CN102241776B - RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof - Google Patents
RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein and preparation method and application thereof Download PDFInfo
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- CN102241776B CN102241776B CN 201110135019 CN201110135019A CN102241776B CN 102241776 B CN102241776 B CN 102241776B CN 201110135019 CN201110135019 CN 201110135019 CN 201110135019 A CN201110135019 A CN 201110135019A CN 102241776 B CN102241776 B CN 102241776B
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Abstract
The invention provides an RANKL-TNF (Receptor Activator of Nuclear Factor Kappa-B Ligand-Tumour Necrosis Factor) sample region fusion protein, and in particular relates to a fusion protein of an RANKL-TNF sample region protein and a Th2 cell epiposition segment with broad host range property. An experiment shows that an antibody produced by an RANKL-TNF sample region fusion protein immune mouse has the effect of combining RANKL with TNF-alpha, can be used for treating diseases such as rheumatoid arthritis and the like and can play effects of antagonistic bone destruction and inflammation inhibition. The RANKL-TNF sample region fusion protein disclosed by the invention can be used as a therapeutic vaccine and provides a new therapeutic approach and thinking for the diseases such as rheumatoid arthritis and the like. According to the invention, the RANKL-TNF sample region fusion protein is expressed in vitro, and therefore the method has the advantages of high expression yield, strong controllability and relatively low production cost;, and the product has good immunogenicity and is easy to realize mass production.
Description
Technical field
The present invention relates to biomedicine field, be specifically related to a kind of RANKL-TNF sample district fusion rotein, the invention still further relates to preparation method and the purposes in the treating rheumatoid arthritis vaccine of this fusion rotein.
Technical background
Rheumatoid arthritis (rheumatoid arthritis, RA) be a kind of chronic general autoimmune disease, take arthritis mutilans as feature, lesion characteristic is the aggressiveness synovitis, and the joint cartilage that causes thus and destruction of bone, finally cause joint deformity and disable.High due to the morbidity of RA, disability rate is high, the cause of disease is illustrated not yet fully, particularly RA has caused heavy losses to social economy, labor force, therefore, the research of RA treatment is had important medical science and social and economic significance.And RA is a kind of refractory disease, although existing treatment means is varied, all not satisfactory.The present invention is directed to osteoclasia and the key pathology problem of these two RA that are associated of chronic inflammatory reaction in RA morbidity, development RANKL-TNF sample district fusion rotein is for a kind of new thinking has been opened up in the treatment of RA.
RANKL is the member of tumor necrosis factor superfamily (tumor necrosis factor superfamily, TNFSF), and gene is positioned at No. 13 chromosome 13q14s of people, 316 amino acid of total length, and molecular size range is 35～45kD, minute three hypotype: RANKL
1And RANKL
2Be transmembrane tripolymer (trimeric transmembrane form), RANKL
3Be solvable type monomer (solublemonomer), two types all can lose biological activity with bait acceptor osteoprotegerin (osteoprotegerin, OPG) combination, and human-mouse homologous is 77%.RANKL is I type transmembrane protein, by in born of the same parents, born of the same parents are outer and the cross-film section forms, core texture be 158 amino acid of the outer carboxyl terminal of born of the same parents, is folded by several β types to consist of, and is TNF family homology structural domain.RANKL mainly is distributed in immunity system, Skeletal system and the recycle system.The RANKL that participates in osteoclasia is mainly secreted by T cell, articular synovial cells and the mononuclear macrophage of skeletonization/marrow stromal cell, activation, is combined with acceptor RANK by autocrine and paracrine mode to play a role.The expression of RANKL is subject to the regulation and control of cytokine profiles, as bone resorbing factor, glucocorticosteroid, 1 α, and 25 (OH)
2-VD
3, IL-1, IL-6, IL-11, TNF-α, PGE
2With PTH etc., but these factors induced osteogenesis cell expressing RANKL all, thus promote bone resorption.The discovery of RANKL makes us to osteoclast (osteoclast, OC) differentiation, activation, propagation, further understanding arranged.The acceptor RANK of RANKL (receptor activatorof nuclear factor) is tumor necrosis factor receptor super family (tumor necrosis factor receptorsuperfamily, TNFRSF) member, the assignment of genes gene mapping is in 18q22.1,616 amino acid whose peptide sections, belong to I type transmembrane protein, have one short to contain C-terminals in 21 amino acid whose cells of wearing cytolemma group and 383 amino-acid residues, human-mouse homologous is 60%.Mainly express at mononuclear phagocyte system, comprise osteoclast and precursor cell, lymphocyte, endotheliocyte, inoblast.RANKL increases and is combined with the RANK on osteoclast and precursor cell surface thereof at RA patient's joint cavity expression amount, (TNF receptor-associated factor 6 TRAF6) makes its activation after the combination for RANK born of the same parents' internal area conformational change and its adaptin TNF (tumor necrosis factor) receptor associated factor 6; Protein kinase (the mitogen-activated protein kinase that the TRAF6 of activation activates transcription factor NF-KB or mitogen as a multi-functional second messenger (second messager), MAPK) activate, wherein MAPK has comprised again stress activated protein kinase (c-Jun N-terminalkinases/stress-activated protein kinase, JNK) and two approach of extracellular signal-regulated kinase (extracellular signal-regulated kinases, ERK).JNK can further activate c-Jun and activated protein 1 (AP-1), affects the activity of osteoclast and T nucleus intrinsic factor.ERK is the regulatory factor whether c-Fos expresses, and the latter is important transcription factor in differentiation of osteoclast.NF-κ B is entered the kB site of karyon identification DNA sequence dna as a dimer transcription factor by endochylema after activating; T cell c1 (the nuclear factor ofactivated T cell c1 of calcium signal enabling nf activation, NFATc1) and with promoter region be combined, make differentiation of osteoclast genes involved transcriptional expression, broken bone precursor cell propagation, differentiation, ripe for the osteoclast of function is arranged increase bone resorption.Simultaneously, also there is RANK in the osteoclast surface, makes brokenly the bone increased functionality after RANKL is combined.Meanwhile, express at the RANKL on the T cell of activation and be combined with RANK on self or DC, the energy activating immune system.Experiment in vitro proves, the RANKL that the T cell produces can directly start the propagation of osteoclast; Ctla4
-/-The clpp gene deratization can suppress activating T cell to the destruction of bone density.Vicious cycle between immunity system and skeletal system is the reason of RA osteoclasia sustainable existence.
RA is the systemic inflammation of chronic progress, and many inflammatory factors have participated in rheumatoid pathologic process, and TNF-α is one of inflammatory cytokine that holds the central place in the RA pathogeny, and it has participated in the carcinogenesis of human of RA.But TNF-α inducing endothelial cell is expressed adhesion molecule (inter-cellularadhesion molecule, ICAM), promotes white corpuscle and blood vessel endothelium to stick infiltration and causes local inflammation.In addition, TNF-α stimulates synovial membrane fibroblast and chondrocyte to produce PGE
2(prostaglandin E
2, PGE
2) and collagenase, promote destruction of bone and the absorption of bone and the hyperplasia of fibroblast, suppress the synthetic of osso-albumin.TNF-α also can promote human chondrocytes eccrine fiber albumen lyase activator, make proplasmin become Tryptase and accelerate arthritic loss process, TNF-α has increased the release of synovial membrane endotheliocyte and fibroblast growth factor and has promoted the formation of pannus.TNF-α also can impel synovial cell, scavenger cell, fibroblast and chondrocyte to produce IL-1, IL-8, in addition TNF-α itself and increase the weight of tissue injury.The scholars such as Saxne report that in 50% RA patients serum, the TNF-alpha levels raises, and TNF-α raise mostly be the fast active period patient of joint obvious tumefaction, tenderness and erythrocyte sedimentation rate.Prompting RA arthritis is heavier, and the TNF-alpha levels is higher.TNF-α participates in three kinds of main pathologic processes in reactivity RA: 1. activate vascular endothelial cell, strengthen the expression of endotheliocyte ICAM, blood middle leukocytes is pooled to joint cavity by the interaction with ICAM just when sacroiliitis.2. stimulate phoirocyte and polymorphonuclear cell to produce the small molecules inflammatory mediators such as PGE.3. by stimulating synovial cell and chondrocyte, make osteoclast reduce glycoprotein and synthesize, increase the glycoprotein degraded, and produce collagenase and other neutral proteases, release bone calcium etc., thus the destruction that causes bone and cartilage.Soluble TNF R can alleviate the pathological change of adjuvant-induced arthritis effectively.
Research is found, take RANKL as the destruction that the medicine of blocking target can alleviate RA bone and cartilage, can significantly improve the symptom of the molten bone of metastases and postmenopausal osteoporosis with RANKL monoclonal antibody or RANK-Fc.In an Argentine random double blind test, use studies show that of humanized monoclonal antibody AMG-162 treatment postmenopausal osteoporosis patient, the RANKL monoclonal antibody can significantly increase the bone amount, alleviate the symptoms such as ostalgia, have no obvious adverse reaction, but shortcoming is invalid to struvite synovitis, must with other preparation combined utilization of TNF alpha antibody.The TNF-alpha-2 antagonists is to suppress clinically the sure preparation of RA inflammatory reaction curative effect, but has 1/3 patient invalid to three kinds of antagonists of present listing, and because the price comparison of TNF-alpha-2 antagonists is expensive, is difficult to be widely used.
Summary of the invention
First purpose of the present invention is to provide a kind of RANKL-TNF sample district fusion rotein for above-mentioned deficiency.
Second purpose of the present invention is to provide the preparation method of above-mentioned RANKL-TNF sample district fusion rotein.
The 3rd purpose of the present invention is to provide the purposes of above-mentioned RANKL-TNF sample district's fusion rotein in the biotechnological formulation for the treatment of rheumatoid arthritis/vaccine preparation.
RANKL-TNF sample of the present invention district fusion rotein, it is RANKL-TNF sample district albumen and the fusion rotein with promiscuous Th2 cell epitope fragment.
RANKL and TNF-α are the key factors of RA bone injury and inflammation sustainable existence, simultaneously, RANKL is the TNFSF family member, the TNF sample district of RANKL and the aminoacid sequence of TNF-α have the similarity of height, the present invention with in RANKL with TNF-α similarity high two sections zones candidate's epi-position as polypeptide vaccine, but RANKL is from the body composition, so is difficult to produce antibody under native state.The present invention replaces with promiscuous Th2 cell epitope by the non-similar area with RANKL and TNF-α, thereby has strengthened its immunogenicity.The gene order GAATGGGAGTTCGTAAACACCCCACCTCTCGTC (aminoacid sequence EWEFVNTPPLV) of the BRU strain reversed transcriptive enzyme RT (414-424aa) that this promiscuous Th2 cell epitope can be HIV-I, or other general host's property Th2 cell epitope, such as: OVA, BSA, PPD etc.
Term " general host's property " refers to host's adaptability widely.
In embodiments of the present invention, aminoacid sequence such as SEQ ID No.2 or 4 of described RANKL-TNF sample district fusion rotein; Those skilled in the art can not affect under its active prerequisite according to this fragment, replace, lack and/or add one or several amino acid, obtain the mutant nucleotide sequence of described albumen.For example, the Serine of the 6th is replaced with tyrosine, perhaps with the Methionin disappearance of the 77th, perhaps add die aromatischen Aminosaeuren at the 118th.Therefore, RANKL-TNF sample district fusion rotein comprises that also aminoacid sequence shown in SEQ ID No.2 or 4 is substituted, replaces and/or increases one or several amino acid, have with isoreactivity by SEQ ID No.2 or the 4 derivative protein that obtain.
The present invention also comprises the gene of the above-mentioned fusion rotein of encoding.In embodiments of the present invention, encode the nucleotide sequence of this antigen-4 fusion protein gene as shown in SEQ ID No.1 or 3.Should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.
Said gene of the present invention can be operably connected with expression vector, obtain to express the recombinant expression vector of fusion rotein of the present invention, and then this expression vector can be imported host cell, obtain expressing the genetic engineering bacterium of RANKL-TNF sample district fusion rotein.Expression vector used in the present invention can be selected various known prokaryotic expression carriers.
The present invention also provides a kind of method for preparing above-mentioned fusion rotein, comprise the steps: that gene clone with coding RANKL-TNF sample district fusion rotein is in expression vector, with described expression vector transformed host cell, screening positive clone, after yeast culture and abduction delivering, separation and purification obtains this fusion rotein.
In embodiments of the present invention, by building recombination, be connected with the pET-28a expression vector, transform e. coli bl21 (DE3), expressed fusion protein, and by methods such as molecular sieve or ion exchange chromatographies, fusion rotein is carried out purifying, adopt SDS-PAGE, Western-blot etc. to carry out physico-chemical property to fusion rotein and identify.
In a preferred embodiment of the present invention, comprise in the step of suitable condition bottom fermentation culturing engineering bacterium:
Recombinant clone plasmid transformation escherichia coli BL21 (DE3) competent cell that order-checking is correct is selected single colony inoculation in 10mL LB substratum (kantlex that contains 30mg/L), and 37 ℃, the 250rpm shaking culture is spent the night.Get the 30mL overnight culture next day and transfer in 1L LB substratum (kantlex that contains 30mg/L), 37 ℃, 250rpm is cultured to OD
600To 0.5, adding IPTG to make it final concentration is 0.4mM, 37 ℃, and centrifugal collection thalline after 250rpm continuation shaking culture 4.5h.
Get the coli somatic of collecting after cultivating, use the PBS of precooling to wash thalline 6 times, ultrasonic resuspended 10min (super 4s stops 6s, and power is 40%).It is resuspended that every gram wet thallus adds the bacterial cellular protein lysate of 10 times of volumes, adds N,O-Diacetylmuramidase to 1mg/mL, adds simultaneously proteinase inhibitor PMSF to 1mM, after placing 30min on ice, the ultrasonic bacterium 10min that splits in ice bath, the centrifugal 10min collecting precipitation of 12,000rpm.Again respectively with TE damping fluid, each washing of 1%trixton-100 once, precipitation is dissolved in ultrasonic resuspended thalline (5mL/g wet thallus) in PBS, slowly drip simultaneously isopyknic 8M urea soln, magnetic stir bar stirs 30min, centrifuged deposit washs 1 time with the 4M urea soln, precipitation is resuspended in the dissolving damping fluid that contains 4M urea, and the centrifuging and taking supernatant obtains to contain the sex change liquid of purpose fusion rotein through the filtering with microporous membrane of 0.45 μ m.Affinity chromatography (Ni-NTAHis prepacked column 5mL) purified fusion protein.According to the BCA albuminimetry with the protein denaturation liquid concentration dilution of purifying to 0.5mg/mL.Prepare renaturation solution (4M urea by progressively the successively decrease method of (4M-3M-2.5M-2M-1.5M-1.2M-0.8M-0.5M-0.2M-0M) of urea concentration, 50mM Tris, 0.5mM EDTA, 50mM NaCl, the 3mM reduced glutathion, the 1mM Sleep-promoting factor B, 10% (V/V) glycerine, 1% glycine, PBS, pH7.6), the protein denaturation liquid that dilutes is placed in dialysis tubing, is not more than 4 ℃ of slow dialysis renaturations that stir of ratio of 1: 20 in the inside and outside liquid proportional of renaturation.Whether separate out and what of the amount of separating out are determined best renaturation dialysis conditions according to albumen in dialysis procedure, approximately changed the dialyzate that urea concentration reduces gradually in 4 hours, last in the PBS of pH7.6 solution dialysed overnight, change PBS solution therebetween one time, protein denaturation liquid is carried out dialysis renaturation.
Experiment shows, the antibody that RANKL-TNF sample district fusion protein immunization mouse produces has the effect in conjunction with RANKL and TNF-α, can be used for the treatment of the diseases such as rheumatoid arthritis, performance alleviates osteoclasia and inflammation-inhibiting effect, thereby can be for the preparation of the vaccine of rheumatoid arthritis.And then the present invention also comprises a kind of therapeutic vaccine for the treatment of rheumatoid arthritis, and it is made for acceptable carrier on above-mentioned RANKL-TNF sample district's fusion rotein of effective dose and biotechnological formulation/vaccinology.
RANKL-TNF sample of the present invention district fusion rotein provides a kind of new treatment approach and thinking for diseases such as rheumatoid arthritis.The present invention is by vivoexpression RANKL-TNF sample district fusion rotein, and expression amount is high, and controllability is strong, and production cost is relatively low, and expression product has good immunogenicity, easily realizes scale operation.
Description of drawings
Fig. 1 shows the structural representation of the expression vector that the present invention is selected and the rear expression vector of recombinating; Wherein, Fig. 1 (a) is pET-28a (+) plasmid schematic diagram; Fig. 1 (b) is pET-28a (+)-RTFP-1 recombinant plasmid schematic diagram; Fig. 1 (c) is pET-28a (+)-RTFP-2 recombinant plasmid schematic diagram; Fig. 1 (d) is pET-28a (+)-RTFP-3 recombinant plasmid schematic diagram.
Fig. 2 is RANKL-TNF sample district fusion rotein PCR product agarose gel electrophoretogram; Wherein, Fig. 2 (a) is the structure result of fusion rotein RTFP-1 recombinant expression plasmid, swimming lane M
1, M
2: molecular weight Marker; Full gene cloning plasmid (BC117286, the 1097bp of swimming lane 1:RANKL; PDONR223,5004bp); Swimming lane 2,3: the AB of goal gene, CD fragment; Swimming lane 4: goal gene total length (443bp); Swimming lane 5: the double digestion fragment of goal gene total length (Sal I, Nde I); Swimming lane 6:pET-28a (+) plasmid; Swimming lane 7:pET-28a (+) plasmid double digestion (Sal I, Nde I); Swimming lane 8: the recombinant expression plasmid (pET-28a (+)-RTFP-1) that contains goal gene; Swimming lane 9,10: the PCR of recombinant expression plasmid identifies (pET-28a (+), pET-28a (+)-RTFP-1); The double digestion of swimming lane 11:pET-28a (+)-RTFP-1 recombinant expression plasmid is identified (Sal I, Nde I);
Wherein, Fig. 2 (b) is the structure result of fusion rotein RTFP-2 recombinant expression plasmid, swimming lane M
1, M
2: molecular weight Marker; Full gene cloning plasmid (BC117286, the 1097bp of swimming lane 1:RANKL; PDONR223,5004bp); Swimming lane 2,3,4: the AB of goal gene, CD, CE fragment; Swimming lane 5: goal gene total length (443bp); Swimming lane 6: the double digestion fragment of goal gene total length (Sal I, Nde I); Swimming lane 7::pET-28a (+) plasmid; Swimming lane 8:pET-28a (+) plasmid double digestion (Sal I, Nde I); Swimming lane 9: the recombinant expression plasmid (pET-28a (+)-RTFP-2) that contains goal gene; Swimming lane 10,11: the PCR of recombinant expression plasmid identifies (pET-28a (+), pET-28a (+)-RTFP-2); The double digestion of swimming lane 12:pET-28a (+)-RTFP-2 recombinant expression plasmid is identified (Sal I, Nde I);
Wherein, the structure result of Fig. 2 (c) fusion rotein RTFP-3 recombinant expression plasmid, swimming lane M
1, M
2: molecular weight Marker; Swimming lane 1,2: the total length of goal gene (410bp); Swimming lane 3:pET-28a (+) plasmid; Swimming lane 4,5: the recombinant expression plasmid (pET-28a (+)-RTFP-3) that contains goal gene.
Fig. 3 is RANKL-TNF sample district fusion protein S DS-PAGE electrophoretogram; Wherein, swimming lane M
1: molecular weight Marker; Swimming lane 1,3,5,7: do not add recombinant expression plasmid (pET-28a (+), pET-28a (+)-RTFP-1, pET-28a (+)-RTFP-2, pET-28a (+)-RTFP-3 that IPTG induces; 37 ℃, 250rpm); Swimming lane 2,4,6,8: the recombinant expression plasmid (pET-28a (+) that adds IPTG to induce, pET-28a (+)-RTFP-1 (17.9KDa), pET-28a (+)-RTFP-2 (19.9KDa), pET-28a (+)-RTFP-3 (16.6KDa); 37 ℃, 250rpm, IPTG 0.4mM).
Fig. 4 shows is that Western-blot analyzes RANKL-TNF sample district fusion rotein;
Wherein, figure a is for detecting fusion rotein, swimming lane 1:RTFP-1 fusion rotein with anti-human RANKL polyclonal antibody; Swimming lane 2:RTFP-2 fusion rotein; Swimming lane 3:RTFP-3 fusion rotein;
Wherein, figure b is for detecting fusion rotein, swimming lane 1: positive control (rhTNF-α, 17.5KDa) with anti-humanTNF-α's polyclonal antibody; Swimming lane 2: negative control (pET-28a (+) plasmid); Swimming lane 3:RTFP-1 fusion rotein; Swimming lane 4:RTFP-2 fusion rotein; Swimming lane 5:RTFP-3 fusion rotein.
RANKL-TNF sample district fusion protein S DS-PAGE electrophoretogram after Fig. 5 purification renaturation; Wherein, swimming lane M
1: molecular weight Marker; Swimming lane 1:RTFP-1 fusion rotein; Swimming lane 2:RTFP-2 fusion rotein; Swimming lane 3:RTFP-3 fusion rotein.
Fig. 6 shows is that the security of RANKL-TNF sample district fusion rotein is identified;
Wherein, Fig. 6 (a) is that osteoclast generates test, wherein A. positive controls: (M-CSF10ng/mL+RANKL 40ng/mL); B. fusion rotein RTFP-1 experimental group: (M-CSF10ng/mL+ fusion rotein RTFP-1 30 μ g/mL); C. fusion rotein RTFP-2 experimental group: (M-CSF 10ng/mL+ fusion rotein RTFP-2 30 μ g/mL);
Wherein, Fig. 6 (b) is the test of L929 apoptosis, wherein A. blank group: (L929); B. positive controls: (L929+TNF-α 20ng/mL); C. fusion rotein RTFP-1 experimental group: (L929+ fusion rotein RTFP-1 30ug/mL); D. fusion rotein RTFP-2 experimental group: (L929+ fusion rotein RTFP-2 30ug/mL);
Wherein, Fig. 6 (c) is that fusion rotein is to the measurement result of L929 apoptosis activity.
What Fig. 7 showed is titre and the time length that RANKL-TNF sample district's fusion rotein stimulation body produces antibody, and wherein Fig. 7 (a) detects NAT and the time length that produces in immune serum as envelope antigen with rhRANKL; Fig. 7 (b) detects NAT and the time length that produces in immune serum as envelope antigen with rhTNF-α.
Fig. 8 shows is that RANKL-TNF sample district fusion rotein stimulates body to produce in antibody and the ability of rhTNF-α and rhRANKL, and wherein Fig. 8 (a) is that the survival rate of respectively organizing of the cachexy mouse due to rhTNF-α detects; Fig. 8 (b) is that the calcium level of respectively organizing mouse after discharging with bone calcium due to rhRANKL is measured.
What Fig. 9 showed is that RANKL-TNF sample district's fusion rotein stimulation body produces antibody to the provide protection assessment of collagen-induced sacroiliitis (Collagen-Induced Arthritis, CIA) mouse, and wherein Fig. 9 (a) is the sickness rate of respectively organizing mouse; Fig. 9 (b) is that the swollen joint expansibility of respectively organizing mouse is measured; Fig. 9 (c) is the joint scoring of respectively organizing mouse; Fig. 9 (d) is that the bone erosion degree of respectively organizing mouse Micro-CT changes.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
According to RANKL-TNF sample district recombinant fragment (RANKL P171-234, P246-309, the BRU strain reversed transcriptive enzyme RT (414-424aa) of HIV-1) the characteristic Design primer (seeing the following form) of gene order and pET-28a (+) expression vector, (Gen Bank NO:BC117286,1097bp take the RANKL full-length cDNA as template; PDONR223,5004bp), kept the His label that pET-28a carries, adopt the method connection three's of recombinant PCR gene fragment, wherein the gene order of the BRU strain reversed transcriptive enzyme RT (414-424aa) of HIV-I is inserted in the centre of two RANKL-TNF sample district gene fragments, building and obtaining expression vector is pET-28a (+)-RTFP-1; The gene order of the BRU strain reversed transcriptive enzyme RT (414-424aa) of HIV-I is inserted in the end of two RANKL-TNF sample district gene fragments, it is pET-28a (+)-RTFP-2 that structure obtains expression vector, and is pET-28a (+)-RTFP-3 with the expression vector that the wild-type mutant of above-mentioned two RANKL-TNF sample district gene fragment direct splicing obtains in contrast.Corresponding fusion gene sequence is as shown in SEQ ID No.1 or 3,5.The PCR reaction conditions: 98 ℃ of denaturation 30s, circulate 30 times by following parameter: 96 ℃ of sex change 5s, 55-65 ℃ of annealing 15s, 72 ℃ are extended 15s, and last 72 ℃ are extended 10min.The PCR product carries out its size of electrophoresis observation in 1% sepharose, PCR by recombinant expression plasmid identifies and the double digestion evaluation, see Fig. 2, pcr amplification goes out the purpose band of expection size, and double digestion identifies that the goal gene fragment that recombinant plasmid contains is the expection size, illustrates that the present invention has successfully built the prokaryotic expression carrier of RANKL-TNF sample district fusion rotein.
Table 1 relevant primer
The abduction delivering of embodiment 2 RANKL-TNF sample district's fusion roteins in intestinal bacteria
Recombinant clone plasmid transformation escherichia coli BL21 (DE3) competent cell that order-checking is correct is selected single colony inoculation in 10mL LB substratum (kantlex that contains 30mg/L), and 37 ℃, the 250rpm shaking culture is spent the night.Get the 30mL overnight culture next day and transfer in 1L LB substratum (kantlex that contains 30mg/L), 37 ℃, 250rpm is cultured to OD
600To 0.5, adding IPTG to make it final concentration is 0.1mM, 37 ℃, and centrifugal collection thalline after 250rpm continuation shaking culture 4.5h, SDS-PAGE and Western-blot the analysis showed that after 0.4mMIPTG induces can obtain a large amount of fusion roteins, sees Fig. 3,4.
Purifying and the renaturation of embodiment 3 RANKL-TNF sample district fusion roteins
The positive colony bacterial strain is in 0.4mM IPTG, and after 37 ℃ of inducing culture 4.5h, 4 ℃, the centrifugal 15min of 6000rpm collects thalline, uses the PBS of precooling to wash thalline 6 times, ultrasonic resuspended 10min (super 4s stops 6s, and power is 40%).It is resuspended that every gram wet thallus adds the bacterial cellular protein lysate of 10 times of volumes, adds N,O-Diacetylmuramidase to 1mg/mL, adds simultaneously proteinase inhibitor PMSF to 1mM, after placing 30min on ice, the ultrasonic bacterium 10min that splits in ice bath, the centrifugal 10min collecting precipitation of 12,000rpm.Again respectively with TE damping fluid, each washing of 1%trixton-100 once, precipitation is dissolved in ultrasonic resuspended thalline (5mL/g wet thallus) in PBS, slowly drip simultaneously isopyknic 8M urea soln, magnetic stir bar stirs 30min, centrifuged deposit is washed 1 time with the 4M urea soln, precipitation is resuspended in the dissolving damping fluid that contains 4M, and the centrifuging and taking supernatant obtains to contain the sex change liquid of purpose fusion rotein through the filtering with microporous membrane of 0.45 μ m.Affinity chromatography (Ni-NTAHis prepacked column 5mL) purifying target protein.According to the BCA albuminimetry with the protein denaturation liquid concentration dilution of purifying to 0.5mg/mL.Prepare renaturation solution (4M urea by progressively the successively decrease method of (4M-3M-2.5M-2M-1.5M-1.2M-0.8M-0.5M-0.2M-0M) of urea concentration, 50mM Tris, 0.5mM EDTA, 50mM NaCl, the 3mM reduced glutathion, the 1mM Sleep-promoting factor B, 10% (V/V) glycerine, 1% glycine, PBS, pH7.6), the protein denaturation liquid that dilutes is placed in dialysis tubing, is not more than 4 ℃ of slow dialysis renaturations that stir of ratio of 1: 20 in the inside and outside liquid proportional of renaturation.Whether separate out and what of the amount of separating out are determined best renaturation dialysis conditions according to albumen in dialysis procedure, approximately changed the dialyzate that urea concentration reduces gradually in 4 hours, last in the PBS of pH7.6 solution dialysed overnight, change PBS solution therebetween one time, protein denaturation liquid is carried out dialysis renaturation, RANKL-TNF sample district fusion protein S DS-PAGE electrophorogram after renaturation is seen Fig. 5, illustrates that this fusion rotein of employing aforesaid method can successful renaturation.
The Differentiation of Bone Marrow Cells of foundation take M-CSF and RANKL as DIF is the inducing culture system of osteoclast.Get the BABL/c female SPF mouse of 6-15W, medullary space with irrigation with syringe mouse bones of limbs under aseptic condition obtains medullary cell, and (slide counting cells density is 2.5 * 10 to make single cell suspension with RPMI1640 substratum and the resuspended medullary cell of 10%FBS after 70nm cell sieve filters
6/ mL), in 25cm
2In culturing bottle at 37 ℃, 5%CO
2Former culture bone marrow cells in mice under the saturated humidity condition adds M-CSF to cultivate 24h after final concentration is 25ng/mL, and next day, the collecting cell suspension, be the non-tack myelomonocyte of M-CSF dependency.1000rpm, the centrifugal 5min of normal temperature abandons supernatant, and PBS washes twice.With RPMI1640 substratum and 10%FBS re-suspended cell, counting and adjust cell concn is 2.5 * 10
6/ mL is inoculated in cell respectively in 6 orifice plates and 96 orifice plates.Generate test by the osteoclast that promotes myelomonocyte to be divided into osteoclast, experiment is divided into RANKL-TNF sample district's recombinant polypeptide group (the fusion rotein RTFP-1/2 that contains the M-CSF+30 μ g/mL of 25ng/mL) and natural human RANKL positive controls (the M-CSF+40ng/mL RANKL that contains 25ng/mL), every group of 12 samples, can fusion rotein is observed in TRAP dyeing stimulate myelomonocyte to be divided into osteoclast, result shows, fusion rotein is seen Fig. 6 (a) without the biologic activity of the promotion osteoclast generation of natural RANKL.
Set up L929 apoptosis culture system, L929 clone is the present of cytobiology teaching and research room of the Capital University of Medical Sciences, conventional recovery cell, and with RPMI1640 substratum and 10%FBS re-suspended cell, and counting adjustment cell concn is 1 * 10
6/ mL is inoculated in cell respectively in 6 orifice plates and 96 orifice plates, at 37 ℃, 5%CO
2Adherent culture 24h under the saturated humidity condition, experiment is divided into RANKL-TNF sample district's recombinant polypeptide group (fusion rotein that contains 30 μ g/mL), natural humanTNF-α's cytokine group (the rhTNF-α that contains 20ng/mL), and experiment contrast group (only cultivating with above-mentioned perfect medium), add respectively above-mentioned fusion rotein and cytokine to continue to cultivate 12h, can the multiple hole of 63 of every group of orifice plates be observed fusion rotein and be promoted the apoptotic metamorphosis of L929 under light microscopic; 96 12 of every group of orifice plates samples are measured apoptosis rate with crystal violet staining assay under the 570nm wavelength, result shows, compares with natural humanTNF-α's cytokine group, and fusion rotein is seen Fig. 6 (b) without promoting the apoptotic activity of L929.
Though above-mentionedly experiment showed, that fusion rotein is the variant in RANKL-TNF sample district, without the osteoclasia effect of natural RANKL and the apoptosis-promoting effect of natural TNF-α, safety performance is good, can be further used in vivo test.
The female BABL/c mouse of fusion protein immunization 6～8w (SPF level), by body weight, mouse is divided into experiment contrast group and fusion rotein RTFP-1, RTFP-2, RTFP-3 group, (the initial immunity postscript is 0d to subcutaneous abdomen multiple spot immune mouse, respectively at 21d, 42d, 152d booster immunization), every each immunizing dose of mouse is 200ug (100 μ l), complete Freund's adjuvant (CFA) emulsification of initial immunity with immunogen equivalent, booster immunization equivalent incomplete Freund's adjuvant (IFA) emulsification.Different time points (0d, 32d, 49d, 56d, 63d, 70d, 84d, 112d, 142d, 163d, 170d, 177d) eye socket detects with indirect elisa method the ability that produces neutralizing antibody in Mice Body after getting blood, use respectively rhTNF-α (200ng/mL) and rhRANKL (200ng/mL) to be coated with elisa plate, 4 ℃ are spent the night, and seal 1h with 1%BSA37 ℃, serum to be checked dilution in 1: 1000, the antibody of HRP mark 1: 20,000 dilution.The serum antibody titer measurement result shows, contains the fusion rotein group antibody generation level of Th2 cell epitope apparently higher than the experiment contrast group, and apparently higher than the fusion rotein control group (data as shown in Figure 7) without the Th2 cell epitope.
Neutralization experiment in embodiment 7 antibody bodies
With reference to the described method of embodiment 6,1W after the 42d booster immunization, RTFP-2 fusion rotein group and experiment contrast group mouse tail vein give rhTNF-α (20ug/ only), and injection, continuous three times, copy mouse cachexy model every other day.After the injection, the survival rate of mouse is respectively organized in every day monitoring first, and result shows, contains the survival rate of RTFP-2 fusion rotein group mouse of Th2 cell epitope apparently higher than experiment contrast group [data are as shown in Fig. 8 (a)]; RTFP-2 fusion rotein and experiment contrast group mouse tail vein give rhRANKL (10ug/ only), inject continuously 3d, 7d eye socket blood sampling after 3h and injection first before injection and after per injection, measure the serum calcium ion level, result shows, but contains calcium level due to the RTFP-2 fusion rotein group mouse antagonism rhRANKL of Th2 cell epitope raise [data are as shown in Fig. 8 (b)].
The provide protection experiment of embodiment 8 vaccines to collagen-induced sacroiliitis mouse
The male DBA-1 mouse of fusion protein immunization 5W (SPF level), method and immune flow process are with reference to embodiment 6.1W after the last immunity, except experiment contrast group mouse, each is organized mouse and contains the emulsification (200ugCII of the CFA of 4mg/mLBCG apart from the root of the tail 1.5cm of section place's intradermal immunization ox Type Ⅱ collagen (CII) and equivalent, 100ul/ only), same procedure booster immunization (100ugCII+IFA after 3W, 100ul/ is only), copy the CIA mouse model.After booster immunization, the sickness rate of mouse is respectively organized in monitoring every other day, and [standards of grading: 0=is without arthritic symptom to carry out the joint scoring; 1=foot pawl or toe swollen and (or) red; 2=involve the swollen of two joints and (or) red; The plural swollen joint of 3=and (or) red; The arthritic symptom that 4=is serious involves whole sufficient pawl or toe], the swelling of measuring extremities joint with the vernier callipers of 0.02mm range simultaneously is changed to 50d after the last immunity; After last immunity CII, the disconnected neck of 50d is put to death mouse, the Micro-CT tomoscan, and the bone erosion degree of mouse is respectively organized in assessment.Result shows, contain the RTFP-2 fusion rotein group mouse invasion rate reduction of Th2 cell epitope, and disease symptoms is light, bone injury not obvious (p＜0.05, data are as shown in Figure 9).
Claims (8)
1.RANKL-TNF sample district fusion rotein, it is RANKL-TNF sample district albumen and the fusion rotein with promiscuous Th2 cell epitope fragment, and described fusion rotein is the albumen that is comprised of aminoacid sequence shown in SEQ ID No.4.
2. the gene of coding claim 1 described fusion rotein.
3. gene as claimed in claim 2, its nucleotide sequence is as shown in SEQ ID No.3.
4. the expression vector that contains the described fusion rotein encoding gene of claim 2 or 3.
5. the host cell that contains the described fusion protein expression vector of claim 4.
6. method for preparing the described fusion rotein of claim 1, it comprises the steps: the described gene clone of claim 2 or 3 to expression vector, with described expression vector transformed host cell, screening positive clone, after yeast culture and abduction delivering, separation and purification obtains this fusion rotein.
7. the application of fusion rotein claimed in claim 1 in the vaccine of preparation treatment rheumatoid arthritis.
8. vaccine for the treatment of rheumatoid arthritis, it is made for acceptable carrier on the fusion rotein claimed in claim 1 of effective dose and biotechnological formulation/vaccinology.
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| RANKL和骨保护素在类风湿关节炎中的作用;郑召岭等;《徐州医学院学报》;20081231;第28卷(第4期);全文 * |
| 刘俊丽等.重组骨保护素(rhOPG)药物的研究现状及应用展望.《中国骨质疏松杂志》.2008,第14卷(第1期),全文. |
| 郑召岭等.RANKL和骨保护素在类风湿关节炎中的作用.《徐州医学院学报》.2008,第28卷(第4期),全文. |
| 重组骨保护素(rhOPG)药物的研究现状及应用展望;刘俊丽等;《中国骨质疏松杂志》;20080131;第14卷(第1期);全文 * |
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