CN103405779A - Long-acting artesunate drug for preventing schistosome infection and preparation method thereof - Google Patents
Long-acting artesunate drug for preventing schistosome infection and preparation method thereof Download PDFInfo
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- CN103405779A CN103405779A CN2013103043235A CN201310304323A CN103405779A CN 103405779 A CN103405779 A CN 103405779A CN 2013103043235 A CN2013103043235 A CN 2013103043235A CN 201310304323 A CN201310304323 A CN 201310304323A CN 103405779 A CN103405779 A CN 103405779A
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Abstract
The invention discloses a long-acting artesunate drug for preventing schistosome infection and a preparation method thereof. According to the method, a low immunogenicity GST-NSP protein is adopted, a chemical coupling method is employed to couple artesunate (AS) having an effect with the GST-NSP protein so as to prepare an (AS) n-GST-NSP conjugate. While maintaining the adolescent schistosome killing activity of the AS, the conjugate prolongs the half-life period of the AS in a host, not only has an early treatment effect on schistosome infection, and also has a function of preventing schistosome infection, thus providing a new means for preventing schistosome infection of human and a variety of reservoir animal hosts, and controlling the epidemics of schistosomiasis. The drug provided in the invention has good application prospects the pharmacy field, including treatment and prevention of schistosomiasis.
Description
Technical field
The invention belongs to the depot drug product preparing technical field, be specifically related to long-acting artesunate medicine of a kind of prevention schistosomicide and preparation method thereof.
Background technology
Schistosomicide is a kind of infecting both domestic animals and human parasitic disease of serious harm health.At present worldwide, this disease mainly is distributed in Africa, Asia and Latin America, approximately has 600,000,000 populations of 74 countries and regions to be subject to the threat of schistosomicide, has nearly 200,000,000 people to infect schistosomicide.China popular be schistosomiasis japanica, according to the schistosomiasis in China epidemic situations of 2011 circular, China still has the 454Ge county that schistosomiasis endemic is arranged, patient people more than 280,000 is arranged, ill cattle 510,000 bulls, schistosomiasis endemic still exists in China with harm, even can continue the quite a long time.
" concomitant immunity " characteristics of schistosomicide; make the infected be difficult to obtain the permanent anti-infectious immunity protective effect continued; when the process chemotherapy; after being eliminated, the schistosomicide polypide of the infected in its body namely lose soon schistosomicide infection immunity power; when Human Water Contact again can infect schistosomicide again, repeated infection is the epidemiology typical characteristic of schistosomicide.Another key character of schistosomiasis endemic is that nature exists a large amount of Schistosoma japonicum animal reservoir hosts, Schistosoma japonicum is except infecting the people, can also infect many other vertebratess, as: pig, cattle, sheep, Canis familiaris L., rabbit and Mus etc., at nature, can form " natural epidemic foci " of schistosomiasis endemic, poultry has amassed a large amount of sources of infection, and the bulky stool that bovine livestock produces is the main source that waters, epidemic-stricken area worm's ovum pollutes.The epidemic-stricken area crowd is because producing the needs with life, Human Water Contact repeatedly, thereby be very easy to occur schistosomicide and infect again, and the past, the phenomenon of " ten male nine diseases " was very general in epidemic-stricken area.The harm of the pathology of schistosomicide is mainly that its worm's ovum is deposited on worm's ovum granuloma and the hepatic insufficiency due to the persistence worm's ovum inflammatory reaction that liver causes.If can employing prevention property means, schistosomicide namely is killed after infection host maybe can not develop into adult and lay eggs, can effectively prevent or reduce the infringement of schistosomicide to health, be the basic strategy of controlling the harm of schistosomicide and blocking its propagation to put prevention first.
Owing to there is no effective schistosomicide vaccine, present stage is controlled schistosomiasis endemic and still mainly depends on the treatment of chemicals, this means of prevention that depends on chemicals mainly comprises two aspects: the one, by medicine, kill already present imago of blood fluke in the patient body, and reach the purpose for the treatment of schistosomicide; The 2nd, in the high-risk group, carry out prophylactic, reduce the generation of schistosomicide.
Prevent the harm of schistosomicide to host health, must kill the virgin worm entered in host.Schistosomicide medicine praziquantel commonly used is very micro-because of the killing action to schistosomulum, the effect of not preventing schistosomicide.The action effect of artemisinin-based drug is different from praziquantel, what it was mainly killed is the schistosomicide of virgin worm phase, be mainly used in the schistosomicide early treatment, to enter in host the effect of virgin worm best and artesunate is killed, most of or whole female worm is killed before laying eggs, subtract female worm rate and can reach 90%-100%, thereby make host's liver avoid the infringement of worm's ovum.But the shortcoming of artesunate is that its half-life is shorter, half-life in the rat body is about 10min, the average blood plasma removing half-life is 34.2min in healthy human body, the active drug concentration in body be can not maintain for a long time, thereby the crowd of those prolonged and repeated Human Water Contacts and the prevention that domestic animal schistosome infects are not suitable for.But, if extend the artemisine half-life of small-molecule drug in patient body, can make it in host, maintain the time lengthening of valid density, improve the therapeutic effect of medicine, if given epidemic-stricken area crowd and the long-acting medicine of killing schistosomulum of domestic animal preventive use, can also reach the purpose of prevention schistosomicide, in the situation that current, effectively not prevent the schistosomicide vaccine, should be to reach one of optimal strategy of prevention schistosomicide.
At present the strategy of prolong drug half-life mainly contains two classes: the one, change the feature of medicine itself, and namely in the situation that do not affect the medicine pharmacological property, by the whole bag of tricks, change the structure of medicine, affect its internal metabolism speed; The 2nd, research and develop new dosage form (as slow releasing agent) and change route of administration, with prolong drug dispose procedure in vivo.So, which method can be used for extending the half-life of this micromolecular chemicals of artesunate? artesunate is as a kind of micromolecule chemicals, with its various metabolites very easily in vivo by glomerular filtration, so theoretically, we can extend its half-life by the hydrodynamics volume (such as by artesunate and certain macro-molecular protein coupling) that increases medicine.
Summary of the invention
Goal of the invention: the objective of the invention is provides a kind of new tool for China Schistosomiasis Epidemic masses prevent schistosomicide, develop a kind of schistosomicide prophylactic agent with reduced immunogenicity, low side effect, on the basis that keeps artesunate drug effect characteristic, extend its half-life, make it to become a new generation's prevention and the depot drug product for the treatment of schistosomicide.
Technical scheme: the long-acting artesunate medicine of a kind of prevention schistosomicide of the present invention, comprise artesunate AS, also comprise the carrier protein with the coupling of artesunate phase, described carrier protein be to man and animal have no side effect, without or the protein molecular of reduced immunogenicity.The main pharmacologically active of AS is realized by " peroxide bridge " in its chemical constitution, therefore on AS various " modifications ", all under the prerequisite that does not affect " peroxide bridge " structure, carry out, the present invention select to adopt mixed anhydride method on AS is long-armed carboxyl and the free amine group in the GST-NSP protein structure carry out coupling, preparation (AS)
n-GST-NSP conjugate, in theory, various macromole serum albumin all can be used as the carrier protein with the coupling of AS phase, but natural albumen all has immunogenicity, except entering inside and outside host of the same race, as enter in other different plant species body, can cause strong immune rejection, thereby not only can affect the effect of medicine, even also may cause serious safety issue, so, for this prevention and treatment with Amphixenosis of multiple reservoir host of schistosomicide, just need to avoid host's immunne response, use all can not produce the carrier of serious immunoreactive reduced immunogenicity protein as depot drug product in different plant species.
Described carrier protein is comprised of GST and NSP two parts, and described GST is the Schistosoma japonicum glutathione transferase molecule of engineered reduced immunogenicity, and described NSP is the reduced immunogenicity non-structural protein of artificial design.
Described GST Argine Monohydrochloride sequence and sequence SEQ ID NO:1 maintain at least 90% homology, and described GST pyrenoids nucleotide sequence is encoded to SEQ ID NO:2.
Described NSP albumen is comprised of alanine, glycine, proline, glutamic acid, serine and threonine, described NSP Argine Monohydrochloride sequence and sequence SEQ ID NO:3 maintain at least 90% homology, and described NSP pyrenoids nucleotide sequence is encoded to SEQ ID NO:4.
Described carrier protein aminoacid sequence and sequence SEQ ID NO:5 maintain at least 90% homology, and described carrier protein is nucleotide sequence coded is SEQ ID NO:6.
A kind of preparation method of preventing the long-acting artesunate medicine of schistosomicide, its step is as follows:
(a) structure of the encoding gene of GST-NSP albumen and recombinant expression plasmid: at first according to synthetic its DNA sequence of the coding NSP gene of design, and introduce respectively restriction enzyme site BamH1 and Sal1 at its 5 ' end and 3 ' end, by will the encode genetic fragment of NSP albumen of these two restriction enzyme sites, be inserted into the downstream of expression plasmid gst gene, the gene order generation frame endomixis of NSP protein gene and upstream, BamH1 site coding GST albumen makes to encode, make GST and NSP albumen can in same reading frame, carry out amalgamation and expression, form the recombiant plasmid system of GST-NSP protein fusion expression.
(b) expression of GST-NSP albumen: by recombinant plasmid transformed Gram-negative Host Strains, containing on the LB solid medium of kanamycin screening and containing the transformant engineering bacteria of recombiant plasmid, the engineering bacteria that will contain recombiant plasmid is inoculated in the LB fluid medium to be cultivated, adopt IPTG to induce, obtain the expression product of GST-NSP fusion rotein.
(c) separation and purification of GST-NSP albumen: select glutathione agarose gel 4B post, the GST-NSP albumen in expression product is carried out to affinity purification.
(d) coupling of artesunate and carrier protein: after AS is dissolved with DMSO, with the free carboxy in isobutyl chlorocarbonate activation AS structure, then it is combined with GST-NSP albumen free amine group, forms (AS)
n-GST-NSP, n are the AS molecular number be coupled on the GST-NSP protein molecular, remove unconjugated AS molecule by dialysis.
In described step (a), expression plasmid is pET41a (+), is adapted at expressing in the escherichia coli host engineering bacteria.But the carrier protein in the present invention also can be selected to utilize other plasmid to express, these expression plasmids include, but are not limited to the plasmid that protokaryon, eukaryotic expression system etc. are commonly used.
Particularly, this expression plasmid contains suitable promoter, in order to control the expression of fusion rotein.These promoteres include, but are not limited to the following stated promoter T7Promoter, and preferred promoter is T7Promoter.
In described step (b), the Gram-negative Host Strains is e. coli bl21.
The present invention also provides a kind of mensuration (AS)
nThe method of AS coupling rate in-GST-NSP conjugate.Particularly, adopt the ultraviolet scanning method to measure respectively AS, (AS)
nThe highest absworption peak wavelength of-GST-NSP, GST-NSP solution, then the absorbance of three kinds of solution of mensuration under the highest absworption peak wavelength, calculate AS coupling rate in conjugate according to Bell's law (A=ε bc).
The present invention also provides a kind of mensuration (AS)
n-GST-NSP conjugate is in the method for the virgin worm drug activity of external schistosomicide.Particularly, schistosomulum is transformed to the virgin worm of preparation through machinery, then virgin worm is placed in and contains (AS)
nIn the RPMI1640 culture medium of-GST-NSP conjugate, cultivate, arrange simultaneously without (AS)
n-GST-NSP conjugate culture medium negative control and the positive control that is added with AS, measure (AS) by the survival condition of observing different incubation time schistosomulums
n-GST-NSP conjugate external kills virgin worm effect.
The present invention also provides a kind of detection (AS)
n-GST-NSP conjugate is at the assay method of mice Half-life in vivo.Particularly, by the GST-NSP albumen of purification and freund adjuvant mixed immunity mice, prepare anti-(AS) of purification
n-GST-NSP conjugate specific antibody IgG, by anti-(AS) of purification
n-GST-NSP conjugate specific antibody IgG and horseradish peroxidase (HRP) combination, anti-(AS) of preparation HRP labelling
n-GST-NSP conjugate specific antibody IgG conjugate, and measure its work and tire.To resist (AS)
n-GST-NSP conjugate specific antibody IgG is the capture antibody coated elisa plate, with anti-(AS) of HRP labelling
n-GST-NSP conjugate specific antibody IgG conjugate, for detecting antibody, is set up the DASELISA immunization that detects AS, and sets up the standard curve that detects AS.By the tail vein injection method by (AS)
nIn-GST-NSP conjugate input mouse blood, and point is taked serum sample through eye socket venous blood collection method between difference is advanced.Adopt above-mentioned double antibody sandwich method to detect after administration the content of AS in the different time points mouse vein, calculate (AS)
nThe half-life of-GST-NSP conjugate.
The present invention also provides a kind of detection (AS)
nThe immunogenic method of-GST-NSP conjugate.Particularly, by (AS)
n-GST-NSP conjugate, through subcutaneous injection method direct immunization mice, through the week for 7 weeks immunity, then through venous blood collection, is measured anti-(AS) in serum
nThe antibody horizontal of-GST-NSP conjugate is tired, to observe the immunogenicity of this conjugate.
The present invention also provides a kind of mensuration (AS)
n-GST-NSP conjugate is killed the method for schistosomulum effect in Mice Body.Particularly, by the infection by cercariae of schistosoma mice, when schistosomicide is grown to lung phase child worm, through the tail vein injection drug treatment, dissect mice after 35 days after 7 days, check imago of blood fluke number in Mice Body, the counting worm reduction rate, observe (AS)
n-GST-NSP conjugate therapeutic effect in vivo.
The present invention also provides a kind of mensuration (AS)
nThe detection method of-GST-NSP conjugate prevention schistosomicide effect.Particularly, through the mouse tail vein injection administration, after administration, through mouse part skin, infected schistosomulum in 7 days, 14 days, 21 days respectively, and dissect mice after infecting 35 days, check imago of blood fluke number in Mice Body, the counting worm reduction rate, observe (AS)
n-GST-NSP conjugate, observe and dissect mice, checks imago of blood fluke number in Mice Body, and the counting worm reduction rate, observe (AS)
nThe effect of-GST-NSP conjugate prevention schistosomicide.
Beneficial effect: the present invention will have artesunate and the reduced immunogenicity carrier protein GST-NSP albumen coupling of killing the schistosomulum effect, prepare (AS)
n-GST-NSP conjugate.This conjugate is in the insecticidal activity that keeps AS, extended AS in host half-life and maintain in vivo time of effective drug effect concentration, not only has the effect of schistosomicide being carried out to the early treatment, the function that also has the prevention schistosomicide, for schistosomicide, the control schistosomiasis endemic of the prevention mankind and many animals reservoir host provides a kind of new tool, comprise aspect the treatment of schistosomicide and prevention that at pharmaceutical field good application prospect is arranged.
The accompanying drawing explanation
Fig. 1 is recombiant plasmid NSP-pET43.1a of the present invention and GST-NSP/pET41a restriction analysis figure,
Wherein: Isosorbide-5-Nitrae-DNA molecular amount mark; The NSP-pET43.1a plasmid of 2-restructuring is through BamH I/Sal I double digestion product; 3-recombiant plasmid NSP-pET43.1a; 5-plasmid pET41a; 6-recombiant plasmid GST-NSP/pET41a; The GST-NSP/pET41a plasmid of 7-restructuring is through BamH I/Sal I double digestion product.
Fig. 2 is the recombinate expression product electrophoretogram of GST-NSP fusion rotein of the present invention,
Wherein: 1-molecular weight of albumen standard; 2-contains the supernatant of the e. coli bl21 expression product of plasmid GST-NSP/pET41a; 3-contains the supernatant of the e. coli bl21 expression product of plasmid pET41a; The supernatant of 4-e. coli bl21 expression product.
Fig. 3 is the recombinate purification figure of GST-NSP albumen of the present invention,
Wherein: 1-protein molecular weight mark; The restructuring GST-NSP albumen of 2-purification.
Fig. 4 is that AS of the present invention, GST-NSP reach (AS)
nThe uv absorption spectra of-GST-NSP.
Fig. 5 is AS concentration of the present invention and 450nm absorbance canonical plotting.
Fig. 6 is that the present invention injects merely after AS the concentration map of AS in the different time points mice serum.
Fig. 7 is that the present invention injects conjugate (AS)
nThe concentration map of AS in the different time points mice serum after-GST-NSP.
The specific embodiment
In order to deepen the understanding of the present invention, the invention will be further described below in conjunction with embodiment and accompanying drawing, and this embodiment only, be used to explaining the present invention, does not form the restriction to protection domain of the present invention.
Embodiment 1:(AS)
nThe preparation of-GST-NSP conjugate
(a) structure of GST-NSP recombinant expression plasmid: adopting pET41a (+) is the expression vector plasmid, first, to expressing vector plasmid pET41a (+), carry out BamH1, Sal1 double digestion, actual conditions is as follows, expression vector plasmid pET41a (+), 10 μ L; BamH11 μ L, Sal11 μ L, enzyme cutting buffering liquid 5 μ L; Distilled water 33 μ L, cumulative volume is 50 μ L, in 37 ℃ of thermostat water baths, reaction is 3 hours, by agarose gel electrophoresis, reclaim linearizing pET41a (+) plasmid DNA, the NSP/pET43.1a plasmid DNA that contains the NSP genetic fragment is made same double digestion, by agarose gel electrophoresis, reclaim the NSP genetic fragment of the about 1368bp of molecular weight, as shown in Figure 1.
The NSP genetic fragment reclaimed is connected with the expression plasmid pET41a (+) of above-mentioned double digestion and builds recombinant expression plasmid GST-NSP/pET41a.The linked system volume is generally 10 μ L, and pET41a (+) plasmid vector of double digestion and the mol ratio of double digestion NSPDNA are 1:3, DNA ligase buffer 1 μ L, and DNA ligase 1 μ L, adding sterilized water to cumulative volume is 10 μ L.In 16 ℃ of thermostat water baths of coupled reaction, reaction is 16 hours.Connect product and transform the bacillus coli DH 5 alpha competent cell, then be applied on the LB flat board that contains kanamycin and cultivate.
The evaluation of positive colony is that picking is containing the single bacterial clone of growing on the LB flat board of kanamycin, its plasmid DNA of extracting.With restricted enzyme BamH1, Sal1, recombiant plasmid is carried out to restricted enzyme cutting analysis.In the centrifuge tube of a 0.5mL, add recombiant plasmid GST-NSP/pET41a30 μ L, BamH12 μ L, Sal12 μ L, enzyme cutting buffering liquid 5 μ L, deionized water 11 μ L, cumulative volume are 50 μ L, 37 ℃ of water bath heat preservations 3 hours.The enzyme action product is carried out to 1% agarose gel electrophoresis, the dyeing of forming sediment of bromination second, observe the enzyme action result under uviol lamp.The result confirmation, the NSP gene correctly is inserted in expression plasmid pET41a (+), as shown in Figure 1.
(b) expression of GST-NSP albumen: recombiant plasmid GST-NSP/pET41a is transformed to e. coli bl21, containing on the LB solid medium of kanamycin screening and containing the transformant engineering bacteria of recombiant plasmid, the engineering bacteria that will contain recombiant plasmid GST-NSP/pET41a is inoculated in the 50mLLB fluid medium, 37 ℃ of overnight incubation, second day is transferred in the 1LLB culture medium (1:100 dilution) while continuing to be cultured to OD600nm=2.4 by the 10mL culture, adding final concentration is that the IPTG of 0.5mM carried out abduction delivering 4 hours again, expression product carries out the SDS-PAG electrophoresis, found that 2 times of body GST-NSP fusion rotein of the engineering bacteria energy expression ratio anticipated molecular value large 2 times (120kDa) that contains recombiant plasmid GST-NSP/pET41a, as shown in Figure 2.
(c) separation and purification of GST-NSP albumen:
The expression product antibacterial is carried out to cracking, high speed centrifugation, supernatant is crossed glutathione agarose 4B glue post, make the GST-NSP protein binding on glue, again by glutathione solution by fusion rotein from eluting on gel, SDS-PAGE analyzes demonstration and has obtained highly purified restructuring GST-NSP albumen, as shown in Figure 3.
(d) coupling of AS and GST-NSP albumen:
1) 46mgAS dissolves with 800 μ L dimethyl formamides, is cooled to 10 ℃, then adds 16 μ L isobutyl chlorocarbonates, stirs 30min under 10 ℃.
2) 12mg is dissolved in to the GST-NSP albumen in PBS, under 10 ℃ of environment, with 50mM sodium carbonate buffer dialysis 2h, dialysis is transferred to the GST-NSP protein solution in one small beaker after finishing, and slowly stirs under 4 ℃ of environment, does not produce bubble as far as possible.
3) reacted AS in step 1) is dropwise slowly added in the small beaker that fills GST-NSP, 4 ℃ of stirring reactions spend the night.
4) with 100mLPBS, dilute conjugate, through the super filter tube ultrafiltration, remove unconjugated AS compound, and to be concentrated to final volume be 1mL.
Embodiment 2: the mensuration of coupling rate
Adopt the ultraviolet scanning method to measure AS, (AS)
nThe highest absorption peak of-GST-NSP and GST-NSP.And measure AS, (AS) by the highest absorbing wavelength
nThe light absorption value of-GST-NSP and GST-NSP.
By formula:
(annotate: ε means molar absorption coefficient; A means absorbance; Ca means the mole percent of AS in conjugate; Cb means the mole percent of carrier protein NSP in conjugate)
Result shows, AS, (AS)
nThe highest absorbing wavelength of-GST-NSP and GST-NSP is: 260nm, 270nm and 280nm.As shown in Figure 4.This result means that conjugate has own unique light wave absworption peak (270nm), shows that conjugate is successfully prepared.Through the coupling rate of calculating AS, be 506:1, namely on GST-NSP albumen coupling 506 AS molecules.
Embodiment 3:(AS)
nExternal virgin worm effect and the half-dead lethal dose of killing and wounding of-GST-NSP conjugate
For measuring (AS)
nWhether-GST-NSP conjugate has retained the insecticidal activity of AS, adopts extracorporeal culture-ing to measure respectively AS, (AS)
nThe activity of-GST-NSP conjugate, concrete grammar is as follows:
1) Snails of getting 30 Schistosoma japonicum is put in the 100mL beaker, cleans for several times with clear water, and nylon wire on the rim of a cup cover, add dechlorination water and cover nylon wire, puts under 25-28 ℃ of environment illumination 2h left and right, effusion cercaria.
2) by 25 * 25mm coverslip with after alcohol-pickled, tap water is cleaned and wiped clean.
3) the RPMI1640 basal medium that ice bath crosses of packing in 15mL cone-shaped glass centrifuge tube, juxtaposition is cold preservation on ice.25 * 25mm coverslip is fractureed, with the equal glass shred of proper (can enter in pipe but can not arrive the pipe end) of tweezers gripping length and width, with tow sides, paste the cercaria on the beaker liquid level respectively, and input has filled in the centrifuge tube of cold RPMI1640 basal medium rapidly, then by centrifuge tube ice bath 10min left and right.
4), with the centrifugal 1min of the rotating speed of 1500 rev/mins, make the cercaria on slide sink to the pipe end.Take out slide, again stickyly get cercaria, then put in centrifuge tube centrifugally, again collect cercaria, so repeat the cercaria of collecting as much as possible q.s.With RPMPI1640 repeated washing 2 times, last centrifugal rear abandoning supernatant, will manage end cercaria and add a small amount of RPMI1640 culture medium and suspend.
5) cleaning mixture that contains cercaria that will collect injects an aseptic culture dish, adds RPMI1640 culture fluid (containing 10%FBS) to be adjusted to the experiment desired density, namely in every milliliter of RPMI1640 culture fluid, contains the about 20-30 bar of cercaria.
6) get 24 porocyte culture plates, draw respectively the RPMI1640 culture fluid that contains cercaria in the 1mL culture dish, add successively in each hole.
7) the 24 porocyte culture plates that added cercaria are placed in to 37 ℃, 5%CO
2CO2 gas incubator in cultivate 12-16h, under inverted microscope, observe successively survival and the de-tail situation of each hole mesocercaria.
8) will be in the virgin worm of the de-tail of external process to have been transferred to the 24 porocyte culture plates that added in advance virgin worm culture fluid, volume of culture is 2mL.Containing 5%CO
237 ℃ of incubators in cultivate.RPMI1640 matched group and 5%NaHCO are set simultaneously
3Matched group.The AS, (AS) that in experimental port, add respectively variable concentrations
n-GST-NSP conjugate, observe the death condition of polypide under inverted microscope respectively at 20min, 4h, 24h, 48h.Polypide death is defined as: virgin worm internal structure is unclear, and pellicle is destroyed, and content becomes radial and overflows; Or internal structure is unclear, pellicle is complete, but has not yet to see the activist after observing 30s.Experiment repeats 2～3 times, and calculates median lethal dose(LD 50) (LD50 value).
Result: (AS)
n-GST-NSP conjugate has retained the pharmaceutically active of the virgin worm of schistosomicide of AS, can kill schistosomulum external, as shown in table 1.But it kills virgin worm activity and with AS, compares decline to some extent, median lethal dose(LD 50) LD50 is about 4-5 times of AS median lethal dose(LD 50), as shown in table 2.
Embodiment 4:(AS)
n-GST-NSP conjugate immunogenicity determining
For observing (AS)
n-GST-NSP conjugate immunogenicity, the present invention is with (AS) of purification
n-GST-NSP conjugate and GST-NSP protein immunization mice, observe the variation of mouse antibodies reaction.Concrete grammar is as follows:
Get 20 mices, be divided at random two groups, group one is recombiant protein GST-NSP immune group, and group two is conjugate (AS)
n-GST-NSP immune group.Every group of 10 mices, through subcutaneous antigen inoculation (every mouse inoculation 3g specifies antigen, does not add any immunological adjuvant), weekly, 6 weeks of continuous immunity.Every mice before immunity, after immunity 4 weeks and 6 weeks through tail vein blood, separation of serum ,-30 ℃ save backup.
Adopt indirect enzyme-linked immunosorbent assay (ELISA) method to detect, the restructuring GST-NSP fusion rotein of take is antigen, detects anti-restructuring GST-NSP albumen and conjugate (AS)
n-GST-NSP immune serum antibody titer.Operating procedure is as follows:
1) wrapper sheet: with 100 μ LGST-NSP protein liquids or 100 μ L conjugates (AS)
n-GST-NSP solution (0.1MNaHCO
3, pH8.6,10 μ g/ml) and coated elisa plate, 4 ℃ are spent the night;
2) inferior daily PBST (0.05%Tween-20) washes plate 3 times, and every hole adds the PBS confining liquid 300 μ L that contain 5% defatted milk powder, 37 ℃ of insulation 1h;
3) discard hole inner sealing liquid, wash plate 3 times with PBST, every hole adds the immune serum 100 μ L by the 1:50 doubling dilution, 37 ℃ of effect 1h;
4) discard solution in hole, after with PBST, washing plate 3 times, every hole adds goat anti-mouse HRP-IgG(1:1000 dilution) 100 μ L, 37 ℃ of effect 1h;
5) discard liquid in hole, PBST washes plate 3 times, exhausts debris, adds substrate TMB50 μ L colour developing, room temperature lucifuge effect 5min;
6) add 2mol/LH
2SO
450 μ L cessation reactions, by the absorption value at 96 microplate reader survey 450nm wavelength places, hole.
7) with 2.1 times of positive judgment thresholds of negative control light absorption value.
Result: restructuring NSP albumen and conjugate (AS)
n-GST-NSP immune mouse after 29 days antibody horizontal reach between 1:1600-1:3200, after 42 days, antibody horizontal reaches between 1:3200-1:6400.GST-NSP albumen and conjugate (AS) are described
n-GST-NSP still has certain immunogenicity, but less immunogenic.
Embodiment 5:(AS)
nThe half-life of-GST-NSP conjugate in blood measured
For measuring (AS)
nThe metabolism situation of-GST-NSP conjugate in host, after the present invention adopts double antibody sandwich method to measure intravenously administrable, (AS)
nThe half-life of-GST-NSP conjugate in mouse blood.Concrete grammar is as follows:
Administration and sample process
AS group: the AS injection that at first configures 3.84 μ g/ μ L.Get 33 mices, every mice is through tail vein injection AS, dosage is every mice 26.8mg/kg, then respectively after administration 2min, 15min, 30min, 1h, 2h, 5h, 12h, 24h, 51h, 72h, 97h mice is carried out to tail vein blood separation of serum, serum, through acetonitrile precipitation (every 10 μ L serum add 30 μ L acetonitriles), is stored in supernatant in-30 ℃ of refrigerators.
(AS)
n-GST-NSP group: at first configuration (AS)
n-GST-NSP injection, concentration are that 2.41 μ g/ μ L. get 45 mices, and every mice is through tail vein injection (AS)
n-GST-NSP conjugate injection, dosage are every mice 12.05mg/kg.2min, 15min, 30min, 1h, 2h, 5h, 12h, 24h, 51h, 72h, 97h carry out tail vein blood separation of serum to mice after administration respectively, are stored in-30 ℃ of refrigerators the mice serum of acquisition standby.
The drafting of AS concentration standard curve
1) wrapper sheet: with the little mouse-anti of 100 μ L (AS)
n-GST-NSP antibody-solutions (0.1MNaHCO
3, pH8.6, little mouse-anti (AS)
n-GST-NSP antibody 10 μ g/ml) coated elisa plate, 4 ℃ are spent the night;
2) inferior daily PBST(PBS contains 0.05%Tween-20) wash plate 3 times, every hole adds the PBS confining liquid 300 μ L that contain 5% defatted milk powder, 37 ℃ of insulation 1h;
3) discard hole inner sealing liquid, wash plate 3 times with PBST, get 3.84 * 10
-3μ g/ μ LAS solution is by with lower volume, adding in each hole: 4.68 μ L, 5.27 μ L, 5.86 μ L, 11.7 μ L, 17.58 μ L, 23.43 μ L, 35.16 μ L, 46.8 μ L, 52.7 μ L, 58.6 μ L(respectively in corresponding each hole the content of AS be 0.0180 μ g, 0.0203 μ g, 0.0225 μ g, 0.0450 μ g, 0.0675 μ g, 0.0900 μ g, 0.1350 μ g, 0.1800 μ g, 0.2025 μ g, 0.2250 μ g), the PBS solution of last each Kong Junyong 1% defatted milk powder mend to final volume be 100 μ L, each concentration is done two multiple Kong Bingzuo blanks, 37 ℃ of effect 1h,
4) discard solution in hole, after with PBST, washing plate 3 times, every hole adds the little mouse-anti of HRP labelling (AS)
n-GST-NSP antibody (1:50 dilution) 100 μ L, 37 ℃ of effect 1h;
5) discard liquid in hole, PBST washes plate 3 times, exhausts debris, adds substrate TMB50 μ L colour developing, room temperature lucifuge effect 5min;
6) add 2mol/LH
2SO
450 μ L cessation reactions, by the absorption value at 96 microplate reader survey 450nm wavelength places, hole.In the different time points mice serum, AS concentration reaches (AS)
nThe mensuration of-GST-NSP concentration
1) collect mouse tail vein injection AS injection or (AS)
nThe serum sample of each time point after-GST-NSP conjugate injection;
2) wrapper sheet: with the little mouse-anti of 100 μ L (AS)
n-GST-NSP antibody-solutions (0.1MNaHCO
3, pH8.6, little mouse-anti (AS)
n-GST-NSP antibody 10 μ g/mL) coated elisa plate, 4 ℃ are spent the night;
3) inferior daily PBST(PBS contains 0.05%Tween-20) wash plate 3 times, every hole adds the PBS confining liquid 300 μ L that contain 5% defatted milk powder, 37 ℃ of insulation 1h;
4) discard hole inner sealing liquid, wash plate 3 times with PBST, respectively at the serum sample of different time points after the mice administration that adds the 1:100 dilution in each hole, establish simultaneously blank, 37 ℃ of effect 1h;
5) discard solution in hole, after with PBST, washing plate 3 times, every hole adds the little mouse-anti of HRP labelling (AS)
n-GST-NSP antibody (1:50 dilution) 100 μ L, 37 ℃ of effect 1h;
6) discard liquid in hole, PBST washes plate 3 times, exhausts debris, adds substrate TMB50 μ L colour developing, room temperature lucifuge effect 5min;
7) add 2mol/LH
2SO
450 μ L cessation reactions, by the absorption value at 96 microplate reader survey 450nm wavelength places, hole.
Result: under different AS concentration, measure respectively its A
450nm, then draw out standard curve, as shown in Figure 5, from Fig. 5 can, linear relationship is preferably arranged between the concentration of AS and light absorption value.
Adopt the ELISA method to measure respectively different time points AS group after administration, (AS)
nThe variation of AS content in-GST-NSP group Mice Body, according to AS concentration and absorbance standard curve, calculate after administration the concentration of AS in each different time points Mice Body, during simple injection AS as can be known, the regularity of distribution of medicine in mice serum is: reach rapidly peak value (2min), but the half-life shorter (being about 30min), with forefathers' result of study, match, as shown in Figure 6.And (AS)
nIn-GST-NSP group Mice Body, the concentration of AS reaches the obvious backward delay of time of summit, is the 15min after administration, and the half-life is 2h after administration, obviously extends, as shown in Figure 7.
Embodiment 6:(AS)
nThe treatment of-GST-NSP conjugate and the effect of preventing schistosomicide
For further observing (AS)
nThe pharmaceutically active of-GST-NSP conjugate in host, the present invention has observed (AS) by the method for intravenously administrable
n-GST-NSP conjugate is killed the effect of virgin worm to schistosomicide in the infecting mouse body.Concrete grammar is as follows:
1) virgin worm therapeutic test in body
Every ICR mice infects 35 of schistosoma japonicum cercariaes through skin of abdomen, and therapeutic dose is AS injection group 150mg/kg, (AS) through the tail vein injection drug treatment the 7th day (lung phase) after schistosomicide
n-GST-NSP conjugate injection group 600mg/kg(dosage herein is the dosage of contained AS in coupling drug).Control group mice is not given any Drug therapy.Infect to cut open in latter 35 days and kill mice, through portal vein perfusion, collect adult counting with the citric acid normal saline.By following formula, calculate worm reduction rate:
The one-tenth borer population of worm reduction rate=(the one-tenth borer population of the average every Mus of one-tenth borer population-experimental group of the average every Mus of matched group)/average every Mus of matched group * 100%.
2) prevention schistosomicide test
70 ICR mices are divided into to seven groups (10 one groups) at random, every mice is all adopted to the tail vein injection administration: group one, group two, group three are AS injection group, and injected dose is 150mg/kg; Group four, group five, group six are (AS)
n-GST-NSP conjugate injection group, injected dose are that 600mg/kg(dosage herein is the dosage of contained AS in coupling drug); Group seven is not injected any medicine, as the blank group.Group one, group four, group seven administration after seven days simulating natural environment infect schistosoma japonicum cercariae, group two, group five simulating natural environment after the administration fortnight infect schistosoma japonicum cercariae, group three, group six administration after 21 days simulating natural environment infect schistosoma japonicum cercariae.
Schistosomicide mice under simulating natural environment: inject dechlorination water in a clear glass cylinder, the depth of water is controlled at 1.5cm, the coverslip that is loaded with 1000 cercarias is put into to the glass jar that fills dechlorination water, rocking gently glass jar is dispersed in dechlorination water cercaria, 10 mices of same group are put into to glass jar together, on glass jar, cover and prevent that mice from escaping from wire gauze, careful taking-up mice after 30min.
Each is organized mice and all in infection, cutd open in latter 35 days and kill, and through portal vein perfusion, collects adult counting with the citric acid normal saline.By following formula, calculate worm reduction rate:
The one-tenth borer population of worm reduction rate=(the one-tenth borer population of the average every Mus of one-tenth borer population-experimental group of the average every Mus of matched group)/average every Mus of matched group * 100%
Result: the interior therapeutic result of the test shows, AS(150mg/kg) with (AS)
n-GST-NSP(600mg/kg) similar to the killing effect of Schistosomula Japonicum in vivo, the worm lotus of two groups is respectively 10.00 ± 1.50 and 10.50 ± 0.52, worm reduction rate is respectively 61.54% and 59.62%, and difference does not have statistical significance (p>0.05) between the two, as shown in table 3.
In the situation that simulation mice natural infection schistosoma japonicum cercariae, the schistosomulum that AS infected after to administration in 7,14 and 21 days does not have lethal effect, matched group (49.00 ± 0.56) is originally identical with the average worm lotus base of 7 days (48.60 ± 0.50), 14 days (48.30 ± 0.73) and 21 days (48.60 ± 0.65) infected group mices, difference does not have statistical significance (p > 0.05) between the two, therefore, AS does not have preventive effect to the schistosomicide that administration occurred in 7 days afterwards at least.(AS)
nThe average worm lotus of the mice number of 7 days postoperative infection groups of-GST-NSP conjugate administration is 32.90 ± 0.53, and with matched group, comparing difference has statistical significance (p<0.05), and worm reduction rate reaches 32.86%.And after administration, the average worm lotus of the 14th day, 21 days infected group mices is respectively 48.80 ± 0.66 and 48.90 ± 0.69, with matched group, to compare, difference does not have statistical significance (p>0.05), shows at administration (AS) in mice serum after the 14th day
n-GST-NSP conjugate has been reduced to invalid concentration, as shown in table 4.
Table 1 AS, (AS)
nThe comparison of-GST-NSP to the Schistosomula Japonicum toxic action
Annotate: (1) dosage herein refers to the AS comprised in conjugate content
* medicine group and RPMI1640,5%NaHCO
3Relatively, P all<0.05 for negative control group
Table 2 AS with (AS)
n-GST-NSP is the LD lethal to virgin worm after different action time
50Value
Table 3 AS with (AS)
nIn-GST-NSP body, kill the effect of schistosomulum relatively
Table 4 AS with (AS)
NThe comparison of-GST-NSP prevention schistosomicide effect
Claims (9)
1. a prevention schistosomicide long-acting artesunate medicine, comprise artesunate AS, it is characterized in that, also comprises the carrier protein with the coupling of artesunate phase, described carrier protein be to man and animal have no side effect, without or the protein molecular of reduced immunogenicity.
2. the long-acting artesunate medicine of a kind of prevention schistosomicide according to claim 1, it is characterized in that, described carrier protein is comprised of GST and NSP two parts, described GST is the Schistosoma japonicum glutathione transferase molecule of engineered reduced immunogenicity, and described NSP is the reduced immunogenicity non-structural protein of artificial design.
3. the long-acting artesunate medicine of a kind of prevention schistosomicide according to claim 2, it is characterized in that, described GST Argine Monohydrochloride sequence and sequence SEQIDNO:1 maintain at least 90% homology, and described GST pyrenoids nucleotide sequence is encoded to SEQIDNO:2.
4. the long-acting artesunate medicine of a kind of prevention schistosomicide according to claim 2, it is characterized in that, described NSP albumen is comprised of alanine, glycine, proline, glutamic acid, serine and threonine, described NSP Argine Monohydrochloride sequence and sequence SEQIDNO:3 maintain at least 90% homology, and described NSP pyrenoids nucleotide sequence is encoded to SEQIDNO:4.
5. the long-acting artesunate medicine of a kind of prevention schistosomicide according to claim 1, it is characterized in that, described carrier protein aminoacid sequence and sequence SEQIDNO:5 maintain at least 90% homology, and described carrier protein is nucleotide sequence coded is SEQIDNO:6.
6. the preparation method of the long-acting artesunate medicine of prevention schistosomicide as claimed in claim 1, is characterized in that, step is as follows:
(a) structure of the encoding gene of GST-NSP albumen and recombinant expression plasmid: at first according to synthetic its DNA sequence of the coding NSP gene of design, and introduce respectively restriction enzyme site BamH1 and Sal1 at its 5 ' end and 3 ' end, by will the encode genetic fragment of NSP albumen of these two restriction enzyme sites, be inserted into the downstream of expression plasmid gst gene, make the to encode gene order generation frame endomixis of upstream, BamH1 site coding GST albumen in NSP protein gene and expression vector, form the recombiant plasmid system of GST albumen and NSP protein fusion expression.
(b) expression of GST-NSP albumen: by recombinant plasmid transformed Gram-negative Host Strains, at the transformant engineering bacteria that contains screening recombiant plasmid on the LB solid medium of kanamycin, the engineering bacteria that will contain recombiant plasmid is inoculated in the LB fluid medium to be cultivated, and obtains expression product GST-NSP albumen through inducing of IPTG.
(c) separation and purification of GST-NSP albumen: select glutathione agarose gel 4B post, the GST-NSP albumen in expression product is carried out to affinity purification.
(d) coupling of artesunate and GST-NSP albumen: after artesunate is dissolved with DMSO, with the free carboxy in isobutyl chlorocarbonate activation artesunate structure, then it is combined with GST-NSP albumen free amine group, forms (AS)
n-GST-NSP conjugate, remove unconjugated artesunate molecule by dialysis.
7. the preparation method of the long-acting artesunate medicine of prevention schistosomicide according to claim 6, is characterized in that, in described step (a), expression plasmid is pET41a (+), and described pET41a (+) contains promoter.
8. the preparation method of the long-acting artesunate medicine of prevention schistosomicide according to claim 7, is characterized in that, described promoter is T7Promoter.
9. the preparation method of the long-acting artesunate medicine of prevention schistosomicide according to claim 7, is characterized in that, in described step (b), the Gram-negative Host Strains is e. coli bl21.
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| CN112876545A (en) * | 2021-03-19 | 2021-06-01 | 中国极地研究中心(中国极地研究所) | Antifreeze protein and preparation method and application thereof |
| CN113045671A (en) * | 2021-01-29 | 2021-06-29 | 长春万成生物电子工程有限公司 | Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof |
| WO2022057696A3 (en) * | 2021-09-08 | 2022-07-28 | 中南大学湘雅医院 | Recombinant protein coding sequence, recombinant protein, and method for preparing monoclonal antibodies |
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| CN1668323A (en) * | 2002-06-06 | 2005-09-14 | 华盛顿大学 | Covalent conjugates between artemisinin-related endoperoxides and iron-carrying proteins and methods of use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113045671A (en) * | 2021-01-29 | 2021-06-29 | 长春万成生物电子工程有限公司 | Recombinant antigen for detecting schistosoma japonicum, preparation method and application thereof |
| CN112876545A (en) * | 2021-03-19 | 2021-06-01 | 中国极地研究中心(中国极地研究所) | Antifreeze protein and preparation method and application thereof |
| WO2022057696A3 (en) * | 2021-09-08 | 2022-07-28 | 中南大学湘雅医院 | Recombinant protein coding sequence, recombinant protein, and method for preparing monoclonal antibodies |
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