Eukaryotic Expression And Purification And Founction Of Four Destruxin A-affinity Proteins

Destruxin A?DA?,a cyclodepsipeptidic mycotoxin isolated from the entomopathogenic fungus,Metarhizium anisopliae,has good insecticidal activity and anti-immune function,but the molecular mechanism of DA affecting target insects is not clear yet.In the preliminary study,a specific protein band was isolated by enzymatic hydrolysis and electrophoresis from the Bm12 cells treated with DA.Then,more than 100proteins were identified by mass spectrometry and molecular docking by MOE2014software,the proteins were suspected to have affinity with DA.In order to further study the interaction between these proteins and DA,four proteins are selected as research object,which are respectively Bm_nscaf2970₀87?P07??Bm_nscaf2204₁40?P20??Bm_nscaf2993₀29?P27?and Bm_nscaf2971₀8?P37?.Firstly,construct the Bac-to-Bac baculovirus expression system,transfect the Spodoptera frugiperda SF-9 cells and separation and purification of the four kinds of silkworm protein by affinity chromatography technique.Meanwhile,in this experiment also study on the interaction between DA and target protein by insect two-hybrid system.The main results were shown as follows:?1?Cloning and expression of silkworm protein genesAlign the silkworm proteins Bm_nscaf2970₀87?P07?,Bm_nscaf2204₁40?P20?,Bm_nscaf2993₀29?P27?and Bm_nscaf2971₀8?P37?from NCBI database,find the appropriate homologous gene sequences,design and synthesis primer,and add His tags.Extract total RNA from Bm12 cells,then synthesis the first chain cDNA.One step cloning method?P20 and P27 protein genes?and traditional double enzyme digestion method?P07 and P37 protein genes?are used to construct recombinant expression vector pFast-Bac^{T M}1.Construct a recombinant insect baculovirus through the Bac-to-Bac baculovirus expression system,then,amplify bacillus strains and transfect SF-9 cells,finally,the target proteins were expressed.The result shows that SF-9 cells have obvious changes in morphology after transfected.After transfected with 24h,the diameter of the cells is increased by 25⁵0%.The nucleus is enlarged and may be filled with the whole cell.After transfected with 24h to 72h,the cells stop growing.The granules appear in the cells,and cells are separated from the culture bottle.After transfected more than 72h,the cells begin to die and monolayer cells began to appear.?2?Isolation,purification and quality controlAmplify cultivate SF-9 cells infected with recombinant baculovirus,and crude extraction of cell protein.Separation and purification of target protein by affinity chromatography technology.To test the isolation and purification of target protein by Western blot using His-antibody.The results show that,P20 and P27 protein bands in conformity with the theoretical value 81626.21Da and 56339.37Da.At present,The P27protein has been separated and purificated for three times,The concentration of P27 protein were respectivly 10 mg·mL^-1,80 mg·mL^-1 and 40 mg·mL^-1,purity were 90%,70%and40%,The gene of P07 protein has been preservated at the pFast-Bac^{T M}1 vector.The purification of P20 and P37 protein are ongoing.?3?Study on the interaction between DA and P27 proteinThrough the analysis of biological information,the P27 p rotein is a scavenger receptor family protein,which interacts with the hemolymph protein HP14.The interaction between DA and P27 protein was studied by insect two-hybrid system.The PCR products of P27and HP14 proteins gene was reacted with TOPO vector to construct entry vectors.The entry vectors were reacted with PIE-AD and PIE-BDB vector in LR enzyme to construct PIE-AD-P27,PIE-DBD-HP14 vector.Then PIE-AD-P27,PIE-DBD-HP14 and PIE-LUC vector with luciferase gene were transfected SF-9 cells.Cells which were transfected with12h were treated with DA with 0.002,0.02,0.2,2,20,50,100 and 200 mg·L^-1 respectively for 48 h.The results showed that the fluorescence of treated group without DA?CK?was significantly higher than that of the control group.There was a negative correlation between the fluorescence value and the DA treatment dose,when the concentration of DA was 200,2,0.02,0.002 and 0 mg·L^-1?CK?,the fluorescence values respectively were 30.7,37.3,61.3,305.3 1237.3.The results suggested that DA affects the interaction betwee n P27protein and HP14 protein,i.e.,there is an interaction between DA and P27 protein,and the P27 is a DA-binding protein.?4?The virulence of Beauveria bassiana on hemocytes of Ostrinia furnacalis and effect of B.bassiana on cellular morphology of hemocyteMTT and CCK-8 were respectively employed to test the toxicity of B.bassiana onhemocytes of O.furnacalis,meanwhile,the optical microscope and scanning electron microscope were used to observe the growth of B.bassiana and morphological changes of hemocytes of O.furnacalis cell,SYSU-O fHe-C.The IC₅₀ vales of B.bassiana against SYSU-OfHe-C cells at 24 h post-treatment were respectively recorded as 2.8×10⁵ and1.4×10⁵ mL^-1 by means of MTT and CCK-8.Furthermore,under real-time monitoring with optical microscope,the encapsulization and phagocytosis were not found when the conidia of B.bassiana were inoculated in the culture o f logarithmic stage SYSU-O fHe-C cells,B.bassiana completed the germination after treatment 10 h and grew the whole visual field.Meanwhile,the numbers reduction and morphological changes of SYSU-O fHe-C cells caused by B.bassiana were observed.The SYSU-OfHe-C cells shape was changed from the approximate circular to irregular with the phenomenu of aggregation,fragmentation,cavitation,protuberance,etc.A few SYSU-O fHe-C cells were penetrated by B.bassiana mycelia.