| As the raw materials of traditional Chinese medicine with the effects of bactericidal,antioxidant,antiviral and improve biological immune function,pine pollen and astragalus contains a large number of nutrients and bioactive substances,which have been widely used in the treatment of a variety of bacterial diseases,have a breakthrough in studying inhibition of tumor cells and human papillomavirus,and have great benefits to the health of human and animals.Nowadays more and more attention has been paid to the study of plant polysaccharides,as a new research object,polysaccharides is recognized by the international scientific community as one of the dominant scientific themes in the 21 st century.Relevant studies have confirmed that bioactive polysaccharides is the most important functional substance in plant cells and its biological activity is mainly reflected in the following aspects: immune regulation,anti-tumor,blood pressure lowering,anti-oxidation,anti-radiation,anti-bacterial,protection of important immune organs,etc.Plant polysaccharides as an active substance with immune regulation have been widely used in many fields.The extraction methods of plant active polysaccharides,such as water boiling and physicochemical methods,are time-consuming,low yield,often used organic chemical reagents and cumbersome operation,making the total yield of plant active polysaccharides low,which greatly affects the extensive application of plant polysaccharides in animal husbandry.In order to solve the above problems in the traditional extraction of plant active polysaccharides,we used a cellulase producing bacteria for the fermentation of polysaccharides,in order to obtain a simple,no organic chemical use,low cost plant polysaccharides extraction method.In this study,Bacillus licheniformis with cellulase production characteristics produced cellulase in the enzymatic fermentation medium to release active polysaccharides in the plant cell walls of pine pollen,astragalus powder and wallbreaker pollen,respectively.Pinus massoniana pollen polysaccharides(PPPS),Astragalus mongholicus polysaccharides(APS)and Broken Pinus massoniana pollen polysaccharides(BPPPS)were extracted from the fermentation broth with 80% ethanol respectively.Three plant extracts of polysaccharides were identified by HPLC,and it was proved that the main components of the three extracts were polysaccharides.The content of polysaccharides in plant extracts of three polysaccharides at different time points was determined by concentrated sulfuric acid-phenol method.The results showed that the maximum extraction volumes of PPPS,APS and BPPPS were 3.08g/100 g,5.85g/100 g and 8.43g/100 g,respectively.1.4 times,2.5 times and 3 times of water formulation respectively;It is 0.7 times,1.3 times and 1.8 times of physical and chemical methods.The results showed that the activity of cellulase changed dynamically at different time points,and the activity of cellulase in different combinations showed different changing rules.In order to test the immunomodulatory effect of the obtained plant polysaccharide fermentation purified products,PRRSV GP5 subunit was selected as the immunogen tool for the experiment,in order to preliminarily explore how the polysaccharide extracts could play an immunomodulatory role in gp5-induced cellular immunity,humoral immunity and other aspects.Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious and infectious disease caused by Porcine reproductive and respiratory syndrome virus(PRRSV),characterized by reproductive disorders in female pigs and respiratory diseases in newborn piglets.But there are still various problems in practical application.According to the PRRSV GP5 protein expression purification method previously constructed in the laboratory,highly pathogenic PRRSV envelope glycoprotein 5(GP5)was conducted prokaryotic expression to gain GP5 protein ACTS as an immunogen,and heterogeneous Pinus massoniana pollen polysaccharides PPPS,Astragalus mongholicus polysaccharides APS and Broken Pinus massoniana pollen polysaccharides BPPPS as an immune adjuvant,made into subunit vaccine.Choosing 44 healthy rabbits,divided randomLy into 11 groups,primary and secondary immunization were performed on day 0 and 14 of the trial,respectively.GP5 protein was purified in group 1.Groups 2-4 were immunized with different concentrations of PPPS(100,200,400 mg/mL)and GP5 protein.Groups 5-7 were immunized with APS(100,200,400mg/mL)and GP5 protein at different concentrations.Groups 8-10 were immunized with different concentrations of BPPPS(100,200,400mg/mL)and GP5 protein.The 11 th group was injected with normal saline(SPSS),and the immunization was strengthened once 2 weeks later.Peripheral blood of each group was taken every week,GP5 antibody titer in serum was detected by ELISA method,peripheral blood lymphocyte conversion rate was detected by MTT method,and il-2 and ifn-gamma cytokines concentrations in peripheral blood were detected by ELISA kit.The results showed that the above immune indexes increased in all the vaccine groups,particularly,compared with GP5 group and SPSS group,the vaccine group with high concentration of polysaccharide as adjuvant showed significant improvement(P<0.05).The test results show that PRRSV GP5 protein induced specific immune response,and PPPS,APS and BPPPS as an adjuvant played a certain role of immunomodulation on GP5 protein.In this experiment,the extraction rate of polysaccharide from three polysaccharide plants fermented by bacillus lichene-producing cellulase was significantly higher than that of water extraction and physical and chemical methods,with the highest multiple reaching 3 times and1.8 times,respectively.Three plant polysaccharide extracts were used as immune adjuvants to act on the subunit vaccine of PRRSV GP5 protein,which could significantly improve the indicators of cell immunity and humoral immunity.It was preliminarily proved that the polysaccharide extracts obtained by this method had a good immune enhancement effect.This study is beneficial for the more efficient and lower cost extraction of plant polysaccharides and provides technical reserves for its extensive application in animal husbandry. |
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