Studies On Expression Profile Of BmAGO3Gene In Bomhyx Mori And Construction Of A Novel BmNPV

A gene was screened from the cDNA library of Bombyx mori, which includs an openreading frame （ORF） of2781bp coding926amino acid residues （Accession number inGenBank is NM₀01104597.2）. Its predicted molecular weight is104.5kDa and the isoelectricpoint is9.0. Using bioinformatics analysis, this protein showed a high similarity with argonaute3protein in other species, so we named it BmAGO3（Bombyx mori AGO3）. Due to the largemolecular weight, it’s difficult to express the whole BmAGO3gene in E.coli BL21（DE3）. Weselected high antigenicity domain of BmAGO3named Kago-3to clone into vector pET-28a forpreparation of antibody. Kago-3contains an ORF of702bp, encoding234amino acids residues,predicted molecular weight of27.34kDa and an isoelectric point of8.88.The Kago-3gene was amplified by PCR and successfully constructed the recombinantvector of pET-28a （+）-（Kago-3）, conversion Eoli. BL21（DE3） competent cells. After inducedexpression, the results of Western Blotting analysis shows that a specific band appears in theposition of about30kDa, which is consistent with the expected value （the weight of His tag is3.56kDa, and Kago-3is27.34kDa）. Using Kago-3fusion protein purified by Ni2+-NTA affinitychromatography, immuned the New Zealand male rabbits. And the serum titer of polyclonalantibody is1:12800which detected by ELISA. Total proteins of different developmental stagesand the fifth instar different organization were extracted respectively for detecting the expressionlevel differences by Western Blotting. The results demonstrated that the BmAGO3gene wasexpressed in silkworm eggs, pupa, moth and fifth instar larval stage but expression levels aredifferent, from high to low: moth, fifth instar larvae, pupae and eggs. The differential expressionlevel was also obvious in the tissues of fifth instar larvae: a higher level in the head and skin,lower level in the fat body and trachea, nothing in other tissues. Because AGO proteins are thekey component of miRNA passway, the results of expression profiling analysis for BmAGO3suggested that there are active miRNA regulations in the the head and skin tissue of fifth instarlarvae as well as moth period. The results of subcellular localization showed that the BmAGO3protein was mainly distributed in the cytoplasm. BmAGO3expression was furtherdown-regulated by RNA interference in BmN cells, and cell viability was compared before andafter RNA interference by MTT assay. the results showed that after RNA interference, cellviability was increased.Additionally, in order to study the infection mechanism of Bombyx mori nucleopolyhedrosisvirus （BmNPV）, a novel recombinant BmNPV was constructed. The enhanced green fluorescntprotein （EGFP） was displayed on its envelope by inserting egfp between sigal peptide（SP）sequence and transmembrane（TM） domain sequence of gp64, and red fluorescnt protein（RFP） was displayed on its nucleocapsid by fusion expressed with major capsid protein VP39. Thus,the envelope of recombinant BmNPV was labeled with EGFP and the nucleocapsid was labeledwith RFP. This provided an useful tool to research the infection mechanism and pathway. Inaddition, this recombinant BmNPV could display two different proteins on envelope andnucleocapsid simultaneously, that meant it could display several antigen proteins for vaccinepreparation.