Expression Of The EupsNPV Polyhedrin Gene, Antibody Preparation And Its Utilization In The Virus Detection

The Baculoviddae is a virus family with large,circular,covalently closed,double-stranded DNA genomes.They are highly pathogenic to arthropods,mainly insects of the orders Lepidoptera,Hymenoptera and Diptera.The family can be divided into two genera, Nucleopolyhedrovirus(NPV)and Granulovirns(GV)by the morphology of occlution bodies(OBs). Most of these baculoviruses we have found belong to Nucleopolyhedrovirus(NPV),We can also identify and distinguish NPV as single nucleocasid NPVs(SNPVs)and multiple nucleocasid NPVs(MNPVs)depending on the number of nucleocapsids packaged into each virion. Baculoviruses play an important part in regulating many insect populations as a pathogenic factor. They are highly specific to their hosts and possess non-polluted,effective for a lasting control on pests and they are useless for the pests in the acquisition of resistance to NPVs.So they are studied more popularly nowadays and the wide application of baculoviruses as bio-pesticides will be implemented in future.The tea tussock,Euproctis pseudoconspersa,is one of the major pests of tea bushes.Euproctis pseudoconspersa nucleopolyhedrovirus(EupsNPV)is highly pathogenic to the tea tussock and has been used to control the tea tussock as a commercial bio-pesticide in the fields.Euproctis pseudoconspersa nucleopolyhedrovirus(EupsNPV)polyhedron was expressed by prokaryotic expression plasmid.The polyclonai antibody against it was prepared and was used to detect EupsNPV bio-insecticide.Thereby,we can provide an accurate and effective way to detect EupsNPV bio-pesticide and it is also appropriate for detecting other NPVs.The results of research are as follows:1.A bioassay was determined the virulence to the 2rd-instar larvae and its regression equation was obtained:y=1.3952x-1.7737,correlation coefficient r=0.9975(p＜0.01).2.The ph fragment 741 bp in length was amplified from EupsNPV genome.Its prokaryotic expression vector pET-28-a- Ep-ph was constructed,then transformed into Escherichia.coli. BL21(DE3)and highly expressed.The fusion protein was isolated on SDS-PAGE and recovered by traditional gel extraction.White rabbit of New Zealand was immunized with the purified fusion protein and antiserum against ph was prepared and its titre was detected 6.4x104.3.Western blotting indicated that the specific detection of polyclonal antibody was sensitive. The pure virus liquid was detected by indirect ELISA and standard curve of determination of concentration of EupsNPV was drawn,its regression equation was obtained: y=0.4152x-0.8229,correlation coefficient r=0.9897(p＜0.01).The final detection results showed that the value of viral bio-insecticide had kept accordance with the standard curve well.