Establishment Of PCR-based Detection Method For Hyphantria Cunea Nuclear Polyhedrosis Virus And Research Of ORF72

Hyphantria cunea Nuclear Polyhedrosis Virus?HycuNPV?,a powerful non-hazardous,highly specialized and environment-friendly biocontrol pathogenic factor,plays an important role in the sustainable control of Hyphantria cunea population.In this paper,establishment of an efficient and rapid quantitative detection method for HycuNPV and study on ORF72 gene of HycuNPV not only provide a feasible monitoring method for the continuous effectiveness of the virus,but also fill the gap in the study of ORF72 functional gene.The research results of this paper are as follows.1.We established a detection method of HycuNPV by fluorescent quantitative PCR using the TaqMan probe.First,we contructed the polyhedrin gene into the pGEM-T-easy vector and prepared the standard plasmid.Then,the recombinant plasmid with concentration of 1.0×10¹¹copies/?L was serially diluted 10-fold,and plasmids with concentrations of 1.0×10⁸,1.0×10⁷,1.0×10⁶,1.0×10⁵,1.0×10⁴,and 1.0×10³copies/?L were used as fluorescence quantitative PCR templates to establish amplification curves and standard curves.According to the standard curve,we calculated the number of HycuNPV copies contained in the larvae at different time points after receiving the poison the content of nuclear polyhedrosis virus.Based on the genomic DNA template of the larvae infected3.0×10⁶PIB/mL virus,we performed the fluorescence quantitative PCR and found that the content of virus had a significant upward trend on the 5^th day on the larvae and reached a maximum on the 6^th day.2.Research on gene of ORF72.A ORF72 gene was sub-cloned into the pGEX-4T-1vector.The protein was over-expressed under different induced conditions and purified by GST-tag affinity chromatography column.SDS-PAGE gel electrophoresis detection showed that ORF72 protein could be integrated with GST-tag protein on the pGEX-4T-1and the size of the expressed fusion protein was about 38.2kDa.The induced conditions in over-expression of the recombinant proteins were optimized.The major recombinant protein was obtained by a feasible condition at 25?with 1.0mmmol/LIPTG for 4h.Moreover,the recombinant protein was expressed to high levels in the two strains of Escherichia coli BL21 and Rosetta,while the expression in the Rosetta strain was higher than BL21.In order to further study its function,the recombinant ORF72 was purified using GST-affinity chromatography.