Expression In E. Coli And Insect Cells Of Apisimin CDNA From Apis Cerana Cerana

Honeybee is regarded as one of the members of insect families and a model organism for studying insect social behaviors.It was always honored as a research hot spot either in foreign and domestic.Especially,the honeybee genome sequences were published in《Nature》on 26^thOct.2006,which confirmed as the landmark in honeybee researching.The following emphasis would be transferred to the studying of the functions of honeybee genes.A cDNA library has been constructed from nurse honeybee heads of Apis cerana cerana.The apisimin cDNA clone was screened from the cDNA library.The cDNA of apisimin was 407 bp and contains an open reading frame(ORF)of 237 bp,encoding 78 amino acid residues,peptide with a signal peptide of 24 amino acids in the N-terminal.The molecular weight of mature apisimin was predicted 5.9 kDa.The similarity of apisimin was 93%and 100%,comparing Apis cerana cerana with Apis mellifera and Apis cerana india,respectively.The apisimin gene was amplified by PCR from the cDNA library.PCR production was linked with pUCm-T vector,pUCm-apisimin was digested by BamHⅠand XhoⅠafter sequencing,and linked with pET30a which was digested by BamHⅠand XhoⅠ, and then transformed into E.coli BL21(DE3).After identification by PCR and double enzyme digestion,the correct prokaryotic expression vector pET30a-apisimin was induced by IPTG,the protein were collected and analysed by SDS-PAGE and Western-blotting analysis.As the products existed mainly as inclusion body in the prokaryotic expression system,the apisimin were then expressed in the Bac-to-Bac system.Apisimin gene was linked with pFastBacHTa vector,and transformed DH10Bac competent cells.The correct virus particle Bacmid-pFastBacHTa-Apisimin is 2543 bp,identified by M13 primer.The silkworm cell were induced into BacmidDNA with Fugen6.The silkworm cells were collected after inducing 72h,and the cytoderm was broken up by the ultrasonic.The results of SDS-PAGE and Western blot analysis comfirmed that the apisimin gene only displayed on specifically expressed band.