| In this paper,we studied vaccine pulsatile delivery system using Newcastle Disease(ND)inactivated vaccine as a model.The goal is to complete fall immunization via a single dose injection and to reach the same immune effect of ordinary inactivated vaccine repeatedly.First we developed the preparation technology of PLGA microsphere using OVA as a model,and studied the releasing characteristics and immune characteristics of OVA-PLGA microsphere.Then we prepared NDV-PLGA microsphere using the same method,and further studied its releasing characteristics in vitro and immune characteristics in chicken.Part Ⅰ:Preparation and quality evaluation of OVA-PLGA microspheresUsing OVA as embedding material and PLGA as carrier material,we explored the preparation process of OVA-PLGA microspheres by double emulsion-solvent evaporation.The OVA-PLGA microspheres was injected subcutaneously into mice and its immune effect was determined by indirect ELISA method.The results showed that the optional preparation conditions for OVA-PLGA microspheres are as follows:water/oil ration is 1/30 in primary emulsion and OVA/PLGA ration is 1/20.We also found that mixing speed in double emulsion is the key factor affecting microspheres size.Under these conditions,5 μm and 20 μm particle size microspheres were prepared and their encapsulation efficiency was measured as 81.6%and 85.2%respectively.They appeared pulsatile release peak in the 28th day and 42nd day in vitro release test.Murine subcutaneous injection of single dose OVA-PLGA antigen microspheres could produce higher level of antibody response which was superior to that of single dose injection of OVA alumium adjuvants antigen.Part Ⅱ:Preparation and quality evaluation of NDV-PLGA microspheresUsing PLGA microspheres preparation method acquired above,we prepared different size PLGA microspheres embedding inactivated NDV and evaluated their qualities.The results showed that NDV-PLGA 50/50 of the 10 μm microspheres has smooth surface,good dispersion and its encapsulation rate is 65%.It appeared a pulsatile release peak in the 35th day in vitro release test.NDV-PLGA 50/50 of the 20μm microspheres has smooth surface,good dispersion and its encapsulation rate is 67.2%.It appeared a pulsatile release peak in the 49th day in vitro release test.NDV-PLGA 75/25 of the 20 μm microspheres has smooth surface,good dispersion and its encapsulation rate is 66.4%.It appeared a pulsatile release peak in the 77th day in vitro release test.NDV-PLGA 75/25 of the 10 μm microspheres has smooth surface,good dispersion and its encapsulation rate is 67.6%.It appeared a pulsatile release peak in the 77th day in vitro release test.Part Ⅲ:Research on immunity effect of NDV-PLGA microsphere vaccineChickens without maternal antibodies against NDV were grouped randomly.They were immuned respectively with NDV-PLGA microsphere vaccine and inactivated ND vaccine.The results showed that NDV-PLGA microsphere vaccine had the effect of pulsatile release in chickens and were able to maintain high and constant antibody level of which the pulsatile release system composed of blending microspheres had the best immune effect. |
