CN103614340A - Cell line for stable expression of chicken interleukin 10 protein, as well as construction method and application thereof - Google Patents
Cell line for stable expression of chicken interleukin 10 protein, as well as construction method and application thereof Download PDFInfo
- Publication number
- CN103614340A CN103614340A CN201310572460.7A CN201310572460A CN103614340A CN 103614340 A CN103614340 A CN 103614340A CN 201310572460 A CN201310572460 A CN 201310572460A CN 103614340 A CN103614340 A CN 103614340A
- Authority
- CN
- China
- Prior art keywords
- chil
- cho
- expression
- chicken
- clone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a cell line for stable expression of a chicken interleukin 10 protein, as well as a construction method and application thereof. The construction method disclosed by the invention comprises the steps of cloning a chicken interleukin 10 encoding gene after removal of a signal peptide into a eukaryotic expression vector containing a neomycin resistance gene in a fixed direction to obtain a recombinant plasmid, transfecting cells of a Chinese hamster ovary cell line CHO-K1, and performing G418 pressurized screening and purification to obtain a cell line CHO-chIL-10M for stable and high-efficient expression of a chIL-10 protein, wherein the microorganism collection number is CGMCC NO. 7659. The cell line CHO-chIL-10M constructed by the construction method disclosed by the invention has extensive application prospects in preparation of recombinant chicken interleukin 10 proteins and chicken interleukin 10 monoclonal antibodies and other aspects.
Description
Technical field
The present invention relates to a strain and express the clone of recombination chicken interleukin, relate in particular to the clone of strain stably express chicken interleukin-2 10 fusion roteins, 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, by white corpuscle produce, the cytokine of mediated cell interphase interaction, in activation, propagation and the differentiation of cell, play regulatory role.Along with molecular biological development, about the research of chicken interleukin-2 gene and function becomes focus gradually, up to now, from chicken, isolated 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 effective prevention and control livestock and poultry can also produce huge economic benefit in livestock industry is produced.
The existing gene engineering method of preparing recombination chicken IL-10 albumen in various degree exist the defects such as expression efficiency is lower, unstable expression, have much room for improvement.
Summary of the invention
One of 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 that builds 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 increase the respectively whole encoding genes of chicken chIL-10 (euchIL-10F) and the chicken number of writing to DNA encoding peptide (euchIL-10M), directed cloning is in the carrier for expression of eukaryon pEGFP-N1 that contains reporter gene eGFP and neomycin resistance gene (neo) respectively, through the enzyme evaluation of cutting and check order, obtain recombinant plasmid N1-euchIL-10F and N1-euchIL-10M.The present invention is by the recombinant plasmid N1-euchIL-10F obtaining and N1-euchIL-10M difference transfection Chinese hamster ovary line CHO-K1 cell, through G418 pressurization screening, purifying, obtain the clone CHO-chIL-10M and the CHO-chIL-10F that express chIL-10 albumen.CHO-chIL-10M and CHO-chIL-10F clone are through continuous 2 mono-clonal purifying, go down to posterity and be cultured to for 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 a little less than; Experimental result shows that 2 kinds of eukaryotic expression recombinant plasmids can be at CHO-K1 cells recombination chicken IL-10 albumen, but clone CHO-chIL-10M expresses the expression efficiency of recombination chicken IL-10 albumen, will higher than CHO-chIL-10F, express the expression efficiency of recombination chicken IL-10 albumen far away, and the stability of clone CHO-chIL-10M expression recombination chicken IL-10 albumen to be far superior to the stability of CHO-chIL-10F.
RT-PCR qualification result shows, the whole encoding genes 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 left and right doubly of the whole encoding genes of chIL-10 in CHO-chIL-10F clone.
Western blot detected result shows, the whole encoding genes 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 go the expression amount of the IL-10 encoding gene of signal peptide will be far away higher than the expression amount of the 10 whole encoding genes of chicken interleukin-2 in CHO-chIL-10F.
IFA detected result shows, mouse-anti chIL-10 serum and clone CHO-chIL-10F and CHO-chIL-10M all present specificity red fluorescence, and the anti-chIL-10 serum of rabbit does not react with any cell strain.
Having no signal peptide is to be difficult in advance expection to the effect of IL family member vivoexpression.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 is found, under other condition same case, do not compare with not containing signal peptide, ChIL-18 has signal peptide can obviously promote efficiency or the expression amount of its vivoexpression; Experiment of the present invention finds very unexpectedly, after the signal peptide of ChIL-18 is removed, and expression efficiency or expression amount that its expression efficiency in cell or expression amount will exceed chicken interleukin-2 10 full-length genes far away.
In view of chicken in CHO-chIL-10M clone goes the expression amount of IL-10 encoding gene of signal peptide or expression efficiency far away higher than expression amount or the expression efficiency of the 10 whole encoding genes of chicken interleukin-2 in CHO-chIL-10F, the present invention submits to the mechanism of patent approval to carry out preservation clone CHO-chIL-10M, and its microbial preservation is numbered: CGMCC NO.7659; Classification And Nomenclature is: Chinese hamster ovary line.Depositary institution: China Committee for Culture Collection of Microorganisms'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;
The constructed clone CHO-chIL-10M of the present invention can be efficient, stable expression recombination chicken IL-10 albumen, at aspects such as preparing recombination chicken IL-10 albumen and chicken interleukin-2 10 monoclonal antibodies, have wide practical use.
the present invention relates to term
Term " polynucleotide " or " gene " mean deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Unless specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in described term, described analogue has and is similar to the binding characteristic of reference nucleic acid and carries out metabolism to be similar to the mode of the Nucleotide of natural generation.Unless other specific limited, otherwise described term also means oligonucleotide analogs, it comprises PNA(peptide nucleic acid(PNA)), in antisense technology DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters etc.) used.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and the clear and definite sequence of appointment.Specific; can through mixing sequence that base and/or Hypoxanthine deoxyriboside residue replace, realize degenerate codon and replace (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing one of them or above selected (or all) codon the 3rd; The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With the people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " reporter gene " is protein that a kind of coding can be detected or the gene of enzyme, is that its expression product is very easy to certified gene.Regulate sequence to merge mutually formation mosaic gene its encoding sequence and genetic expression, or merge mutually with other goal gene, under regulating and controlling sequence is controlled, express, thereby utilize its expression product to demarcate the expression regulation of goal gene, screening obtains transformant.
Term " host cell " means the cell that comprises polynucleotide of the present invention, and no matter use which kind of method to insert to produce recombinant host cell, for example, directly absorb, other known method in transduction, f pairing or affiliated field.Exogenous polynucleotide can remain the nonconformity carrier of plasmid for example 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 that is transcribed into RNA.
Term " carrier for expression of eukaryon ": the DNA vector that one or more transform for realizing eukaryote, comprises eukaryotic cell and transcribe and translate required controlling element, as promotor, poly A tailing signal etc.; The replication site that in addition, can also have selectable marker gene, goal gene and eukaryotic cell to identify.
Accompanying drawing explanation
The formation of Fig. 1 G418 pressurization screening 12d resistance colony (F1,100 *).
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 of Fig. 4 stably express chIL-10CHO cell strain identifies; 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
Below in conjunction with specific embodiment, further describe the present invention, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not formed to any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can the details of technical solution of the present invention and form be modified or be replaced, but these modifications 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, the male new zealand rabbit of clean level, BALB/c mouse are provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture's Experimental Animal Center, and it is black that laboratory animal production licence is numbered SCXK() 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; Dye in advance albumen Marker purchased from safe this biotechnology (Shenzhen) of rich enzyme company limited; Foetal calf serum, RPMI 1640, DMEM/F12 substratum are purchased from HyClone company; Plasmid extracts test kit in a small amount, DNA glue reclaims test kit all purchased from Omega company; G418 vitriol is purchased from Amresco company; The goat anti-rabbit igg of DyLight 594 marks is purchased from EarthOx company, and the sheep anti-mouse igg of DyLight 594 marks is 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 systems
1, experimental technique
The extraction of the separation of 1.1 chicken peripheral blood lymphocytes, cultivation and total RNA
Aseptic wing vein gathers an Adult SPF Chicken Sodium Citrate anticoagulation 10mL, by the separated peripheral blood lymphocyte of chicken lymphocyte separation medium for product description.With the complete RPMI1640 substratum containing 10% foetal calf serum, adjust cell density to 1 * 10
6/ mL, joins 6 porocyte plates and cultivates, and adds wherein final concentration to be respectively the LPS of 10ug/mL, 20ug/mL and ConA that final concentration is respectively 10ug/mL, 25ug/mL, 30ug/mL as stimulant, in 37 ℃, and 5%CO
2in incubator, cultivate, every 4h, rock once, in different time collecting cell.With Trizol Reagents total RNA extraction reagent box, extract cell total rna, concrete grammar by specification carries out.
1.2chIL-10 the structure of prokaryotic expression carrier, expression and sero-fast preparation
According to the chIL-10 gene (NM_001004414.2) of GenBank login, 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 ℃ of 5min; 94 ℃ of 30s, 68.3 ℃ of 30s, 72 ℃ of 30s, 32 circulations; 72 ℃ of 10min.Goal gene and pET-30a (+) with restriction enzyme EcoR I and Xho I after to amplification purifying carry out double digestion, and enzyme is cut product and use T after glue is reclaimed test kit purifying
4dNA Ligase connects, transformed competence colibacillus DH5 α cell.With PCR method, restriction endonuclease analysis and determining nucleic acid sequence, identify the RT-PCR expression plasmid of gained after transforming, 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, difference collecting precipitation and supernatant, SDS-PAGE analysis purposes protein expression form.A large amount of abduction delivering recombinant bacteriums, cut target protein place adhesive tape, grind, resuspended with appropriate PBS, and immune new zealand rabbit and BALB/c mouse, prepare rabbit anti-serum and Mouse Antisera respectively.
The structure of 1.3chIL-10 gene eucaryon expression plasmid
According to the gene order of NM_001004414.2, design 2 pairs of eukaryotic expression primers, the chIL-10 total length that increases respectively (being called euchIL-10F) and go signal peptide (being called euchIL-10M) sequence.
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)（SEQ?ID?NO.6）。
Goal gene with Xho I and Pst I after to amplification purifying and the carrier for expression of eukaryon pEGFP-N1 that contains reporter gene eGFP carry out double digestion, and enzyme is cut product and use T after glue is reclaimed test kit purifying
4dNA Ligase connects, and transforms DH5 α competent cell.With PCR method, restriction endonuclease analysis and determining nucleic acid sequence, identify the positive colony of gained after transforming.Spend intracellular toxin a small amount of plasmid extraction kit and extract plasmid from identify correct clone bacterium.Resulting eukaryotic expression recombination plasmid 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
CHO-K1 passage, in 96 porocyte plates, when cell reaches 80%, is added to the DMEM/F12 nutrient solution that contains different concns (100-1200mg/L) G418 and 10% foetal calf serum, and cultured continuously approximately 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.After 24h, with the DMEM/F12 complete culture solution containing final concentration 800mg/L G418, carry out resistance screening.With limiting dilution assay to the continuous mono-clonal of transfection positive cell; Set up the cellular control unit strain (CHO-N1) of 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 resistance colony; Fig. 2 is scanning cofocal microscope figure after transfection 24h.Result from Fig. 3, CHO-chIL-10M and CHO-chIL-10F cell are through continuous 2 mono-clonal purifying, go down to posterity and be cultured to for 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 weak (Fig. 3), show that 2 kinds of eukaryotic expression recombinant plasmids can express 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 will significantly be better than expression efficiency and the expression stability of CHO-chIL-10F.Experimental result explanation, after the signal peptide of ChIL-18 is removed, expression efficiency or expression amount that its expression efficiency in cell or expression amount will exceed chicken interleukin-2 10 full-length genes far away.
The evaluation of the clone of embodiment 2 stably express chicken interleukin-2 10 albumen
1. experimental technique
1.1RT-PCR detect
With Trizol Reagents, extract test kit and extract constructed CHO-chIL-10F and the CHO-chIL-10M cell total rna of embodiment 1, concrete grammar is undertaken by test kit specification sheets.Get reverse transcription product cDNA, through pcr amplification, identify chIL-10 gene, response procedures is: 95 ℃ of 5min; 94 ℃ of 30s, 64 ℃ of 30s, 72 ℃ of 40s, 30 circulations; 72 ℃ of 10min.
1.2IFA detect
By the CHO-chIL-10F after clone purification and CHO-chIL-10M cell strain with 1 * 10
4individual/hole is inoculated in 96 orifice plates, with the negative contrast of CHO-N1 cell, CHO-K1 cell is as blank, chIL-10 rabbit anti-serum and the Mouse Antisera of preparing of take is respectively primary antibodie (1:50), take DyLight 594 mark goat anti-rabbit iggs and DyLight 594 mark sheep anti-mouse iggs as two anti-(1:200), carry out IFA detection.
1.3Western blot detects
By CHO-chIL-10F and collection after PBS washing for CHO-chIL-10M cell, with RIPA cell pyrolysis liquid, extract total protein of cell.After SDS-PAGE separation, be transferred to pvdf membrane, rabbit anti-serum (1:50), Mouse Antisera (1:50), the anti-GFP label monoclonal antibody (1:1000) of preparing of take is respectively primary antibodie, with IRDye
tMthe goat anti-rabbit igg of 700DX mark and IRDye
tMthe goat anti-mouse IgG of 700DX mark (1:5000) is two anti-, carries out Western blot detection.Utilize the double-colored laser imaging system scanning of Odyssey near infrared.With the negative contrast of CHO-N1 cell of empty carrier transfection, using CHO-K1 cell as blank simultaneously.
2 experimental results
2.1RT-PCR qualification result
Respectively CHO-chIL-10F, CHO-chIL-10M, CHO-N1 and Chinese hamster ovary celI are extracted to total RNA.With two couples of primer euChIL10F-U/euChIL10F-L and euchIL-10M-U/euchIL-10M-L, carry out RT-PCR amplification respectively, result amplifies respectively the band of 533bp and 473bp 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 all do not have corresponding amplified production, illustration purpose gene has obtained stably express (Fig. 4) in cell; Expression of results shows, the chIL-10 that removes signal peptide (being called euchIL-10M) expression amount in vitro has approximately exceeded 4-6 doubly than the expression amount of chIL-10 total length (being called euchIL-10F).
2.2Western blot detected result
Western blot detected result shows (Fig. 5), utilize GFP specific monoclonal antibody and mouse polyvalent antibody the band of 46ku and 44ku size can in the protein cleavage product of CHO-chIL-10F and CHO-chIL-10M, 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.
2.3IFA detected result
IFA detected result shows, mouse-anti chIL-10 serum and CHO-chIL-10F and CHO-chIL-10M all present specificity red fluorescence, and the anti-chIL-10 serum of rabbit does not react (Fig. 6) with any cell strain.
Claims (5)
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. a method that builds clone described in claim 1, is characterized in that, comprises the following steps: will go the chicken interleukin-2 10 encoding gene directed clonings of signal peptide in the carrier for expression of eukaryon that contains neomycin resistance gene, obtain recombinant plasmid; By the Transfected Recombinant Plasmid Chinese hamster ovary line CHO-K1 cell obtaining, through the clone CHO-chIL-10M of G418 pressurization screening, purifying, acquisition stably express chIL-10 albumen.
3. it is characterized in that in accordance with the method for claim 2: described carrier for expression of eukaryon is pEGFP-N1.
4. the purposes of clone claimed in claim 1 in preparing recombination chicken IL-10 albumen.
5. the purposes of clone claimed in claim 1 in preparing chicken interleukin-2 10 monoclonal antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310572460.7A CN103614340B (en) | 2013-11-15 | 2013-11-15 | The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310572460.7A CN103614340B (en) | 2013-11-15 | 2013-11-15 | The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103614340A true CN103614340A (en) | 2014-03-05 |
| CN103614340B CN103614340B (en) | 2015-09-02 |
Family
ID=50165060
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310572460.7A Expired - Fee Related CN103614340B (en) | 2013-11-15 | 2013-11-15 | The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103614340B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
| CN114805537A (en) * | 2022-04-26 | 2022-07-29 | 华南农业大学 | Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180959A (en) * | 2011-03-17 | 2011-09-14 | 天津康莱森生物科技集团有限公司 | Improved chook Interleukin-2 protein and preparation method thereof |
-
2013
- 2013-11-15 CN CN201310572460.7A patent/CN103614340B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180959A (en) * | 2011-03-17 | 2011-09-14 | 天津康莱森生物科技集团有限公司 | Improved chook Interleukin-2 protein and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| DIAN-CHANG ZHANG 等: "Cloning, Characterization and Expression Analysis of Interleukin-10 from the Zebrafish (Danio rerion)", 《JOURNAL OF BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
| LISA ROTHWELL 等: "Cloning and Characterization of Chicken IL-10 and Its Role in the Immune Response to Eimeria maxima", 《THE JOURNAL OF IMMUNOLOGY》 * |
| 龚婷 等: "鸡IL-2全基因和去信号肽基因的酵母表达研究", 《郑州牧业工程高等专科学校学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114369152A (en) * | 2022-01-24 | 2022-04-19 | 扬州大学 | Recombinant chicken interleukin-9 protein and preparation and application of antibody thereof |
| CN114369152B (en) * | 2022-01-24 | 2023-11-21 | 扬州大学 | Preparation and application of recombinant chicken interleukin-9 protein and antibody thereof |
| CN114805537A (en) * | 2022-04-26 | 2022-07-29 | 华南农业大学 | Recombinant plasmid for expressing canine interleukin 6, cell strain for stably expressing canine interleukin 6 protein, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103614340B (en) | 2015-09-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10538786B2 (en) | Recombinant arterivirus replicon systems and uses thereof | |
| CN110981968B (en) | Fusion protein containing rabies virus G protein, preparation method, application and vaccine thereof | |
| EP2952581A1 (en) | Recombinant virus-like particles encoded by multi-gene vector | |
| Singh et al. | Expression of rabies glycoprotein and ricin toxin B chain (RGP–RTB) fusion protein in tomato hairy roots: a step towards Oral vaccination for rabies | |
| CN116554305A (en) | Recombinant humanized III-type collagen, preparation method and application thereof | |
| CN104725515A (en) | Elastin-like protein polypeptide-coxsackie adenovirus receptor fused protein as well as preparation method and application thereof | |
| CN106755089B (en) | Cell line for expressing goat lymphocyte activating molecules and construction method and application thereof | |
| GB2605895A (en) | Detection of optimal recombinants using fluorescent protein fusions | |
| CN103614340B (en) | The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application | |
| CN106497973B (en) | A kind of mankind and other mammalian cells adhere to body expression vector, expression system, preparation method and application | |
| RU2009124471A (en) | VIRAL VECTOR AND ITS APPLICATION | |
| D'Adamo et al. | Prospects for viruses infecting eukaryotic microalgae in biotechnology | |
| CN104131022A (en) | Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof | |
| CN107653266B (en) | Application of recombinant expression vector containing lepidoptera virus enhancer in expressing protein in diptera insect cells | |
| CN109055398A (en) | A kind of tuberculosis DNA vaccines comprising codon optimization CFP10 gene | |
| CN105886531B (en) | The construction method of molecular labeling swine fever virus attenuated vaccine | |
| CN102239261A (en) | Artificial chromosome vector | |
| CN101343638B (en) | Toxoplasma transgenosis carrier based on piggyback transposon and construction method | |
| CN103352048A (en) | Microalgae chloroplast carrier for efficient cloning and expression and application thereof | |
| CN114107253B (en) | System and method for gene editing by using engineering cells | |
| CN104450781B (en) | A kind of cell line of overexpression CIAPIN1 albumen and its preparation method and application | |
| CN115433741A (en) | Construction method of recombinant virus taking Gatas virus as vector to express reporter protein | |
| CN110872358B (en) | HA-Fc fusion protein, preparation method thereof and vaccine | |
| CN103266121A (en) | Replicon DNA (deoxyribonucleic acid) vaccine vector, and construction method and application thereof | |
| CN106754722A (en) | Vero cell line of stabilization expression bovine trypsinogen and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150902 Termination date: 20211115 |