalian cell to have a preference for is designed the E gene order of optimization, synthetic then this gene (shown in SEQ ID NO:1) that obtains.
2. the clone of people's IgG antibody 1 Fc fragment gene: separate the B cell from human peripheral, the extracting cell total rna uses reverse transcription (RT)-polymerase chain reaction (PCR) clone to obtain human IgG1 Fc fragment gene (shown in SEQ ID NO:2) then.
3. the structure of fores encephalitis virus coating E and human IgG1 Fc section fusion gene: the method for cutting, being connected with enzyme is spliced into one section fusion gene (shown in SEQ IDNO:3) with fores encephalitis virus coating E gene and human IgG1 Fc section.E gene carboxyl terminal is the BamHI restriction enzyme site, and IgG1 Fc section aminoterminal also is the BamHI restriction enzyme site, and there are 6 Nucleotide GGATCC in this site, two fragment genes is merged latter two BamHI restriction enzyme site become one.
4. the structure of E of tick borne encephalitis virus gene and E-IgG1 Fc fusion gene expression plasmid: respectively E gene and E-IgG1 Fc fusion gene are inserted the expressing cho cell plasmid pCIDA-GS-neo with independent intellectual property right and (see the Chinese patent ZL 200610024202.5 that the applicant has authorized, denomination of invention is: a kind of method with mammal cell with high efficient secretion expression hepatitis C virus envelope protein E 2, invention is artificial: Zhao Ping, Liao Xiaoling, Cao Jie, Qi Zhongtian.), obtain E albumen and E-Fc fusion protein expression plasmid.
5. structure and the cell cultures and the protein purification of the Chinese hamster ovary celI strain of stably express E albumen and E-IgG1 Fc fusion rotein: respectively with above-mentioned two kinds of expression plasmid transfection CHO-K1 cells, detect E albumen in the cells and supernatant with western blotting method with liposome.Screen the CHO-K1 cell clone that obtains stable transfection with methionine sulfoxide (MSX), carry out serum-free culture then.The collecting cell culture supernatant, centrifugal, nickel post affinity purification E albumen is used in ultrafiltration then, with staphylococcus aureus protein A resin affinity purification E-IgG1 Fc fusion rotein.
6. immune mouse and rabbit: respectively with E albumen and E-IgG1 Fc fusion rotein combined immunization adjuvant ISA720, immune mouse and rabbit, immunity three times.
7. mouse and rabbit anteserum antibody test: with the E antibody in enzyme linked immunosorbent assay (ELISA) detection mouse and the rabbit immune serum.
8. detect mouse boosting cell external to the proteic specific reaction of E: put to death mouse, the aseptic mouse spleen of getting, the single splenocyte of separation and Culture stimulates with recombinant expressed E albumen, detects mouse boosting cell excretory cytokine IFN-γ, IL-2 and IL-12 with ELISA.
The result shows: E of tick borne encephalitis virus gene and human IgG1 Fc fragment gene merge can significantly improve the proteic expression level of E, and fusion rotein inductive E antibody horizontal is significantly higher than the protein induced E antibody horizontal of E.Fusion rotein can carry out the high-performance affinity chromatography purifying with staphylococcal protein A or streptococcal protein G link coupled resin.This fusion rotein has application prospect as the Teck-borne Encephalitis Vaccine target antigen.
The present invention has proved that human IgG1 Fc section and fores encephalitis virus envelope E protein merge the method purifying E albumen that not only helps with affinity chromatography, can also improve the proteic expression level of E, and the proteic immunogenicity of enhancing E, significantly improve protein induced antibody of E and cellullar immunologic response, for exploitation recombinant subunit Teck-borne Encephalitis Vaccine provides a kind of effective candidate antigens.
Description of drawings
Fig. 1 is the structure of plasmid pCIDA-E-Fc
Wherein: PCMV is a cytomegalovirus CMV promotor, and introD is the intron donor sequences, and GS is the glutaminase synthase gene, introA is the intron receptor sequence, E is a forest encephalitis coating E gene, and Fc is people's IgG antibody 1 Fc fragment gene, and polyA is a SV40 virus polyadenylic acid sequence.Fig. 2 is the structure of plasmid pCIDA-E
Wherein: PCMV is a cytomegalovirus CMV promotor, and introD is the intron donor sequences, and GS is the glutaminase synthase gene, and introA is the intron receptor sequence, and E is a forest encephalitis coating E gene, and polyA is a SV40 virus polyadenylic acid sequence.
Fig. 3 is that the proteic immunoblotting of E albumen and E-Fc detects
With the E albumen in the Chinese hamster ovary celI culture supernatant of immunoblotting detection plasmid pCIDA-E, pCIDA-E-Fc transfection, the Chinese hamster ovary celI culture supernatant of usefulness untransfected plasmid in contrast.
Fig. 4 is the proteic polyacrylamide gel electrophoresis analysis of reduced form E and E-Fc.
Fig. 5 is the proteic polyacrylamide gel electrophoresis analysis of reduced form E-Fc albumen and oxidized form E-Fc.
Fig. 6 is an E detection of antibodies in the mice serum
Respectively with E, E-Fc and bovine serum albumin associating ISA720 adjuvant immunity mouse, in 0,2,4 each immunity of week once, detect E antibody (serum dilution in 1: 200, the mean value of 10 mice serum detected values) in the mice serum with ELISA in different time points.
Fig. 7 is an E detection of antibodies in the rabbit anteserum
Respectively with E, E-Fc and bovine serum albumin associating ISA720 adjuvant immunity rabbit, in 0,2,4 each immunity of week once, detect E antibody (serum dilution in 1: 200, the mean value of 10 rabbit anteserum detected values) in the rabbit anteserum with ELISA in different time points.
Fig. 8 is that ELISA detects mouse boosting cell at the secreted cytokine of E albumen stimulation
For the third time 2 weeks of immunity back, put to death mouse, the separation and Culture splenocyte stimulates with E albumen, detects cytokine IL-2, IL-12 and IFN-γ (mean values of 5 mouse boosting cell detected values) in the cells and supernatant with ELISA after three days.
Embodiment
Below in conjunction with embodiments of the invention and accompanying drawing enforcement of the present invention is elaborated; following examples are to be to implement under the prerequisite with the technical solution of the present invention; provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Synthesizing of embodiment 1. China gloomy strain fores encephalitis virus coating E gene:
Aminoacid sequence (GenbankAccession:AY182009) according to China's gloomy strain E of tick borne encephalitis virus coded by said gene, the codon that utilizes mammalian cell to have a preference for is designed the E gene order (shown in SEQ ID NO:1) of optimization, its 5 ' end contains NheI restriction enzyme site GCTAGC and initiator codon ATG, 3 ' end contains BamHI restriction enzyme site GGATCC, entrust Shanghai JaRa Bioisystech Co., Ltd to synthesize this gene with conventional overlap extension pcr, this gene is inserted cloning vector T carrier (U.S. Promega company product), called after pGEM-E.
The clone of embodiment 2. people's IgG antibody 1 Fc fragment gene:
Separate the B cell with the magnetic bead of CD19 monoclonal antibody mark (German Miltenyibiotec company product) from 20ml Freshman blood plasma, operation is consulted and used explanation and is carried out.With Trizol Reagent (American I nvitrogen product) extracting cell total rna, operation is consulted and used explanation and is carried out.The cell total rna that obtains is dissolved in the sterilization distilled water that DEPC handles.Use reverse transcription reaction (RT)-polymerase chain reaction (PCR) amplification human IgG1 Fc fragment gene then.Agents useful for same is a U.S. Promega company product.Get RNA2 μ l (about 5 μ g), place in one 0.5 milliliters of centrifuge tubes, add following each component again: 5 μ l AMV reverse transcription reaction damping fluids, 1 μ l dNTP (concentration 10mM), 1 μ l oligo (dT) 15 (concentration 5pmol/ μ l), in 70 ℃ of hot water baths 5 minutes, place ice-water bath more rapidly, add following component: 2 μ l RNA enzyme inhibitorss (30U/ μ l), 1 μ l AMV reversed transcriptive enzyme (5U/ μ l), the sterilization redistilled water that adds the DEPC processing is to final volume 25 μ l.Mixing, in 42 ℃ of water-baths 40 minutes, 95 ℃ of 5 minutes deactivation reversed transcriptive enzymes were again got 5 μ l reverse transcription products immediately and are placed in one 50 μ l centrifuge tube, add then primers F cF (5 '-
GGATCCGAACCCAAATCTTGTGACA-3 ', underscore are the BamHI restriction enzyme site) and FcR (5 '-
GTCGACAACTCATTTACCCGGAGACAGGGAGAG-3 ', underscore is the SalI restriction enzyme site) each 0.1 μ l, and 5 μ l Pfu polymeric enzyme reaction damping fluid, 1 μ l dNTP (concentration 10mM) and 1 μ l Pfu polysaccharase (3U/ μ l), replenish two aqua sterilisas that steam to total reaction volume 50 μ l, carry out thermal cycle reaction with the PCR instrument, totally 30 circulations, add Taq enzyme 1U then, 72 ℃ 10 minutes, temperature is reduced to 4 ℃ of end reactions then, reaction product is carried out agarose gel electrophoresis, and the purpose band is reclaimed, and is connected with PCR product cloning carrier T carrier (U.S. Promega company product), connecting the clone who obtains entrusts Shanghai JaRa Bioisystech Co., Ltd to carry out dna sequencing evaluation, the correct clone's called after pGEM-Fc that obtains.The Fc gene order is seen shown in SEQ ID NO:2.
The structure of embodiment 3. fores encephalitis virus coating E and human IgG1 Fc section fusion gene expression plasmid:
With NheI and BamHI digested plasmid pGEM-E, agarose gel electrophoresis reclaims the E gene fragment of 1400 base pairs.With BamHI and SalI digested plasmid pGEM-Fc, agarose gel electrophoresis reclaims the Fc gene fragment of 700 base pairs.(construction process is referring to the Chinese patent ZL 200610024202.5 that sees that the applicant has authorized to cut the expressing cho cell carrier pCIDA-GS-neo with independent intellectual property right that the inventor makes up with NheI and SalI enzyme, denomination of invention is: a kind of method with mammal cell with high efficient secretion expression hepatitis C virus envelope protein E 2, invention is artificial: Zhao Ping, Liao Xiaoling, Cao Jie, Qi Zhongtian.), agarose gel electrophoresis reclaims linearized vector, the linearized vector and the E gene that reclaim are connected with the Fc gene, the plasmid that obtains is cut evaluation with NheI and SalI enzyme earlier, entrust Shanghai JaRa Bioisystech Co., Ltd to carry out dna sequencing then and identify the plasmid called after pCIDA-E-Fc that sequence is correct.
The E-Fc fusion sequence is seen shown in SEQ ID NO:3.Plasmid pCIDA-E-Fc structure is as shown in Figure 1: PCMV is a cytomegalovirus CMV promotor, introD is the intron donor sequences, GS is the glutaminase synthase gene, introA is the intron receptor sequence, E is a forest encephalitis coating E gene, Fc human IgG1 Fc fragment gene, polyA are SV40 virus polyadenylic acid sequence.
The structure of embodiment 4. E of tick borne encephalitis virus gene expression plasmids:
With plasmid pGEM-E is template, pcr amplification E gene, contain in the upstream primer NheI restriction enzyme site (5 '-
GCTAGCCGCCACCATGGAGAGCGTGGTGACCCGCGTG-3 ', underscore are the NheI restriction enzyme site), add encoding sequence and the terminator codon and the SalI restriction enzyme site of 6 Histidines in the downstream primer, sequence is 5 '-
GTCGACAACTCA
GTGGTGGTGGTGGTGGTGGAAGGCGCCG CCCAGCACGG-3 ', underscore are the encoding sequence of SalI restriction enzyme site and 6 Histidines), PCR reaction system and thermal cycle conditions are with reference to described in the embodiment 2.The PCR product is connected with cloning vector T carrier, connects the clone who obtains and entrusts Shanghai JaRa Bioisystech Co., Ltd to carry out the dna sequencing evaluation, obtains right-on clone, called after pGEM-E6H.Cut pGEM-E6H with NheI and SalI enzyme, cut out the E gene and insert expressing cho cell carrier pCIDA-GS-neo, with the enzyme evaluation plasmid of cutting, check order, called after pCIDA-E, structure as shown in Figure 2: PCMV is a cytomegalovirus CMV promotor, and introD is the intron donor sequences, GS is the glutaminase synthase gene, introA is the intron receptor sequence, and E is a forest encephalitis coating E gene, and polyA is a SV40 virus polyadenylic acid sequence.
Embodiment 5: the structure and the protein purification of the Chinese hamster ovary celI strain of stably express E albumen and E-Fc fusion rotein:
Go down to posterity with the DMEM nutrient solution that contains 10% foetal calf serum and to cultivate the CHO-K1 cell, with 0.25% trypsin digestion and cell, be inoculated in the 35mm Tissue Culture Dish, next day is respectively with 4 μ g pCIDA-E plasmids and pCIDA-E-Fc plasmid and 10 μ l lipofectamine Lipofectamine (American I nvitrogen product) mixings, transfection CHO-K1 cell, operation is carried out according to operation instruction.Add the DMEM nutrient solution that 1ml contains 10% foetal calf serum after 8 hours, continue to cultivate 48 hours.With the E albumen in the immunoblotting detection culture supernatant, operation is carried out with reference to " the molecular cloning experiment guide third edition " (Science Press, 2002).Culture supernatant is mixed with the sample-loading buffer that contains β mercaptoethanol (destroying the disulfide linkage that is formed by two halfcystines), boil, carry out polyacrylamide gel electrophoresis then, then with the albumen electrotransfer in the gel to nylon membrane, with the phosphate buffered saline buffer room temperature reaction that contains 10% skim-milk 1 hour, ((Changchun Biological Products Institute produces, and lot number is: 20070901-2 with forest encephalitis purifying inactivated vaccine with protein E of tick borne encephalitis virus antibody then.) immunizing rabbit, immunity three times, use the affinitive layer purification rabbit igg then, with the phosphate buffer 1 that contains 10% skim-milk: 1000 dilutions) room temperature reaction is 1 hour, then with anti-rabbit igg (the U.S. Sigma company product) room temperature reaction of horseradish peroxidase-labeled 1 hour, with chemoluminescence method detection reaction band, operation is carried out with reference to " the molecular cloning experiment guide third edition " (Science Press, 2002).
The result all can detect E albumen in shown in Figure 3 in the Chinese hamster ovary celI culture supernatant of two kinds of plasmid transfections, and the E molecular weight of albumen is between 50kDa and 70kDa, and the E-Fc molecular weight of albumen is between 70kDa and 100kDa, and is consistent with expection.The E protein content of E protein content in the cell conditioned medium of pCIDA-E-Fc plasmid transfection in the cell conditioned medium of pCIDA-E plasmid transfection.
With the Chinese hamster ovary celI of transfection with 0.25% tryptic digestion, be inoculated in 5 100mm Tissue Culture Dishs, GMEM-S nutrient solution (U.S. Sigma company product) with the foetal calf serum that contains 10% dialysis is cultivated, add methionine sulfoxide (MSX) (U.S. Sigma company product) to final concentration 25 μ mol/L, cell begins death after 3-4 days, the clone that the back culture dish formation of two weeks is formed by individual cells propagation, choose single clone and be inoculated in 96 orifice plates, continue to cultivate with the GMEM-S nutrient solution that contains 25 μ mol/L MSX, culture supernatant detects the proteic expression of E2 with immune marking method after one week, to the highest clone of expression level with tryptic digestion, limiting dilution is inoculated 96 orifice plates, continue to cultivate with the GMEM-S nutrient solution of 25 μ mol/L MSX, after two weeks, there is monoclonal hole inner cell to be transferred to 24 orifice plates length, continue to cultivate, cell is paved with orifice plate and is transferred to amplification culture in the little square vase later on, use serum free medium (U.S. Sigma company product) to substitute the GMEM-S substratum gradually subsequently, make cell adapt to complete serum free medium gradually, change suspension growth into from adherent growth.Use suspension cell instead shake-flask culture, volume of culture increases gradually, when increasing to 100 milliliters, volume is transferred in the WAVE bio-reactor (U.S. GE company product) to cultivate, and amplification culture volume to 4 liter gradually.The harvested cell culture supernatant, centrifugal removal cell, concentrate with ultrafiltration again, close the method purifying E albumen of chromatography subsequently with the nickel ion metal-chelate, method purifying E-Fc albumen with the staphylococcal protein A affinity chromatography, can obtain 35 milligrams in E albumen in each hundred milliliters of nutrient solution, 0 milligram of E-Fc protein 21.Fig. 4 is the polyacrylamide gel electrophoresis analytical results of reduced form E and E-Fc albumen (contain the β mercaptoethanol in the sample-loading buffer, destroy disulfide linkage).Thereby two Fc monomers can form intermolecular disulfide bond by halfcystine and form dimer, oxidized form (does not contain the β mercaptoethanol in the sample-loading buffer, do not destroy disulfide linkage) and reduced form (contain the β mercaptoethanol in the sample-loading buffer, the destruction disulfide linkage) polyacrylamide gel electrophoresis analysis revealed E-Fc albumen is dimer, consistent with expection, as shown in Figure 5.
Embodiment 6: use E and E-Fc protein immunization mouse and rabbit:
Respectively recombinant expressed E and E-Fc albumen are adjusted concentration to 0.25mg/ml with phosphate buffered saline buffer, with the abundant mixing of equal-volume oil/water and milk formulation adjuvant ISA720 (French Seppic company product), room temperature was placed 30 minutes, then in female BALB/c mouse of subcutaneous multi-point injection immunity (body weight 20 grams) and male rabbit (2.5 kilograms of body weight), every each immunizing dose of mouse is amounted to E or E-Fc albumen 5 μ g, every each immunizing dose of rabbit is amounted to E or E-Fc protein 20 μ g, two weeks, once immunity was three times altogether.With same dosage bovine serum albumin (U.S. Sigma company product) associating ISA720 adjuvant immunity mouse and rabbit in contrast.15 of every group of mouse, 10 of every group of rabbit.
Antibody test in embodiment 7. mouse and the rabbit anteserum:
Each immune the day before yesterday, (time was respectively for 0,2,4 weeks, blood sampling time was decided to be for 0 week first) and the last immunity after every two weeks (totally 7 times, time was respectively for 6,8,10,12,14,16,18 weeks) get blood from mouse orbit and rabbit auricular vein, with the E antibody in enzyme-linked immunosorbent assay (ELISA) detection mouse and the rabbit anteserum.
E antibody test: the E albumen of purifying is diluted to concentration 0.05mg/ml with phosphoric acid salt, add enzyme mark microwell plate, every hole 0.1ml, place 4 ℃ of refrigerator overnight, absorb the E protein solution next day, every hole is washed once with the 0.2ml phosphate buffered saline buffer, every then hole adds phosphate buffered saline buffer (confining liquid) 0.1ml that contains 3% bovine serum albumin bletilla 0.05%Tween 20, room temperature was placed 2 hours, absorb confining liquid then, every hole adds mouse or the rabbit anteserum 0.1ml with the confining liquid dilution, room temperature was placed 40 minutes, absorb serum then, with the phosphate buffered saline buffer that contains 0.05%Tween 20 (washing lotion) hole flushing 5 times, every then hole adds goat-anti mouse or goat anti-rabbit igg (the U.S. Sigma company product) 0.1ml with the HRP mark of confining liquid dilution, room temperature was placed 40 minutes, absorb antibody then, with washing lotion hole flushing 5 times, every hole adds the substrate solution 0.1ml that contains TMB, the room temperature lucifuge was placed after 10 minutes, added 2M sulfuric acid and each 0.05ml of 30% superoxol, mixing, with the absorbance value of microplate reader detection 450nm wavelength, reference wavelength 630nm.Under the identical extent of dilution, it is antibody positive that E and E-Fc protein immunization serum absorbance value surpass 2.1 times of BSA immune serum average light absorption value.
Result: in two weeks of back of immunity for the first time, the positive rate of E antibody in E and the E-Fc mice immunized serum (positive rate when serum dilutes with minimum extent of dilution at 1: 50) is respectively 70% and 100%, and the positive rate of E antibody is respectively 80% and 100% in the rabbit anteserum of E and E-Fc immunity.To the 18th week, the E antibody positive rate is 100% in E and E-Fc mice immunized and the rabbit anteserum subsequently.According to ELISA detected result (Fig. 6,7), the mouse of E-Fc protein immunization and the E antibody antibody horizontal in the rabbit anteserum all are higher than the mouse and the rabbit of E protein immunization, show that IgG1 Fc section can significantly improve the protein induced antibody response of E.
Two weeks of back of immunity for the third time, E albumen and E-Fc mice immunized are respectively got 5, mouse is put to death in the cervical vertebra dislocation, and the aseptic mouse spleen of getting grinds on nylon wire, using lymphocyte separation medium (it is company's product that Shenzhen reaches section) then is medium, isolate single lymphocyte with gradient centrifugation, be suspended in the RPMI RPMI-1640 that contains 15% foetal calf serum, be inoculated in 24 orifice plates and cultivate, every hole nutrient solution 1ml contains 10
5Cell.Add recombinant expressed E albumen simultaneously, concentration 20 μ g/ml.In 37 ℃ of CO
2Incubator is cultivated, and gets supernatant after three days, detects cytokine IL-2, IL-12 and IFN-γ in the supernatant with ELISA, and detection reagent is that to reach section be company's product in Shenzhen.
Result: as shown in Figure 8, the secreted cytokine of mouse boosting cell that these three kinds of cytokine levels of the mouse boosting cell excretory of E-Fc fusion protein immunization all are significantly higher than the E protein immunization shows with the fusion of human IgG1 Fc section to strengthen the protein induced cellullar immunologic response of E.
Claims (4)
1. fusion rotein, it is characterized in that this fusion rotein is the Fc section that has merged people's IgG antibody 1 at the carboxyl terminal of fores encephalitis virus envelope E protein, wherein encode the gene order of fores encephalitis virus envelope E protein shown in SEQ ID NO:1, and the gene order of the Fc section of coding people IgG antibody 1 such as SEQ ID NO:2 institute are not.
2. fusion rotein, the gene order of this fusion rotein that it is characterized in that encoding is shown in SEQ ID NO:3.
3. fusion rotein, it is characterized in that this fusion rotein obtains by the following method: at first structure contains just like the expression of gene plasmid shown in the SEQ ID NO:3, transfection Chinese hamster ovary cell then, this fusion rotein of secreting, expressing, and separation and purification.
4. according to claim 1, the application of 2 or 3 described fusion roteins in the preparation Teck-borne Encephalitis Vaccine.
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| CN104330556A (en) * | 2014-10-22 | 2015-02-04 | 中国检验检疫科学研究院 | Detection test paper of forest encephalitis antibody, test paper preparation method and detection kit of detection test paper |
| CN106519041A (en) * | 2016-11-24 | 2017-03-22 | 唐山怡安生物工程有限公司 | Establishing method of pig immunoglobulin Fc fragment-swine classical fever E2 fusion protein in CHO cell strain, as well as preparation method and application of fusion protein |
| CN109705222A (en) * | 2018-12-27 | 2019-05-03 | 中国科学院武汉病毒研究所 | The fusion protein and the preparation method and application thereof of flaviviridae envelope protein |
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| CN104330556A (en) * | 2014-10-22 | 2015-02-04 | 中国检验检疫科学研究院 | Detection test paper of forest encephalitis antibody, test paper preparation method and detection kit of detection test paper |
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| CN106519041B (en) * | 2016-11-24 | 2019-07-05 | 唐山怡安生物工程有限公司 | The construction method and its preparation method and application of pig immune globulin Fc segment and swine fever E2 fusion protein in Chinese hamster ovary celI strain |
| CN109705222A (en) * | 2018-12-27 | 2019-05-03 | 中国科学院武汉病毒研究所 | The fusion protein and the preparation method and application thereof of flaviviridae envelope protein |
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