Establishment Of A Novel Method For Determination Of Bculovirus Titer And BmNPV/Silkworm Expression System

BmNPV/silkworm expression system express foreign protein in silkworm,which is the only economic insects that can artificial feeding on a large scale.Compared with the insect cell expression system,it show higher expression level,and cheaper cost.It has great application prospect.It is important to determine titer of virus stock for arriving at an optimal multiplicity of infection that maximizes recombinant protein expression.A number of methods have been reported,however,most of them are tedious,time-consuming,and can only detect AcMNPV.VP39 is the major nucleocapsid of baculovirus.The homology of amino acid sequences between AcMPV and BmNPV is 93.7%.In this study,a method for detecting both AcMNPV and BmNPV based on VP39 monoclonal antibody was created.The method is very useful to perfect BmNPV/silkworm expression system,which was also established in this paper.We firstly constructed a expression vector pTO-T7-vp39-Ac/Bm which contains 16-903bp of VP39 sequence.When transformed into E.coli,the bacteria expressed a 32kD recombinant protein.VP39-Ac/Bm mainly expressed in inclusion body which was dissolved in 8M urea.VP39-Ac/Bm was purified by electroelution and the final purity is above 97%.The purified VP39-Ac/Bm was used to immunize BALB/c mice. 17 monoclonal antibodies were purified,and 8 had good activty to both baculovirus by Western Blotting and immunofluorescence.The monoclonal antibodies presented high sensitivity by CONFOCAL assay,which showed the antibodied could interact with infected cells after 16 hours.Then we utilized Elispot reader to futher evaluate the antibodies.Finally,we found that one antibody was comparable to titers determined by traditional methods.The result indicated that a precise,quick and high throughput method which can detect both AcMNPV and BmNPV is established.In this study,the BmNPV/silkworm was firstly established using the red fluorescent protein(DsRFP).The body of silkworm infected by Bv-DsRFP showed red coulour after 5-6 days.The RFP was transiently expressed in the 5^th day.We constructed a fragment of HEV ORF2(112-606aa),which is believed to contain nutralization epitope.We compared several factors in this system,finally,we choiced silkworm larve as infected host,not pupae;homogenation as material of purification,not haemolymph;l0mL buffer/3.8g silkworm was optimal for homogenate.Then the HEV ORF2 protein was purified through repeated freezing and thawing,ammonium sulfate precipitation and DEAE aion-exchange chromatography.The final purity was over 90%by SDS-polyacrylamide gel electrophoretic analysis,and it could form virus like particles(VLP) autonomously by transmission electron microscopy investigation.The VLP showed high immunogenicity.And it meaned that utilizing BmNPV/silkworm system to produce the diagnostic reagent of HE antigen was valid,which provided a new solution to reasearch and development of engineered vaccine of HE.The protein also presented good antigenicity.In this study,we initially established an intact and effective technology road of BmNPV/silkworm expression system.Also we provided a helpful reference to the problem of difficult purification of silkworm homogenate.