CN101089180A - Recombination LFA3 gene, and its mixed gene and product - Google Patents
Recombination LFA3 gene, and its mixed gene and product Download PDFInfo
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Abstract
The invention relates to recombination LFA3 gene having sequence as illustrated as SEQ ID no.1, and discloses fusion gene containing recombination LFA3 gene and Fc fragment of human Ig antibody fused therewith, and also protein product of said fusion gene. This invention also provides recombination expression vactor containing said fusion gene, host cells, and the application of said recombination LFA3-Ig fusion gene for preparing medicine to treat autoimmune disease. This invented recombination LFA3 fusion gene has higher expression level. This invention has important economic velue for commercial production of said protein.
Description
Technical field
The invention belongs to field of immunology, specifically, relate to a kind of reorganization LFA3 gene, and fusion gene and product.
Background technology
People recognize in recent years, the lymphocytic activation of T needs at least 2 signals, first signal by T cell antigen receptor (TCR) and antigen presenting cell surface MHC/ antigenic peptide complexes in conjunction with being produced, the combination of these two kinds of parts needs specific recognition, with immunoreactive specificity substantial connection is arranged.Second signal is that other accessory molecule of T cell surface combines generation with the respective ligand on antigen presenting cell surface, and this signal is called costimulatory signal again, has had been found that at present multiple part is right.Do not have the stimulation of subsidiary signal if the T cell only is subjected to the stimulation of first signal, the T cell not only is not activated, but also might cause this t cell proliferation, causes clonal anergy (anergy) in vivo.
T cell, antigen presenting cell surface participate in producing altogether, and the molecule of hormesis is referred to as costimulatory molecules.The common stimulation path of generally acknowledging comprises CD2:LFA3 approach, B7:CD28 approach and CD11a:ICAM-1 approach etc. at present.These signal transduction paths are having a very important role aspect the immunologic function adjusting of T cell, and the blocking-up of any signal transduction path all might cause the immunologic function downward modulation.
Principle can be developed autoimmune disease such as psoriatic medicine whereby.From the genetically engineered angle, the strategy of developing this class medicine mainly contains three kinds.A kind of is the monoclonal antibody of development signalling channel associated protein, and a kind of is the receptor protein of development signalling channel associated protein, and a kind of is the antagonist that utilizes the signalling channel associated protein.These three kinds of strategies all are to utilize a kind of (or multiple) to come the disabling signal conductive process with signalling channel protein-specific bonded albumen.
CD2 claims LFA 2 again, and (lymphocyte function associated antigen 2 LFA-2) or sheep red blood cell (SRBC) (SRBC) acceptor, for striding the film single chain molecule, is expressed in T cell, thymocyte and NK cell.The about 50kDa of molecular weight, its maturation protein is made up of 332 amino acid, and its precursor is by 19 signal peptides that amino acid is formed.The part of people CD2 molecule is CD58 (LFA-3), and its structure is similar to CD2, and also for striding the film strand, its precursor protein has 28 amino acid whose signal peptides, and maturation protein is made up of 233 amino acid.It is above-mentioned that both all belong to the Ig superfamily member.CD58 is distributed more widely, is expressed in T, B cell, monocyte, dendritic cell, neutrophil leucocyte, thrombocyte, red corpuscle and some non-hematopoietic cell surface.CD2 combines with CD58, can strengthen between T cell and APC or target cell to adhere to, and promotes the T cell to antigen recognition and CD2 institute Mediated Signal Transduction.
U.S. BIOGEN company has developed a kind of FLA3-Ig fusion rotein (trade(brand)name Amevive), because this fusion rotein has kept and part CD2 bonded FLA3 film outskirt, after the T cells contacting of CD2 molecule being arranged with expression, can combine with CD2, the LFA3 molecule on blocking-up antigen presenting cell (APC) surface combines with CD2, thereby the activation of blocking t cell, propagation and differentiation suppress IL-2, production of cytokines such as IFN-γ, IL-8.On the other hand, the Fc section of fusion rotein can with the Fc receptors bind of cell surface, make monocyte, NK cell to T cell aggregation, by the cell-mediated cytotoxicity (ADCC) that antibody relies on, cause the apoptosis of T cell.Be used for the treatment of psoriatic after the Amevive listing, and obtained good therapeutic action.But also have many psoriatics that the treatment of Amevive is shown as anergy, therefore, need the substitute products that a specific specificity is stronger, avidity is higher clinically.
Summary of the invention
First technical problem that the present invention need solve provides a kind of new reorganization LFA3 gene, has the structure of natural LFA3 gene-joint-natural LFA3 gene.
Second technical problem that the present invention need solve provides a kind of new fusion gene, the people Ig antibody Fc fragment gene that contains above-mentioned reorganization LFA3 gene and merge with it.
The 3rd technical problem that the present invention need solve provides a kind of recombinant expression vector that contains this fusion gene.
The 4th technical problem that the present invention need solve provides a kind of host cell that contains this recombinant expression vector.
The 5th technical problem that the present invention need solve provides a kind of purposes of protein expressioning product in preparation treatment autoimmune disease medicine of the LFA3-Ig of reorganization fusion gene.
Inventive concept of the present invention is such:
Utilize engineered method, specific receptors protein molecular and immunoglobulin (Ig) (antibody) artificially that is positioned at surface of cell membrane originally can be combined, form fusion rotein.Generally be to get the cell outskirt of coding membranin and the dna sequence dna of antibody constant region, connect into fusion gene, in appropriate carriers and cell, express, obtain non-existent fusion protein molecule under the native state, its existing cell surface receptor part bonded original characteristic with it, be shla molecule as antibody again, and long half time, action time is comparatively lasting.Compare with humanized antibody, the avidity of acceptor (part) is stronger, and all protein ingredient all is that human body is intrinsic, and therefore humanized degree can not cause immune response in vivo than the antibody height of coming from the antibody transformation of mouse source.At present existing multiple soluble receptors is produced out, and the different cell-membrane receptor cell outskirts according to they contain can produce various specific functions.The ligand L FA-3 of people CD2 molecule then can combine with the CD2 molecule competitively, and this stimulates path altogether thereby block CD2/LFA3.
Simultaneously, for avoiding in fusion rotein occurring non-natural aminoacid sequence, we do not design restriction enzyme site at LFA3 with the junction of Fc section and are connected, but employing Overlaping PCR method connects.
In the present invention, " LFA3-Ig fusion rotein " is meant by people LFA3 soluble part and people's antibody Fc fragment and merges formed fusion rotein.
Technology contents of the present invention is as follows:
The invention discloses a kind of reorganization LFA3 gene, it is characterized in that, described reorganization LFA3 gene has the structure of natural LFA3 gene-joint-natural LFA3 gene, described reorganization LFA3 gene has the sequence shown in the SEQID NO:1, and the aminoacid sequence of the sequence encoding of described joint is (Ala
3Ser
2)
3, that is SEQID NO:2.One of dna sequence dna of this aminoacid sequence is: 5 '-GCTGC TGCTT CTTCT GCCGCTGCTT CTTCT GCTGC CGCTT CTTCT-3 ' (SEQ ID NO:3).
The invention also discloses a kind of fusion gene, it is characterized in that, the people Ig antibody Fc fragment gene that described fusion gene contains above-mentioned reorganization LFA3 gene and merges with it, its sequence is shown in SEQ ID NO:4.
The protein of this fusion gene comprises reorganization LFA3 and people Ig antibody Fc fragment, and its sequence is shown in SEQ IDNO:5.
The present invention also provides a kind of recombinant expression vector, and this recombinant expression vector contains the LFA3-Fc fusion gene, but transfection mammalian cell and realize high expression level.Described carrier can be the expression vector of various suitable transfection mammalian cells well known in the art.
The invention also discloses a kind of host cell, it is characterized in that, described host cell contains above-mentioned recombinant vectors.Described host cell is selected from Chinese hamster ovary celI, Hela cell, various Mammals engineering cell strains commonly used such as mouse Ltk-(4) cell and monkey kidney VERO cell.
Reorganization LFA3 gene provided by the present invention, because this gene is formed by connecting by the joint sequence of two natural LFA3 genes by artificial design, had two CD2 combining sites, increased avidity, can combine with CD2 by the surperficial LFA3 molecule of more effective blocking-up antigen presenting cell (APC), thereby the activation of blocking t cell, propagation and differentiation suppress IL-2, production of cytokines such as IFN-γ, IL-8.Recombinant vectors by making up makes the protein product expression amount of this LFA3-Ig fusion gene keep higher level simultaneously.
The present invention also provides the purposes of protein product in preparation treatment autoimmune disease medicine of this reorganization LFA3-Ig fusion gene, autoimmune disease comprises rheumatoid arthritis, ankylosing spondylitis, graft-rejection, psoriatic, systemic lupus erythematous, mouthful xerophthalmia scheorma syndrome, scleroderma, polyarteritis nodosa, Wegener granulomatosis etc., preferred psoriatic here.
Description of drawings
Fig. 1: several signalling channels of APC cell and T cell interaction.
Fig. 2: LFA3-Ig fusion rotein structural representation, connect with joint between two LFA3, LFA3-Ig utilizes the extracellular region of the LFA3 that recombinant DNA technology makes up and the fusion rotein of people's IgG antibody Fc section, its N-end contains the extracellular section of people LFA3, follows heavy chain hinge area, CH2 and CH3 district by the human IgG1.
Fig. 3: the amplification synoptic diagram of LFA3-Ig fusion gene, wherein, 1,2,3,4,5,6 represent primer 1,2,3,4,5,6 respectively.
Fig. 4: fusion rotein of the present invention and Amevive are to the inhibition result of mixed lymphocyte reacion.
Embodiment
Below in conjunction with the present invention of embodiment further detailed description, yet should be appreciated that enumerating these embodiment is for an illustration, and be not to be used for limiting the present invention.
The design and the preparation of embodiment 1 reorganization LFA3 fusion gene
1, design of primers
Primer 1:5 '-GCTAGCGCCGCCACCATGGTTGCTGGGAGCGACGCGGGG-3 ' (SEQ ID NO:6)
Primer 2: 5 '-AGCAGAAGAAGCAGCGGCAGAAGAAGCAGCAGCATTAGTCAATGCACA-3 ' (SEQID NO:7)
Primer 3:5 '-TCTGCCGCTGCTTCTTCTGCTGCCGCTTCTTCTTTTTCCCAACAAATA (SEQ IDNO:8)
Primer 4:5 '-
TGTGTGAGTTTTGTCAGTTAGTGTGGGAGA-3 ' (SEQ ID NO:9)
Primer 5:5 '-TCTCCCACACTAACT
GACAAAACTCACACA-3 ' (SEQ ID NO:10)
Primer 6:5 '-TACTCGAGTCATTTACCCGG-3 ' (SEQ ID NO:11)
Primer 4,5 is complementary fully, and 5 are 5 '-adopted primer is arranged, and 4 is 3 '-antisense primer, the codon of non-underscore part and the goal gene amino acid whose cDNA sequence in LFA3 ending of encoding; The codon of underscore part and the goal gene IgG that encodes wherein
1The complementary sequence of the start-up portion amino acid cDNA of Fc section;
2, the design and the preparation of reorganization LFA3 gene
Get normal people's peripheral blood 10ml, separate mononuclearcell, get 1 * 10 with the Ficoll density gradient method
7Cell with PBS washing back centrifugation, is abandoned supernatant, with TRIZAL reagent extracted total RNA.
The operation steps that requires according to test kit is a template with total RNA of PBMC, and primer 1,2 and primer 3,4 carry out RT-PCR respectively according to ordinary method.
The product that obtains with above-mentioned RT-PCR is a template, and primer is 1,4 to carry out Overlap-PCR, and reaction conditions carries out according to a conventional method.Obtaining single band that molecular size is about 801bp on agarose gel electrophoresis carries out gel and reclaims.PCR product cloning test kit with Promega company inserts the cDNA that is obtained in the pGEM-T carrier, and ordinary method transformed into escherichia coli TG1 bacterial strain is just selected candidate clone through blue hickie screening, and PCR identifies and confirms positive colony, carries out the cDNA order-checking then.Sequencing result shows that the gene order and the implementation sequence that obtain are in full accord.
The reorganization LFA3 gene that is obtained is to be connected by joint sequence between two LFA3 genes, and the dna sequence dna of this joint sequence is SEQ ID NO:3.Its amino acid sequence coded is (Ala
3Ser
2)
3(SEQ IDNO:2).One of dna sequence dna of this aminoacid sequence is: 5 '-GCTGC TGCTT CTTCT GCCGCTGCTT CTTCT GCTGC CGCTT CTTCT-3 ' (SEQ ID NO:3).
3, the segmental acquisition of human IgG1 Fc:
With the carrier pMG18 that contains human IgG1's constant region gene is lamina membranacea, and primer 5,6 carries out PCR by following method:
The composition of reaction system:
| Composition | Quantity (μ l) |
| 10X PCR reaction buffer 25 mMMg 2SO 4Pfu archaeal dna polymerase (5U/ μ l)) primer (2 μ g/ μ l) template DNA (1.8 μ g/ μ l) sterilization tri-distilled water | 10 10 2 each 3 μ l amount to 6 μ l, 1 to 100 μ l |
Reaction conditions:
Pre-sex change: 94 ℃, 2 minutes;
Major cycle: 94 ℃, 1 minute; 55 ℃, 1 minute; 72 ℃, 3 minutes;
Cycle number: 20
Extend the back: 72 ℃, and 5 minutes.
The PCR process is carried out on PROGENE GENIUS thermal cycler.
Obtain the Fc section of IgG1.The PCR product carries out gel glue and reclaims, PCR product cloning test kit with Promega company inserts the cDNA that is obtained in the pGEM-T carrier, ordinary method transformed into escherichia coli TG1 bacterial strain, just select candidate clone through blue hickie screening, PCR identifies and has confirmed positive colony, carries out the cDNA order-checking then.Sequencing result shows that gene order of obtaining and theoretical sequence are in full accord.
4, the acquisition of LFA3-Ig fusion gene
With above-mentioned steps 2 and 3 fragments that obtain is template, and primer is 1,6, carries out PCR by the method for above-mentioned steps 3, amplifies 1-6 fragment (LFA3)
2-Ig.After finishing product is identified on 0.8% agarose gel electrophoresis that finding has the single band of a molecular weight about 1455bp in the product, consistent with 1455 sizes of expection.PCR product cloning test kit with Promega company inserts the cDNA that is obtained in the pGEM-T carrier, ordinary method transformed into escherichia coli TG1 bacterial strain, just select candidate clone through blue hickie screening, PCR identifies and confirms positive colony, carry out the cDNA order-checking then, confirm the right-on clone of sequence, be LFA3-Ig.
1, the structure of integrative gene expression vector
Use Nhe I and Xho I (Phamacia) (10U/ul) to digest pUC 18 and expression plasmid pMSG (Phamacia) respectively the product LFA3-Ig gene of embodiment 1.37 ℃ of reactions of endonuclease reaction are spent the night.Enzyme is cut product and carry out isolation identification on 0.8% agarose electrophoresis, with the big or small correct fragment of size that is about of gel recovery test kit recovery of Promega company.
Use T4 dna ligase (10U/uI) LFA3/ joint/Fc to be connected to the Nhe I and the Xho I site of pMSG carrier respectively.
According to ordinary method (seeing Sambrook etc., 1989) connection gained reorganization pMSG transformed into escherichia coli DH5a bacterial strain, and on the IPTG/X-Gal plate culture medium, carry out blue hickie screening.Respectively get 10 of hickies and be inoculated in and extract plasmid after 37 ℃ of cultivations of LB liquid nutrient medium and carry out enzyme as previously mentioned and cut evaluation, every kind of fusion gene obtains 4 segmental clones of insertion with correct size at least, as candidate clone.
Carry out dna sequencing with two in the above-mentioned candidate clone.According to sequencing result, confirm to insert the in full accord of fragments sequence and design respectively.
2, the conversion of Chinese hamster ovary celI and expression
Get the coli strain that has above-mentioned reorganization pMSG, be inoculated in the 2xYT liquid nutrient medium that 500mI has added penbritin respectively, 37 ℃, the 260rpm concussion was cultivated 16 hours.With " Ultrapure Plasmid Purification Kit " extracting plasmid DNA of Qiagen company, extractive process is carried out according to the specification sheets that producer provides.
Use the liposome transfection Chinese hamster ovary celI, the transfection reagent box available from Invitrogene company (lipofectamine2000 transfection reagent, 11668-027).Get pMSG plasmid 100 micrograms of above-mentioned purifying during transfection and as the DNA sample Chinese hamster ovary celI is carried out transfection, the transfection schedule of operation is carried out according to the specification sheets of producer.
Chinese hamster ovary celI after the transfection is through continuous 3 months methotrexate (MTX) screening, and to 10 μ M, per two weeks increase a concentration to its concentration from 0.05 μ M, and the consumption of each MTX is about previous 2 times, concrete visual cell's growing state and deciding.Cell cultures is carried out according to routine, and substratum is: 15% foetal calf serum (Gibco)+RPMI 1640/DEME, and in 37 ℃, 5%CO
2Cultivate in the incubator.Carry out mono-clonalization according to routine extreme dilution method then.Use the ELISA method and detect its Expression of Fusion Protein amount respectively, obtain cloning 89 altogether.
The conventional cultivation in the DEME substratum studied through the expression intensity of the above-mentioned part clone of ELISA LFA3-Ig, and concrete data are as follows:
| Tested clone's quantity | 44 |
| Average expression level (mg/L supernatant) | 25.2±0.33 |
Above data declaration, reorganization LFA3-Ig fusion gene expression level of the present invention is higher, and this proteinic scale production is had important economic value.
People T lymphoma cell Jurkat surface expression CD2, we have utilized Flow cytometry recombinant human LFA3-Ig fusion rotein and Amevive
Combine the ability of Jurkat surface C D2 with anti-CD2 monoclonal antibody TS2/18 (ATCC HB195) competition.
1, test principle
People T lymphoma cell line Jurkat (ATCC TIB152) surface of cell membrane is expressed CD2, LFA3 and anti-CD2 monoclonal antibody all can with the CD2 specific combination.Fluorescently-labeled anti-CD2 monoclonal antibody and cell bonded amount effect curve should be serpentine, and promptly along with the increase of marker concentrations, fluorescence intensity also increases gradually, until reach capacity (binding curve); If there be of the competition of LFA3-Ig fusion rotein to FITC-CD2 mAb, under the condition of FITC-CD2 mAb concentration fixed (the sub-saturated point of binding curve), increase along with the LFA3-Ig fusion rotein concentration that adds, cell bonded FITC-CD2 mAb amount reduces gradually, fluorescence intensity weakens, so amount effect curve is anti-serpentine or " second " font; Under the target cell condition identical with the FITC-fusion rotein, the EC50 value is more little, and then the ability of the LFA3-Ig fusion rotein of explanation adding and traget antibody competition is strong more, and promptly the avidity of LFA3-Ig fusion rotein is high more.
2, material and reagent
1) survey the viable cell strain: HTL Jurkat cell (ATCC TIB152), going down to posterity is incubated at RPMI1640/DMEM (1: the 1) nutrient solution that contains 10% new-born calf serum;
2) culture condition: 37 ℃, 5%CO2, saturated humidity;
3) contain the PBS (PBSS) of 1%NBS;
4) recombinant human LFA3-Ig fusion rotein of the present invention;
5) Amevive, Biogen company, lot number P21020;
6) the anti-CD2 monoclonal antibody (dialysis method mark) of fluorescein isothiocyanate (FITC) mark;
7) flow cytometer: the FACScan of U.S. Becton Dickinson company.
3, test method
1) Amevive is diluted to 2000 μ g/mL with PBSS, adds the FITC-CD2 mAb of equal-volume 4 μ g/mL.
2) rhLFA3-Ig fusion rotein is diluted to 200 μ g/mL with PBSS, adds the FITC-CD2 mAb of equal-volume 4 μ g/mL.
3) use continuous 2 doubling dilution Amevive of FITC-CD2 mAb and the rhLFA3-Ig fusion rotein of the present invention of 2 μ g/mL then respectively.
4) the Jurkat cell of logarithmic phase, PBSS adjusts cell density to 1 * 10
6/ mL divides cell suspension then to the streaming dedicated pipe, the 0.1mL/ pipe.
5) 200g is centrifugal 5 minutes, abandons supernatant.
6) the serial dilution liquid with Amevive and rhLFA3-Ig fusion rotein of the present invention adds in the streaming pipe of step 5,0.1ml/ pipe, each concentration is established 2 multiple pipes, establish 3 groups of contrasts in addition, 1. the FITC-CD2mAb that only adds 2 μ g/mL is a blank, 2. add the negative contrast of cell suspension of 2 μ g/mL FITC-CD2 mAb and 1000 μ g/mL homotype antibody, the cell suspension that 3. only adds PBSS is similar to positive control, and 2 multiple pipes are established in every group of contrast.Ice bath lucifuge 45 minutes.
7) the PBSS washed cell twice, and centrifugal 5 minutes of 200g is resuspended among the 300 μ L PBSS.
8) go up machine testing.
4, interpretation of result
Adopt Origin6.0 match typical curve: X-coordinate is Amevive or rhLFA3-Ig fusion rotein concentration of the present invention, and ordinate zou is the mean number of fluorescence intensity geometric mean, and curve is anti-serpentine, selects Logistic4 parametric equation regression model for use.
The regression equation that this software provides is:
Y＝(A-B)/[1+(X/C)D]+B
According to this equation, during X=+ ∞, Y=B, i.e. the highest limit; When X=0, Y=A, i.e. minimum; And when X=C, Y=(A+B)/2, promptly half is effective.Therefore the numerical value of C is medium effective concentration (EC50).
5, result
Table 1:Amevive and rhLFA3-Ig fusion rotein of the present invention competition are summed up in conjunction with test-results
| Experiment numbers | EC50(μg/ml) | Relative affinity | |
| The rhLFA3-Ig fusion rotein | |
||
| 1 | 1.1 0 | 10.34 | 9.4 |
| 2 | 1.17 | 11.62 | 9.9 |
| 3 | 1.12 | 10.05 | 9.0 |
| 4 | 1.08 | 11.14 | 10.3 |
| 5 | 1.02 | 10.30 | 10.1 |
Relative affinity is the ratio of rhLFA3-Ig fusion rotein avidity and Amevive, and the data from table 1 can see that the avidity of LFA3-Ig provided by the present invention is about 10 times of reference substance Amevive.
Embodiment 4 rhLFA3-Ig fusion roteins are to the inhibition of mixed lymphocyte reacion
1, test principle
Mixed lymphocytes is cultivated (mixed lymphocyte culture, MLC) or claim mixed lymphocyte reacion (mixed lymphocyte reaction, MLR) be to derive from the lymphocyte of two individualities when external mixed culture, because the DR in the cell surface HLA-II class antigen is different with DP antigen, can stimulate the other side's lymphocyte transformation (being called two-way MLC) mutually, and produce multiple lymphokine, promote killer cell activities such as NK, CTL, LAK.If side's lymphocyte that will stimulate is handled with ametycin (mitomycine C) or radiation exposure earlier, make this cell lose multiplication capacity, but still keep stimulating the test of the other side's lymphopoiesis ability to be called unidirectional MLC; As not adding above-mentioned processing, then both sides stimulate mutually, are called two-way MLC.The MLC reaction can be by the multiplication capacity of 3H-TdR incorporation efficiency or morphologic detection method and mtt assay detection reaction cell.Stimulate mutually in the process of conversion at lymphocyte, mutually combining between costimulatory molecules plays an important role, and CD2/LFA-3 is that important costimulatory molecules is right, if add exogenous LFA3, then can compete bind lymphocytes surface C D2, thereby suppresses MLR.
2, material and reagent
1) glass fiber filter and negative pressure leaching device, scintillation solution and liquid scintillation counter;
2) desk centrifuge: Eppendorf company product;
3) Biohazard Safety Equipment: prosperous company product is made in Taiwan;
4) cell culture incubator: U.S. Revco product;
5) flow cytometer: the FACScan of U.S. Becton Dickinson company;
6) culturing bottle, centrifuge tube, transfer pipet, pipettor, aseptic suction nozzle;
7) 3H-TdR: Shanghai nuclear research institute of Chinese Academy of Sciences product, specific radioactivity 30Ci/mmol, radioactive concentration 1mCi/mL.
8) lymphocyte separation medium
9) heparin
10) cell culture reagent, calf serum, Hangzhou folium ilicis chinensis bio-engineering research institute product; The RPMI1640 substratum, GIBCO company product.
11) phosphate buffered saline (PBS) and contain the PBS (PBSS) of 1% calf serum.
12) the fresh peripheric venous blood of healthy volunteer.
3, test method
Get two healthy volunteers (A, B) anticoagulant heparin venous blood 10mL respectively, lymphocyte layering liquid density gradient centrifugation separates peripheral blood mononuclear cell (PBMC), serum-free RPMI1640 washing 3 times, each centrifugal 10min of 1000rpm.
Count, and adjust cell concn to 2.0 * 10 with the RPMI1640 complete culture solution that contains 20%FCS
5/ mL.
The rhLFA3-Ig fusion rotein is diluted to 4000ng/mL with the RPMI1640 complete culture solution that contains 20%FCS;
With the RPMI1640 complete culture solution dilution rhLFA3-Ig fusion rotein that contains 20%FCS, to concentration be 4000,2000,1000,500,250 and 125ng/mL (note: sample also will dilute 1 times when adding in isopyknic cell suspension in subsequent step, thus final concentration be respectively 2000,1000,500,250,125 and 62.5ng/mL).
Amevive is diluted to 40000ng/mL with the RPMI1640 complete culture solution that contains 20%FCS;
With the RPMI1640 complete culture solution dilution Amevive that contains 20%FCS, to concentration be 40000,20000,10000,5000,2500 and 1250ng/mL (note: sample also will dilute 1 times when adding in isopyknic cell suspension in subsequent step, thus final concentration be respectively 20000,10000,5000,2500,1250 and 625ng/mL).
The lymphocyte suspension of A, two Different Individual of B detects two-way MLR, and each hole adds each 50 μ L of two kinds of cell suspensions in 96 porocyte culture plates, and the most weak reaction pair adds the mononuclearcell 100 μ L of same individuality according to (min).
RhLFA3-Ig fusion rotein and Amevive diluent are added above-mentioned 96 porocyte culture plates, 100 μ L/ holes, the strongest reaction pair adds the RPMI1640 complete culture solution that contains 20%FCS according to (max) hole (be respectively and contain two kinds every group different lymphocytic holes), 100 μ L/ holes, the most weak reaction pair is according to also adding the RPMI1640 complete culture solution that contains 20%FCS, and 100 μ L/ holes are provided with 3 multiple holes, 37 ℃, the 5%CO2 incubator is cultivated 6d.
The every hole of 20h adds 0.5Ci 3H-TdR before stopping cultivating.
With cell harvestor cell is adsorbed on the glass fiber filter paper sheet, uses the physiological saline thorough washing, flush away free 3H-TdR; After the filter paper oven dry, put into the plastics tubing that fills scintillation solution respectively successively by its order with tweezers; The automatic working sample 1min of β scintiloscope.
4, interpretation of result
The calculating stimulation index (Stimulator Index, SI):
SI=(add sample mix lymphocyte reaction cpm average-the most weak reaction pair and look after the cpm average)/(the strongest reaction pair is looked after cpm average-the most weak reaction pair and looked after the cpm average).
Table 2:rhLFA3-Ig fusion rotein is to the restraining effect of MLR
| Concentration (ng/ml) | The rhLFA3-Ig fusion rotein | |
| 3H-TdR incorporation (cpm) | SI(％) | |
| /(A) /(B) | 1383±212 1405±112 | / / |
| 2,000 1,000 500 250 125 63 the strongest contrasts | 4363±182 4577±259 8442±224 11651±725 17185±714 20784±451 21423±919 | 14.9±0.91 15.9±1.29 35.2±1.12 51.2±3.62 78.9±3.56 96.8±2.25 100 |
Table 3:Amevive is to the restraining effect of MLR
| Concentration (ng/ml) | Amevive | |
| 3H-TdR incorporation (cpm) | SI(％) | |
| / (A)/(B) 20,000 10,000 5,000 2,500 1,250 625 the strongest contrasts | 1383±212 1405±112 4012±273 4115±207 7572±324 10436±220 15420±263 18433±243 21423±919 | / / 13.1±1.36 13.6±1.03 30.9±1.62 45.2±1.10 70.0±1.31 85.1±1.21 100 |
Draw the rejection ratio illustration 4 of rhLFA3-Ig fusion rotein and Amevive, the rhLFA3-Ig fusion rotein can obviously suppress MLR, and the inhibition ability is 10 times of Amevive.
Above result shows that rhLFA3-Ig fusion rotein of the present invention all significantly is being better than Amevive aspect avidity, the specificity.
Sequence table
＜110〉Shanghai CP Guojian Pharmaceutical Co.,Ltd
＜120〉a kind of reorganization LFA3 gene, and fusion gene and product
<150>CN200610026070.X
<151>2006-04-26
<160>11
<170>PatentIn version 3.0
<210>1
<211>771
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(771)
＜223〉reorganization LFA3 nucleotide sequence
<400>1
ATGGT TGCTG GGAGC GACGC GGGGC GGGCC CTGGG GGTCC TCAGC GTGGT CTGCC TGCTG 60
CACTG CTTTG GTTTC ATCAG CTGTT TTTCC CAACA AATAT ATGGT GTTGT GTATG GGAAT 120
GTAAC TTTCC ATGTA CCAAG CAATG TGCCT TTAAA AGAGG TCCTA TGGAA AAAAC AAAAG 180
GATAA AGTTG CAGAA CTGGA AAATT CTGAA TTCAG AGCTT TCTCA TCTTT TAAAA ATAGG 240
GTTTA TTTAG ACACT GTGTC AGGTA GCCTC ACTAT CTACA ACTTA ACATC ATCAG ATGAA 300
GATGA GTATG AAATG GAATC GCCAA ATATT ACTGA TACCA TGAAG TTCTT TCTTT ATGTG 360
CTTGA GTCTC TTCCA TCTCC CACAC TAACT TGTGC ATTGA CTAAT GCTGC TGCTT CTTCT 420
GCCGC TGCTT CTTCT GCTGC CGCTT CTTCT TTTTC CCAAC AAATA TATGG TGTTG TGTAT 480
GGGAA TGTAA CTTTC CATGT ACCAA GCAAT GTGCC TTTAA AAGAG GTCCT ATGGA AAAAA 540
CAAAA GGATA AAGTT GCAGA ACTGG AAAAT TCTGA ATTCA GAGCT TTCTC ATCTT TTAAA 600
AATAG GGTTT ATTTA GACAC TGTGT CAGGT AGCCT CACTA TCTAC AACTT AACAT CATCA 660
GATGA AGATG AGTAT GAAAT GGAAT CGCCA AATAT TACTG ATACC ATGAA GTTCT TTCTT 720
TATGT GCTTG AGTCT CTTCC ATCTC CCACA CTAAC TTGTG CATTG ACTAA T 771
<210>2
<211>15
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(15)
＜223〉joint aminoacid sequence
<400>2
Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser
1 5 10 15
<210>3
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(45)
＜223〉joint nucleotide sequence
<400>3
GCTGC TGCTT CTTCT GCCGC TGCTT CTTCT GCTGC CGCTT CTTCT 45
<210>4
<211>1455
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(1455)
＜223〉fusion gene nucleotide sequence
<400>4
ATGGT TGCTG GGAGC GACGC GGGGC GGGCC CTGGG GGTCC TCAGC GTGGT CTGCC TGCTG 60
CACTG CTTTG GTTTC ATCAG CTGTT TTTCC CAACA AATAT ATGGT GTTGT GTATG GGAAT 120
GTAAC TTTCC ATGTA CCAAG CAATG TGCCT TTAAA AGAGG TCCTA TGGAA AAAAC AAAAG 180
GATAA AGTTG CAGAA CTGGA AAATT CTGAA TTCAG AGCTT TCTCA TCTTT TAAAA ATAGG 240
GTTTA TTTAG ACACT GTGTC AGGTA GCCTC ACTAT CTACA ACTTA ACATC ATCAG ATGAA 300
GATGA GTATG AAATG GAATC GCCAA ATATT ACTGA TACCA TGAAG TTCTT TCTTT ATGTG 360
CTTGA GTCTC TTCCA TCTCC CACAC TAACT TGTGC ATTGA CTAAT GCTGC TGCTT CTTCT 420
GCCGC TGCTT CTTCT GCTGC CGCTT CTTCT TTTTC CCAAC AAATA TATGG TGTTG TGTAT 480
GGGAA TGTAA CTTTC CATGT ACCAA GCAAT GTGCC TTTAA AAGAG GTCCT ATGGA AAAAA 540
CAAAA GGATA AAGTT GCAGA ACTGG AAAAT TCTGA ATTCA GAGCT TTCTC ATCTT TTAAA 600
AATAG GGTTT ATTTA GACAC TGTGT CAGGT AGCCT CACTA TCTAC AACTT AACAT CATCA 660
GATGA AGATG AGTAT GAAAT GGAAT CGCCA AATAT TACTG ATACC ATGAA GTTCT TTCTT 720
TATGT GCTTG AGTCT CTTCC ATCTC CCACA CTAAC TTGTG CATTG ACTAA TGACA AAACT 780
CACAC ATGCC CACCG TGCCC AGCAC CTGAA CTCCT GGGGG GACCG TCAGT CTTCC TCTTC 840
CCCCC AAAAC CCAAG GACAC CCTCA TGATC TCCCG GACCC CTGAG GTCAC ATGCG TGGTG 900
GTGGA CGTGA GCCAC GAAGA CCCTG AGGTC AAGTT CAACT GGTAC GTGGA CGGCG TGGAG 960
GTGCA TAATG CCAAG ACAAA GCCGC GGGAG GAGCA GTACA ACAGC ACGTA CCGTG TGGTC 1020
AGCGT CCTCA CCGTC CTGGG GCAGG ACTGG CTGAA TGGCA AGGAG TACAA GTGCA AGGTC 1080
TCCAA CAAAG CCCTC CCAGC CCCCA TCGAG AAAAC CATCT CCAAA GCCAA AGGGC AGCCC 1140
CGAGA ACCAC AGGTG TACAC CCTGC CCCCA TCCCG GGATG AGCTG ACCAA GAACC AGGTC 1200
AGCCT GACCT GCCTG GTCAA AGGCT TCTAT CCCAG CGACA TCGCC GTGGA GTGGG AGAGC 1260
AATGG GCAGC CGGAG AACAA CTACA AGACC ACGCC TCCCG TGCTG GACTC CGACG GCTCC 1320
TTCTT CCTCT ACAGC AAGCT CACCG TGGAC AAGAG CAGGT GGCAG CAGGG GAACG TCTTC 1380
TCATG CTCCG TGATG CATGA GGCTC TGCAC AACCA CTACA CGCAG AAGAG CCTCT CCCTG 1440
TCTCC CGGTA AATGA 1455
<210>5
<211>456
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(456)
＜223〉fusion gene aminoacid sequence
<400>5
Phe Ser Gln Gln Ile Tyr Gly Val Val Tyr Gly Asn Val Thr Phe His
1 5 10 15
Val Pro Ser Asn Val Pro Leu Lys Glu Val Leu Trp Lys Lys Gln Lys
20 25 30
Asp Lys Val Ala Glu Leu Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser
35 40 45
Phe Lys Asn Arg Val Tyr Leu Asp Thr Val Ser Gly Ser Leu Thr Ile
50 55 60
Tyr Asn Leu Thr Ser Ser Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro
65 70 75 80
Asn Ile Thr Asp Thr Met Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu
85 90 95
Pro Ser Pro Thr Leu Thr Cys Ala Leu Thr Asn Ala Ala Ala Ser Ser
100 105 110
Ala Ala Ala Ser Ser Ala Ala Ala Ser Ser Phe Ser Gln Gln Ile Tyr
115 120 125
Gly Val Val Tyr Gly Asn Val Thr Phe His Val Pro Ser Asn Val Pro
130 135 140
Leu Lys Glu Val Leu Trp Lys Lys Gln Lys Asp Lys Val Ala Glu Leu
145 150 155 160
Glu Asn Ser Glu Phe Arg Ala Phe Ser Ser Phe Lys Asn Arg Val Tyr
165 170 175
Leu Asp Thr Val Ser Gly Ser Leu Thr Ile Tyr Asn Leu Thr Ser Ser
180 185 190
Asp Glu Asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr Asp Thr Met
195 200 205
Lys Phe Phe Leu Tyr Val Leu Glu Ser Leu Pro Ser Pro Thr Leu Thr
210 215 220
Cys Ala Leu Thr Asn Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
225 230 235 240
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
245 250 255
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
260 265 270
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
290 295 300
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
305 310 315 320
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
325 330 335
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
340 345 350
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
355 360 365
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
370 375 380
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
385 390 395 400
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
405 410 415
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
420 425 430
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
435 440 445
Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210>6
<211>39
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(39)
＜223〉primer nucleotide sequence
<400>6
GCTAG CGCCG CCACC ATGGT TGCTG GGAGC GACGC GGGG 39
<210>7
<211>48
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(48)
＜223〉primer nucleotide sequence
<400>7
AGCAG AAGAA GCAGC GGCAG AAGAA GCAGC AGCAT TAGTC AATGC ACA 48
<210>8
<211>48
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(48)
＜223〉primer nucleotide sequence
<400>8
TCTGC CGCTG CTTCT TCTGC TGCCG CTTCT TCTTT TTCCC AACAA ATA 48
<210>9
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(30)
＜223〉primer nucleotide sequence
<400>9
TCTCC CACAC TAACT GACAA AACTC ACACA 30
<210>10
<211>30
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(30)
＜223〉primer nucleotide sequence
<400>10
TGTGT GAGTT TTGTC AGTTA GTGTG GGAGA 30
<210>11
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)...(20)
＜223〉primer nucleotide sequence
<400>
TACTC GAGTC ATTTA CCCGG 20
Claims (10)
1. a reorganization LFA3 gene is characterized in that described reorganization LFA3 gene has the structure of natural LFA3 gene-joint-natural LFA3 gene.
2. the described reorganization of claim 1 LFA3 gene is characterized in that, described reorganization LFA3 gene has the sequence shown in the SEQ ID NO:1.
3. the described reorganization of claim 1 LFA3 gene is characterized in that, the aminoacid sequence of the sequence encoding of described joint is (Ala
3Ser
2)
3, that is SEQ ID NO:2.
4. the described reorganization of claim 3 LFA3 gene is characterized in that, the dna sequence dna of described joint is SEQ ID NO:3.
5. a fusion gene is characterized in that, the people Ig antibody Fc fragment gene that described fusion gene contains claim 1 described reorganization LFA3 gene and merges with it, and its sequence is shown in SEQ IDNO:4.
6. the protein of the described fusion gene of claim 5 comprises reorganization LFA3 and people Ig antibody Fc fragment, and its sequence is shown in SEQ ID NO:5.
7. a recombinant expression vector is characterized in that, described recombinant expression vector contains the described gene order of claim 5.
8. a host cell is characterized in that, described host cell contains the described recombinant vectors of claim 7.
9. the described host cell of claim 8 is characterized in that, described host cell is selected from Chinese hamster ovary celI, Hela cell, one of mouse Ltk-(4) cell and monkey kidney VERO cell.
10. the purposes of the protein of the described fusion gene of claim 6 in the medicine of preparation treatment autoimmune disorder.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710101482 CN101089180A (en) | 2006-04-26 | 2007-04-23 | Recombination LFA3 gene, and its mixed gene and product |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610026070 | 2006-04-26 | ||
| CN200610026070.X | 2006-04-26 | ||
| CN 200710101482 CN101089180A (en) | 2006-04-26 | 2007-04-23 | Recombination LFA3 gene, and its mixed gene and product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101089180A true CN101089180A (en) | 2007-12-19 |
Family
ID=38942681
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710101482 Pending CN101089180A (en) | 2006-04-26 | 2007-04-23 | Recombination LFA3 gene, and its mixed gene and product |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101089180A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177613A (en) * | 2017-07-18 | 2017-09-19 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity |
| WO2019190984A1 (en) * | 2018-03-29 | 2019-10-03 | Pfizer Inc. | Lfa3 variants and compositions and uses thereof |
-
2007
- 2007-04-23 CN CN 200710101482 patent/CN101089180A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177613A (en) * | 2017-07-18 | 2017-09-19 | 哈尔滨紫霞生物科技有限公司 | A kind of method for improving restructuring Porcine interferon-gamma fusion protein antiviral activity |
| WO2019190984A1 (en) * | 2018-03-29 | 2019-10-03 | Pfizer Inc. | Lfa3 variants and compositions and uses thereof |
| CN112236446A (en) * | 2018-03-29 | 2021-01-15 | 弗埃塞股份有限公司 | LFA3 variants and compositions and uses thereof |
| AU2019242451B2 (en) * | 2018-03-29 | 2024-05-09 | Pfizer Inc. | LFA3 variants and compositions and uses thereof |
| US12103958B2 (en) | 2018-03-29 | 2024-10-01 | Pfizer Inc. | Lymphocyte function-associated antigen 3 (LFA3) variant polypeptides and methods of use thereof to treat CD2-mediated immune diseases, disorders or conditions |
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