| AcMNPV(Autographa californica multiple nucleopolyhedro virus) ODV-E25 (E25) is one of the envelope proteins of budded virus and occlusion-derived virus. We have used the two-hybrid GAL4 system screening several positive clones interacting with ODV-E25 such as rpl41, p21, adh and p44. This study we use various of experimental methods to verify the interaction between RPL41(or P21) and E25.1) Subcellular colocalization analysis of AcMNPV E25 and P21.We constructed new virus with the Bac-to-Bac recombinant technology with the expression of the fusion protein E25 and EGFP, as well as the fusion protein P21 and RFP. The observation by confocal microscopy showed that two fluorescence distribution areas overlapped, which suggested the interaction of E25 and P21.2)Bimolecular fluorescence complementation experiment of AcMNPV E25 and RPL41 (or P21)We constructed different virus with the Bac-to-Bac recombinant technology with the expression of E25, C155, N173 and P21. By confocal microscopy some false positive were observed.3)Co-Immunoprecipitation analysis of the interaction between E25 and P21(or RPL41).We put the antibody of P21 (or RPL41) into Sf9 cell lysis solution which was infected by AcMNPV virus. Then we use the antibody of E25 and P21(or RPL41) to test. Co-Immunoprecipitation analysis indicated that E25 was co-precipitated with P21(or RPL41) which suggested the interaction of E25 and P21(or RPL41).4)GST-Pull Down analysis of the interaction between E25 and ADH (or eIF3G).Firstly we construct virus which can express the fusion protein GST-tagged protein and ADH(or eIF3G). Secondly we put GST affinity resin into Sf9 cell lysis solution which was infected by the recombinant virus. Then we use the antibody of E25 to test. GST-Pull Down analysis indicated the interaction between E25 and GST-tagged protein.5)The construction of odv-e25 (e25) truncated mutants and back-cross experience.Several kinds of e25 truncated mutants were constructed. Truncated segments of different length were cloned into pGBKT7. Through the back-cross experience in the yeast two hybrid test, we attempted to probe the area of E25 which have an interaction of the four host proteins. |
