Study On Isolation And Purification Of Sargassum Polysaccharide And Its Resistance To Prrsv In Vitro

Sargassum polysaccharides（SP）are natural polysaccharides extracted from the brown algae Sargassum,which have been shown to have various biological activities such as immunomodulation,antioxidant,anti-inflammatory and anti-viral actions.The typical clinical symptoms of PRRSV infection are reproductive failure of sows and severe respiratory diseases and growth performance of piglets.There is no drug against PRRSV,and there are still many deficiencies in vaccine immunization.The present study was carried out to conduct isolation and purification of Sargassum polysaccharide and analysis of their component purity,molecular weight,and investigate anti-PRRSV infection of the components of the polysaccharide in MARC-145 cells and provide theoretical basis for utilization of Sargassum polysaccharide,especially in the prevention of PRRSV..（1）Isolation,purification and molecular weight determination of Sargassum polysaccharideThe crude polysaccharide of Sargassum arborescens was extracted by enzymatic hydrolysis and alcohol precipitation,and the protein was removed by trichloroacetic acid method;the polysaccharide content in the crude polysaccharides of Sargassum was detected by anthrone sulfuric acid method and fucose was used as a control;DEAE-Sepharose Fast Flow Ion exchange column chromatography was used to separate and purify fractions from the crude Sargassum polysaccharide,and the purified components of the polysaccharide were analyzed by Molish reaction,iodine-potassium iodide reaction,ultraviolet-visible spectroscopy and ninhydrin reaction.The purity and molecular weight of the fractions were determined by Sephacryl S200HR gel column chromatography.The results showed that the yield of crude Sargassum polysaccharide was17.13%;the content of reduced sugar in the crude polysaccharide was 45.04%;There are four elution fractions obtained after separation and purification of crude Sargassum polysaccharide were performed by DEAE-Sepharose Fast Flow ion exchange chromatography,including distilled water eluted component named SP1,0.5M Na Cl eluted component named SP2,1.0 M Na Cl eluted component named SP3,1.5 M Na Cl eluted component named SP4,the four components were positive for Molish reaction,which indicated they contain carbohydrates;Iodine-potassium iodide reaction is negative,indicating that there is no starch in the fractions;Ninhydrin reaction is negative,indicating that there is no amino acid in the components;UV spectrum has no absorption peaks at 260 nm,280 nm,indicating no protein or nucleic acid;The qualitative analysis of the four components is consistent with a single curve of Sephacryl S200HR gel chromatography elution.the molecular weights of SP1 and SP2 are 31048 Dalton（31.08KD）and 177699 Dalton（177.699KD）.（2）Establishment of qRT-PCR detection method for PRRSVPCR method was used to amplify the PRRSV nucleocapsid N gene sequence,and the cloning method was used to construct the N gene recombinant plasmid with p MD-18T as the vector;the sequence of the positive plasmid obtained by using scientific methods were analyzed and identified;the recombinant N gene plasmid was diluted 10-fold as a template,and the standard curve of PRRSV and its linear regression equation were established by qRT-PCR;MARC-145 cells was infected with 100 TCID₅₀virus concentration,PCR was used to detect nucleic acid of PRRSV.The results showed that the PRRSV nucleocapsid N gene sequence was successfully amplified,and the amplified fragment had good specificity.The gene fragment of PRRSV positive recombinant plasmid sequence is consistent with the expected gene fragment,with 100%homology.qRT-PCR detection method can effectively amplify PRRSV standard plasmid,the detection range is 8.396×10²～8.396×10⁹ copies/μL,and the standard curve has good linear relationship and strong specificity,which can be used for following PRRSV quantitative analysis.The 100TCID₅₀ virus concentration can successfully infect MARC-145 cells,which can be used as the late virus infection concentration.（3）The anti-PRRSV experiments of Sargassum polysaccharide components SP1and SP2 in vitro.The anti-PRRSV effect and mechanism of SP1 and SP2 were investigated in vitro,CCK8 method was used to evaluate the toxic effects of SP1 or SP2 on cells;Through the four methods of action:drug treatment before virus infection,drug treatment after virus infection,simultaneous viral infection and drug treatment,and drug and virus incubation together before addition to cells.The qRT-PCR method is used to determine effects of SP1 or SP2 on the amplification of the PRRSV virus nucleocapsid N gene;then the inhibitory effects of SP1 and SP2 on different stages of PRRSV proliferation adsorption,replication and release were observed,and the mechanism of anti-PRRSV action of SP1 and SP2 was preliminarily clarified.The results showed that SP1 co-incubated with MARC-145 cells at a concentration of≤100μg/mL had no effect on cells within 48 hours,and SP2co-incubated with MARC-145 cells at a concentration of≤800μg/mL had no effect on cells within 48 hours;The four action methods of of SP2 and SP2 can significantly inhibit the replication of the PRRSV nucleocapsid N gene（P<0.01）,and are superior to ribavirin.SP1 and SP2 at low,medium and high dose inhibited the adsorption of PRRSV on MARC-145 cells,and the inhibition ability of PRRSV adsorption is positively correlated with concentration;SP1at dose of25μg/mLor 50μg/mL revealed anti-PRRSV replication effect,which were better that of ribavirin at 100μg/mL.SP1 component at 25μg/mL could not inhibit the release of PRRSV,while SP1at 50μg/mL and 100μg/mL significantly inhibited the virus release（P<0.01）.SP2 at 25μg/mL,50μg/mL or 100μg/mLsignificantly inhibited the replication and release of PRRSV in MARC-145 cells（P<0.01）.（4）Effect of SP1 and SP2 on expression of CD163 receptor and viral valenceA relative qRT-PCR method was used to detect expression of CD163receptor in MARC-145 cells treated with SP1 and SP2 at25μg/mL,50μg/mL or100μg/mL for 24 hours after 100TCID₅₀ infection for 2h.The viral valence values were determined in in viral infected MARC-145 cells treated SP1 and SP2 at 25μg/mL,50μg/mL or 100μg/mL.The results showed that SP1 at 50μg/mL or 100μg/mL except 25μg/mL and SP2 at 25μg/mL,50μg/mL or 100μg/mL significantly inhibited expression of CD163 receptor of PRRSV（P<0.01）.SP1 and SP2 at 25μg/mL,50μg/mL or 100μg/mL have the effect of inhibiting the virus titers of progeny virus.;Conclusion:SP1 and SP2 from Sargassum polysaccharide interfered with the adsorption,replication,and release of PRRSV,and reduce virulence values of progeny virus and decreased expression of CD163 receptor in MARC145 cells,thereby inhibiting PRRSV infection in MARC-145 cells.