CN103550763A - Nano vaccine of LAMP-2 epitope peptide and preparation method of nano vaccine - Google Patents
Nano vaccine of LAMP-2 epitope peptide and preparation method of nano vaccine Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and relates to a nano vaccine of LAMP-2 epitope peptide and a preparation method of the nano vaccine. The nano vaccine comprises an effective amount of polypeptide with the amino acid sequence as shown in SEQ IN DO:1, or a polypeptide mixture with the amino acid sequence as shown in SEQ IN DO:1 and SEQ IN DO:2, the nano vaccine can be prepared through the synergistic effect of a polylactic acid-glycolic acid copolymer with the polypeptide, and the nano vaccine can be applied to preparation of a nano vaccine for anti-neutrophile granulocyte cytoplasm antibody relevancy vasculitis and anti-LAMP-2 antibody relevancy pauci-immune complex crescent glomerulonephritis. Due to adoption of the nano vaccine, spontaneous immune response of autoantigen characteristics is eliminated, and immune response to other antigens is not interfere, and a novel method is provided for treatment on anti-neutrophile granulocyte cytoplasm antibody relevancy vasculitis and anti-LAMP-2 antibody relevancy pauci-immune complex crescent glomerulonephritis.
Description
Technical field
The invention belongs to biomedicine field, relate to the nano vaccine of LAMP-2 epitope peptide, with and concrete application form and preparation method.
Background technology
Few immune complex patients with crescent nephritis (Pauci-immune crescentic glomerulonephritis, PICGN) be ANCA (antineutrophil cytoplasmic autoantibodies, ANCA) the vasculitic topical manifestations of dependency, take hematuria, albuminuria, edema, hypertension is main clinical manifestation, can cause rapid progress, irreversible renal failure.Be characterized in GCW seldom or there is no an immune complex deposit, glomerular capillary button loop necrosis and diffuse crescent formation, neutrophilic granulocyte directly causes the damage of tiny blood vessels endothelium and glomerule.Course advancement is dangerous, and the case fatality rate in diagnosis after 1 year is up to 15%, even if adopt timely, strong immunosuppressant therapy, in 5 years, mortality rate is still up to more than 35%.PICGN is external modal patients with crescent nephritis, accounts for 66%; In China, be only second to II type patients with crescent nephritis, account for 25%.Sickness rate has rapid rising trend, serious threat human health in recent years.Enter fast after end stagerenaldisease, need to carry out maintenance hemodialysis or renal transplantation, for society and family bring heavy financial burden and burden on society.
With regard to the current treatment of PICGN, high dose glucocorticoid associating Clinical Observation of CTX Pulse Therapy is classical scheme, but curative effect is limited, and toxic and side effects is large, and using dosage and the course for the treatment of are all restricted.The neotype immunosuppressant adopting in recent years, as mycophenolate, ciclosporin A, FK506 and Tumor necrosis factor-alpha antibody etc., curative effect is limited, also has the great risk of opportunistic infection simultaneously, even fatal complication.Therefore, the new method of specific treatment in the urgent need to finding effectively.
ANCA dependency vasculitis belongs to typical autoimmune disease.The ideal effect for the treatment of autoimmune disease is to eliminate the special spontaneous immunne response of autoantigen, and does not disturb the immunne response for other antigens.Therapeutic vaccine for specific target antigen meets this principle.At present, MS patient's Cop1 vaccine has been benefited from patient, and has also obtained very large clinical treatment progress for the curative synthetic peptide vaccine research of myasthenia gravis and insulin dependent diabetes mellitus (IDDM).
The mechanism of autoimmune disease is mainly that pathogen is broken immunologic tolerance by molecular simulation (Molecular Mimicry) mechanism: the structure of some epitope of pathogen and host tissue albumen is same or similar, and the immunne response that causes pathogen to stimulate body to produce directly acts on self component of structural similarity.In ANCA dependency vasculitis, the target antigen that certified ANCA is relevant has neutrophilic granulocyte myeloperoxidase (MPO) (myeloperoxidase, but fail to find the positive evidence that infection is relevant to MPO or PR3 MPO) and protease 3 (protease3, PR3), always.Anti-lysosome membrane albumen-2(Lysosomal membrane protein-2, LAMP-2) antibody is the ANCA new subtype of just finding recently, this antibody is almost present in all PICGN patients, with the dependency of pathological changes be antigen MPO-ANCA and PR3-ANCA 2 times.In neutrophilic granulocyte, LAMP-2 can shuttle back and forth between lysosome membrane and after birth, provide direct path, and PR3 and MPO does not have this function for circulating antibody enters in born of the same parents.At present, anti-LAMP-2 antibody be considered to ANCA dependency vasculitis more special and effectively sign.
In drug delivery system field, nanoparticle size is defined between 1-1000nm.Medicine is dispersed, seals, is adsorbed on polymer particle, different according to preparation method, can be made into nanosphere and nanocapsule etc., and polymer used is natural or synthetic macromolecular material.Because nanoparticle is little at particle diameter, specific surface area is large, can strengthen medicine rate of dissolution and oral administration biaavailability in vivo, tissue or cell are had targeting and extend it in the action time of site of action, nano medicament carrying system has become the important research direction of domestic and international medicine and pharmacology.
Polylactic acid (polylactic acid, PLA) and Poly(D,L-lactide-co-glycolide (poly (lactide-co-glycolide) acid, PLGA) be nontoxic Bioegradability, by lactic acid, lactic acid and hydroxyacetic acid, be polymerized respectively, it is a kind of degradable functional polymer organic compound, there is good biological degradability, biocompatibility and nontoxic feature, by FDA approval, gone on the market and be widely used in the preparation of polymer nanoparticle.
The present invention is directed to blank and " bottleneck " problem of the selectively targeted medicine research of ANCA dependency vasculitis both at home and abroad at present, from immunological molecule, start with, use nano vaccine technology, immunologic new development of modern times and pharmaceutics are organically combined, for the deficiencies in the prior art, carried out improving invention.
Summary of the invention
In view of this, the invention provides a kind of nanometer formulation of LAMP-2 epitope peptide, it has the vasculitic effect for the treatment of ANCA dependency, especially for the immunization therapy of the few immune complex patients with crescent nephritis of anti-LAMP-2 antibody dependency.
For achieving the above object, technical scheme of the present invention is:
The application of LAMP-2 epitope peptide in preparing the few immune complex patients with crescent nephritis of ANCA (ANCA) dependency vasculitis and anti-LAMP-2 antibody dependency nano vaccine, the LAMP-2 epitope peptide that it contains effective dose; Described LAMP-2 epitope peptide is the polypeptide of the aminoacid sequence as shown in SEQ IN DO:1, or is the mixtures of polypeptides of the aminoacid sequence as shown in SEQ IN DO:1 and SEQ IN DO:2.
On the basis of existing technology, by molecular simulation, test and learn: the 72nd to 80 amino acids residue polypeptide of bacterial flagellum FimH albumen (are abbreviated as P
72-80) (be abbreviated as P with people LAMP-2 epi-position the 41st to 49 amino acids residue polypeptide
41-49) there is 100% homology, when body passes through identification antibacterial FimH antigen, also there is to cross reaction in the LAMP-2 albumen of self.After prompting bacterial invasion human body, the autoimmune starting for LAMP-2 by FimH is the pathogenetic key of LAMP-2-ANCA dependency PICGN.Confirm after deliberation the P of LAMP-2 albumen
41-49polypeptide (sequence is: HGTVTYNGS, see sequence table SEQ IN DO:1) and P
331-341polypeptide (sequence is: QGKYSTAQDCS, see sequence table SEQ IN DO:2) be the pathogenic epi-position of the LAMP-2 target antigen of people PICGN.
The present invention is by the pathogenic epitope peptide of finding: the aminoacid sequence shown in SEQ IN DO:1 and SEQ IN DO:2 is combined with nanotechnology, can overcome polypeptide and protein half-life short, be subject to enzymolysis impact, be difficult to by problems such as biological barriers, and route of administration is expanded to pulmonary's suction, oral administration, percutaneous dosing, drug administration by injection etc.Described LAMP-2 epitope peptide can business be bought acquisition, also can adopt Fmoc solid-phase polypeptide technology to complete preparation.The nanometer formulation that LAMP-2 epitope peptide is relevant can be prepared by general formulation method, as emulsion polymerization method, emulsifying-solvent evaporation method, emulsion-solvent siffusion method, the nanometer sedimentation method, the even method of high pressure breast, supercritical fluid technology etc.By the selection of adjuvant and the modification on nanoparticle surface, the polypeptide of effective dose can be delivered to target organ or target site, reach the effect that strengthens drug effect or slow release.
Specific to the nano vaccine of LAMP-2 epitope peptide, the invention also discloses the selection of its utilization and adjuvant thereof, this utilization provides new approaches for vasculitis and few immune complex patients with crescent nephritis.
Another object of the present invention is to provide the few immune complex patients with crescent nephritis of a kind of LAMP-2 antibody dependency and the vasculitic nano vaccine of ANCA dependency, this vaccine is under the synergism of adjuvant, through pharmacological evaluation, confirm to there is good therapeutic effect.
For achieving the above object, technical scheme of the present invention is:
The few immune complex patients with crescent nephritis of LAMP-2 antibody dependency and the vasculitic nano vaccine of ANCA dependency, prepared by LAMP-2 epitope peptide and the Poly(D,L-lactide-co-glycolide of effective dose; Described LAMP-2 epitope peptide is the polypeptide of the aminoacid sequence as shown in SEQ IN DO:1, or is the mixtures of polypeptides of the aminoacid sequence as shown in SEQ IN DO:1 and SEQ IN DO:2.
By research, find (nanometer formulation Evaluation of Biocompatibility progress, Shenyang Pharmaceutical University's journal, the 27th the 12nd phase of volume, 987-992), different carrier materials all has stimulation in various degree to organism, need to carry out rational purification to these carrier materials, change consumption, the methods of disposal such as outside modification reduce its side effect or are effectively converted into therapeutical effect, in preparation process, can further improve toxicity and the safety of nanometer formulation.
Poly(D,L-lactide-co-glycolide (poly (lactide-co-glycolide) acid, PLGA) be the outstanding adjuvant that a kind of human tolerance is very large, it is hydrolyzed metabolism by polyester in vivo, is first degraded to lactic acid and hydroxyacetic acid, is finally degraded to carbon dioxide and water.PLGA applies to the present invention, can prepare the nanoparticle that form is good, envelop rate is high, with LAMP-2 epitope peptide synergism, shows good therapeutic effect again.
Further, the mass ratio of described LAMP-2 epitope peptide and Poly(D,L-lactide-co-glycolide is 1:2-5.
Further, the copolymerization ratio LA:GA=50:50 of lactic acid and hydroxyacetic acid in described Poly(D,L-lactide-co-glycolide.
Further, described nano vaccine is the W/O/W type nanoparticle making by solvent evaporation method, and nanoparticle particle diameter is 100-1000nm.
The present invention also provides a kind of simple method for preparing of above-mentioned nano vaccine, and the method is simple to operate, and equipment requirements is low, and the nanoparticle particle diameter and the envelop rate that make are all more satisfactory.This preparation method comprises the following steps:
First LAMP-2 epitope peptide is scattered in the organic facies containing Poly(D,L-lactide-co-glycolide, forms water-in-oil type colostrum; Redispersion, in water, forms W/O/W type emulsion, and organic solvent is removed in volatilization, obtains; Quality/the mg of described Poly(D,L-lactide-co-glycolide and organic facies and volume/ml is than being 15-50:1; The volume ratio of described organic facies and water is 1:5-14.
Further, described organic facies is one or more in dichloromethane, chloroform, acetone, ethyl acetate, Nitrocarbol., oxolane and trichloroethylene, preferably dichloromethane.
Further, described water is the phosphate buffer containing mass fraction 0.2%-2.5% polyvinyl alcohol.Polyvinyl alcohol is surfactant, at this, plays the effect of emulsifying agent, and other materials also can be used for the present invention as gelatin, Polysorbate, poloxamer etc.Described water pH is 4-7.This water preferably has the solution of buffer substance and controls pH, as used phosphate buffer.
Further, described LAMP-2 epitope peptide with containing the organic facies of Poly(D,L-lactide-co-glycolide, by eddy mixer, stirs formation water-in-oil type colostrum, or form water-in-oil type colostrum second by ultrasonic emulsification 20-60; Colostrum adds after water, and ultrasonic emulsification 20-60 forms W/O/W type emulsion second; Described ultrasonic power is 90W, within ultrasonic every 3 seconds, pauses 1 second.
Further, the step of described removal organic solvent comprises: the water dilution that the W/O/W type emulsion system of formation is doubly measured with 3-4, under room temperature, stir 3-4 hour.
When above-mentioned employing solvent evaporation method is prepared this nano vaccine, described PLGA molecular weight, PLGA concentration, oily watr-proportion and preparation condition, all affect the effect of the nanoparticle of preparation, and the copolymerization ratio of PLGA also affects bioavailability.In preparation process, can use eddy mixer and ultrasound wave and carry out emulsifying, emulsification times can be adjusted according to the power of instrument, and the preparation effect of nanoparticle can judge by methods such as microscopic examination, envelop rate tests.Before organic solvent is removed in volatilization, available water further carries out after dilution again, and stirring and suitable intensification can be accelerated the volatilization of organic solvent.
Prepared liquid W/O/W type nano vaccine can be further centrifugal, collecting precipitation, gained precipitation relaunders, disperses with distilled water, be placed in low-temperature freeze-drying machine (80 ℃) dry 40 hours, or drying under reduced pressure after centrifugalize, obtain the nanoparticle of dryness, be convenient to transportation, preserve.
Use Poly(D,L-lactide-co-glycolide as adjuvant, to prepare the method for nano vaccine, also comprise spray drying method.As LAMP-2 epitope peptide and Poly(D,L-lactide-co-glycolide being dissolved in to the mixed solvent of dichloromethane and chloroform, spraying is drying to obtain.
In addition, the present invention also utilizes polypeptide and the complete Freund's adjuvant synergism of the aminoacid sequence shown in SEQ IN DO:1, success is induced and has been set up vasculitis renal damage model, polypeptide and complete Freund's adjuvant synergism that aminoacid sequence shown in SEQ IN DO:1 is described, can be used as the derivant of setting up vasculitis renal damage model.
Useful technique effect of the present invention is:
The present invention is based on LAMP-2 target antigen and carry out the exploitation of polypeptide vaccine, specificity induction is for the immunologic tolerance of LAMP-2 target antigen, use nano vaccine technology, the nanometer formulation that can prepare the multiple LAMP-2 of containing epitope peptide, for the treatment of the few immune complex patients with crescent nephritis of ANCA dependency vasculitis and anti-LAMP-2 antibody dependency provides new method.Utilize the synergism of Poly(D,L-lactide-co-glycolide and LAMP-2 epitope peptide formulations, through experiment, confirm, the nano vaccine of preparation can be eliminated the special spontaneous immunne response of autoantigen, and do not disturb the immunne response for other antigens, further confirmed to use LAMP-2 epitope peptide at the effectiveness for the treatment of ANCA dependency vasculitis and the immunity of PICGN disease.
Accompanying drawing explanation
Fig. 1 is the nano vaccine scanning electron microscope (SEM) photograph of embodiment 2 preparations;
Fig. 2 is the content analysis figure that complete Freund's adjuvant associating polypeptide P41-49 induces the serum creatinine CREA of the vasculitis renal damage model of setting up;
Fig. 3 is the content analysis figure that complete Freund's adjuvant associating polypeptide P41-49 induces the blood uric acid UA of the vasculitis renal damage model of setting up;
Fig. 4 is the content analysis figure of the serum creatinine CREA of model after nano vaccine immunity;
Fig. 5 is the content analysis figure of the blood uric acid UA of model after nano vaccine immunity;
Fig. 6 is the 7th day and the 14th day twenty-four-hour urine protein content analysis chart of the WKY P of Rats ICGN model of FimH associating Titermax gold adjuvant foundation;
Fig. 7 is the content analysis figure of the serum creatinine CREA of the WKY P of Rats ICGN model set up of FimH associating Titermax gold adjuvant;
Fig. 8 is the content analysis figure of the blood uric acid UA of the WKY P of Rats ICGN model set up of FimH associating Titermax gold adjuvant.
The specific embodiment
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in preferred embodiment, condition is carried out routinely.
Document (Nat Med.2008,14 (10): 1088-96) that the pathogenic epi-position of LAMP-2 target antigen of people PICGN is published from Kain etc.Kain etc. adopt PICGN patient's serum, analyze the combination epi-position of having found two autoantibodys by SPOTscan, by sequence analysis, turn out to be the pathogenic epitope sequences P of LAMP-2 target antigen
41-49and P (HGTVTYNGS)
331-341(QGKYSTAQDCS).
Embodiment 1 is loaded with LAMP-2 epitope peptide P
331-341the preparation of nano vaccine
Electronic balance takes 5mg LAMP-2 epitope peptide P
331-341(purity >95%, Zhongtai Bio-Chem. Co., Ltd., Hangzhou) and 15mg Poly(D,L-lactide-co-glycolide (LA:GA=50/50, sigma), be placed in 10ml centrifuge tube, add 0.6ml dichloromethane, eddy mixer stirs into even mixed liquor, add 6ml to contain the phosphate buffer (pH is 6.5) of 1% polyvinyl alcohol, be placed in ultrasonic 20s(power 90W under ultrasonic probe, time 20s, every 3s dwell interval 1s).By emulsion pour into be equipped with 20ml containing in the phosphate buffer (pH is 6.5) of 0.3% polyvinyl alcohol, put into stirrer, at room temperature, magnetic stirrer 3h, makes organic solvent volatilization.Emulsion after organic solvent volatilization is poured in EP pipe, put into centrifuge tube centrifugal (15000rpm, 10min), outwell supernatant, add distilled water, use suction pipe purge nanoparticle 3 times, obtain.
Embodiment 2 is loaded with LAMP-2 epitope peptide P
41-49the preparation of nano vaccine
Electronic balance takes 5mg LAMP-2 epitope peptide P
41-49(purity >95%, Zhongtai Bio-Chem. Co., Ltd., Hangzhou) and 15mg Poly(D,L-lactide-co-glycolide (LA:GA=50/50, sigma), be placed in 10ml centrifuge tube, add 0.6ml dichloromethane, eddy mixer stirs into even mixed liquor, add 6ml to contain the phosphate buffer (pH is 6.5) of 1% polyvinyl alcohol, be placed in ultrasonic 20s(power 90W under ultrasonic probe, time 20s, every 3s dwell interval 1s).By emulsion pour into be equipped with 20ml containing in the phosphate buffer (pH is 6.5) of 0.3% polyvinyl alcohol, put into stirrer, at room temperature, magnetic stirrer 3h, makes organic solvent volatilization.Emulsion after organic solvent volatilization is poured in EP pipe, put into centrifuge tube centrifugal (15000rpm, 10min), outwell supernatant, add distilled water, use suction pipe purge nanoparticle 3 times, obtain.
The evaluation of experimental example 3 nano vaccines
(1) morphologic observation of the nano vaccine of preparing
By evenly spreading upon on mica sheet after embodiment 1 and the dilution of 2 washed nanoparticle suspensions, after natural drying, by scanning electron microscope (S-3400N electron microscope), carry out morphologic observation.Test result shows, the nanoparticle balling-up of preparing with emulsion-solvent evaporation method is good, and form is good, and particle diameter is even.As shown in Figure 1, the nano vaccine scanning electron microscope (SEM) photograph of preparing for embodiment 2.
(2) the particle diameter test of the nano vaccine of preparing
Embodiment 1 and 2 washed nanoparticles, with packing in particle instrument (Malvern company) sample cell after distilled water dilution, are measured to particle size distribution.Test result shows, is loaded with the particle diameter of nanoparticle of epitope peptide between 100-1000nm, and particle size distribution is more even; According to integration statistics, the mean diameter of embodiment 1 nanoparticle is 237nm, and the mean diameter of embodiment 2 nanoparticles is 238nm.
(3) the envelop rate test of the nano vaccine of preparing
Adopt Sephedex-G100 gel chromatography, collect the free LAMP-2 epitope peptide P of embodiment 2
41-49, and by spectrophotometric determination A280 value.Meanwhile, prepare variable concentrations LAMP-2 epitope peptide P
41-49standard protein sample, by spectrophotometric determination its shading value, and draw the standard curve of concentration and shading value.Take standard curve as foundation, calculate free LAMP-2 epitope peptide P in nano-particle
41-49content, be calculated as follows envelop rate:
Envelop rate=(W
always-W
trip)/W
always* 100%
Wherein, W
alwayslAMP-2 epitope peptide P used during for preparation
41-49total amount, W
tripfor free LAMP-2 epitope peptide P
41-49amount.
Result demonstration, nanoparticle is sealed respond well, and wherein, the envelop rate of embodiment 1 and embodiment 2 nano vaccines is respectively 65% and 62%.The nanoparticle suspension of embodiment 2 is placed 6 months in 4 ℃, without layering and deposited phenomenon.
(4) the vivo effect evaluation of nano vaccine
By each 9 of 18 WKY rat male and female, be divided at random 6 groups, 3 every group, carry out in the steps below the vivo effect evaluation of nano vaccine.The meaning that the code name of following steps represents is as follows:
A, complete Freund's adjuvant associating polypeptide P
41-49vasculitis renal damage model is set up in induction
Method and test: model group (CFA+Pep) is set, with 300ul complete Freund's adjuvant+300ug LAMP-2 epitope peptide P
41-49, after emulsator emulsifying, select large leg outer side subcutaneous injection, metabolism of rat cage is collected urine, and monitoring twenty-four-hour urine albumen changes; Matched group (CFA) is set, wherein with distilled water, replaces polypeptide, other treatment steps are same.Within 47 days, put to death afterwards, blood biochemistry index (serum creatinine CREA, blood uric acid UA) detection and kidney are carried out in rat heart blood sampling, pathologic detects (H & E, PAS, Mas dyeing).
Each test result of organizing twenty-four-hour urine albumen is as shown in table 1; The content analysis figure of serum creatinine CREA as shown in Figure 2; The content analysis figure of blood uric acid UA as shown in Figure 3.
The test result of the twenty-four-hour urine albumen of table 1 renal damage model
Note: Mean is meansigma methods; SEM is standard error.
Analyze: as shown in Table 1, model group rat kidney twenty-four-hour urine albumen is apparently higher than matched group.From Fig. 2 and Fig. 3, model group serum creatinine, blood uric acid are apparently higher than matched group (p is respectively 0.0430 and 0.0434, p < 0.05, has statistical significance).On pathology, nephridial tissue pathology detection shows, the hyperemia of model group glomerule, and petechial hemorrhage is downright bad, proliferation of mesangial cells, kidney interstitial Tubular epithelial cell partly comes off, inflammatory cell infiltration; Pathologic detects and shows, bronchial mucosa congestion and edema, and alveolar wall hypertrophy is subsided, part fracture, interstitial inflammatory cell infiltration.
Conclusion: based on LAMP-2 epitope polypeptide P
41-49associating complete Freund's adjuvant has successfully built the scorching kidney of rat aorta, lung damage model.
The immunological effect of the nano vaccine of B, embodiment 2 preparations
Method and test: vaccine intervention group (Vac+CFA+Pep) is set, at animal model, causes a disease first 7 days and 3 days, with 100ul, be loaded with LAMP-2 epitope peptide P respectively
41-49nano vaccine carry out subcutaneous vaccination, and in the time of 0 day with 300ul complete Freund's adjuvant+300ug LAMP-2 epitope peptide P
41-49, carry out subcutaneous injection.Metabolism of rat cage is collected urine, and monitoring twenty-four-hour urine albumen changes.Rat heart blood sampling is carried out blood biochemistry index (serum creatinine CREA, blood uric acid UA) and is detected, and compares with the model group (CFA+Pep) in A step.
Each test result of organizing twenty-four-hour urine albumen is as shown in table 2; The content analysis figure of serum creatinine CREA as shown in Figure 4; The content analysis figure of blood uric acid UA as shown in Figure 5.
The test result of the twenty-four-hour urine albumen of table 2 immunological effect
Analyze: as shown in Table 2, vaccine intervention group rat obviously alleviates compared with model control group Rat 24 h urine protein; From Fig. 4 and Fig. 5, vaccine intervention group serum uric acid level is starkly lower than matched group (P=0.0244), but serum creatinine does not have significant difference.
Conclusion: through LAMP2 polypeptide P
41-49after/PLGA nano vaccine is intervened, Rat 24 h urine protein quantitation, serum uric acid level alleviate.Show to be loaded with LAMP-2 epitope peptide P
41-49nano vaccine can eliminate the special spontaneous immunne response of autoantigen.
The effect of the WKY P of Rats ICGN model that the nano vaccine of C, embodiment 2 preparations is set up at FimH associating Titermax gold adjuvant
Method and test: Titermax gold adjuvant matched group (Titermax) is set, FimH model group (FimH+Titermax), FimH causes a disease and vaccine intervention group (Vac+FimH+Titermax).150ug FimH fusion rotein+150ul Titermax gold adjuvant for FimH model group, after emulsator emulsifying, select large leg outer side subcutaneous injection, metabolism of rat cage is collected urine, monitoring twenty-four-hour urine albumen changes, and Titermax gold adjuvant matched group is only injected 300ul Titermax gold adjuvant.FimH causes a disease and vaccine intervention group is caused a disease first 7 days and 3 days at animal model, respectively with being loaded with LAMP-2 epitope peptide P
41-49nano vaccine carry out subcutaneous vaccination, and with 300ul Titermax gold adjuvant, carried out subcutaneous injection in the time of 0 day.Metabolism of rat cage is collected urine, and monitoring twenty-four-hour urine albumen changes.Blood biochemistry index is carried out in rat heart blood sampling, and (serum creatinine (serum creatinine CREA, blood uric acid UA) detects and nephridial tissue pathology detection (H & E dyeing).
Each test result of organizing twenty-four-hour urine albumen is as shown in table 3, and the 7th day and the 14th day twenty-four-hour urine protein content analysis chart are as shown in Figure 6; The content analysis figure of serum creatinine CREA as shown in Figure 7; The content analysis figure of blood uric acid UA as shown in Figure 8.
The test result of the twenty-four-hour urine albumen of table 3 WKY P of Rats ICGN model
Analyze: from table 3 and Fig. 6, in 14 days, FimH model group Rat 24 h urine protein is apparently higher than matched group (P=0.0290) and vaccine intervention group, however after 14 days, FimH cause a disease with vaccine intervention group and FimH model group urine protein quantitation on but no difference of science of statistics (P=0.0576).As shown in Figure 7 and Figure 8, serum creatinine, serum uric acid level do not have significant difference.On pathology, the visible diffuse crescent formation of FimH model group glomerule, but vaccine intervention group glomerule degree of necrosis is lighter than disease model group.
Conclusion: FimH fusion rotein associating Titermax gold adjuvant has successfully built PICGN model; After nano vaccine is intervened, glomerule degree of necrosis alleviates.Show to be loaded with LAMP-2 epitope peptide P
41-49nano vaccine do not disturb the immunne response for other antigens, also alleviated the degree of necrosis of glomerule simultaneously, improved vasculitic situation.
Finally explanation is, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
- The application of 1.LAMP-2 epitope peptide in preparing the few immune complex patients with crescent nephritis of ANCA dependency vasculitis and anti-LAMP-2 antibody dependency nano vaccine, the LAMP-2 epitope peptide that it contains effective dose; Described LAMP-2 epitope peptide is the polypeptide of the aminoacid sequence as shown in SEQ IN DO:1, or is the mixtures of polypeptides of the aminoacid sequence as shown in SEQ IN DO:1 and SEQ IN DO:2.
- 2. the few immune complex patients with crescent nephritis of anti-LAMP-2 antibody dependency and the vasculitic nano vaccine of ANCA dependency, is characterized in that: prepared by LAMP-2 epitope peptide and Poly(D,L-lactide-co-glycolide by effective dose; Described LAMP-2 epitope peptide is the polypeptide of the aminoacid sequence as shown in SEQ IN DO:1, or is the mixtures of polypeptides of the aminoacid sequence as shown in SEQ IN DO:1 and SEQ IN DO:2.
- 3. nano vaccine according to claim 2, is characterized in that: the mass ratio of described LAMP-2 epitope peptide and Poly(D,L-lactide-co-glycolide is 1:2-5.
- 4. nano vaccine according to claim 2, is characterized in that: the copolymerization ratio LA:GA=50:50 of lactic acid and hydroxyacetic acid in described Poly(D,L-lactide-co-glycolide.
- 5. nano vaccine according to claim 2, is characterized in that: described nano vaccine is the W/O/W type nanoparticle making by solvent evaporation method, and nanoparticle particle diameter is 100-1000nm.
- 6. the preparation method of the nano vaccine described in claim 2 to 5 any one, is characterized in that, comprises the following steps: first LAMP-2 epitope peptide is scattered in the organic facies containing Poly(D,L-lactide-co-glycolide, forms water-in-oil type colostrum; Redispersion, in water, forms W/O/W type emulsion, and organic solvent is removed in volatilization, obtains;Quality/the mg of described Poly(D,L-lactide-co-glycolide and organic facies and volume/ml is than being 15-50:1;The volume ratio of described organic facies and water is 1:5-14.
- 7. preparation method according to claim 6, is characterized in that: described organic facies is one or more in dichloromethane, chloroform, acetone, ethyl acetate, Nitrocarbol., oxolane and trichloroethylene.
- 8. preparation method according to claim 6, is characterized in that: described water is the phosphate buffer containing mass fraction 0.2%-2.5% polyvinyl alcohol, and water pH is 4-7.
- 9. preparation method according to claim 6, it is characterized in that: described LAMP-2 epitope peptide with containing the organic facies of Poly(D,L-lactide-co-glycolide, by eddy mixer, stirs formation water-in-oil type colostrum, or form water-in-oil type colostrum second by ultrasonic emulsification 20-60;Colostrum adds after water, and ultrasonic emulsification 20-60 forms W/O/W type emulsion second;Described ultrasonic power is 90W, within ultrasonic every 3 seconds, pauses 1 second.
- The collaborative application for setting up vasculitis renal damage model as derivant of the polypeptide of the aminoacid sequence shown in 10.SEQ IN DO:1 and complete Freund's adjuvant.
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| CN111663008A (en) * | 2020-07-21 | 2020-09-15 | 广西壮族自治区兽医研究所 | Double LAMP (loop-mediated isothermal amplification) detection kit for avian nephritis viruses and H9 subtype avian influenza viruses and primer group thereof |
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| CN102485274A (en) * | 2010-12-01 | 2012-06-06 | 吉林大学 | Preparation method and use of poly(lactic-co-glycolic acid) (PLGA) microspheres as nucleic acid vaccine vectors |
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| Title |
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| MOLECULAR MIMICRY IN PAUCI-IMMUNE FOCAL NECROTIZING GLOMERULONEP: "Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis", 《NAT MED.》, vol. 14, no. 10, 31 October 2008 (2008-10-31), pages 1088 - 1096 * |
| 高小玲等: "生物可降解聚酯和壳聚糖在疫苗制剂中的应用", 《中国医药工业杂志》, vol. 36, no. 1, 31 December 2005 (2005-12-31), pages 46 - 51 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111663008A (en) * | 2020-07-21 | 2020-09-15 | 广西壮族自治区兽医研究所 | Double LAMP (loop-mediated isothermal amplification) detection kit for avian nephritis viruses and H9 subtype avian influenza viruses and primer group thereof |
| CN111663008B (en) * | 2020-07-21 | 2023-07-14 | 广西壮族自治区兽医研究所 | Double LAMP (loop-mediated isothermal amplification) detection kit for avian nephritis virus and H9 subtype avian influenza virus and primer group thereof |
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