CN103614340B - The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application - Google Patents
The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application Download PDFInfo
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Abstract
The invention discloses the clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application.The present invention will go the chicken interleukin-2 10 encoding gene directed cloning of signal peptide in containing in the carrier for expression of eukaryon of neomycin resistance gene, obtain recombinant plasmid by its transfected Chinese hamster ovary cell line CHO-K1 cell, pressurizeing through G418, screening, purifying, acquisition are stablized, the clone CHO-chIL-10M of high expression chIL-10 albumen, and its microbial preservation number is CGMCC NO.7659.Clone CHO-chIL-10M constructed by the present invention has wide practical use preparing in recombination chicken IL-10 albumen and chicken interleukin-2 10 monoclonal antibody etc.
Description
Technical field
The present invention relates to the clone that recombination chicken interleukin is expressed in a strain, particularly relate to the clone of strain stably express chicken interleukin-2 10 fusion rotein, the invention further relates to construction process and the application thereof of this clone, belong to the preparation field of recombination chicken IL-10 albumen.
Background technology
Interleukin-is called for short interleukin, is produced, the cytokine of mediated cell interphase interaction by white corpuscle, plays regulatory role in the activation of cell, propagation and differentiation.Along with molecular biological development, about the research of chicken interleukin-2 gene and function becomes focus gradually, up to now, from chicken, isolate multiple interleukin, such as: IL-1, IL-2, IL-6, IL-10, IL-12, IL-16, IL-18 etc., these interleukins can be applied to the aspects such as immunotherapeutic agent, immunostimulant, structure novel gene engineered vaccine, utilize the cytokines such as interleukin to strengthen or regulate the non-specific immunity of body, not only can effective prevention and control livestock and poultry, huge economic benefit can also be produced in Animal husbandry production.
The existing gene engineering method preparing recombination chicken IL-10 albumen in various degree there is the defects such as expression efficiency is lower, unstable expression, have much room for improvement.
Summary of the invention
An object of the present invention is to provide the clone of strain stably express chicken interleukin-2 10 albumen;
Two of object of the present invention is to provide the method for the clone building described stably express chicken interleukin-2 10 albumen;
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention increases the whole encoding gene of chicken chIL-10 (euchIL-10F) and the chicken number of writing to DNA encoding peptide (euchIL-10M) respectively, directed cloning is in the carrier for expression of eukaryon pEGFP-N1 containing reporter gene eGFP and neomycin resistance gene (neo) respectively, to cut through enzyme and check order qualification, obtain recombinant plasmid N1-euchIL-10F and N1-euchIL-10M.Recombinant plasmid N1-euchIL-10F and the N1-euchIL-10M transfected Chinese hamster ovary cell line CHO-K1 cell respectively that the present invention will obtain, through G418 pressurization screening, purifying, obtain the clone CHO-chIL-10M and CHO-chIL-10F that express chIL-10 albumen.CHO-chIL-10M and CHO-chIL-10F clone is through continuous 2 mono-clonal purifying, Secondary Culture is to the 26th generation, under FITC light-filtering channels, CHO-chIL-10M still can observe the strong green fluorescence that reporter gene eGFP albumen excites, and CHO-chIL-10F fluorescence is more weak; Experimental result shows that 2 kinds of eukaryotic expression recombinant plasmids can at CHO-K1 cells recombination chicken IL-10 albumen, but the expression efficiency of clone CHO-chIL-10M expression recombination chicken IL-10 albumen will express the expression efficiency of recombination chicken IL-10 albumen far away higher than CHO-chIL-10F, and the stability of clone CHO-chIL-10M expression recombination chicken IL-10 albumen will be far superior to the stability of CHO-chIL-10F.
RT-PCR qualification result shows, the whole encoding gene of chIL-10 (euchIL-10F) and the chicken number of writing to DNA encoding peptide (euchIL-10M) obtain stably express respectively in CHO-chIL-10F and CHO-chIL-10M clone, but in CHO-chIL-10M clone the chicken number of writing to DNA encoding peptide expression intensity be the 4-6 doubly left and right of the whole encoding gene of chIL-10 in CHO-chIL-10F clone.
Western blot detected result shows, the whole encoding gene of chIL-10 (euchIL-10F) and the chicken number of writing to DNA encoding peptide (euchIL-10M) all obtain correction in cell, but in CHO-chIL-10M clone, chicken goes the expression amount of the IL-10 encoding gene of signal peptide will far away higher than the expression amount of the whole encoding gene of chicken interleukin-2 in CHO-chIL-10F 10.
IFA detected result shows, mouse-anti chIL-10 serum and clone CHO-chIL-10F and CHO-chIL-10M all present specificity red fluorescence, and rabbit anti-chIL-10 serum does not react with any cell strain.
The effect of no signal peptide to IL family member vivoexpression is had to be difficult to expection in advance.Lee's row (Lee's row, the foundation of stably express ChIL-18 albuminous cell system and the preparation of ChIL-18 monoclonal antibody, 2012, Master's thesis) research discovery, under other identical conditions, with not containing compared with signal peptide, ChIL-18 has signal peptide obviously can promote efficiency or the expression amount of its vivoexpression; Experiment of the present invention finds very unexpectedly, and after being removed by the signal peptide of chicken IL-10, its expression efficiency in cell or expression amount will exceed expression efficiency or the expression amount of chicken interleukin-2 10 full-length gene far away.
In view of chicken in CHO-chIL-10M clone removes the expression amount of the IL-10 encoding gene of signal peptide or expression efficiency far away higher than expression amount or the expression efficiency of the whole encoding gene of chicken interleukin-2 in CHO-chIL-10F 10, clone CHO-chIL-10M submits to the mechanism of patent accreditation to carry out preservation by the present invention, and its microbial preservation is numbered: CGMCC NO.7659; Classification And Nomenclature is: Chinese hamster ovary line.Depositary institution: China Microbiological preservation management committee's common micro-organisms center; The preservation time is on May 29th, 2013: preservation address: No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China institute, Institute of Microorganism, Academia Sinica;
Clone CHO-chIL-10M constructed by the present invention can be efficient, stable expression recombination chicken IL-10 albumen, have wide practical use preparing in recombination chicken IL-10 albumen and chicken interleukin-2 10 monoclonal antibody etc.
the present invention relates to term
Term " polynucleotide " or " gene " mean the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside, ribonucleoside or ribonucleotide and polymkeric substance thereof.Except nonspecific restriction, otherwise the nucleic acid of the known analogue containing natural nucleotide contained in described term, and described analogue has the binding characteristic that is similar to reference nucleic acid and carries out metabolism in the mode of the Nucleotide being similar to natural generation.Unless other specific restriction, otherwise described term also means oligonucleotide analogs, and it comprises PNA (peptide nucleic acid(PNA)), DNA analogue used in antisense technology (thiophosphatephosphorothioate, phosphamide acid esters etc.).Unless otherwise, otherwise the specific nucleic acid sequence sequence that also impliedly contains its conservative varient (including, but is not limited to degenerate codon replace) of modifying and complementary sequence and clearly specify.Particularly, the 3rd sequence replaced through mixing base and/or deoxyinosine residue by producing one of them or more than one selected (or all) codon replaces to realize degenerate codon (people such as Batzer, Nucleic Acid Res.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); The people such as Rossolini, MolCell.Probes 8:91-98 (1994)).
Term " reporter gene " is the gene of a kind of coding protein that can be detected or enzyme, is that its expression product is very easy to certified gene.Its encoding sequence is merged formation mosaic gene mutually with Gene expression and regulation sequence, or merge mutually with other goal gene, express under regulating and controlling sequence controls, thus utilize its expression product to demarcate the expression regulation of goal gene, screening obtains transformant.
Term " host cell " means the cell comprising polynucleotide of the present invention, and no matter uses which kind of method to carry out inserting to produce recombinant host cell, such as, directly absorb, transduce, known other method in f pairing or affiliated field.Exogenous polynucleotide can remain the non-integrated vector of such as plasmid or can be integrated in host genome.
Term " conversion ": heterology DNA sequence dna is incorporated into host cell or organic method.
Term " expression ": endogenous gene or transgenosis transcribing and/or translating in cell.
Term " encoding sequence ": the nucleotide sequence being transcribed into RNA.
Term " carrier for expression of eukaryon ": one or more DNA vectors transformed for realizing eukaryote, comprises eukaryotic cell and transcribes and translate required controlling element, as promotor, poly A tailing signal etc.; In addition, the replication site that selectable marker gene, goal gene and eukaryotic cell can also be had to identify.
Accompanying drawing explanation
Fig. 1 G418 pressurizes and screens the formation (F1,100 ×) of 12d resistant colonies.
Scanning cofocal microscope figure after Fig. 2 transfection 24h.
The result of Fig. 3 chIL-10 gene stably express (100 ×) in CHO-K1 cell.
The RT-PCR qualification of Fig. 4 stably express chIL-10CHO cell strain; 1:CHO-chIL-10F; 2:CHO-N1; 3:CHO; 4: water; 5:CHO-chIL-10M; M:DNA molecular mass standard.
The Chinese hamster ovary celI strain Western blot of Fig. 5 stably express chIL10 fusion rotein analyzes; 1:CHO-N1; 2:CHO; 3:CHO-chIL-10F; 4:CHO-chIL-10M; 5:pET-chIL-10; M:DNA molecular mass standard; A: the anti-chIL-10 serum of rabbit; B: mouse source anti-GFP label monoclonal antibody; C: mouse-anti chIL-10 serum.
The Chinese hamster ovary celI strain IFA of Fig. 6 stably express chIL-10 identifies (100 ×); A:CHO-chIL-10F; B:CHO-chIL-10M; C:CHO-N1; D:CHO.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
1 experiment material
1.1 experiment material
Eukaryon expression plasmid pEGFP-N1 and CHO-K1 cell are preserved by Harbin Veterinary Medicine Inst., China Academy of Agriculture's laboratory animal group; No-special pathogen (SPF) chicken, cleaning grade Male New Zealand rabbits, BALB/c mouse are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center, and laboratory animal production licence is numbered SCXK (black) 2011-007.
1.2 main agents
Ex Taq, EcoR I, Xho I, Pst I and competence bacillus coli DH 5 alpha are purchased from precious biotechnology Dalian company limited; Lipofectamine
tM2000 transfection reagents are purchased from Invitrogen company; Pre-dyed albumen Marker is purchased from safe this biotechnology (Shenzhen) company limited of rich enzyme; Foetal calf serum, RPMI 1640, DMEM/F12 substratum are purchased from HyClone company; Plasmid Mini Kit, DNA glue reclaim test kit all purchased from Omega company; G418 vitriol is purchased from Amresco company; DyLight 594 mark goat anti-rabbit igg purchased from EarthOx company, DyLight 594 mark sheep anti-mouse igg purchased from Jackson company, IRDye
tMthe goat anti-rabbit igg of 700DX mark and IRDye
tMthe mountain sheep anti-mouse igg (1:5000) of 700DX mark is purchased from Rockland company, and anti-GFP label monoclonal antibody is purchased from Rockland company; Chicken lymphocyte separation medium is purchased from BD company; Lipopolysaccharides (LPS) and concanavalin A (ConA) are purchased from Invitrogen company, and other reagent is domestic analytical pure.
The structure of embodiment 1 stably express chicken interleukin-2 10 albuminous cell system
1, experimental technique
The extraction of the separation of 1.1 chicken peripheral blood lymphocytes, cultivation and total serum IgE
Aseptic wing venous collection Adult SPF Chicken Sodium Citrate anticoagulation 10mL, is separated peripheral blood lymphocyte by product description chicken lymphocyte separation medium.With the complete RPMI1640 substratum adjustment cell density containing 10% foetal calf serum to 1 × 10
6/ mL, joins 6 porocyte plates and cultivates, and adds final concentration wherein and be respectively the LPS of 10ug/mL, 20ug/mL and final concentration is respectively the ConA of 10ug/mL, 25ug/mL, 30ug/mL as stimulant, in 37 DEG C, and 5%CO
2cultivate in incubator, rock once every 4h, in different time collecting cell.Extract cell total rna with Trizol Reagents total RNA extraction reagent box, concrete grammar by specification carries out.
The structure of 1.2 chIL-10 prokaryotic expression carriers, expression and sero-fast preparation
According to the chIL-10 gene (NM_001004414.2) that GenBank logs in, the gene of design prokaryotic expression primer amplification coding chIL-10 maturation protein;
Upstream primer sequence is:
proChIL10-F：5'-CG
GAATTCTTGGAGCCCACCTGCCTG-3'(EcoR I)(SEQ ID NO.1)，
Downstream primer sequence is:
proChIL10-R：5'-CC
CTCGAGTCACTTCCTCCTCCTCATCAGC-3'(Xho I)(SEQ ID NO.2)。
PCR response procedures: 95 DEG C of 5min; 94 DEG C of 30s, 68.3 DEG C of 30s, 72 DEG C of 30s, 32 circulations; 72 DEG C of 10min.Carry out double digestion with restriction enzyme EcoR I and Xho I to the goal gene after amplification also purifying and pET-30a (+), digestion products reclaims after kits through glue and uses T
4dNA Ligase connects, transformed competence colibacillus DH5 α cell.With PCR method, restriction endonuclease analysis and determining nucleic acid sequence, the RT-PCR expression plasmid of gained after qualification transforms, called after pET-chIL-10.By pET-chIL-10 Transformed E .coli BL21 (DE3), 0.1mmol/L IPTG abduction delivering.Collect thalline, ultrasonic disruption in ice bath, respectively collecting precipitation and supernatant, SDS-PAGE analysis purposes protein expression form.A large amount of abduction delivering recombinant bacterium, cuts target protein place adhesive tape, grinding, resuspended with appropriate PBS, and immunize New Zealand rabbit and BALB/c mouse, prepare rabbit anti-serum and Mouse Antisera respectively.
The structure of 1.3 chIL-10 gene eucaryon expression plasmids
According to the gene order of NM_001004414.2, design 2 pairs of eukaryotic expression primers, increase chIL-10 total length (being called euchIL-10F) and go signal peptide (being called euchIL-10M) sequence respectively.
The primer sequence of euchIL-10F is:
EuChIL10F-U:5'-
cTCGAGaCCATGCAGACCTGCTGCCAAG-3'(Xho I) (wherein ACC is Kozak sequence) (SEQ ID NO.3);
EuChIL10F-L:5'-AA
cTGCAGcTTCCTCCTCCTCATCA-3'(Pst I) (removing terminator codon) (SEQ ID NO.4).
The primer sequence of euchIL-10M is:
euchIL-10M-U：5'-
CTCGAGGCCATGTTGGAGCCCACCT-3'(Xho I)(SEQ ID NO.5)；
euchIL-10M-L：5'-AA
CTGCAGCTTCCTCCTCCTCATC-3'(Pst I)(SEQID NO.6)。
Carry out double digestion with Xho I and Pst I to the goal gene after amplification also purifying and the carrier for expression of eukaryon pEGFP-N1 containing reporter gene eGFP, digestion products reclaims after kits through glue and uses T
4dNA Ligase connects, and transforms DH5 α competent cell.With PCR method, restriction endonuclease analysis and determining nucleic acid sequence, the positive colony of gained after qualification transforms.Spend intracellular toxin mini-scale plasmid extraction test kit and extract plasmid from identifying correct clone bacterium.The eukaryotic expression recombination plasmid obtained is called after N1-chIL-10F and N1-chIL-10M respectively.
1.4 set up the CHO-K1 clone of stably express chIL-10 gene
By CHO-K1 passage in 96 porocyte plates, when cell reaches 80%, add the DMEM/F12 nutrient solution containing different concns (100-1200mg/L) G418 and 10% foetal calf serum, cultured continuously about 2 weeks, determines G418 the smallest cell lethal quantity.Reference reagent box specification sheets, utilizes Lipofectamine
tM2000 transfection CHO-K1 cells, the same transfection of carrier pEGFP-N1 is as negative control.Resistance screening is carried out with the DMEM/F12 complete culture solution containing final concentration 800mg/L G418 after 24h.With limiting dilution assay to the continuous mono-clonal of transfection positive cell; Set up the cellular control unit strain (CHO-N1) expressing empty carrier pEGFP-N1 simultaneously.Conventional trysinization, continuous passage culturing cell.
2 experimental results
Fig. 1 is the formation result (F1,100 ×) of G418 pressurization screening 12d resistant colonies; Fig. 2 is scanning cofocal microscope figure after transfection 24h.From the result of Fig. 3, CHO-chIL-10M and CHO-chIL-10F cell is through continuous 2 mono-clonal purifying, Secondary Culture is to the 26th generation, under FITC light-filtering channels, CHO-chIL-10M still can observe the strong green fluorescence that reporter gene eGFP albumen excites, and CHO-chIL-10F fluorescence more weak (Fig. 3), show that 2 kinds of eukaryotic expression recombinant plasmids can be expressed in CHO-K1 cell, but there is highly significant difference in the expression intensity of reporter gene or efficiency, the expression efficiency of CHO-chIL-10M and expression stability significantly will be better than expression efficiency and the expression stability of CHO-chIL-10F.Experimental result illustrates, after being removed by the signal peptide of chicken IL-10, its expression efficiency in cell or expression amount will exceed expression efficiency or the expression amount of chicken interleukin-2 10 full-length gene far away.
The qualification of the clone of embodiment 2 stably express chicken interleukin-2 10 albumen
1. experimental technique
1.1 RT-PCR detect
Extract CHO-chIL-10F and the CHO-chIL-10M cell total rna constructed by test kit extraction embodiment 1 with Trizol Reagents, concrete grammar is undertaken by test kit specification sheets.Get reverse transcription product cDNA, through pcr amplification qualification chIL-10 gene, response procedures is: 95 DEG C of 5min; 94 DEG C of 30s, 64 DEG C of 30s, 72 DEG C of 40s, 30 circulations; 72 DEG C of 10min.
1.2 IFA detect
By CHO-chIL-10F and the CHO-chIL-10M cell strain after clone purification with 1 × 10
4individual/hole is inoculated in 96 orifice plates, with CHO-N1 cell for negative control, CHO-K1 cell is as blank, respectively with preparation chIL-10 rabbit anti-serum and Mouse Antisera for primary antibodie (1:50), marking goat anti-rabbit igg and DyLight 594 with DyLight 594, to mark sheep anti-mouse igg be two anti-(1:200), carries out IFA detection.
1.3 Western blot detect
Collect after CHO-chIL-10F and CHO-chIL-10M cells rinsed with PBS, extract total protein of cell with RIPA cell pyrolysis liquid.Through SDS-PAGE be separated after, be transferred to pvdf membrane, respectively with preparation rabbit anti-serum (1:50), Mouse Antisera (1:50), anti-GFP label monoclonal antibody (1:1000) for primary antibodie, with IRDye
tMthe goat anti-rabbit igg of 700DX mark and IRDye
tM700DX mark goat anti-mouse IgG (1:5000) be two resist, carry out Western blot detection.Utilize the double-colored laser imaging system scanning of Odyssey near infrared.Simultaneously with the CHO-N1 cell of empty carrier transfection for negative control, using CHO-K1 cell as blank.
2 experimental results
2.1 RT-PCR qualification results
Respectively total serum IgE is extracted to CHO-chIL-10F, CHO-chIL-10M, CHO-N1 and Chinese hamster ovary celI.RT-PCR amplification is carried out respectively with two couples of primer euChIL10F-U/euChIL10F-L and euchIL-10M-U/euchIL-10M-L, result amplifies the band of 533bp and 473bp respectively from CHO-chIL-10F and CHO-chIL-10M, with total length and go the chIL-10 gene theory size of signal peptide consistent, and CHO-N1 and CHO does not all have corresponding amplified production, illustration purpose gene has obtained stably express (Fig. 4) in cell; Expression of results shows, goes the chIL-10 of signal peptide (being called euchIL-10M) expression amount in vitro approximately to exceed 4-6 doubly than the expression amount of chIL-10 total length (being called euchIL-10F).
2.2 Western blot detected results
Western blot detected result display (Fig. 5), utilize GFP specific monoclonal antibody and mouse polyvalent antibody in the protein cleavage product of CHO-chIL-10F and CHO-chIL-10M, the band of 46ku and 44ku size can be detected respectively respectively, meet the fusion rotein theoretical value of eGFP gene and chIL-10 gene, but utilize rabbit anti-serum band not detected.Show total length and go the chIL-10 gene of signal peptide all in cell, to obtain correction, but there is the species variation of rabbit body and mouse body in the immunogenicity of recombinant protein.
2.3 IFA detected results
IFA detected result shows, mouse-anti chIL-10 serum and CHO-chIL-10F and CHO-chIL-10M all present specificity red fluorescence, and rabbit anti-chIL-10 serum does not react (Fig. 6) with any cell strain.
Claims (3)
1. the clone CHO-chIL-10M of stably express chicken interleukin-2 10 albumen, is characterized in that, its microbial preservation is numbered: CGMCC NO.7659.
2. clone CHO-chIL-10M according to claim 1 is preparing the purposes in recombination chicken IL-10 albumen.
3. clone CHO-chIL-10M according to claim 1 is preparing the purposes in chicken interleukin-2 10 monoclonal antibody.
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