Expression And Identification Of Cystatin From Snake Venom In Bac-to-Bac Baculovirus Expression System

ObjectiveTo investigate the expression of cystatin from snake venom in Bac-to-Bacbaculovirus expression system.MethodsAccording to amino acid sequence of cystatin from snake venom , the full lengthof cystatin gene was artificially synthesized by slowly annealing PCR . The cystatingene was amplified by PCR . The recombinant donor plasmid was constructed byinserting cystatin gene sequence into the plasmid pFastBacHTc . The plasmidpFastBacHTc-cystatin was transformed into DH10Bac competent E.coli cells toconstruct the recombinant shuttle vector Bacmid-cystatin by the site-specifictransposition . Sf9 cells were transfected with Bacmid-cystatin via Cellfectin togenerate the recombinant baculovirous . After the amplification of the recombinantbaculovirous , the viral titer was determined by the viral plaque assay . Time-analysisand MOI-choice of recombinant fusion protein expression were performed byWestern-blot . Total cellular protein was analyzed by SDS-PAGE . The recombinantfusion protein was identified by Western-blot .Results1,The recombinant donor plasmid pFastBacHTc-cystatin was constructed ,which was identified by digestion with two different endonucleases . Then thepositive plasmid was sent to measure its DNA sequence . It was tested that the cystatingene was successfully inserted into the vector pFastBacHTc . 2,The recombinant shuttle vector Bacmid-cystatin was constructed . The cystatingene was successfully inserted into Tn7 attachment site of the shuttle vector , whichwas identified by PCR . 3,It has been shown that the suitable time and MOI of the recombinant fusionprotein expression was 96 h post-infection and 10 , respectively . The expressedprotein was 6×His-cystatin fusion protein identified by SDS-PAGE and Western-blotanalysis , whose molecular weight was about 15000 Dalton similar with the theoreticmolecular weight .Conclusion The recombinant fusion cystatin protein was successfully expressed inBac-to-Bac baculovirus expression system .