| As a typical retrovirus,ALV-J has broken out many times in chicken flocks and caused great losses to the breeding industry in recent years.At present,the purification methods for ALV-J prevention and control have the disadvantages of large investment and long cycle,so it is urgent to find new ways for effective prevention and control.Previous studies have shown that pine pollen polysaccharide has certain activity in antiALV-J test.However,due to the lack of in-depth research,the material basis of inhibiting ALV-J replication by pine pollen polysaccharide is still unclear,which cannot explain its anti ALV-J mechanism,and it is difficult to evaluate and control its activity and quality.At the same time,as a kind of macromolecular polysaccharide,it is difficult to be absorbed through the gastrointestinal mucosa in animals,and it is easy to be recognized by the immune system,resulting in metabolic inactivation.Therefore,it is of great scientific significance and research value to study the material basis of inhibiting ALV-J replication,to explain its structure-activity relationship,to further construct an appropriate drug delivery system,to improve its bioavailability and anti-ALV-J activity in vivo,and to provide a new method for the prevention and treatment of ALV-J.1.Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigationSingle factor experiment was used to optimize the extraction and purification process,and the optimum extraction conditions were as follows: extraction temperature 90?,p H 9.0,liquid-solid ratio 30:1.The final extraction rate of total polysaccharides was 6.5±0.19%.Three fractions(PPP-1,PPP-2 and PPP-3)were isolated by DEAE ion exchange column and Sephadex G-200 molecular exclusion column.CCK-8 test showed that none component had obvious cytotoxicity below 800 ?g/m L.In vitro antiviral test showed that the anti-ALV-J activity of the three components was PPP-2 > PPP-3 >> PPP-1.Their molecular weight,monosaccharide composition and triple helix conformation were further analyzed to further study the structure-activity relationship of the three components against ALV-J.The results showed that the molecular weights of the three components with significant differences were463.70 k Da,99.41 k Da and 26.97 k Da,respectively.The monosaccharide compositions of PPP-2 and PPP-3 were similar,the contents of glucuronic acid and galacturonic acid were higher than those of PPP-1,but the glucose content was significantly lower;Congo red test showed that only PPP-2 had triple helix structure,and its activity decreased significantly after destroying the triple helix structure of PPP-2.The structure-activity relationship of polysaccharide against ALV-J was preliminarily speculated as follows: the composition was the main factor affecting the activity of polysaccharide,the monomer containing acid monosaccharide showed better anti-ALV-J activity,and the spatial conformation was also an important factor affecting its activity.The maintenance of triple helix structure could improve the anti-ALV-J effect of PPP-2,and molecular weight may also affect its activity.In this part,we studied the material basis of inhibiting ALV-J replication and elucidated its structure-activity relationship,which provided material basis for further study of anti-ALV-J activity of pine pollen polysaccharides.2.Preparation of pine pollen polysaccharide chitosan nanoparticles and its anti ALV-J activity investigationIn order to improve the metabolic inactivation of pine pollen polysaccharide in vivo,the pine pollen polysaccharide chitosan nano drug was prepared by the electrostatic interaction between pine pollen polysaccharide and chitosan and the self-assembly principle of chitosan nanoparticles(CS-PPPs NPs)to prolong the action time of pine pollen polysaccharide in chickens and improve the anti-ALV-J activity.The optimum preparation technology was obtained by screening the conditions of CS-PPPs NPs by the encapsulation rate,drug loading and particle size.The optimum preparation technology was obtained: the concentration of chitosan was 2 mg/m L,the ratio of sodium polyphosphate and chitosan was 1:8,the concentration of polysaccharide accounted for 10.00% of chitosan,and the encapsulation rate of CS-PPPs NPs was 91.2%,the drug loading was 8.2%,the average particle size was 264 nm,PDI= 0.21.In vitro release experiments showed that the cumulative release rate of CS-PPPs NPs was 90% at 96 h,which had a good sustained-release effect.Cell experiments showed that the nanoparticles could carry drugs into cells and release slowly.There was no obvious toxic effect on DF-1 cells when the concentration was lower than 400 ?g/m L.The results of in vitro anti-ALV-J activity showed that CS-PPPs NPs prolonged the action time and improved the antiviral effect through sustained release.The results of further experiments showed that the blank chitosan nanoparticles had no obvious antiviral effect,and the pine pollen polysaccharide solution had better antiviral effect on ALV-J replication in the short term,but in the long-term experiment,the metabolic excretion effect of PPPs group decreased significantly.CS-PPPs NPs showed a good sustained-release effect in vivo,and had a good effect in the long-term anti-ALV-J replication,which could protect the chicken body for a long time and improve the immunity and growth rate of chickens.3.Enhancement of anti-ALV-J activity of pine pollen polysaccharides by antibody targeting chitosan nano drug delivery systemIn order to improve the antiviral activity and reduce the toxicity to animals,pine pollen polysaccharides(PPPs)were used as the model drug and JE9 antibody was used as the indicator of anti-ALV-J,a novel targeting anti-ALV-J nano drug delivery system(Ab-CS-PPPs DDS)was constructed.The results showed that JE9 antibody was successfully linked with chitosan.The particle size,entrapment efficiency,drug loading and in vitro release showed that the nanoparticles did not change significantly after JE9 antibody were linked with chitosan.FITC fluorescent probe was used to investigate the virus targeting in cells and animals.The results showed that the cells infected with ALV-J had faster uptake and longer duration of drug nanoparticles linked with JE9 antibody.In vivo experiments showed that the nano drug with JE9 antibody had obvious enrichment in kidney,thymus,spleen and bursa of fabricius of ALV-J chickens.In vivo and in vitro anti-ALV-J experiments showed that the effect of nano drug on inhibiting ALV-J replication was improved after coupling antibody,which indicated that nano-DDS had good distribution and inhibition targeting for ALV-J.Toxicological studies showed that when the concentration was lower than 400 ?g/m L,there was no obvious toxic effect on DF-1 cells;in vivo metabolism showed that the nano-DDS still had a good sustained-release effect after the antibody was coupled;in vitro antiviral experiment results showed that at the concentration of 100 ?g/m L,Ab-CS-PPPs NPs could continuously reduce the replication of ALV-J,showing a good antiviral effect in vitro.At last,the chicks infected with ALV-J were injected with CS-PPPs,NPs and Ab-CS PPPs intramuscularly.The results showed that in the early stage of virus infection,through drug intervention,Ab-CS-PPPs NPs could release slowly,maintain a stable drug concentration,significantly reduce the viral load in tissues,and promote the growth and development of ALV-J infected chickens.Through the isolation and identification of monosaccharide and monosaccharide composition in pine pollen polysaccharide,the anti-ALV-J active components were studied,and the structure-activity relationship was preliminarily analyzed.With the help of electrostatic interaction and self-assembly principle,the chitosan nano drug with high encapsulation efficiency and drug loading,sustained-release and long-term effect was prepared.At the same time,the chitosan nano-DDS based on antibody targeting was constructed,which further improved the anti-ALV-J activity of the polysaccharide,and provided a new idea and method for the prevention and treatment of ALV-J.It also provides a reference for the development and evaluation of other targeted antiviral drugs. |
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