Cloning And Expression Analysis Of The Gene CDNA Encoding Epidermal Growth Factor In Silkworm, Bombyx Mori.

Epidermal growth factor(EGF)was decomposed as an epidermal and cells from proEGF,.EGF has been widely used in moisting skin and repairing trauma experiment based on its activity to promote cell reproductive,differentiation and wound healing.The gene cDNA which encoded Epidermal Growth Factor in Bombyx mori was cloned and expressed in this research.The specific primers that were designed according to the informations from public B.mori genomic and NCBI databases could clone the BmEGF cDNA sequence.The BmEGF cDNA sequence was 654 bp and encoded a 217-aa polypeptide.The were by using biological information software analyzed the molecular characteristics of BmEGF.Phylogenetic tree which based on Neighbor-Joining(NJ)showed that the distance between the silkworm B.mori and the Danaus plexippus was the nearest,while silkworm B.mori was far away from Trichuris suis and Trichimella spiralis.The total RNA of Bombyx mori was extracted to analyze the expression of epidermal growth factor in different developmental stages and tissues,then reversely transcribed and synthesized the first strand cDNA by M-MLV.The 18 S RNA gene of Bombyx mori was used as a reference gene.That the BmEGF expressed difference in different stages and tissues was analyzed by real time quantitative PCR.The results showed that the BmEGF was expressed in all stages and tissues,but its expression level was different,such as the expression of BmEGF was higher in silkworm eggs incubation of 9th day and was highest in the period of larvae moulting,was higher in testicle and ovary in 7th day of the fifth instar ages,was up-regulated in development period of metamorphosis.While its expression was higher in fat body of pupation of 6th day and in testis and ovary of the moth.The BmEGF transcription in epidermis was investigated by using real-time PCR technology after the silkworm treated with microbials including NPV,B.bassiana,E.coli and M.luteus.Results showed that the BmEGF gene expression pattern can increase firstly and then decrease in the epidermis of the fifth instar 3th day silkworm.The BmEGF transcriptional level peak appeared at 3h in epidermis induced with E.coli,however the peak appeared at 6h induced with other 3 microbials.So BmEGF is relatively sensitive to E.coli and is response slowly to NPV in the four pathogens.The result suggests that BmEGF may involve in immune response of silkworm larvae.That the ORF connected with pET-28a(+)vector could carry out prokaryotic expression by IPTG induction.Western blot analysis indicted that the expression of recombinant BmEGF protein was successfully in E.coli Transetta(DE3),and its expressipon level was differnt induced by IPTG concentrations differentiy.This experiment will contribute to further study about eukaryotic expression and function of BmEGF gene.