CN104013648A - Long-noded pit viper extract and application thereof - Google Patents

Abstract

The invention discloses a long-noded pit viper extract and application thereof. The long-noded pit viper extract is prepared by the following steps: adding IPTG (isopropyl-beta-d-thiogalactoside) into fermentation liquor obtained by performing fermentation culture on recombinant bacterium of a glucocorticoid-containing receptor gene for performing induction culture to obtain induction culture liquor; centrifuging the induction culture liquor, collecting precipitate, ultrasonically smashing, and extracting proteins to obtain glucocorticoid receptor proteins, wherein the nucleotide sequence of the glucocorticoid receptor gene is shown as SEQ ID NO:1; coupling the glucocorticoid receptor proteins to a Ni-NTA Agarose column for serving as a stationary phase, making long-noded pit viper decoction flow through the Ni-NTA Agarose column as a flowing phase, washing twice by using a PBS (Phosphate Buffer Solution) washing column, adding IgG ElutionBuffer elution for eluting, and collecting effluent to obtain the long-noded pit viper extract. By using the prepared long-noded pit viper extract, the mutation action between a glucocorticoid receptor and NF-kB can be enhanced, and a basis is laid for the substituents of glucocorticoids; the prepared long-noded pit viper extract has a potential anti-inflammatory effect.

Description

A kind of Agkistrodon extract and application thereof

(1) technical field

The present invention relates to a kind of Agkistrodon extract, particularly from Agkistrodon, extract material and the application thereof that can be combined with glucocorticoid receptor (GR).

(2) background technology

Agkistrodon belongs to the dry body of Viperidae Agkistrodon [Agkistrodon ac μ t μ s (G μ enther)], and sweet in the mouth is salty, warm in nature, has and dispels the wind, dredging collateral, effect of relieving convulsion.Serpentis is used as medicine and has it from ancient times, and " Kaibao Bencao " carries: " main apoplexy arthralgia chiefly caused by damp pathogen is meciless, spasm of muscles and vessels, and actinal surface Wai is oblique, hemiplegia, arthralgia, strong wind mange and storm wind pruritus, flaccidity of the lower limbs can not standing for a long time ".Chemical constitution study shows, Agkistrodon main chemical compositions has fat, lipid, volatile oil, fatty acid, vitamins, phospholipid and aminoacid and micromolecule compounds, also contain in addition 3 kinds of toxalbumin and a large amount of rare amino acid classes, and containing (Wang Guijin such as hyaluronidase, arginine esterase and anticoagulants, Sun Jiaming, wangdan, etc. the HPLC-MS/MS of inosine qualification and assay in Agkistrodon halys meat. the modernization of World Science technology-Chinese medicine, 2007.9(5) 68-71).

Modern study thinks, Serpentis tissue has strong biological activity, applies properly, and determined curative effect, has unique effect aspects such as pain relieving, antiinflammatory, righting.Effect of all Serpentiss is seemingly how close, we utilize the clinical practice of rheumatism research platform to find, Agkistrodon can effectively reduce the use amount of glucocorticoid in treatment rheumatism, play the effect of parahormone sample, and have no side effect that (Si Tuzhong, thanks to Computer Associates International Inc., Xie Zhijun, model rises Experience in Treating anemofrigid-damp arthralgia experience forever. Zhejiang University of Traditional Chinese Medicine's journal, 2008. 32:(2) and, 300-301).The reports such as Fang Ping Fu, by Chinese medicine worm, Serpentis class Drug therapy rheumatic arthritis 50 examples, total effective rate be respectively 90% and 97% (Fang Ping Fu, rheumatoid arthritis treatment status and prospects. practical tcm internal medicine magazine, 1998.12:(2), 13-14).The reports such as Zhang Hai, adopt white Serpentis shenfu decoction treatment patient with rheumatoid arthritis 52 examples, obtain a satisfactory effect (sea, the routine observation of curative effect of white Serpentis shenfu decoction treatment rheumatoid arthritis 52, China's Chinese medicine magazine, 2008.23 (7), 651-652).In addition, pass through mouse test, we find that the water of Agkistrodon, alcohol extract all have obvious antiinflammatory action, and Agkistrodon extract obviously suppresses adjuvant and collagen-induced rat paw edema (Zhang Jida, Deng, the impact of Agkistrodon water extract on C-II induced arthritis in rats TNF-α, IL-6 and IL-10, China's Chinese medicine magazine, 2012.27 (5), 1407-1409. paddy perseverance is deposited, etc., the immunoregulation effect of Agkistrodon water extraction liquid to adjuvant arthritis rats, China's Chinese medicine magazine, 2012.27 (10), 2676-2678).

At present for rheumatism patient, using glucocorticoid (gl μ cocorticoid) is conventional Therapeutic Method, but the glucocorticoid of life-time service high dose easily increase infection risk and cause osteoporosis and osteonecrosis etc. ( h, et al, Mechanisms involved in the side effects of gl μ cocorticoids.Pharmacology & Therape μ tics, 2002.96, (1), 23-43).Little and the significant glucocoricoid medicine of therapeutical effect of screening side effect and to improve interior therapeutic index be a very promising job.Reichardi etc. observe, glucocorticoid receptor (GR) (gl μ cocorticoid receptors, GR) transgenic mice (GRdim/dim) that Dimerized region aminoacid is undergone mutation can not form dimer (Reiehar dt HM, Kaestner KH, T μ ekermann J, et al.DNA bingding of the gl μ cocorticoid for receptor is not necessary for s μ rvival.cell, 1998.93 (4), 531-541), but dexamethasone is in the inflammatory model of GRdim/dim mice and normal mouse, all show good anti-inflammatory activity.Inspired by this, everybody starts to find and exploitation selectivity glucocorticoid receptor modulator (SGRMS).SGRMS with GR specific binding after, main performance suppresses nuclear factor (n μ clear transcription factor, NF-κ B) and activator protein (activator protein-1, the proinflammatory inflammation factor Transcription such as AP-1 (non genome effect), to relating to glyconeogenesis, the transcriptional activation of the related genes such as fat and amino acid metabolism weakens (genome effect), and then in producing anti-inflammatory effect, reduce its side effect (Cindy Stahn & Frank B μ ttgereit Genomic and nongenomic effects of gl μ cocorticoids Nat μ re Reviews Rhe μ matology2008.7 (4), 525-533).The dosage that Ravindran etc. use glucocorticoid and therapeutic effect and safety are added up, while showing clinical use high dose glucocorticoid antiinflammatory, mainly work with non genome effect, but can not suppress the genome effect of glucocorticoid, necessity (the Vinod Ravindran that exploitation only retains the novel glucocorticoid of non genome effect is proposed, Newer gl μ cocorticoids:Overcoming mechanistic h μ rdles.Indian Jo μ rnal of Rhe μ matology, 2012.7 (4), 221 – 225).After this; the appearance in succession of the compounds such as AL-4388, LGD5552, ZK216348 and CpdA; the low toxic and side effects glucocoricoid composition of prompting exploitation has very large feasibility (Schacke H; et al; Dissociation of transactivation from transrepression by a selective gl μ cocorticoid receptor against leads to separation of therape μ tic effects from side effects; Proc Natl Acad Sci Μ SA; 2004.101 (1), P227-32; Miner JN, et al, Antiinflammatory gl μ cocorticoid receptor ligand with red μ ced side effects exhibits an altered protein-protein interaction Proc Natl Acad Sci Μ SA, 2007.104 (49), 19244-19249).Inventor's standby GR affinity column of drawing up, according to receptor-ligand theory from Agkistrodon folk prescription enrichment can with the ligand component of the similar glucocorticoid of GR specificity combination.

It is to adopt the modes such as silicagel column separation, qualification, pharmacological screening that the material base of natural drug is studied conventional method, and these class methods exist the shortcomings such as reagent consumption is large, target component is indefinite.In the present invention, inventor utilizes can be in conjunction with the Agkistrodon extract of glucocorticoid receptor (GR) in GR affinity column specificity screening Agkistrodon water extraction liquid, so the clearer and more definite and medical material amount that needs of target still less, also for the acquisition of other Effective Component of Chinese Medicines provides a kind of new thinking.

(3) summary of the invention

The object of the invention is to provide a kind of new screening can be combined with glucocorticoid receptor (GR) from Agkistrodon material and application; It is clear and definite that the method is filtered into partial objectives for, and the medical material amount of use is few; The Agkistrodon extract obtaining can be fast for the Screening target of the glucocorticoid receptor (GR) selective modulator of high-efficiency low-toxicity.Compared with the isolation identification of conventional compounds, the specificity of the compound filtering out and effectiveness are all stronger.

The technical solution used in the present invention is:

The invention provides a kind of Agkistrodon extract, the preparation method of described Agkistrodon extract is using glucocorticoid receptor (GR) as immobile phase, Agkistrodon water decoction is as mobile phase, collecting the Agkistrodon extracting solution composition that can be combined with glucocorticoid receptor (GR) in Agkistrodon water decoction is Agkistrodon extract, concrete described Agkistrodon extract is prepared as follows: (1) glucocorticoid receptor protein expression: be isopropylthiogalactoside to adding IPTG(in the fermentation liquid obtaining through fermentation culture containing the recombination engineering of glucocorticoid receptor gene) carry out inducing culture (preferably the final concentration of IPTG is 1mM), obtain inducing culture liquid, by centrifugal inducing culture liquid, collecting precipitation extracts albumen after ultrasonication, obtains glucocorticoid receptor (GR) albumen, the nucleotides sequence of described glucocorticoid receptor gene is classified as shown in SEQ ID NO:1, (2) preparation of Agkistrodon extract: glucocorticoid receptor (GR) albumen coupling prepared by step (1) is to Ni-NTA Agarose post as immobile phase, flow through again the Ni-NTA Agarose post of coupling sugar cortical hormone receptor 4 body protein using Agkistrodon water decoction as mobile phase, in Agkistrodon water decoction, the material of energy and glucocorticoid receptor (GR) combination can be attached on Ni-NTA Agarose post, then twice, PBS washing Ni-NTA Agarose post, add IgG Elution Buffer eluent (preferably purchased from 21004PIERCE company) to carry out eluting and carry out eluting, directly collect effluent, obtain Agkistrodon extract, described Agkistrodon water decoction is prepared as follows: after Agkistrodon powder is mixed with mass ratio 1:100 with distilled water, decoct to 5% of raw material volume, filter, the phosphate buffer that is 7.0 with pH value after filtrate lyophilization dissolves that (volumetric usage of buffer is how many not to be affected the present invention, conventionally fixed according to the amount of required Agkistrodon water decoction), filter, filtrate is Agkistrodon water decoction.

Agkistrodon water decoction of the present invention is preparation as follows specifically: Agkistrodon dry body 20g, smash, adding distilled water 2000ml(is 2000g), medicine decocting tank decocts to 100ml, after filtration by solution-20 DEG C of lyophilizations, be ground into pressed powder, the phosphate buffer that is 7.0 with pH value dissolve, filter (preferably using 0.25 μ M filtering with microporous membrane), filtrate is Agkistrodon water decoction.Further, the method of the described recombination engineering fermentation culture of step (1) is: the recombination engineering containing glucocorticoid receptor gene is seeded in LB fluid medium, under 37 DEG C, 200rpm condition, cultivate 12h, obtain seed liquor, seed liquor is seeded in LB fluid medium with the inoculum concentration of volumetric concentration 1%, then adding final concentration is the ampicillin (Ampicillin, Amp) of 100 μ g/ml, under 37 DEG C, 200rpm condition, is cultured to OD ₆₀₀reach 0.6～0.8, obtain fermentation liquid.

Further, the condition of the described fermentation liquid inducing culture of step (1) is: to the IPTG that adds final concentration 1mM in fermentation culture, inducing culture 12h under 15 DEG C, 200rpm condition, obtains inducing culture liquid.

Further, the described protein extracting method of step (1) is: by centrifugal inducing culture liquid, collecting precipitation is made bacteria suspension with the phosphate buffer that pH value is 7, in bacteria suspension, add Phenylmethanesulfonyl fluoride (the Phenylmethanes μ lfonyl fl μ oride of final concentration 1mM, PMSF), at power 300W, ultrasonication under 4 DEG C of conditions of temperature, ultrasonic 10s, interval 10s, ultrasonic 10min, centrifugal, abandoning supernatant, precipitation adds the consumption of carbamide lysate and PMSF(carbamide lysate can dissolution precipitation, be generally 10 times of centrifugal acquisition precipitation volume, the consumption of PMSF is the final concentration 1mM in carbamide lysate conventionally), vibration after piping and druming evenly, ice is put 5min, repeating vibration and ice puts 4 times, centrifugal, get precipitation, obtain glucocorticoid receptor (GR) albumen, the compound method of the every 50mL of described carbamide lysate is: carbamide 24.04g, and sodium chloride 0.8766g, sodium phosphate 0.9503g, adding distil water is settled to 50mL.Further, Agkistrodon extract of the present invention is prepared as follows: (1) glucocorticoid receptor (GR) albumen preparation: the recombination engineering containing glucocorticoid receptor gene is seeded in LB fluid medium, under 37 DEG C, 200rpm condition, cultivate 12h, obtain seed liquor, seed liquor is seeded in LB fluid medium with the inoculum concentration of volumetric concentration 1%, then adding final concentration is the Amp of 100 μ g/ml, under 37 DEG C, 200rpm condition, is cultured to OD ₆₀₀reach 0.6～0.8, to the IPTG that adds final concentration 1mM in culture fluid, inducing culture 12h under 15 DEG C, 200rpm condition, obtains inducing culture liquid; By centrifugal inducing culture liquid, collecting precipitation is made bacteria suspension with the phosphate buffer that pH value is 7.0, adds the PMSF of final concentration 1mM in bacteria suspension, ultrasonication 10s under power 300W, 4 DEG C of conditions of temperature, interval 10s, ultrasonic 10min, centrifugal, abandoning supernatant, precipitation adds carbamide lysate and PMSF, vibration after piping and druming evenly, ice is put 5min, repeats vibration and ice and puts 4 times, centrifugal, get supernatant, obtain glucocorticoid receptor (GR) albumen; The compound method of the every 50mL of described carbamide lysate is: carbamide 24.04g, and sodium chloride 0.8766g, sodium phosphate 0.9503g, adding distil water is settled to 50mL; (2) Agkistrodon extract: glucocorticoid receptor (GR) albumen coupling prepared by step (1) is to Ni-NTA Agarose post as immobile phase, flow through Agkistrodon water decoction as the mobile phase Ni-NTA Agarose post of protein-coupled receptor, then twice, PBS washing pillar, add IgG Elution Buffer eluent to carry out eluting, collect effluent, obtain Agkistrodon extract; Described Agkistrodon water decoction is prepared as follows: after Agkistrodon powder is mixed with mass ratio 1:100 with distilled water, decoct to 5% of raw material volume, filter, filtrate is ground into powder after-20 DEG C of lyophilizations, then the phosphate buffer that is 7 with pH value dissolves, with 0.25 μ M filtering with microporous membrane, filtrate is Agkistrodon water decoction.

The present invention also provides a kind of described Agkistrodon extract in the application of preparing in glucocorticoid antiinflammatory drugs.Glucocorticoid is mainly by being combined with glucocorticoid receptor (GR), activating glucocorticoid receptor (GR) and NF-κ B does mutually, thereby play antiinflammatory action, glucocorticoid receptor (GR) mainly by with the interactions such as NF-κ B and AP-1, suppress NF-κ B and the proinflammatory inflammation factor Transcription of AP-1, thereby produce anti-inflammatory effect.Detect and show that Agkistrodon extract that the present invention separates can significantly strengthen the interaction of glucocorticoid receptor (GR) and NF-κ B, points out this conjugate to have anti-inflammatory activity by cytologic experiment.

The preparation flow of Agkistrodon extract of the present invention is:

(1) based on glucocorticoid receptor (GR) complete sequence on NCBI website, adopt the full gene of method amplification glucocorticoid receptor (GR) of RNA reverse transcription, and carry out nucleotide transformation, make its applicable prokaryotic expression system, improved nucleotides sequence is classified as shown in SEA ID NO:1, and the aminoacid sequence of translating is shown in SEA ID NO:2.

(2) complete gene constructed in prokaryotic expression carrier by what synthesize in step (1).

(3) prokaryotic expression carrier building is transformed to escherichia coli, IPTG induces glucocorticoid receptor protein expression, screens the prokaryotic expression system of high efficient expression glucocorticoid receptor (GR).

(4) prepare the nickel post of coupling sugar cortical hormone receptor.

(5) the nickel post of coupling as immobile phase, Agkistrodon water decoction is as mobile phase, the compound that enrichment can be combined with glucocorticoid receptor (GR), adopt albumen eluent (being IgG Elution Buffer eluent) to carry out eluting, collect Agkistrodon extract, the Agkistrodon extract obtaining carries out structural analysis through LC-MS chromatograph, and result shows all to have peak to occur in the time of 10min and 11min.

The cloning process of the full gene of glucocorticoid receptor (GR) described in step of the present invention (1) is techniques well known, can adopt dependent merchandise test kit to carry out clonal expansion, but we make it more appropriate to prokaryotic expression for clone's nucleotide sequence by the mode of synthetic splicing in the present invention, obtain the sequence shown in SEQ ID NO:1.

Compared with prior art, beneficial effect of the present invention is mainly reflected in: the present invention relates to a kind of taking Agkistrodon water decoction as mobile phase, taking the Ni-NTA Agarose post of coupling sugar cortical hormone receptor 4 body protein as immobile phase, using IgG Elution Buffer eluent as eluant, prepare the method for Agkistrodon extract, the method combines albumen pronucleus expression technology and chromatographic column separating effective ingredient, can more effectively separate the target component that can be combined with glucocorticoid receptor (GR) in Agkistrodon water decoction; Glucocorticoid, mainly by being combined with glucocorticoid receptor (GR), activates glucocorticoid receptor (GR) and NF-κ B and does mutually, thereby play antiinflammatory action.Agkistrodon extract prepared by the present invention, can strengthen glucocorticoid receptor (GR) and NF-κ B and interact, and has potential anti-inflammatory effect, for the succedaneum of glucocorticoid provides foundation.

(4) brief description of the drawings

Fig. 1: the agarose gel electrophoresis figure of the full gene of glucocorticoid receptor (GR) (GR α) pcr amplification product, swimming lane 1 is the full gene PCR amplified production of glucocorticoid receptor (GR), swimming lane M is Marker.

Fig. 2: glucocorticoid receptor (GR) nucleotide sequence (being structured in cloning vehicle P Μ C57, through order-checking qualification) and the former nucleotide sequence comparison chart spectrum of genetic modification.

Fig. 3: extract the full genophore plasmid of glucocorticoid receptor (GR) agarose gel electrophoresis figure, swimming lane 1-3 is for extracting the full genophore plasmid enzyme restriction qualification of glucocorticoid receptor (GR), swimming lane 1:BamH I/EcoR I enzyme action, expection band 1502bp, 746bp; Swimming lane 2:Hind III/Xho I enzyme action, expection band 1071bp, 554bp, 368bp; Swimming lane 3:BamH I/Xho I enzyme action, cuts out the about 2360bp of GR α CDS total length; M1:Marker.

Fig. 4: the SDS-PAGE electrophoretogram of the glucocorticoid receptor (GR) prokaryotic expression of qualification IPTG induction, swimming lane M:Marker, swimming lane 1:E4 is unloaded to express, swimming lane 2:IPTG induces unloaded, swimming lane 3:E4-GR α, swimming lane 4:IPTG induction E4-GR α, albumen Marker molecular weight is followed successively by under upper: 170KD, 130KD, 100KD, 70KD, 55KD, 40KD, 35KD and 25KD.

Fig. 5: the glucocorticoid receptor (GR) protein SDS-PAGE electrophoretogram of purification Identification, swimming lane 1 is BSA (2.0 μ g), the glucocorticoid receptor (GR) albumen that swimming lane 2 is ni-sepharose purification; Swimming lane M1 is Marker, and albumen Marker molecular weight is followed successively by under upper: 94KD, 66KD, 36KD, 14KD.

Fig. 6: the glucocorticoid receptor (GR) protein electrophoresis figure of protein immunoblot purification Identification, swimming lane M is Marker; The glucocorticoid receptor expression albumen that swimming lane 1 is ni-sepharose purification; Marker molecular weight is followed successively by under upper: 120KD, 85KD, 60KD, 40KD.

Fig. 7: the Agkistrodon extract collection of illustrative plates of being combined with glucocorticoid receptor (GR) in mesolow chromatographic isolation Agkistrodon water decoction, peak 1 is carbamide peak, peak 2 and 3 is respectively the eluting peak of Agkistrodon extract.

Fig. 8: the Mass Spectrometric Identification collection of illustrative plates of No. 2 eluting peaks.

Fig. 9: the Mass Spectrometric Identification collection of illustrative plates of No. 3 eluting peaks.

Figure 10: the glucocorticoid receptor (GR) that Agkistrodon extract regulates and the interactional protein immunoblot figure of NF-κ B.The dosage that adds from left to right Agkistrodon extract in the cell culture fluid of 6 orifice plates is respectively 0 μ l, 1 μ l, 2 μ l, 5 μ l, 10 μ l, 100 μ l, and A(is IB:NF-κ B) what represent is the NF-κ B content of being combined with glucocorticoid receptor (GR).B(is IB:GR α) what show is the content of glucocorticoid receptor (GR), shows that each group is consistent.C(is IB:NF-κ B) what show is NF-κ B expression.Result shows to add 10 μ l Agkistrodon extracts and can strengthen the interaction of glucocorticoid receptor (GR) and NF-κ B.

(5) detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

As do not specialize part, in the list of references that can quote according to the handbook such as " molecular cloning experiment guide " ((U.S.) M.R. Green //J. Pehanorm Brooker Science Press), " LC-MS analysis technical standard compilation/analysis and testing technology series standard compilation " (China Standards Press, 2013) that those skilled in the art were familiar with and the present invention, listed method is implemented.In addition, the material using in embodiment unless otherwise stated, all can be bought by commercial sources from the market.

Embodiment 1: Agkistrodon extract in the method enrichment Agkistrodon water decoction based on affinity chromatograph

1, the clone of the full gene of glucocorticoid receptor (GR) (gl μ cocorticoid reccptor, GR)

Lymphocyte separation medium (LTS1077 TBD company) separates normal human blood leukocyte, and Trizol method is extracted the total RNA of leukocyte.Then total RNA reverse transcription is become to cDNA, the system that reverse transcription provides taking the AMV reverse transcriptase of TaKaRa company is standard (TaKaRa RNA LA PCRTM KiT (AMV) Ver.1.1Code No:DRR012A).According to the serial number NM_001020825.1 of the glucocorticoid receptor (GR) providing on GeneBank, design a pair of far-end primer amplification containing GR α CDS with Primer5.0 and obtain about 2.8kb fragment, upper and lower primer sequence is: 5-TGTGCGAGAATGGGGAGG-3; 5-GGGCACTGGTGGTTTAGG-3.PCR reaction condition: 94 degree 3 minutes, (94 degree 35 seconds, 59 degree 30 seconds, 72 degree 3 minutes) 30 circulations, 72 degree 10 minutes, 16 degree are preserved; The full gene of glucocorticoid receptor (GR) α that pcr amplification obtains identifies through nucleic acid electrophoresis, and size is correct, and as shown in Figure 1, arrow indicating positions is increased genes of interest to electrophoresis result, the PCR product that No. 1 swimming lane is GR α, and the swimming lane of labelling M is Marker.PCR product is linked in cloning vehicle P Μ C57, checked order.Order-checking completes in Shanghai Sheng Gong biological engineering company limited, and the sequence on sequencing result and NM_001020825.1 is compared, and detects the accuracy of clone's gene.To check order No. 15 plasmids of pMD18-T-GR α correct as template, use primer amplification to obtain the GR α open reading frame (aminoacid sequence is shown in SEQ ID NO:2) of about 2.4kb, primer sequence is: 5-ATAcgcggatcc GCACTGATGGACTCCAAAGAATC-3;

5-ATACCGctcgag CCATTCTTATTAAGGCAGTCAC-3; PCR reaction condition: 94 degree 2 minutes, (94 degree 15 seconds, 64 degree 15 seconds, 72 2 points of degree 30 seconds) 30 circulations, 72 degree 10 minutes, 16 degree are preserved;

The correct gene of clone is carried out to nucleic acid transformation, make it more appropriate to prokaryotic expression, improved nucleotide sequence (shown in SEQ ID NO:1) utilizes DNAman software to compare with the difference of former nucleotide sequence, and compare of analysis result as shown in Figure 2.

Nucleotide sequence SEQ ID NO:1:

SEA ID NO:2 is:

MDSKESLTPSSKEIPSDVLGSERRKVIGFYKTLRGGATAKVSASSPSLAAAAQSDSKQRRLLVDFPKGSGSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDFRLLEESIANLSRSTSVSENPMSSSSAVSGTPTEELPQTQSDVSSEQQNLKGQAGTSGGNVKLYPADQSTFDILQDLEFSSGSPGKETNESPWRPDLLMDENCLLSPLAGEDDPFLLEGNSSEDCKPFILPDTKPKIKDNGDVILSSPNSVPLPQVKTEKEDFIELCTPGVIKQEKLGPVYCQASFSGANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVSSENWNRCQGSGDDNLTSLGTMNFPGRSVFSNGYSSPGMRPDVSSPPSSSSTATGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQDTSENLNKTVVPATLPQLTPTLVSLLEVIEPEVLYAGYDASVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFKNLHLDDQMILLQYSWMFLMAFALGWRSYRQSSGNLLCFAPDLVVNEQRMTLPCMYDQCKHMLYVSSELKRLQVSYEEYLCMKTLLLLSSVPKEGLKSQELFDEIRMTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQLPKYSNGNIKKLLFHQK。

2, the structure of glucocorticoid receptor (GR) prokaryotic expression carrier

Expression motif is as follows,

Expression Vector E4 skeleton: Trx label+His sequence label+TEV cleavage site+glucocorticoid receptor (GR);

Expressing destination protein molecular weight is 102961.2 dalton, and pI is 6.27.Sequence is as follows,

MSDKIIHLTD?DSFDTDVLKA?DGAILVDFWA?EWCGPCKMIA

PILDEIADEY?QGKLTVAKLN?IDQNPGTAPK?YGIRGIPTLL

LFKNGEVAAT?KVGALSKGQL?KEFLDANLAG?SGSGHMHHHH

HHSSGLVPRG SGMKETAAAK FERQHMDSPD LGTENLYFQG+ glucocorticoid receptor (GR) aminoacid sequence.

Experiment basic process is divided into PCR product and cuts glue recovery, and double digestion, reclaims double digestion product, is connected to corresponding prokaryotic expression carrier.Connect product and transform escherichia coli, choose positive colony amplification culture, extract plasmid, adopt the middle restriction enzyme site of two ends restriction enzyme site and sequence jointly to detect.

PCR product is cut glue and is reclaimed employing DNA gel recovery test kit recovery PCR product.Reclaiming test kit description by AxyPrep DAN gel carries out.In 1.5mL centrifuge tube, add double digestion system to prepare as table 1, then 37 DEG C of water-bath 3h.In table 1, restricted enzyme used is all purchased from TakaRa company.

Table 1 double digestion system

Double digestion product is clean to be reclaimed with the clean recovery of PCR test kit recovery double digestion product.Undertaken by TaKaRa Code:DV807A description.The PCR product of double digestion is connected with the prokaryotic expression carrier that carries out equally double digestion, and linked system is as shown in table 2, and then 16 DEG C of circulator baths spend the night.

Table 2 linked system

Connect product transformed competence colibacillus bacillus coli DH 5 alpha, the positive single colony inoculation of picking is to containing in 10mL LB culture fluid (containing the Amp of final concentration 100 μ g/ml in culture fluid) culture tube, and 200 revs/min of 37 DEG C of constant-temperature tables, spend the night.Collect thalline, obtain the recombination engineering containing glucocorticoid receptor gene, extract plasmid.Extracting test kit description in a small amount by AxyPrep plasmid DNA carries out.In the middle of adopting two ends restriction enzyme site and sequence, restriction enzyme site detects jointly.Result shows that Expression Vector E4 expression vector is the prokaryotic expression system of high efficient expression glucocorticoid receptor (GR) albumen, Fig. 3 shows the enzyme action identification and analysis that is structured in the GR prokaryotic expression carrier in E4 prokaryotic expression carrier, and result shows that vector construction is correct.M1 swimming lane is Marker, and swimming lane 1 is BamH I/EcoR I enzyme action product, expection band 1502bp, 746bp; Swimming lane 2 is Hind III/Xho I product enzyme action, expection band 1071bp, 554bp, 368bp; Swimming lane 3 is BamH I/Xho I enzyme action, cuts out the about 2360bp of GR α CDS total length.Under similarity condition, with pGS21a, Expression Vector E3 carrier is contrast, and result shows to determine that through examination Expression Vector E4 expression vector is optimal expression system.With pGS21a, the expression vector that Expression Vector E3 is framework construction, the destination protein content of expressing after induction is very low.

3, the abduction delivering of glucocorticoid receptor (GR) albumen

(1) induction: the recombination engineering that the Expression Vector E4 carrier containing glucocorticoid receptor gene is transformed is seeded in LB fluid medium, under 37 DEG C, 200rpm condition, cultivate 12h, obtain seed liquor, after adding respectively 35mL LB culture fluid in 2 50mL centrifuge tubes, add again 175 μ l Amp and 350 μ l seed liquor to shake up, seal up sealed membrane.Above-mentioned two pipe culture fluid are put to 37 DEG C of shaking table 200rpm of constant temperature and shaken bacterium 6-8h, make OD ₆₀₀reach 0.6-0.8.In super-clean bench toward the IPTG (Merck CB420322) that adds respectively final concentration 1mM in two pipe culture fluid, sealer, 15 DEG C, 200rpm shakes bacterium 12 hours.

(2) protein extraction: shake the two pipe culture fluid that spend the night at 4 DEG C, 12000rpm, centrifugal 5min, abandons waste liquid.Be 7.0 by 3mL PBS(pH value respectively) piping and druming precipitation, merge into a pipe after evenly, make bacteria suspension.In above-mentioned bacteria suspension, add final concentration 1mM Phenylmethanesulfonyl fluoride (Phenylmethanes μ lfonyl fl μ oride, PMSF) to carry out ultrasonication cell, power 300W is set, ultrasonic 10s interval 10s, ultrasonic time 10min, 4 DEG C of temperature.By the bacteria suspension 12000rpm after fragmentation, 4 DEG C, centrifugal 10min.In precipitation, add 6mL carbamide lysate (carbamide 24.04g, sodium chloride 0.8766g, sodium phosphate 0.9503g, adding distil water is settled to 50mL) and working concentration PMSF (the green skies that are 1mM, ST506), piping and druming is evenly vibrated, and then ice is put 5min.Vibration ice is put 4 circulations.4 DEG C afterwards, 12000rpm, centrifugal 10min.Supernatant after centrifugal is the total protein of the recombination engineering transforming containing the E4 carrier of glucocorticoid receptor gene after IPTG induction, get 20 μ l supernatant and add isopyknic 2 × sample-loading buffer, detect the abduction delivering situation of albumen by SDS-PAGE method.

Under similarity condition, with the recombination engineering that do not transform containing the E4 carrier of glucocorticoid receptor gene, without the e. coli total protein of IPTG induction, the recombination engineering not transforming containing the E4 carrier of glucocorticoid receptor gene, the recombination engineering transforming through the e. coli total protein of IPTG induction with containing the E4 carrier of glucocorticoid receptor gene, without the e. coli total protein of IPTG induction in contrast.

The abduction delivering situation of SDS-PAGE as shown in Figure 4, after result shows IPTG induction, express obviously by destination protein.Remaining supernatant will be for the preparation of glucocorticoid receptor (GR) affinity column in step 4.M swimming lane represents albumen Marker, and albumen Marker molecular weight is followed successively by under upper: 170KD, 130KD, 100KD, 70KD, 55KD, 40KD, 35KD, 25KD.No. 1 swimming lane is the e. coli total protein without IPTG induction that contains E4 zero load.No. 2 swimming lanes are that IPTG induces the e. coli total protein that contains E4 zero load.No. 3 swimming lanes are the e. coli total protein without IPTG induction that contains E4-GR alpha expression carrier.No. 4 swimming lanes are the e. coli total protein of IPTG induction containing E4-GR alpha expression carrier.

4, glucocorticoid receptor (GR) protein purification and chromatographic column preparation

First by ni-sepharose purification glucocorticoid receptor (GR) albumen, detect the joint efficiency of nickel post and glucocorticoid receptor (GR) albumen.Process is as follows, (1) nickel post pretreatment: prepare nickel post, open the sub-lid of void column, add 1ml filler (Ni-NTA Agarose, QIAGEN30210), then open the lower cover of pillar, the approximately 800 μ L that flow away, add 2 times of column volume binding buffer liquid (formula of every 50mL is: carbamide 24.04g at once, sodium chloride 0.8766g, sodium phosphate 0.9503g, imidazoles 0.0344, adding distil water is settled to 50mL) balance.Above filler, lower cover is covered when approximately 500 μ L.

(2) glucocorticoid receptor (GR) albumen coupling Ni-NTA Agarose post: the supernatant that contains destination protein of preparation in step 3, after the membrane filtration of 0.45 μ m, is got to filtrate and is added in pretreated nickel post.By nickel post be placed on vertical shaking table, 4 DEG C, 70 rpms place 1.5h.With 1mL PBS(pH value be 7.0) 2 pillars of washing, obtain the Ni-NTA Agarose post of coupling sugar cortical hormone receptor 4 body protein.

(3) checking of coupling protein: (formula of every 50mL is: carbamide 24.04g to use respectively the binding buffer liquid of 500 μ L and 1mL, sodium chloride 0.8766g, sodium phosphate 0.9503g, imidazoles 1.72g, adding distil water is settled to 50mL) the Ni-NTA Agarose post of eluting coupling sugar cortical hormone receptor 4 body protein, collect eluent and be placed in 1.5mL centrifuge tube.Get 20 μ l eluents and add 20 μ l2 × sample-loading buffers (the green skies of P0015B biotechnology research institute) to carry out SDS-PAGE qualification, detect protein purification efficiency, as shown in Figure 5, result shows that the purity of protein eluting is high to result.Bovine serum albumin (BSA) sample that in figure, No. 1 swimming lane is 2.0 μ g.No. 2 swimming lanes are the glucocorticoid receptor (GR) total protein through ni-sepharose purification eluting, and arrow indicating positions is destination protein.M1 is albumen Marker swimming lane, and albumen Marker molecular weight is followed successively by under upper: 94KD, 66KD, 36KD, 14KD.In order to identify the correctness of sugared cortex receptor protein of prokaryotic expression, select the primary antibodie (μ sc-1002, Santa Cr μ z company) of glucocorticoid receptor (GR), whether the albumen reclaiming by protein immunoblot technical appraisement is glucocorticoid receptor (GR).Result shows that the albumen of purification is correct as Fig. 6, and M is albumen Marker swimming lane, and albumen Marker molecular weight is followed successively by under upper: 120KD, 85KD, 60KD, 40KD.1 is the glucocorticoid receptor expression of ni-sepharose purification, shows that through glucocorticoid receptor (GR) Identification of the antibodies the albumen of prokaryotic expression is glucocorticoid receptor (GR).Nickel post can reclaim use, use respectively lavation buffer solution (carbamide 24.04g, sodium chloride 0.8766g, sodium phosphate 0.9503g, imidazoles 1.72g, adding distil water is settled to 50mL) 1mL and binding buffer liquid (carbamide 24.04g, sodium chloride 0.8766g, sodium phosphate 0.9503g, imidazoles 0.0344, adding distil water is settled to 50mL) 1mL washs once, then adds binding buffer liquid 1mL balance to prepare loading again.

5, the preparation of Agkistrodon extract

Agkistrodon dry body 20g, smashes, and adds distilled water 2000ml, and medicine decocting tank decocts to 100ml, after filtration by filtrate-20 DEG C of lyophilizations, be ground into pressed powder, be divided into 10 grades and divide as sample.Getting a duplicate samples, to add PBS(pH value be 7.0) dissolve, cross 0.25 μ M microporous filter membrane, filtrate flows through as mobile phase the Ni-NTA Agarose post that step 4 method is prepared coupling sugar cortical hormone receptor, PBS buffer (pH value is 7.0) washed twice.Then carry out eluting (21004PIERCE company) with 200 μ l IgG Elution Buffer eluents, collect effluent, acquisition can with the Agkistrodon extract of glucocorticoid receptor (GR) combination.This extract part is carried out activity analysis by cytology's method, sees step 7.A part, for further qualification, is shown in step 6.

6, liquid matter qualification Agkistrodon extract component

Instrument model is Agilent6520Q-TOF/MS, chromatographic condition: mobile phase A is volumetric concentration 0.1% aqueous formic acid, and B is volumetric concentration 0.1% formic acid acetonitrile solution, and after 20% B isocratic elution, be 3min running time.It is 35 DEG C that column oven temperature is set, and gets the Agkistrodon extract of preparation in step 5, sample size 5 μ l, and auto injection actuator temperature is 5 DEG C.Result, as Fig. 7 shows, detects two detached peakses, is labeled as No. 2 peaks (10min place) and No. 3 peaks (11min place) in figure.

Mass spectrum condition: use respectively ESI positive ion mode image data.Data acquisition mass charge ratio range is: 50-1000m/z, dry gas is N ₂dry gas temperature is 350 DEG C, positive ion mode capillary voltage (Capillary Voltage) is 4000V, cracked voltage (Framentor Voltage) is 180V, taper hole voltage (Skimmer Voltage) is respectively 60V, nebulizer pressure (Neb μ lizer press μ re) is 40psi, uses Centroid pattern to preserve mass spectrometric data, and data acquisition rate is 2spectr μ m/s.Result as shown in Figure 8,9.

7, Agkistrodon extract strengthens the interaction of glucocorticoid receptor (GR) and Nuclear Factor kappa B

The interpolation of cell transfecting and Agkistrodon extracting solution: cultivate HEK293T cell (GNHu43 in the DMEM culture medium of adding 10% hyclone, Chinese Academy of Sciences's Shanghai cell bank), first 24 hours of transfection, is seeded to 6 orifice plates by cell, when Growth of Cells is paved with bottom 80%, prepare transfection.Before transfection, prepare GR α-Myc carrier for expression of eukaryon and NF-κ B-Flag expression vector plasmid.The amount of the plasmid that the rotaring redyeing system of six orifice plates is used is 1 μ g, liposome 3 μ l, transfection step is carried out with reference to transfection reagent S μ perFecting TMII Vitro DNA Transfection Reagent (2102-100 Pu Fei Bioisystech Co., Ltd) description.1ml fresh culture is added in transfection after 24 hours, each hole is added respectively Agkistrodon extract 0 μ l, 1 μ l, 2 μ l, 5 μ l, 10 μ l, 100 μ l, continued to cultivate after 24 hours collecting cell simultaneously.

Co-immunoprecipitation and Western-blot analyzing proteins are done mutually: in the cell of having collected, (cell concentration is approximately two the hole sums of six blocks of plates that cover with cell) adds the IP lysate (the green skies of P0013 Bioisystech Co., Ltd) of 500 μ l, 4 DEG C of rotation 1h make its abundant cracking, 20, the centrifugal 20min of 000g, get supernatant, add primary antibodie (1 μ g/500 μ l), spend the night by 4 DEG C of mixing, then add the 20 green skies of μ l Protein A/G beads(P2012 biotechnology) to mix 4-6h.Afterwards with IP lysate washing ProteinA/G beads once, then with PBS washing beads tri-times.Finally add 20 μ l2 × SDS albumen sample-loading buffers and boil after 10min centrifugally, sucking liquid also carries out SDS-PAGE electrophoresis.Then carry out protein immunoblot qualification, the method is known technology.Primary antibodie used and two resists: NF κ B p65C-20Rabbit Polyclonal Antibody (sc-372Santa Cruz company), GRmouse monoclonal antibody(sc-393232Santa Cruz company), the sheep anti-mouse antibody (926-32210, LI-COR company) of the goat anti-rabbit antibody (926-32221LI-COR company) of two anti-IRDy680 labellings and two anti-IRDy680 labellings.

As shown in figure 10, the Agkistrodon extract that the method obtains can strengthen the interaction of glucocorticoid receptor (GR) and NF-κ B to result.The interactional effect that the Agkistrodon extract that wherein adds 10 μ l in 6 orifice plates can strengthen glucocorticoid receptor (GR) and NF-κ B is the most obvious.

Claims (7)

1. an Agkistrodon extract, it is characterized in that described Agkistrodon extract prepared as follows: the preparation of (1) glucocorticoid receptor (GR) albumen: in the fermentation liquid obtaining through fermentation culture to the recombination engineering containing glucocorticoid receptor gene, add IPTG to carry out inducing culture, obtain inducing culture liquid; By centrifugal inducing culture liquid, collecting precipitation extracts albumen after ultrasonication, obtains glucocorticoid receptor (GR) albumen; The nucleotides sequence of described glucocorticoid receptor gene is classified as shown in SEQ ID NO:1; (2) Agkistrodon extract: glucocorticoid receptor (GR) albumen coupling prepared by step (1) is to Ni-NTA Agarose post as immobile phase, the Ni-NTA Agarose post of flowing through Agkistrodon water decoction as mobile phase again, then twice, PBS washing pillar, add IgG Elution Buffer eluent to carry out eluting, collect effluent, obtain Agkistrodon extract; Described Agkistrodon water decoction is prepared as follows: after Agkistrodon powder is mixed with mass ratio 1:100 with distilled water, decoct to 5% of raw material volume, filter, the phosphate buffer that is 7 with pH value after filtrate lyophilization dissolves, and filters, and filtrate is Agkistrodon water decoction.

2. Agkistrodon extract as claimed in claim 1, the method that it is characterized in that the described recombination engineering fermentation culture of step (1) is: the recombination engineering containing glucocorticoid receptor gene is seeded in LB fluid medium, under 37 DEG C, 200rpm condition, cultivate 12h, obtain seed liquor, seed liquor is seeded in LB fluid medium with the inoculum concentration of volumetric concentration 1%, then adding final concentration is the ampicillin of 100 μ g/ml, under 37 DEG C, 200rpm condition, is cultured to OD ₆₀₀reach 0.6～0.8, obtain fermentation liquid.

3. Agkistrodon extract as claimed in claim 2, is characterized in that the condition of the described fermentation liquid inducing culture of step (1) is: to the IPTG that adds final concentration 1mM in fermentation liquid, inducing culture 12h under 15 DEG C, 200rpm condition, obtains inducing culture liquid.

4. Agkistrodon extract as claimed in claim 1, it is characterized in that the described protein extracting method of step (1) is: by centrifugal inducing culture liquid, collecting precipitation is made bacteria suspension with the phosphate buffer that pH value is 7, in bacteria suspension, add the Phenylmethanesulfonyl fluoride of final concentration 1mM, at power 300W, ultrasonication 10s under 4 DEG C of conditions of temperature, interval 10s, ultrasonic 10min, centrifugal, abandoning supernatant, precipitation is vibrated after adding carbamide lysate and Phenylmethanesulfonyl fluoride piping and druming evenly, ice is put 5min, repeating vibration and ice puts 4 times, centrifugal, get supernatant, obtain glucocorticoid receptor (GR) albumen, the compound method of the every 50mL of described carbamide lysate is: carbamide 24.04g, and sodium chloride 0.8766g, sodium phosphate 0.9503g, adding distil water is settled to 50mL.

5. Agkistrodon extract as claimed in claim 1, it is characterized in that the described Agkistrodon water decoction of step (2) prepared as follows: after Agkistrodon powder is mixed with mass ratio 1:100 with distilled water, decoct to 5% of former reactant liquor volume, filter, filtrate is made powder after-20 DEG C of lyophilizations, then the phosphate buffer that is 7 with pH value dissolves, with 0.25 μ M filtering with microporous membrane, filtrate is Agkistrodon water decoction.

6. Agkistrodon extract as claimed in claim 1, it is characterized in that described Agkistrodon extract prepared as follows: (1) glucocorticoid receptor (GR) albumen: the recombination engineering containing glucocorticoid receptor gene is seeded in LB fluid medium, under 37 DEG C, 200rpm condition, cultivate 12h, obtain seed liquor, seed liquor is seeded in LB fluid medium with the inoculum concentration of volumetric concentration 1%, then adding final concentration is the ampicillin of 100 μ g/ml, under 37 DEG C, 200rpm condition, is cultured to OD ₆₀₀reach 0.6～0.8, to the IPTG that adds final concentration 1mM in culture fluid, inducing culture 12h under 15 DEG C, 200rpm condition, obtains inducing culture liquid; By centrifugal inducing culture liquid, collecting precipitation is made bacteria suspension with the phosphate buffer that pH value is 7, in bacteria suspension, add the Phenylmethanesulfonyl fluoride of final concentration 1mM, ultrasonication under power 300W, 4 DEG C of conditions of temperature, ultrasonic 10s, interval 10s, ultrasonic 10min, centrifugal, abandoning supernatant, precipitation adds carbamide lysate and Phenylmethanesulfonyl fluoride, vibration after piping and druming evenly, ice is put 5min, repeat vibration and ice and put 4 times, 12000rpm, centrifugal 10min, after centrifugal, get supernatant, obtain glucocorticoid receptor (GR) albumen; The compound method of the every 50mL of described carbamide lysate is: carbamide 24.04g, and sodium chloride 0.8766g, sodium phosphate 0.9503g, adding distil water is settled to 50mL; (2) preparation of Agkistrodon extract: glucocorticoid receptor (GR) albumen coupling prepared by step (1) is to Ni-NTA Agarose post as immobile phase, flow through Agkistrodon water decoction as the mobile phase Ni-NTA Agarose post of protein-coupled receptor, then twice, PBS washing pillar, add IgG Elution Buffer eluent to carry out eluting, collect effluent, obtain Agkistrodon extract; Described Agkistrodon water decoction is prepared as follows: after Agkistrodon powder is mixed with mass ratio 1:100 with distilled water, decoct to 5% of raw material volume, filter, filtrate is ground into powder after-20 DEG C of lyophilizations, then the phosphate buffer that is 7 with pH value dissolves, with 0.25 μ M filtering with microporous membrane, filtrate is Agkistrodon water decoction.

Described in a claim 1 Agkistrodon extract in the application of preparing in glucocorticoid antiinflammatory drugs.