CN102260351A - Preparation of influenza virus M2e protein and application of influenza virus M2e protein in resisting influenza virus A - Google Patents
Preparation of influenza virus M2e protein and application of influenza virus M2e protein in resisting influenza virus A Download PDFInfo
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Abstract
The invention provides fusion protein combined by human serum albumin (HSA) and influenza virus M2e protein, a preparation method of the protein, and an application of the protein in studies of resisting influenza virus A.
Description
Invention field
The present invention relates to fused protein and preparation thereof, particularly relate to the fusion rotein that human serum albumin (HSA) and influenza m 2 e protein binding form, and preparation method thereof and the application in anti-influenza A virus research.
Background of invention
M2 albumen is one of main membranin of influenza virus.Since being separated to the first strain influenza virus A hominis strain A/WS/33 (H1N1) in 1933, although experienced worldwide several times being very popular, (Extracellular domainof M2 protein M2e) does not find to exist notable difference to the M2 protein extracellular.Discover, to have and in cell cultures, suppress the function that influenza virus is duplicated at the monoclonal antibody of M2 albumen born of the same parents ectodomain.The more important thing is that the antibody that M2e albumen induces can produce protective immunity.Because all influenza A viruss of popular in the crowd at present, the proteic extracellular region M2e of its M2 are all found to have notable difference, think that M2e is high conservative between strains of influenza viruses.Therefore, M2e albumen is considered to stimulate body can both produce the protective antigen with cross protection effect to different influenza variants.
Human serum albumin is an albumen the abundantest in the human plasma, accounts for about 60% of blood plasma total protein.Except in blood plasma, containing the HSA, in the juice of tissue, skin, geode and health, also contain HSA.HSA also is an important function albumen in the blood, has the blood of keeping oncotic pressure, in conjunction with and transportation endogenous and exogenous material, remove free radical, anticoagulation and influence physiological function such as arterial vascular perviousness.
The nonglycosylated globular protein of strand that human serum albumin is made up of 585 amino-acid residues.Simultaneously, HSA is a kind of inert protein, and is more stable in vivo, itself is the carrier of many castle's intrinsic factors and external source medicine.The structure of HSA shows, its N end and C end are in outstanding position opposite on the albumen, the fusion of N end or C end all needn't be inserted catenation sequence in the middle of cDNA, being very suitable for carrying out albumen merges, after medicine and the HSA combination, can reduce its bioavailability, be increased in the intravital transformation period, thereby can improve curative effect.Produced the human serum albumin integration technology thus, this technology is widely used at present.Simultaneously, HSA can efficient secretory expression in pichia spp, and supernatant foreign protein content is less, and purification ratio is more convenient, more easy realization of large-scale production.
The present invention prepares first and a kind of fusion rotein that is formed by human serum albumin (HSA) and influenza m 2 e protein binding is provided, and preparation method thereof and the application in anti-influenza A virus is studied.
Goal of the invention
An object of the present invention is to provide a kind of fusion rotein that forms by human serum albumin and influenza m 2 e protein binding.
In fusion rotein of the present invention, M2e albumen is positioned at the C-end of human serum albumin fusion proteins.
According to the preferred embodiments of the invention, said fusion rotein produces with the DNA recombinant technology.
According to the preferred embodiments of the invention, wherein employed recombinant expression system is a yeast expression system, the nucleotide sequence of code book invention fusion rotein can be cloned in the Yeast expression carrier.The preferred pichia pastoris phaff expression system of the present invention can be to express in the born of the same parents, also can be secreting, expressing, most preferably pichia spp X-33.
The formed fusion rotein of the present invention had both kept the activity of human serum albumin, had kept the proteic activity of M2e again.With influenza A virus H1N1 and H3N2 the mouse collunarium is attacked, fusion rotein HSA/M2e can reduce losing of mouse body weight significantly and reduce mortality of mice, and reduces lung's virus titer, the attack of protection mouse opposing influenza A virus.
Method well known in the art be can use, the polynucleotide of coding human serum albumin and the polynucleotide of encoding influenza virus M2e obtained as methods such as PCR, RT-PCR method, artificial synthesis.Can derive from any tissue, cell and library etc. of containing corresponding mRNA or cDNA as pcr template with the mRNA or the cDNA that are used for the construction cDNA library, also can obtain with artificial synthetic method.The codon that can select for use the host to have a preference for during synthetic is so as to improving the expression efficiency of product.Method by PCR, read separately under the constant prerequisite of frame in maintenance, suitable restriction endonuclease sites is introduced in both sides at encoding sequence, cut the generation sticky end by enzyme, and under the dna ligase effect, the sticky end of realizing coding human serum albumin and the polynucleotide of encoding influenza virus M2e is connected, and obtains the gene of code book invention fusion rotein.The molecular cloning method of used standard is referring to " molecular cloning test guide " third editions such as J. Sa nurse Brooker, Science Press, 2002.
After obtaining carrying the recombinant expression vector of antigen-4 fusion protein gene, can use usual method, as method transformed host cells such as lithium salts method, PEG method and electroporations.Wherein, the preferred method for transformation of the present invention is an electroporation.Can be identified by the technology that people know,, be extracted DNA, be identified by successful cell transformed with PCR method then, promptly be contained the cell of DNA construct of the present invention as collecting and lysing cell.Perhaps, the albumen in the proteic antibody test cells and supernatant of available AHS's albumin or influenza m 2 e.
Can use and shake bottle or bio-reactor, cultivate the host cell that contains DNA construct of the present invention, to produce fusion rotein of the present invention.Employed substratum should be able to provide thalline (or cell) growth and product to express required material, comprises nitrogenous source, carbon source, pH regulator and becomes to grade, and culture medium prescription should be according to different Objects of Development, by the test acquisition.Cultivation can be divided into two stages, and the fs is mainly used in thalline (or cell) growth, and subordinate phase is mainly used in the synthetic of product.
Can in bacterium that contain DNA construct of the present invention or cell culture, separate with the method for various albumen sepn, purified fusion protein, as saltout, the combination of technology such as organic solvent deposit, ultrafiltration, liquid chromatography (LC) and these technology.Wherein liquid chromatography (LC) can be used molecular sieve, affine, ion-exchange, chromatographic technique such as hydrophobic, anti-phase.
Fusion rotein among the present invention and derivative thereof or its pharmaceutical composition can be by any known methods, comprise injection (as subcutaneous or muscle), transdermal, suction, method administration such as oral.Preferable methods is for sucking or drug administration by injection.Treatment is included in and uses single dose or compound dosage in for some time.
The invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 shows that the PCR of recombinant plasmid pPICZ alpha C-HSA/M2e transformed yeast bacterium DNA identifies electrophoretogram.Wherein swimming lane 1～5 is the PCR product of different yeast transformant genomic dnas; Swimming lane M is DNA marker; Swimming lane 6 is the PCR product of the yeast genomic dna of empty plasmid conversion.
Fig. 2 show the HSA/M2e fusion rotein sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result (coomassie brilliant blue staining).Wherein swimming lane 1 is a Mono S elutriant; Swimming lane 2 is a ultrafiltration and concentration liquid; Swimming lane 3 is the MonoQ elutriant; M is molecular weight of albumen marker.
Fig. 3 shows the Western immunoblotting assay result of HSA/M2e fusion rotein.Wherein swimming lane 1,2,3 and 4 is HSA/M2e samples that the present invention prepares.Figure A is HSA/M2e fusion rotein and the hybridization of HSA antibody; Figure B is HSA/M2e fusion rotein and the hybridization of M2e antibody.
Fig. 4 is the variation that utilizes mouse T lymphocyte quantity after the Flow cytometry immunity, and figure A is the PBS negative control group; Figure B is a HSA/M2e fusion rotein group; Figure C is the split vaccine group.
After Fig. 5 is influenza viruse attack, the result of variations of mouse lung histopathology.Figure A is that PBS group mouse is attacked the pathological change result of poison lung after 6 days; Figure B, C are respectively the pathological change result that HSA/M2e fusion rotein group mouse is attacked poison lung after 6 days; Figure D attacks the pathological change result of poison lung after 6 days for the split vaccine group.
Summary of the invention
The present invention relates to fusion rotein HSA/M2e, its preparation method and application, particularly relate to the application of fusion rotein HSA/M2e in anti-influenza A virus.Below by embodiment preferred forms of the present invention is described.These embodiment are intended to further illustrate for example the present invention, rather than limit the await the reply scope of claim of the present invention by any way.
Embodiment
Embodiment 1: the clone of human serum albumin HSA gene and amplification
1, the preparation of RNA
Embryo's (aborted fetus, 4～8 weeks deriving from The Second Hospital of Jilin Universtiy, through volunteer's informed consent) hepatic tissue of fresh separated is cut into little, put into the liquid nitrogen quick-frozen.(USA) single stage method is extracted RNA for Invitrogen Corp.SanDiego, California to use Trizol reagent.Concrete steps are as follows:
Take out freezing little block organization and put into the mortar that fills liquid nitrogen, tissue is pulverized.Tissue after will pulverizing moves in the 50mL centrifuge tube, adds about 4mL Trizol.Use polytron homogenizer high-speed homogenization 1 minute in room temperature.Add 800 μ L chloroforms (200 μ L/mLTrizol), fully vibration shakes up, and places 5 minutes under room temperature.4 ℃ centrifugal (12000r/min) 15 minutes.Upper water is moved in another centrifuge tube mutually.Add the equal-volume Virahol, vibration shakes up, precipitation at room temperature 15 minutes.4 ℃ centrifugal (13000r/min) 15 minutes abandons supernatant.Add 75% ethanol 1mL washing precipitation, 4 ℃ centrifugal (12000r/min) 5 minutes.Repeat above-mentioned steps, evaporation of residual ethanol under the room temperature.Total RNA precipitation is dissolved in 100 μ L in the DEPC treated water, and-80 ℃ of preservations are standby.Get 1 100 times of μ L diluted samples after ultraviolet spectrophotometer is measured OD
260And OD
280, rna content calculates by following formula: RNA (μ g/mL)=40 * OD
260* extension rate, OD
260/ OD
280=1.8～2.0 expression purity are qualified, with agarose gel electrophoresis method, observe the integrity of RNA.
2, the clone of human serum albumin HSA gene (RT-PCR method)
Get the total RNA 1 μ g of human liver tissue that as above extracts, set up reverse transcription (RT) reaction system in following ratio: the total RNA 1 μ g of human liver tissue; 10 * RNAPCR damping fluid, 2 μ L; MgCl
2(25mmol/L) 4 μ L; RNA enzyme inhibitors (40U/ μ L) 0.5 μ L; DNTPs (each 10mmol/L) 2 μ L; AMV reversed transcriptive enzyme (200U/ μ L) 1 μ L; Oligo dT (20pmol/ μ L) 1 μ L; No RNA enzyme sterilization ultrapure water is added to final volume 20 μ L.42 ℃ of reactions are 60 minutes on the PCR instrument, and 99 ℃ made the reversed transcriptive enzyme deactivation in 5 minutes then.Behind the synthetic cDNA, set up the PCR reaction system according to following ratio:
Above-mentioned cDNA reaction solution 20 μ L; MgCl
2(25mmol/L) 6 μ L; 10 * LAPCR damping fluid, 8 μ L; Upstream primer: 5 '-CAGGAATTCGGCACAATGAGTGGGT-3 ' 1 μ L; Downstream primer: 5 '-GCGCTCGAGGTAGATGTTATAAGCCT-3 ' 1 μ L; TaKaRa LA Taq (5U/ μ L) 1 μ L; Add the sterilization ultrapure water to final volume 100 μ L.The PCR reaction conditions is: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute 30 seconds, totally 30 circulations, then 72 ℃ 10 minutes.Amplified production is carried out 1.0% agarose gel electrophoresis analysis, observe clip size, determine whether to obtain correct goal gene.
3, the structure of cloning vector, conversion and amplification
The people HSA gene fragment of RT-PCR amplification is carried out 1.0% agarose gel electrophoresis analysis and recovery.After the recovery, people HSA gene fragment and pMD18-T carrier be connected for 16 ℃ spend the night.The ligation system comprises: people HSA gene RT-PCR product 0.1～0.3pmol; PMD18-T carrier 0.03pmol; Solution I (contain dna ligase and be connected damping fluid, see Takara company's T support agent box) 5 μ L; The sterilization ultrapure water is to final volume 10 μ L.
According to ordinary method, from 37 ℃ of XL that cultivate 16 hours
1The last single bacterium colony of picking of the negative culture plate of-Blue (not containing antibiotic LB agar plate) (diameter 2～3mm) is inoculated in the 1L culturing bottle that contains 100mL LB substratum, with preparation competence Bacillus coli cells, and frozen standby in-80 ℃.
Get a frozen competent cell, melt the back and add above-mentioned connection product, carry out routine and transform.Get the competent cell that 200 μ L have transformed then and coat on the LB plate culture medium of X-Gal (40 μ g/mL), IPTG (25 μ g/mL) and penbritin (100 μ g/mL), be inverted for 37 ℃ and cultivate 16h.
4, the evaluation of recombinant vectors
White single bacterium colony (" indigo plant is screened in vain ") after picking transforms at random is inoculated in the LB substratum that contains penbritin (100 μ g/mL), 37 ℃ of strong concussion overnight incubation, the conventional then plasmid DNA of extracting.Get that 1 μ L dissolved DNA throw out carries out the agarose gel electrophoresis quantitative analysis and enzyme is cut evaluation.Identify digestion 3 hours with the PstI double digestion down, be accredited as correct clone according to the inscribe zymogram behind 1.0% agarose gel electrophoresis for 37 ℃.Select enzyme and cut the correct clone of evaluation, extract plasmid, be used for the dna sequencing analysis.
The structure of embodiment 2:pPICZ α C-HSA/M2e yeast expression vector
1, the structure of pPICZ α C-HSA plasmid
This research adopts the PCR method to make up pPICZ α C-HSA expression vector, get above-mentioned order-checking and identify the plasmid 100ng that correctly contains the HSA gene, set up the PCR reaction system in following ratio in the 0.2mLEP pipe, (employed primer is a upstream primer: 5 '-GCG TTC GAA ATG AAG TGG GTA ACC TTT ATT TCC C-3 ' and downstream primer: 5 '-GTC GGT ACC TTA TAA GCC TAA GGC AGC TTG AC-3 ') BstBI, KpnI restriction enzyme site are introduced in amplification: contain HSA recombinant plasmid 100ng by PCR; DNTP (10mmol/L) 4 μ L; Contain Mg
2+10 * LA PCR damping fluid, 5 μ L; Upstream primer (20pmol/ μ L) 1 μ L; Downstream primer (20pmol/ μ L) 1 μ L; LA Taq enzyme (5U/ μ L) 0.5 μ L adds the sterilization ultrapure water to final volume 50 μ L.On the PCR instrument, carry out cyclic amplification.Amplification program is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 1 minute 30 seconds, totally 30 circulations, then 72 ℃ 10 minutes.Amplified production reclaims pcr amplification product through 1.0% agarose gel electrophoresis according to the explanation of dna fragmentation purifying/recovery test kit.
2, the evaluation of pPICZ α C-HSA plasmid
Digest the PCR product and the carrier pPICZ α of above-mentioned recovery with BstBI, KpnI, agarose gel electrophoresis quantitatively after, 16 ℃ of connections are spent the night.The ligation system is as follows: goal gene 0.1～0.3pmol; Carrier pPICZ α 0.03pmol after enzyme is cut; 10 * connection damping fluid, 1 μ L; T4DNA ligase enzyme (350U/ μ L) 1 μ L; The sterilization ultrapure water is to final volume 10 μ L.
To connect product and competent cell XL1-blue mixing gently, ordinary method transforms.Get the Bacillus coli cells that 200 μ L have transformed and coat on the less salt LB agar plate that contains Zeocin (25 μ g/mL), 37 ℃ of incubators were cultivated 12～16 hours.The clone that picking is different, overnight incubation in the LB liquid nutrient medium is extracted plasmid then, and carries out XbaI enzyme cutting and identify.Choose enzyme and cut the correct clone of evaluation, extract plasmid, carry out determined dna sequence with plasmid rapid extraction test kit.
3, the structure of pPICZ α C-HSA/M2e expression vector
General gene order and corresponding pichia spp preference codon according to the influenza m 2 e that reports, design two oligonucleotide strands, simultaneously hold to have added respectively to help Eco81I and the KpnI restriction enzyme site that follow-up sequence is connected at 5 ' and 3 ' of the normal chain of M2e oligonucleotide chain and minus strand encoding sequence.Normal chain is that 5 '-TTA GGC TTA TCT TTG TTG ACC GAG GTTGAG ACT CCA ATT AGA AAC GAG TGG GGT TGT AGA TGT AAC GAC TCC TCT GACTAA GGT AC-3 ' minus strand is 5 '-CTT AGT CAG AGG AGT CGT TAC ATC TAC AAC CCC ACTCGT TTC TAA TTG GAG TCT CAA CCT CGG TCA ACA AAG ATA AGC C-3 ', article two, chain is respectively got 1 μ g and 0.5mol NaCl sets up 40 μ L systems, 99 ℃ were boiled 10 minutes, 50 ℃ were boiled 10 minutes, and slowly cooled to room temperature.Complementary strand annealing back forms the joint that has Eco81I and KpnI sticky end two strands.
With Eco81I and KpnI digested plasmid pPICZ α C-HSA, reclaim go forward side by side row agarose gel electrophoresis quantitatively after, 16 ℃ of connections are spent the night.The ligation system is as follows: annealed M2e gene 0.1～0.3pmol; PPICZ α C-HSA 0.03pmol; 10 * connection damping fluid, 1 μ L; T4DNA ligase enzyme (350U/ μ L) 1 μ L; The sterilization ultrapure water is to final volume 10 μ L.
To connect product and competent cell XL1-blue mixing gently, ordinary method transforms.Get the competent cell that 200 μ L have transformed and coat on the less salt LB agar plate that contains Zeocin (25 μ g/mL), 37 ℃ of incubators were cultivated 12～16 hours.The clone that picking is different, overnight incubation in the LB liquid nutrient medium is extracted plasmid then and is carried out identifying with the XbaI double digestion.
Choose enzyme and cut the correct clone of evaluation, extract plasmid with plasmid rapid extraction test kit.Carry out determined dna sequence.
The foundation and the screening of embodiment 3:HSA/M2e Pichia anomala expression system
Get the correct cultivation bacterium liquid of order-checking, extract plasmid DNA and carry out quantitative analysis with agarose gel electrophoresis method by the plasmid extraction kit specification sheets.Get 15～20 μ g plasmid pPICZ α C-HSA/M2e, after PmeI enzymic digestion (linearizing) with phenol/chloroform extracting and use ethanol sedimentation.Linearizing plasmid is standby on ice with 10 μ L ultrapure waters dissolving postposition.
(not containing antibiotic YPD Agar flat board) single bacterium colony of picking from the negative culture plate of the YPD of pichia spp X-33 is inoculated in the 5mLYPD substratum, 250r/min, and 30 ℃ of concussions were cultivated 18 hours, and ordinary method prepares the yeast competent cell.Get the above-mentioned competent cell of 80 μ L then, mix, move in the 0.2cm electricity conversion cup and carry out the electricity conversion with the linearizing recombinant expression plasmid of 15～20 μ g.The bacterium liquid of getting after 50～100 μ L transform is coated on the YPD flat board that contains Zeocin (100 μ g/mL), and 30 ℃ of incubators were cultivated 2～3 days, observed the upgrowth situation of transformant.
Use PCR method (employed primer is a upstream primer: 5 '-GCG TTC GAA ATG AAG TGG GTAACC TTT ATT TCC C-3 ' and downstream primer: 5 '-GTC GGT ACC TTA TAA GCC TAA GGC AGCTTG AC-3 ', reaction conditions is the same) screening transformed yeast bacterium then.Behind the centrifugal recovery thalline, with boil-freeze-cooking method extracts the pastoris genomic dna performing PCR of going forward side by side and identifies, capable 1.0% agarose gel electrophoresis of amplified production observes whether obtaining expecting the gene fragment of size.Qualification result as shown in Figure 1.
Embodiment 4: the expression of fusion rotein HSA/M2e and purifying
1, the expression of fusion rotein HSA/M2e
Get above-mentioned qualification result male clone and be inoculated in 10mL BMGY (pH6.0) substratum, 30 ℃ of concussions were cultivated 24 hours, to OD
600Reach 2.0～6.0 o'clock collecting cells.With equal-volume (10mL) BMMY (pH6.0) re-suspended cell precipitation, abduction delivering is cultivated in 30 ℃ of concussions.Induce in the process, replenished a methyl alcohol to final concentration 0.5% in per 24 hours, replenish the sterilization ultrapure water simultaneously, the fermented liquid cumulative volume is remained unchanged.Respectively get the 0.5mL fermented liquid at the 0th, 24,48,72,96,120, the 144 and 168 hour equi-time point of cultivating, centrifugal collection supernatant is used for protein analysis (SDS-PAGE, WestemBlot and n terminal amino acid sequential analysis).
2, the purifying of fusion rotein HSA/M2e
(1) solution and reagent: solution A: HAc-NaAc damping fluid 200mmol/L (pH3.0); Solution B: 20mmol/LHAc-NaAc damping fluid (pH 3.0); Solution C: solution B contains 1mol/LNaCl; Solution D: 200mmol/LTris-HCl damping fluid (pH 8.0); Solution E: 20mmol/L Tris-HCl damping fluid (pH 8.0) gets final product the solution D dilution for 10 times; Solution F: solution E contains 1mol/L NaCl.
(2) Mono S cation seperation column chromatography
With 5 times of column volume solution B balance Mono S positively charged ion chromatography posts.Adjusting fermented supernatant fluid pH value with 1mol/L NaOH is 3.0, and centrifugal (12000r/min) 10 minutes removes particulate matter, and then supernatant adds 1/10 volume solution A.With the speed application of sample of 0.4mL/min to Mono S resin cation (R.C.), wavelength 280nm on-line monitoring.Application of sample washes to A with solution B after finishing
280Value is reduced to baseline.With solution B and solution C gradient elution, and the fraction collection protein peak.Determine the elution peak of HSA/M2e with SDS-PAGE and Western Blot.
(3) Mono Q anion column chromatography
With 5 times of column volume solution E balance Mono Q anion chromatography posts.The elutriant pH value of adjusting after Vivaflow 50 ultrafiltration desalinations with 1mol/L NaOH is 8.0, and centrifugal (12000r/min) 10 minutes removes particulate matter, and then supernatant adds 1/10 volume solution D.With the speed application of sample of 0.4mL/min to Mono Q resin anion(R.A), wavelength 280nm on-line monitoring.After application of sample finishes, wash to the A280 value with solution E and to reduce to baseline.With solution E and solution F gradient elution, and the fraction collection protein peak.Determine the elution peak of HSA/M2e with SDS-PAGE and Western Blot.With the elution peak of HSA/M2e through Vivaflow 50 desalination and concentration by ultrafiltration once more.
The 10%SDS-PAGE analytical results shows, can reach about 95% through the HSA/M2e of this method purifying purity, and the result as shown in Figure 2.
Embodiment 5: the physico-chemical property of fusion rotein HSA/M2e is identified
1, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Carry out protein molecule flow measurement and preliminary quantitative analysis with the SDS-PAGE method.Method is as follows: prepare 10% separation gel, 5% concentrates glue.Get per 24 hours fermented liquid culture supernatant respectively and add 5 * SDS sample buffer, behind the mixing, boiled 5 minutes in 4: 1 ratios.Get above-mentioned sample, be cooled to room temperature after, centrifugal (8000r/min) 30 seconds gets the every hole of supernatant application of sample 60 μ L.The 60V electrophoresis is adjusted voltage to 120V to concentrating glue and separation gel intersection, continues the constant voltage electrophoretic separation.After coomassie brilliant blue staining and the decolouring, the observation analysis result.
2, the Western engram analysis of expression product
According to a conventional method with fermented liquid supernatant behind SDS-PAGE, be transferred on nitrocellulose (NC) film drying at room temperature 30 minutes.Above-mentioned NC film is placed plate, add 30mL confining liquid (TTBS that contains 0.2%BSA), 4 ℃ of sealings are spent the night.Then, the NC film is put into hybridization bag by 0.1mL antibody-solutions/cm
2Film adds mouse-anti people's HSA antibody and mouse-anti people M2e antibody, room temperature vibration 3 hours.After the TTBS rinsing 3 times, dilute the sheep anti-mouse antibody to 1 of horseradish peroxidase-labeled with antibody diluent: 500, add in the hybridization bag, with NC film room temperature vibration 3 hours.Add 1mL 0.3% (W/V) NiCl or CoCl
2And 10 μ L 30%H
2O
2Solution.After washing film, film is moved in the colour developing liquid, color reaction is shaken and observed to lucifuge gently under the room temperature.The result as shown in Figure 3.
3, the amino acid sequence analysis of expression product
Proteinic primary structure is the basis of its higher structure, during with the gene recombination technology expression secreted protein, signal peptide mistake cutting phenomenon appears in the heterologous protein of expressing easily in the processing ripening process, influence the higher structure and the biologic activity of expression product then.Therefore, determine under the correct situation of molecular weight, tackle expressed recombinant protein and carry out protein N-terminal amino acids sequence mensuration, to determine the exactness of its primary structure at SDS-PAGE.Our sequencing result shows to have and the aminoacid sequence of expecting according to HSA/M2e fusion rotein of the present invention.
Embodiment 6: immunity and immune property thereof research in the fusion rotein HSA/M2e mouse body
The fusion rotein HSA/M2e that the utilization of this example is expressed combines with Freund's complete adjuvant and Freund respectively, observes the variation that mouse immune is learned state after three immunity.Behind the H1N1 and H3N2 influenza infection mouse with lethal quantity; observation is to the influence of body weight change and the mortality ratio of mouse lung virus titer and mouse; and compare, thereby judge and the protection effect of the HSA/M2e protein vaccine that definite the present invention is produced with control group.
1, fusion rotein HSA/M2e of the present invention is to the immunization experiment of mouse
With 27 of the male BALB/c mouse in 6～8 weeks, be divided into 3 groups at random, 9 every group, be respectively PBS negative control group, influenza virus cracking vaccine positive controls, recombinant protein and add the adjuvant immunity group.Every two weeks, eyeball of mouse is got blood, separates polyvalent antibody, puts-20 ℃ of refrigerators and preserves.Concrete steps are as follows: the 1st week: recombinant antigen and complete Freund's adjuvant equal-volume mix, and 100 μ g/ only; PBS blank group 100 μ L/ only; Positive controls influenza virus cracking vaccine 25 μ L/, abdominal injection.The 3rd week: recombinant antigen and incomplete Freund's adjuvant equal-volume mix, and 100 μ g/ only; Blank and positive controls are the same, abdominal injection.The 5th week: recombinant antigen and incomplete Freund's adjuvant equal-volume mix, and 100 μ g/ only; Blank and positive controls are the same, abdominal injection.The 6th～10 week: eyeball is got blood and is prepared polyvalent antibody, carries out the ELISA experiment.Mouse was put to death in cervical vertebra dislocation simultaneously after last all eyeball was got blood, and aseptic its spleen of getting prepares splenocyte suspension, was used for cellular immunization index detection-t lymphocyte subset class quantitative analysis, lymphocyte transformation experiment and IFN-γ-ELISPOT and detected.
(1) ELISA of immune serum experiment
As antigen coated elisa plate, blank adds 200 μ L/ holes bag and is cushioned liquid with synthetic M2e polypeptide 2 μ g/mL, puts wet box and spends the night for interior 4 ℃.PBST washes plate, and 3 times, each 3 minutes.The sealing of 2% calf serum is put in the wet box, hatches 2 hours for 37 ℃.PBST washes plate, and 3 times, each 3 minutes.Add the mice serum to be measured 100 μ L of 10 times of PBS dilutions, put 37 ℃ and hatched 2 hours, do negative control with the normal mouse serum of the identical multiple of dilution simultaneously.PBST washes plate 3 times, each 3 minutes.The goat anti-mouse igg that adds the HRP mark is put 37 ℃ and was hatched 2 hours.Wash plate 3 times, each 3 minutes.Take by weighing 4mg OPD and be dissolved in the 5mL substrate diluent, face with preceding adding 10 μ L 30%H
2O
2, mixing.100 μ L/ holes, room temperature lucifuge reaction 15 minutes.Add 2mol/L H
2SO
4Termination reaction, 50 μ L/ holes.In wavelength 490nm place, working sample optical density value.
The ELISA detected result is as shown in table 1 below, compares with the PBS group, recombinant protein group and influenza split vaccine group 1 week after first immunisation, promptly detects antibody in the serum.And antibody horizontal raises gradually in the 2nd time and the 3rd time immunity back, reaches peak value to immunity back the 8th all antibody horizontals, descend a little thereafter, and the PBS group can not induce corresponding antibodies.The prompting recombinant protein can induce humoral immunoresponse(HI), and its antibody titer is between 1: 800～1: 1000.
The different immunity times of table 1 respectively organize immune serum detect at the intravital antibody horizontal of mouse (n=3,
)
Compare * * P＜0.01, * P＜0.05 with the PBS group
(2) detection of splenic lymphocyte subclass quantity
The disconnected neck of immune mouse is put to death, and soaks 5 minutes in 75% alcohol, and careful separation subcutis and abdominal muscles expose spleen in the super clean bench, cut off reticular tissue on every side, grinds with double-deck slide, softly blows even mixed liquid.Be filtered to the 50mL centrifuge tube that contains the 4mL lymphocyte separation medium with 200 order gauzes, centrifugal 20 minutes of 1500r/min.Buffy coat is to new 50mL centrifuge tube in the middle of careful the absorption.PBS washing 2 times, centrifugal 5 minutes of 1000r/min.Adding 1mL contains the RPMI-1640 re-suspended cell of 10% serum, and counting is transferred cell count to 1.0 * 10
7Individual/mL is standby.
Get the splenic T lymphocyte suspension about 2 * 10 for preparing
6Individual lymphocyte is in the EP pipe, and the PBS that adds 1mL washs centrifugal 10 minutes of 2000r/min 2 times.The PBS 200 μ L of every pipe adding FITC mark anti-mouse CD4 monoclonal antibody (0.1mg/mL), PE mark anti-mouse CD8 monoclonal antibody (0.1mg/mL), PE-Cy5 mark anti-mouse CD3e (0.2mg/mL) are suspension cell again, and each antibody concentration is all by 0.25 μ g/10
6Individual lymphocyte.The room temperature lucifuge was hatched 30 minutes behind the mixing.Centrifugal 10 minutes of 2500r/min, PBS washing 2 times.Use 300 μ L PBS re-suspended cells at last, sample detection.Preparation is respectively dyeed respectively simultaneously, and the antibody list dyes control tube and blank is managed.FACS detects 1 * 10
6Individual cell obtains CD4 respectively
+, CD8
+The lymphocytic quantity of T, the gained data are carried out statistical analysis.
Fluidic cell result shown in the accompanying drawing 4 shows, the t lymphocyte subset class CD4 of recombinant protein group and split vaccine group
+And CD8
+Ratio all be higher than PBS group.And compare recombinant protein group and split vaccine group CD4 with the PBS group
+The lymphocytic increase of T is apparent in view, has produced with CD4 after the recombinant protein immunity is described
+The T lymphopoiesis is main T lymphocyte reaction, has activated the cellular immunization of body to a certain extent.
(3) splenic T lymphocyte transformation experiment
Lymphocytic propagation and differentiation are the important stages in the immune response process, and the power of lymphopoiesis ability has been represented the height of lymphocyte function to a certain extent.
Every hole adds the ConA (4 μ g/mL) or the inactivated influenza virus virus 50 μ L of the RPMI-1640 dilution that contains 10% serum respectively in 96 orifice plates, and not adding stimulates former cell to make negative control, and 3 multiple holes are all established in various stimulations.Every then hole adds the splenic lymphocyte liquid 50 μ L behind the mixing, and making its cumulative volume is 100 μ L, places 5%CO
2, cultivate after 72 hours in 37 ℃ of incubators, every hole adds 10 μ L MTT, continues to cultivate 4 hours, measures the light absorption value (A) of wavelength 450nm with microplate reader, gets 3 hole mean values, calculates stimulation index (SI).The gained data are carried out statistical analysis.SI=stimulates the hole A value/empty A value of contrast * 100%.
The result is as shown in table 2, compares with the PBS group, and the lymphocyte of recombinant protein group and influenza virus cracking vaccine group all has propagation to a certain degree, and the prompting recombinant protein has been induced cellular immunization.
Table 2 spleen T lymphocyte transformation experimental result (n=3,
)
Compare * * P＜0.01, * P＜0.05 with the PBS group
(4) IFN-γ-ELISPOT detects
Wrap by 96 orifice plates with the PBS that contains 10 μ g/mL rat anti-mouse IFN-gamma antibodies, 100 μ L/ holes, 4 ℃ are spent the night, and PBS washes plate 3 times, each 3 minutes.37 ℃ of sealings of 2% calf serum 2 hours, PBS washes plate 3 times, each 3 minutes.With 2 * 10
6Individual/mL splenic lymphocyte is added in 96 well culture plates, 100 μ L/ holes, and every mouse is established 3 stimulates hole and 3 control wells, stimulates every hole, hole to stimulate with 50 μ L inactivated influenza virus virus, and control wells is added 50 μ L PBS, 5%CO
2, cultivated 24 hours in 37 ℃ of incubators.Abandon liquid in the hole, dry the back and add 100 μ L PBST solution, placed 10 minutes, and then washed plate 3 times, each 3 minutes for 4 ℃ with PBST solution.Every hole adds the biotin labeled anti-IFN-γ two of 100 μ L and resists, and hatches 2 hours for 37 ℃.PBST washes plate 3 times, each 3 minutes.Add 100 μ L enzymes and mark anti-avidin antibody (1: 2500), hatched 1 hour for 37 ℃.PBST washes plate 3 times, each 3 minutes.Every hole adds the BCIP/NBT damping fluid of 100 μ L, and room temperature reaction 10 minutes is observed spot and formed.Water flushing 3 times, termination reaction is dried, conventional phase microscope counting spot number.
The result is as shown in table 3, and the ELISPOT spot number average of recombinant protein and influenza split vaccine immune group is higher than control group.
Table 3ELISPOT method detect each immune group mouse T lymphocyte secretion of gamma-IFN result (n=9,
)
Compare * * P＜0.01 with the PBS group
2, fusion rotein HSA/M2e of the present invention is to the provide protection of mouse
(1) fusion rotein HSA/M2e is to the influence of mouse body weight change and mortality ratio
With 60 of 6～8 weeks, the male BALB/c mouse of 18～20g, be divided into 6 groups at random, 10 every group, wherein PBS negative control group, influenza virus cracking vaccine positive controls, recombinant protein add each two groups of adjuvant immunity groups.(concrete steps are as implied above) be the 7th week after immunity, and PBS negative control group, influenza virus cracking vaccine positive controls, recombinant protein are added the adjuvant immunity group, uses H1N1 and H3N2 viral suspension (TCID respectively for each 1 group
50All be about 1 * 10
-6) attack mouse by nasal drip.Nasal drip can cause influenza virus at mouse lung fast, duplicate widely, makes that immune mouse is dead rapidly in a short time.Detections such as titration of virus are done in 2 eyeball of mouse blood samplings of 6d picked at random after the virus attack behind the cervical vertebra dislocation execution mouse.8 of each group residues, mortality ratio and protection ratio are calculated in body weight change of periodic observation mouse and death in 14 days after the virus attack.Protection ratio=(control group mortality ratio-experimental group mortality ratio)/control group mortality ratio * 100%
The result is shown in table 4, table 5, and virus attack is after 6 days, the body weight bottom out of recombinant protein group and split vaccine group mouse, and PBS group mouse body weight continues to descend until death, the prompting recombinant protein can provide protection to mouse.
Table 4H1N1 influenza virus to the influence of mouse body weight (n=8,
)
Compare * * P＜0.01, * P＜0.05 with the PBS group
Table 5H3N2 influenza virus to the influence of mouse body weight (n=8,
)
Compare * * P＜0.01, * P＜0.05 with the PBS group
Behind influenza virus H1N1 and H3N2 attack immune mouse; by day survival condition of observation mouse; control group mice is almost all dead in 14 days; mortality ratio is respectively 87.5% and 100%; and recombinant protein group and influenza split vaccine group mouse great majority can be survived; recombinant protein group mortality ratio is about 25%, 37.5% respectively; the split vaccine group is about 12.5% and 12.5%; recombinant protein and influenza split vaccine can reach more than 62.5% the dead protection ratio of mouse, the results are shown in Table shown in 6.
After table 6 virus attack recombinant protein to the protection effect of immune mouse (n=8,
)
(2) fusion rotein HSA/M2e influences the mouse lung exponential
Mouse is immersed in 75% medical alcohol sterilization 10 minutes, under aseptic condition, cut off skin with the sterilization scissors, blunt separation subcutis and muscle, skin is opened and exposes chest toward both sides, at lung and tracheae junction whole lung is held out and cut off with scissors with aseptic nipper.
The lung tissue of taking out is put into aseptic plate, removes tissues such as tracheae and hilar lymph node, wash 2 times with stroke-physiological saline solution after, blot surface-moisture with sterilization thieving paper, weigh.By formula calculate lung exponential sum lung index inhibiting rate.Heavy (the mg)/mouse body weight (g) * 100% of lung index=mouse lung
Lung index inhibiting rate=(the average lung index of the average lung index-experimental group of the control group)/average lung index of control group * 100%
The result is as shown in table 7, and behind the influenza viruse attack, the lung index of recombinant protein group and split vaccine group is starkly lower than PBS group, illustrates that recombinant protein and split vaccine can reduce the damage of mouse lung after the virus attack.
Lung exponential sum lung index inhibiting rate after table 7 mouse is attacked with H1N1 and H3N2 (n=8,
)
Compare * * P＜0.01 with the PBS group
(3) the mouse lung virus titer is measured behind the influenza viruse attack
The right side lobe of the lung is ground on gauze, add the 1mL stroke-physiological saline solution then and make in the lung suspension suction centrifuge tube, centrifugal 10 minutes of 2500r/min removes impurity and cell debris, gets supernatant and is the lung suspension.
Mdck cell in the culture dish with 0.25% trysinization, is divided to 24 orifice plates, with containing 10% calf serum and antibiotic growth media is cultivated into monolayer cell.After the growth media sucking-off, wash 24 orifice plates twice with aseptic PBS, remove calf serum as far as possible.The lung suspension for preparing is done 10 times of dilutions of series with aseptic PBS, and the lung suspension that drips dilution rocks 24 orifice plates gently and makes the lung suspension of adding evenly be layered on cell surface in the mdck cell surface.Each extent of dilution is done 3 multiple holes, and every hole drips the about 100 μ L of virus.Cells infected is placed 5%CO
2In the incubator, cultivate after 24 hours the observation of cell pathology for 37 ℃.To cause that cytopathic minimum extent of dilution calculates the virus titer of each sample.
The result is as shown in table 8, and virus infection is after 6 days, gets the mouse lung tissue and makes the lung suspension and measure mouse lung virus titer (TCID with mdck cell
50), after as seen attacking with influenza virus H1N1 and H3N2, lung's virus titer of recombinant protein group and split vaccine group is starkly lower than the PBS group.
Each experimental group lung virus titer of table 8 (n=2,
)
(4) mouse lung is organized om observation behind the influenza viruse attack
The mouse lung tissue is fixed 30 minutes with 4% Paraformaldehyde 96, transparent 30 minutes of dimethylbenzene, paraffin embedding 4 μ m section, the dimethylbenzene dewaxing is 2 times behind the roasting sheet, dehydration of alcohol, distillation washing, brazilwood extract dyeing 10 minutes, the dye liquor that flush away is unnecessary, hydrochloride alcohol color separation 60 seconds, flowing water karyon oil blackeite was gone into Yihong liquid 10 minutes, the kytoplasm red coloration, low dehydration of alcohol is sloughed overstain Yihong, dehydration, and dimethylbenzene is transparent, mounting, opticmicroscope are observed the lung tissue form down.
Result shown in the accompanying drawing 5 shows that behind the influenza viruse attack, PBS group mouse is attacked visible interstitial edema in the alveolar of poison back, and foam sample exudate is arranged in the alveolar, and a large amount of monocyte, lymphocytic infiltrations are also seen the kitchen range pulmonary emphysema, and alveolar structure is by considerable damage (figure A).And recombinant protein (figure B, C) and split vaccine (figure D) mice immunized attack poison back alveolar tissue structure and preserve better, the inflammatory cell of infiltration is less, does not see obvious pathological change or slight pathology is arranged, and points out recombinant protein to provide protection to mouse.
Sequence table
＜110〉the holy first Science and Technology Ltd. in Jilin
＜120〉proteic preparation of influenza m 2 e and the application in anti-influenza A virus thereof
<140>
<141>
<160>6
<210>1
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.
<400>1
CAGGAATTCG?GCACAATGAG?TGGGT
<210>2
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as the structure cloning vector.
<400>2
GCGCTCGAGG?TAGATGTTAT?AAGCCT
<210>3
<211>34
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.
<400>3
GCGTTCGAAA?TGAAGTGGGT?AACCTTTATT?TCCC
<210>4
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the pcr amplification primer that designs according to specific nucleotide sequence as construction of expression vector.
<400>4
GTCGGTACCT?TATAAGCCTA?AGGCAGCTTG?AC
<210>5
<211>86
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the proteic encoding sequence of influenza m 2 e of chemosynthesis.
<400>5
TTAGGCTTAT?CTTTGTTGAC?CGAGGTTGAG?ACTCCAATTA?GAAACGAGTG
GGGTTGTAGA?TGTAACGACT?CCTCTGACTA?AGGTAC
<210>6
<211>79
<212>DNA
＜213〉artificial sequence
<220>
＜223〉the white encoding sequence of influenza m 2 e Chi of chemosynthesis.
<400>6
CTTAGTCAGA?GGAGTCGTTA?CATCTACAAC?CCCACTCGTT?TCTAATTGGA
GTCTCAACCT?CGGTCAACAA?AGATAAGCC
Claims (5)
1. fusion rotein that forms by human serum albumin and influenza m 2 e protein binding.
2. according to the fusion rotein of claim 1, be characterised in that said fusion rotein produces with the DNA recombinant technology.
3. according to the fusion rotein of claim 1, be characterised in that said fusion rotein be the pichia pastoris phaff system expression that uses produce.
4. according to the fusion rotein of claim 1, be characterised in that in the said fusion protein molecule that M2e albumen is positioned at the C-end of human serum albumin fusion proteins.
5. the fusion rotein of claim 1 is the application in the resisiting influenza virus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766640A (en) * | 2012-07-11 | 2012-11-07 | 北京健翔和牧生物科技有限公司 | Method for preparing influenza A virus full-length M2 protein |
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|---|---|---|---|---|
| CN101087808A (en) * | 2004-12-21 | 2007-12-12 | 法克斯因内特公司 | Compositions of influenza viral proteins and methods of use thereof |
| WO2008057550A2 (en) * | 2006-11-07 | 2008-05-15 | Sanofi Pasteur Biologics Co. | Stabilization of vaccines by lyophilization |
| CN101227920A (en) * | 2005-07-19 | 2008-07-23 | 陶氏环球技术公司 | Recombinant flu vaccines |
| CN101402687A (en) * | 2008-11-19 | 2009-04-08 | 中国人民解放军军事医学科学院微生物流行病研究所 | Fusion protein with high immunogenicity and uses thereof |
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2010
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| CN101087808A (en) * | 2004-12-21 | 2007-12-12 | 法克斯因内特公司 | Compositions of influenza viral proteins and methods of use thereof |
| CN101227920A (en) * | 2005-07-19 | 2008-07-23 | 陶氏环球技术公司 | Recombinant flu vaccines |
| WO2008057550A2 (en) * | 2006-11-07 | 2008-05-15 | Sanofi Pasteur Biologics Co. | Stabilization of vaccines by lyophilization |
| CN101402687A (en) * | 2008-11-19 | 2009-04-08 | 中国人民解放军军事医学科学院微生物流行病研究所 | Fusion protein with high immunogenicity and uses thereof |
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| 牟旭鹏等: "甲型流感病毒M2e与人血清白蛋白融合蛋白在毕赤酵母中的分泌表达", 《中国老年学杂志》, vol. 30, no. 5, 31 March 2010 (2010-03-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102766640A (en) * | 2012-07-11 | 2012-11-07 | 北京健翔和牧生物科技有限公司 | Method for preparing influenza A virus full-length M2 protein |
| CN102766640B (en) * | 2012-07-11 | 2013-12-25 | 北京健翔和牧生物科技有限公司 | Method for preparing influenza A virus full-length M2 protein |
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