CN104356220B - A kind of have AntiHIV1 RT activity Aedes albopictus albumen of anticoagulant function and preparation method thereof - Google Patents

A kind of have AntiHIV1 RT activity Aedes albopictus albumen of anticoagulant function and preparation method thereof Download PDF

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CN104356220B
CN104356220B CN201410636317.4A CN201410636317A CN104356220B CN 104356220 B CN104356220 B CN 104356220B CN 201410636317 A CN201410636317 A CN 201410636317A CN 104356220 B CN104356220 B CN 104356220B
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aedes albopictus
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徐学清
李琳
刘叔文
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Southern Medical University
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Abstract

The present invention relates to a kind of AntiHIV1 RT activity Aedes albopictus albumen with anticoagulant function, the aminoacid sequence of this albumen is SEQ ID NO：Sequence shown in 1.AntiHIV1 RT activity Aedes albopictus albumen of the present invention be by Aedes albopictus salivary gland total serum IgE purified, amplify total cDNA, then, again with total cDNA for its gene cloning of template amplification to prokaryotic expression carrier, after folding through IPTG abduction delivering, the inclusion body degeneration to expression and renaturation, gel filtration and ion exchange mechansim purification obtain.The AntiHIV1 RT activity Aedes albopictus albumen of the present invention has the activity that can suppress HIV propagation in the cell and the platelet aggregation of several agonist inductions, and the advantage of no cytotoxicity and AntiHIV1 RT activity high specificity, can be used for preparing the medicine of anti-AIDS and its relevant disease.

Description

A kind of AntiHIV1 RT activity Aedes albopictus albumen with anticoagulant function and its system Preparation Method
Technical field：
The present invention relates to there is peptide more than 20 aminoacid and in particular to a kind of albumen obtaining from animal tissue, should Albumen has the biological activity of AntiHIV1 RT activity.
Background technology：
Acquired immune deficiency syndrome (AIDS) (AIDS) is seriously to damage, with systemic immune system, the infectious disease being characterized, from its be found with Come, seriously threaten the existence of the mankind, the development to population in the world health and social economy produces tremendous influence.Along with many Research over year, has many anti-AIDS drugs at present.Act on the difference of viral targets, the anti-AIDS drugs of listing according to medicine Be broadly divided into glycoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitor, protease inhibitor, entry inhibitor, Integrase inhibitor etc..These medicines suppress respectively inhibition of HIV propagation entrance, fusion, reverse transcription, integration, transcription, translation, Group merge overflow etc. process, make AIDS treatment on make great progress (Infect Dis Rep.2013,5 (Suppl1):e2).At present AIDS sickness rate declines rapidly, and people no longer talk AIDS and complexion changed.Due to above-mentioned all medicines only It is link specific for certain in inhibition of HIV reproduction process, easily induction inhibition of HIV produces variation and forms drug resistance strain, leads Some drugses are caused to lose therapeutic value.The drug resistance problems of inhibition of HIV become a century property difficult problem for puzzlement AIDS treatment.Research is high Effect, the anti-AIDS drugs of low toxicity and method are the focuses of current international research.
Joint antiretroviral therapy (cART) the energy effective control disease process of application multi-medicament, and part solution The drug resistance problems of inhibition of HIV.But after the method treatment, many is lived and HIV relevant disease with the patient that must grow, As, chronic inflammation disease and nervus cognition disorder etc. (J Pathol.2008,10:231-241).Additionally, in HIV person Thrombus in vivo disease, the rate of delivering of cardiovascular disease significantly rise, especially thrombocytopenia, and it is in the urgency of HIV patient Often (Circulation.2008,10 in property infection period and acquired immune deficiency syndrome (AIDS) phase:e36–e40；Curr Cardiol Rev.2008,10:203-218).Therefore it is necessary to research and develop effective treatment meanss while suppressing HIV propagation Control these diseases.
Platelet is a part for inflammation systems, closely related with the pathogenesis of HIV person.On the one hand, blood is little Plate can in conjunction with, phagocytosis, internalization HIV, and can use as after activation virus bin so that virus is bred wherein and be transmitted to Farther organ.On the other hand, the platelet being activated by HIV can be discharged many Inflammatory substances, such as sCD40L and blood Platelet activation factor etc. promotes platelet activation and inflammation to occur further, the process (PLoS of aggravation AIDS and relevant disease One.2012,10:e44454；Blood.2012,10:1334–1343).Therefore, suppression platelet contribute to treat AIDS and its Relevant disease.Many is with regard to anti-blood such as aspirin, adp receptor blocker, GPIIb-IIIa Disintegrin and statins The research of platelet molecule reports them and can reduce HIV person's cardiovascular disease, the thrombotic disease inflammatory disease related with HIV The incidence rate of disease.Among them, Statins can suppress inflammation, weakens platelet activation and because blocking inhibition of HIV granule and rush Infection cellular component between combination and there is direct HIV (human immunodeficiency virus)-resistant activity.Many HIV persons can control after accepting Statins Hyperlipemia (the Curr Opin Endocrinol Diabetes Obes.2011,10 that joint antiretroviral therapy is brought: 144-147；Viral Immunol.2005,10:474–489).Therefore Statins is considered as very promising AIDS treatment Drug candidate.Lack enough datas due to current, this compound may be deposited on the road becoming AIDS medicine In unexpected obstacle it is therefore necessary to develop more AntiHIV1 RT activity and hematoblastic drug candidate be used for treating AIDS and its Relevant disease.
The Nature breeds all things on earth, contains the material that many counteracts each other wherein, gives birth to from Chinese herbal medicine and ocean at present It is isolated to multiple alkaloidss with suppression HIV activity, protide, terpenoid, flavonoid, lignanoids and tonkabean in object Plain class etc..Although currently without the active substance finding HIV (human immunodeficiency virus)-resistant activity in HAEMATOPHAGOUS ARTHROPODS body, the film sugar egg of HIV It is found (Insect in soft ticks Antricola delacruzi and Ornithodorus parkeri salivary gland in vain Biochem Mol Biol.2012,42(5):332-42；Insect Biochem Mol Biol.2008,38(1):1-21). Therefore, HAEMATOPHAGOUS ARTHROPODS is probably the important sources of natural AntiHIV1 RT activity protein drug.
There is long history in China to the medicinal study of zootoxin, but pharmacology in the HAEMATOPHAGOUS ARTHROPODS body such as mosquito is lived Property component research few.Aedes albopictus (Aedes albopictus) are the important insect vectors of China, all have throughout the country Distribution.
Content of the invention：
The technical problem to be solved is to provide one kind to belong to biomedical sector to have anticoagulant The Aedes albopictus albumen of the AntiHIV1 RT activity of function, this albumen has high specificity, the good and cheap and easy to get advantage of activity.
The scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of AntiHIV1 RT activity Aedes albopictus albumen with anticoagulant function, the aminoacid sequence of this albumen is MAPIWNAKNPEQIQYLAARCMEEWSPKAKDPKAAIKNWMEWKIQPSNEEATQCYTKCMIENIGYYEPGEKRIKGVRV MQQWETFNRYQSADRNKVHDITDTFDFIKPLKSSSCSDVFNAYKDVHAKHLETIKAILFCDGKSAEKYYKDKGKNVK QKGESIFVHCEEIHYPVGSPQRNELCKVRKYELGTGKPFENLMECIFKGVRYFNDKNELNIDEIARDFTQVGKKPDA VKAAMENCKSKTKETDPGKKAVEYYKCLLADSKVKKDFMEAFDYRELRSKDYYAQLTGKIKPYSASDVRKEVNDLDS NKCV(SEQ ID NO：1)
Above-mentioned Aedes albopictus albumen is a kind of single chain protein of Aedes albopictus salivary gland DNA recombinant expression, and its molecular weight is 36656.83 dalton, isoelectric point, IP is 8.501.
It is of the present invention that to have anticoagulant function AntiHIV1 RT activity Aedes albopictus albumen be using from the lineae ablicantes sucked blood Yellow-fever mosquito salivary gland DNA recombinant expression obtains, and its concrete preparation method comprises the steps of：
(1) use PROMEGA company after extracting Aedes albopictus salivary gland total serum IgEmRNA Isolation Systems test kit isolates and purifies mRNA and utilizes CLONTECH company CreatorTMSMARTTMcDNA Library Construction Kit Construction of Plasmid cDNA Library test kit amplifies the total cDNA of Aedes albopictus salivary gland, then adopts SEQ ID NO：Forward primer shown in 2 and SEQ ID NO：Downstream primer amplification coding SEQ ID NO shown in 3：Anti- shown in 4 The gene of HIV Aedes albopictus albumen, then first by this gene cloning in pET-17b expression vector, then be transformed into BL21 (DE3) Recombinant expressed in pLysS escherichia coli, and collect inclusion body protein between 28-37kDa for the purpose band；
(2) by collected inclusion body guanidine hydrochloride dissolution and fold after go up successively Sephadex SR-100 solvent resistant column, Hiprep SP cation-exchange chromatography post, Sephadex G75 solvent resistant column and Sephadex G75 gel filtration chromatography, Obtain as SEQ ID NO：The Aedes albopictus salivary gland DNA recombinant expression albumen of sequence shown in 1.
The Aedes albopictus albumen of AntiHIV1 RT activity of the present invention has specific inhibitory effect, IC to HIV difference strain50It is situated between In between 10-35nM and have no toxic side effect, ADP, U can be suppressed46619Platelet aggregation with collagen induction.
Brief description：
Fig. 1 is AntiHIV1 RT activity Aedes albopictus albumen escherichia coli expression electrophoretogram of the present invention, in figure, is purpose peak shown in arrow Position, band 1 is to be not added with negative bacterium comparison, and band 2 induces result for positive bacteria, and band 3 is positive bacteria non-induced result, and band 4 is albumen Matter standard.
Fig. 2 is AntiHIV1 RT activity Aedes albopictus protein purification chromatogram of the present invention, is purpose peak shown in figure arrow, and A, B, C, D scheme It is Sephadex SR-100 gel filtration chromatography, Hiprep SP cation-exchange chromatography column chromatography, Sephadex respectively G75 gel filtration chromatography, Sephadex G75 gel filtration chromatography result figure, in Fig. 2 D, electrophoretogram is final step The electrophoresis result of the Peak Activity of Sephadex SR-100 gel filtration chromatography is it is illustrated that band is purpose albumen.
Fig. 3 is that AntiHIV1 RT activity Aedes albopictus albumen of the present invention suppresses platelet aggregation figure, in figure, and a is negative control；B is 1.85 μM albumen is to Convuxlin, U46619Inhibitory action with Collengen induced platelet aggregation；C is 9 μM of PROTEIN C onvuxlin The inhibitory action of induced platelet aggregation.
Specific embodiment：
There is the preparation of AntiHIV1 RT activity Aedes albopictus albumen and the identification of anticoagulant function：
One) there is the clone of the AntiHIV1 RT activity Aedes albopictus protein gene of anticoagulant function：
I, Aedes albopictus salivary gland Total RNAs extraction：
A. live body Aedes albopictus are placed on 30-60 minute frost anesthesia on ice, dissect 50 Aedes albopictus under the microscope and obtain To salivary gland, add 1m1 Total RNAs extraction buffer (Trizol solution, U.S.'s GIBCOBRL Products), even in 5m1 glass It is homogenized 1 minute in slurry device.
B. add equal-volume phenol/chloroformic solution, acutely mix, room temperature is placed 1 minute, and 4 DEG C, 12000rpm is centrifuged 10 points Clock, reject precipitates.
C. supernatant adds isopyknic isopropanol, and room temperature is placed 4 minutes, and 4 DEG C, 12000rpm is centrifuged 10 minutes, and precipitation is used 75% ethanol is washed once, dries, and ttom of pipe precipitate is Xanthopsyllacheopis salivary gland total serum IgE.
II, the purification of Aedes albopictus salivary gland mRNA：
Aedes albopictus salivary gland mRNA isolates and purifies using PROMEGA company of the U.S.mRNA Isolation Systems test kit.
A. take Aedes albopictus salivary gland total serum IgE 500 μ g to be dissolved in 500 μ l DEPC water, put into 65 DEG C of water-baths 10 minutes, plus Oligo (dT) probe of people 3 μ l and 13 μ l 20 × SSC solution, mix, and place room temperature cooling, referred to as A liquid.
B. the washing of magnetic bead (SA-PMP)：Magnetic bead is flicked mixing, adsorbs 30 seconds to magnetic frame, abandon supernatant, plus 0.5 × SSC0.3m1, to magnetic frame adsorb 30 seconds, finally plus 0.1ml 0.5 × SSC suspend, referred to as B liquid.
C. A liquid is added in B liquid, room temperature is placed 10 minutes, adsorb 30 seconds to magnetic frame, abandon supernatant, washed with 0.1 × SSC Wash 4 times, finally abandon supernatant, plus 0.l ml DEPC aqueous suspension, adsorb 30 seconds to magnetic frame, supernatant is moved to new test tube, then Add 0.15m1DEPC water Eddy diffusion, adsorb 30 seconds to magnetic frame, move supernatant to above-mentioned test tube, be then the white of purification in supernatant Stricture of vagina yellow-fever mosquito salivary gland mRNA.
D. add 1/10 volume 3M sodium acetate, pH 5.2, equal-volume isopropanol, place 30 minutes in -70 DEG C, 4 DEG C, 12000rpm is centrifuged 10 minutes, abandons supernatant, is precipitated and dissolved in 10 μ l DEPC water.
III, the total cDNA of Aedes albopictus salivary gland expand：
Using CLONTECH company CreatorTMSMARTTMCDNA Library Construction Kit plasmid CDNA library builds test kit amplification.
A.cDNA first chain synthesizes (mRNA reverse transcription)：
1. add 1 μ l Aedes albopictus salivary gland mRNA, 1 μ l SMART IV oligonucleotides in the aseptic centrifuge tube of 0.5ml Acid, 1 μ l CDS III/3 ' PCR primer, plus 2 μ l deionized waters make cumulative volume reach 5 μ l.
2. mix the reagent in centrifuge tube and be centrifuged 15 seconds with 12000rpm, 72 DEG C are incubated 2 minutes.
3. centrifuge tube is incubated on ice 2 minutes.
4. add 2.0 μ l 5 × the first chain buffering, 1.0 μ l 20mM dithiothreitol, DTTs, 1.0 μ l in centrifuge tube 10mMdNTP mixture, 1.0 μ l PowerScript reverse transcription.
5. mixing centrifuge tube in reagent and with 12000rpm be centrifuged 15 seconds, 42 DEG C be incubated 1 hour.
6. centrifuge tube is placed in the synthesis stopping the first chain on ice.
7. take cDNA first chain synthesized by 2 μ l standby from centrifuge tube.
B. long end polymeric polymerase chain reaction (LD-PCR) method is adopted to expand the second chain
1.95 DEG C of preheating PCR instrument.
2. 2 μ l cDNA the first chain (mRNA reverse transcription), 80 μ l deionized waters, 10 μ l 10 × Advantage 2PCR are delayed Punching, 2 μ l 50 × dNTP mixture, 2 μ l 5 ' PCR primer, 2 μ l CDS III/3 ' PCR primer and the polymerization of 2 μ l escherichia coli Enzyme centrifuge tube is reacted.
3. press following procedure in PCR instrument to expand：1. 95 DEG C, 20 seconds；2. 22 circulations：95 DEG C, 5 seconds；68 DEG C, 6 Minute
4., after loop ends, the cDNA synthesizing in centrifuge tube double-strand is stripped.
C.PCR product PROMEGA companySV Gel and PCR Clean-Up System test kit enters Row extracting and reclaiming, step is as follows：
1. the cDNA double-strand obtaining by PCR is added the reverse mixing of isopyknic film combination buffering, then by mixed liquor Proceed to centrifugal purification post, room temperature stands 5 minutes, so that DNA is fully combined with pellosil.It is centrifuged 30 seconds with 12000rpm, outwell receipts Waste liquid in collector.
2. add the eluent (containing ethanol) of 700 μ l in centrifugal purification post, be centrifuged 30 seconds with 12000rpm, outwell collection Waste liquid in pipe.
3. repeat step 2,12000rpm is centrifuged 5 minutes.
4. centrifugal purification post is placed in new centrifuge tube, adds 30 μ l ultra-pure waters, stand 5 minutes at room temperature, with 12000rpm is centrifuged 30 seconds, and ttom of pipe solution is the total cDNA of Aedes albopictus salivary gland of purified mistake.
IVth, there is the clone of the AntiHIV1 RT activity Aedes albopictus protein gene of anticoagulant function：
Aedes albopictus salivary gland nucleotide sequence (GenBank according to report:AY826093.1) it is Reference Design primer D7F:5’TGCATATGGCACCTCTGTGGAATGCAAAGAATCC’3(SEQ ID NO：2) and D7R:5’ CCCTCGAGTTAAACACACTTGTTGCTGTCGATATC’3(SEQ ID NO：3).With 100 times of dilution lineae ablicantes of crossing of purification she The total cDNA of mosquito salivary gland has the AntiHIV1 RT activity Aedes albopictus protein gene of anticoagulant function for template amplification.PCR reacts Program is：1. 95 DEG C, 4 minutes；2. 35 circulations：94 DEG C, 30 seconds；56 DEG C, 30 seconds；72 DEG C, 45 seconds；3. 72 DEG C, 10 Minute.By amplified production 1% agarose gel electrophoresiies, cut purpose band, carry out purification with DNA purification kit.Will be pure The purpose fragment changed is connected in pMD19-T carrier, obtains final product connection product.Connection product is transformed into the large intestine of Takara company Bacillus DH5 α competent cell.Appropriate converted product is taken to be applied on the LB flat board containing Amp, the list being formed through 37 DEG C of culture 16h Bacterium colony is the positive colony containing purpose fragment.The picking single bacterium colony size of M13 primer detection Insert Fragment.Select containing purpose The positive plasmid of fragment send company to be sequenced.It is (SEQ ID NO that sequencing result display gene is held to 3 ' terminal sequences from 5 '：4)：
catatggcacctatctggaatgcaaagaatcccgagcaaatccagtacctagctgcccgttgtatggaagaatggtc tccgaaggcaaaggatccgaaagcggctatcaaaaactggatggaatggaaaatccagccatcgaacgaagaagcca ctcagtgttataccaagtgtatgatcgagaacatagggtactatgagccgggtgaaaagcgaatcaagggagttcgt gttatgcaacaatgggaaacgttcaaccgataccagtcagcagatcgaaacaaagttcatgatatcacggatacatt cgacttcataaagccgctgaaatcgtccagctgctcggatgtgttcaacgcctacaaagacgtccatgccaaacatc tggaaacaatcaaagcaattctgttttgcgatggaaagtctgccgagaagtattataaggataagggtaaaaatgtc aagcaaaagggggaatctatctttgtacattgtgaagaaatacactacccagtgggtagtccacagcggaatgaatt atgtaaagttagaaagtatgagttgggcactggaaagccattcgaaaatctcatggaatgcatatttaagggcgtac gctacttcaatgataagaacgagttaaatattgacgaaattgcaagggatttcacccaggttggcaagaaacccgat gcggtgaaagcggcaatggaaaactgcaagtccaaaacgaaagaaaccgacccagggaaaaaggctgttgagtacta caagtgcttgctggccgattcaaaagtaaagaaagacttcatggaagcgttcgactatcgagaactaagatccaagg actattacgctcagctaacgggcaaaatcaaaccctacagtgccagtgatgtgcgcaaggaggtcaatgatctagac agcaacaagtgtgtttaactcgag
The AntiHIV1 RT activity Aedes albopictus protein gene cloning E. with anticoagulant function is to pET-17b expression vector：
1. add the pMD19-T carrier containing HIV protein gene for the 1 μ g, 2 μ l H buffer, 0.5 μ l in microcentrifugal tube Nde I and 0.5 μ l Xhol I restricted enzyme, adding water to cause final volume is 20 μ l；1 μ g pET-17b plasmid adopts Same enzyme action system.Enzyme action condition is：37 DEG C are reacted 2 hours；
2. digestion products are used 1% agarose gel electrophoresiies respectively, cut purpose band, carried out with DNA purification kit Purification.
3. by the purpose fragment of purification according to moles 3:1 (protein gene：PET-17b carrier) ratio be mixed to join It is attached in the ligase buffer solution mixture of amount.Connect and be adjusted to 16 DEG C of reactions 8 hours
4. connection product is transformed into the bacillus coli DH 5 alpha competent cell of Takara company.Appropriate converted product is taken to apply Cloth, to the LB flat board containing Amp, is, through the single bacterium colony that 37 DEG C of culture 16h are formed, the positive colony containing purpose fragment, extracts matter Grain carries out enzyme action and sequencing identification.
Two) there is the escherichia coli expression of the AntiHIV1 RT activity Aedes albopictus albumen of anticoagulant function：
By the expression vector chemical conversion containing coding AntiHIV1 RT activity Aedes albopictus protein gene to BL21(DE3) pLysS large intestine bar Recombinant expressed in bacterium.Access 10ml incubated overnight seed bacterium solution in 1L LB culture medium, cultivate 3 hours, when antibacterial light absorption value for 37 DEG C OD600Reach addition IPTG final concentration of 1mM when 0.8 about, continue culture 3 hours, 8000rpm is collected by centrifugation bacterium in 10 minutes Body, with the 25mM Tris-HCl solution suspension precipitation of 100ml pH 8.0, is then centrifuged for 8000rpm and is centrifuged 10 minutes collection bacterium Body, adds the 25mM Tris-HCl solution of 100ml pH 8.0,4 DEG C of ultrasonic disruption cells, surpasses 30 seconds and stop 30 seconds continuous 4 times, Centrifugation 12000rpm is centrifuged 10 minutes and collects inclusion body precipitation afterwards, obtains Fig. 1, purpose using the expression that SDS-PAGE detects albumen Band should be between 28-37kDa.
Two) folding of the escherichia coli expression inclusion body of AntiHIV1 RT activity Aedes albopictus albumen：
25mM Tris-HCl pH 8.0 solution adding 100ml to contain 0.1%Triton 100 in ultrasonic centrifugation product, Stirring at low speed 1 hour under room temperature.It is then centrifuged for 12000rpm and be centrifuged 10 minutes collection precipitations, with the 25mM Tris- of pH 8.0 HCl solution washing precipitation 4 times, adds 25ml to contain the 25mM Tris-HCl solution of 6M guanidine hydrochloride in last centrifugation, After most of dissolving to be precipitated in 1 hour is stirred at room temperature, 12000rpm is centrifuged 10 minutes, takes and adds DTT in supernatant to final concentration For 10mM, the lower stirring of temperature 1 hour, 12000rpm is centrifuged 10 minutes and collects supernatant.According to 1:Supernatant is slowly added dropwise by 200 ratios To in the 25mM Tris-HCl solution of the pH of arginine containing 300mM 7.6, it is stirred overnight rapidly in 4 DEG C.Using 10kDa flat board Ultrafiltration concentration system concentrates and folds protein solution to 25ml, and it is pure that sample centrifugation 12000rpm is used for separation after being centrifuged 20 minutes Change.
Three) the isolating and purifying and identifying of AntiHIV1 RT activity Aedes albopictus inclusion bodies of protein folded product：
The first step, Sephadex SR-100 gel filtration chromatography.The AntiHIV1 RT activity Aedes albopictus egg obtaining as stated above The supernatant that white inclusion body folded product concentrates after centrifugation is splined on 25mM Tris-HCl 0.15M NaCl pH 8.0 solution equilibria GE company Sephadex SR-100 gel permeation chromatography post.Obtain Fig. 2A with same buffer solution elution, collect as shown in the figure Peak Activity.
Second step, Hiprep SP cation-exchange chromatography column chromatography.AntiHIV1 RT activity Aedes albopictus albumen by molecular sieve purification Dialysed 24 hours in the PBS solution of 10L pH 6.0 with 10kDa bag filter, 12000rpm is centrifuged 20 minutes, by supernatant loading Hiprep SP cation-exchange chromatography chromatographic column in GE company.Hiprep SP cation-exchange chromatography post 20mM Na2HPO4-NaH2PO4The buffer balance of pH 6.0, with the linear ladder from 0% to 100% for the same buffer containing 1M NaCl Degree affords Fig. 2 B, collects Peak Activity as shown in the figure.
3rd step, Sephadex G75 gel filtration chromatography.Hiprep SP cation-exchange chromatography column chromatography obtains Peak Activity is splined on 25mM Tris-HCl 0.15NaCl pH 8.0 buffer balance after the concentration of 10kDa super filter tube Sephadex G75 gel permeation chromatography post.Obtain Fig. 2 C with same buffer solution elution, collect Peak Activity as shown in the figure.
4th step, Sephadex G75 gel filtration chromatography.The work that Sephadex G75 gel filtration chromatography obtains Property peak through 10kDa super filter tube concentration after be splined on 25mM Tris-HCl 0.15NaCl pH 8.0 buffer balance Sephadex G75 gel permeation chromatography post.Obtain Fig. 2 D with same buffer solution elution and isolate and purify figure, collect and live as shown in the figure Property peak.
5th step, obtains Fig. 2 D electrophoretogram after the AntiHIV1 RT activity Aedes albopictus protein electrophoresises that purification obtains, and then transferring film is with automatically Protein Sequencer measures N-terminal aminoacid sequence.
The AntiHIV1 RT activity Aedes albopictus albumen with anticoagulant function prepared by said method is to utilize lineae ablicantes A kind of albumen of yellow-fever mosquito salivary gland DNA recombinant expression, molecular weight 36656.83 dalton, isoelectric point, IP 8.501, particular sequence is such as SEQ IDNO：Shown in 1.
Four) there is the preparation of the AntiHIV1 RT activity Aedes albopictus albumen medical solution of anticoagulant function：
Medicine preparation for the AntiHIV1 RT activity Aedes albopictus albumen of pharmacological experiment：The albumen of recombinant expressed purification is through 10kDa It is made into 10mg/ml solution with PBS solution after super filter tube thickening filtration, add final concentration of 1% Triton X-114 acute immediately After strong vibration mixing ice educates 10min, 20 DEG C of incubation 10min, 10000rpm room temperature is centrifuged 10 minutes, careful in superclean bench Aspirate supernatant, to no thermal source vessel, detects that through tachypleus amebocyte lysate endotoxin is less than after 10EU/ml with normal saline or pH 7-8 PBS solution makes 45 μM of sterile solutions.
There is the pharmacological evaluation of the AntiHIV1 RT activity Aedes albopictus albumen of anticoagulant function：
One) the AntiHIV1 RT activity Aedes albopictus protein-specific suppression HIV with anticoagulant function is in cell proliferation
10000/hole TZM-Bl cell is inoculated in 96 orifice plates, 37 DEG C of 5%CO2Overnight incubation.With the DMEM without serum AntiHIV1 RT activity Aedes albopictus albumen is formulated as the medicinal liquid of variable concentrations, the every hole of 60 μ l, each three multiple holes by culture medium.VSVG and HIV disease After poison dilutes 8 times with the DMEM culture medium without serum, 60 μ l/ holes are added in medicinal liquid, slightly mix, 37 DEG C, 5%CO2 incubation 30 minutes.100 μ l medicines and virus mixture is taken to be added in target cell.After 48 hours, Luciferase method detection AntiHIV1 RT activity is white The suppression ratio that stricture of vagina yellow-fever mosquito albumen replicates to VSV-G and inhibition of HIV.
AntiHIV1 RT activity Aedes albopictus albumen suppresses H5N1The Activity determination of influenza pseudoviruss is as follows：MDCK and 293T cell is cultured In the DMEM culture medium containing L-Glutamine, 10% hyclone.Express H using 293T cell transfecting5N1Type influenza strain A/ Thailand/Kan353/2004(H5N1) HA and NA plasmid to pack H5N1Pseudoviruss.The full 293T cell of 70-80% adopts Polyethyleneimine is in 6 orifice plates every hole cotransfection 1 μ g HA plasmid, 1 μ g NA plasmid and 3 μ g HIV skeleton plasmid (pNL4- 3.luc.R_E_), change liquid after 12 hours.Transfection collected supernatant after 48 hours, and 2000g is centrifuged 10 minutes.For detecting to be detectedization Compound anti-cape horn fever cytotoxic activity, mdck cell (104/ hole) plant plate in 96 orifice plates and overnight incubation.Treat AntiHIV1 RT activity Aedes albopictus albumen with After pseudoviruss are incubated 30 minutes altogether in 37 DEG C, virus-medicinal mixture is transferred to mdck cell, and continues to cultivate 48 hours, PBS After washing cell, Luciferase method detection AntiHIV1 RT activity Aedes albopictus albumen is to H5N1The suppression ratio that influenza pseudoviruss replicate.Will be above-mentioned AntiHIV1 RT activity, VSVG and H5N1Data summarization obtains table 1.
Table 1：The specific inhibitory effect to HIV difference strain for the albumen
As seen from the above table, AntiHIV1 RT activity Aedes albopictus albumen can significantly inhibit propagation on cell for the HIV, and this suppression tool There is very strong specificity it is impossible to suppress VSVG and H5N1Propagation on corresponding cell.This albumen does not have to mammalian cell yet Toxicity.
Two) there is AntiHIV1 RT activity Aedes albopictus albumen suppression Convuxlin, U of anticoagulant function46619With The platelet aggregation of Collengen induction
Fresh and healthy people richness plasma platelet 500rpm under room temperature is centrifuged 5 minutes, and at supernatant discarded night, precipitation is with containing Tyrode's solution (137mM NaCl, 2.7mM KCl, the 4mM NaH of 0.1mM EGTA2PO4,33mM NaHCO3,1mM MgCl2, 5.55mM glucose, 0.35%gelatin, pH 6.5) wash twice under same centrifugal condition, finally washing platelet is hanged Float over containing 2mM CaCl2Tyrode's solution (pH 7.4) in, PC is adjusted to 3 × 108Individual/ml referred to as washs platelet. Healthy human blood platelets diluted plasma is to 2.5 × 108Individual/ml is referred to as rich plasma platelet.To 300 μ l richness plasma platelets or After adding final concentration of 1.85 μM or 9 μM of AntiHIV1 RT activity Aedes albopictus albumen to be incubated 2min in washing platelet, it is separately added into end Dense Convuxlin, 2.5 μM of U for 60pM46619, the Collengen induced aggregation of 1.0 μ g/ml, little with Chrono-Lumi blood Plate is assembled instrument (Chronol-Log, Havertown, PA, USA) and is measured light transmission, observes platelet aggregation in 5min and stirs at 37 DEG C Mix the gathering situation under state, and obtain Fig. 3.
Assemble the calculating of suppression ratio (I)：I=B/A × 100% (A=derivant can reach maximum assemble (herein for Light transmittance, %)；B=adds the maximum gathering that derivant can reach after adding inhibitor.)
Visible as shown in Figure 3,1.85 μM or 9 μM of Aedes albopictus AntiHIV1 RT activity and suppression platelet aggregation bifunctional protein Significantly suppress the Convuxlin of 60pM, 2.5 μM of U with after platelet insulation 2min46619, the Collengen of 1.0 μ g/ml lures The platelet aggregation led.U to 2.5 μM46619It is with the suppression ratio of the platelet aggregation of the Collengen induction of 1.0 μ g/ml 100%, and to the suppression ratio of the Convuxlin of 60pM induction be 1.85 μM be 25% and 9 μM be 50%.Suppression have dosage according to Lai Xing.

Claims (2)

1. aminoacid sequence such as SEQ ID NO：AntiHIV1 RT activity Aedes albopictus albumen shown in 1 is in the medicine of preparation suppression platelet aggregation Application in thing.
2. application according to claim 1 is it is characterised in that described AntiHIV1 RT activity Aedes albopictus albumen is by following methods system ?：
(1) use PROMEGA company after extracting Aedes albopictus salivary gland total serum IgEmRNA Isolation Systems test kit isolates and purifies mRNA and utilizes CLONTECH company CreatorTMSMARTTMcDNA Library Construction Kit Construction of Plasmid cDNA Library test kit amplifies the total cDNA of Aedes albopictus salivary gland, then adopts SEQ ID NO：Forward primer shown in 2 and SEQ ID NO：Downstream primer amplification coding SEQ ID NO shown in 3：Anti- shown in 4 The gene of HIV Aedes albopictus albumen, then first by this gene cloning in pET-17b expression vector, then be transformed into BL21 (DE3) Recombinant expressed in pLysS escherichia coli, and collect inclusion body between 28-37kDa for the purpose band；
(2) by collected inclusion body guanidine hydrochloride dissolution, renaturation fold after go up successively Sephadex SR-100 solvent resistant column, Hiprep SP cation-exchange chromatography post, Sephadex G75 solvent resistant column and Sephadex G75 gel filtration chromatography, Obtain as SEQ ID NO：The Aedes albopictus salivary gland DNA recombinant expression albumen of sequence shown in 1.
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