The Research For Silkworms Silkgland Bioreactor Which Expressing HuIFN-ω

The domesticated silkworm,Bomyx mori,is an important economic insect which has been used for silk production for more than 5000 years.As an important model of lepidoptera insect,the germline transformation in B.mori is highly concerned by the researchers.Following the success in mapping of the draft sequences for the genome of B.mori,the key forces at home and abroad focused on the research of gene function and its applications by using the transgenic technology.Now there were some institutions that have developed the method for silkworm transgenesis using piggyBac transposon,,so to develop the techniques such as directional-improving of main economic traits,material innovation,bioreactor,biological materials,silk fibers with high strength and special function has come to the level of practicability gradually. People pay close attention to the feature of distinctive and high effience capability of protein synthesis in the silkgland of silkworm,which makes it becomes the basic of biology of the silk industry.The silk glands of silkworm have many advantages such as high-speed synthesis of silk protein,post-modified capability,large-scale production with low cost,safety to human and livestock,etc.So it can be used as an ideal bioreactor for production of recombinant proteins.In this study,we constructed a piggyBac-derived vector.Which can express especially in the middle gland of silkworm. Using diapause of silkworm strains Daizao as injection materials,screening with the activity of exogenous protein expression in transgenic strains.The main research contents and results are as follows:1.Cloning and sequence analysis of Human IFN-ωgene.First,downloading human interferon-ωgene sequence at NCBI website,using Oligo software for prediction of its Open Reading Frame(ORF).And designing primers at the two ends of ORF.we construct two restriction sites:BamHⅠand NotⅠat 5'of the primers,as well as a his-tag at the Downstream primer for further purification of the recombinant protein.Using genome of healthy human breed as template,we got a sequence of 776 bp in length,and cloned it into pMD19-T Simple vector for sequencing,It is Certified that fragment is our target sequence.Analyzed by bioinformation method,huIFN-ωgene is 588bp in length,consisting no intron,encoding a protein with 196 amino acids,the protein molecular weight was predicted 20.3 kD,and the isoelectric point is 9.18.Using on-line lookuping in signaIP,we found that the location of 1-22 aa is the singal peptide of huIFN-ω.At webside of SMART,huIFN-ωgene contains one typical domain of located at aa to aa.2.Construction of the PiggyBac-based middle silk gland-specific expression vector.The sericin-special expression vector pBac[ser1-huIFN-ω,3xp3-EGFP]for bioactor of silk gland of silkworms was based on the employments of a piggyBac transposon element derived from Trichoplusia ni as basic vector,huIFN-ωas target gene,then the target gene was digested from the pMD19-T Simple vector,Recycled and connected to the downstream of promoter ser-1, which brought silkworm silk gland-specific expression,meanwhile,enhanced green fluorescence protein(EGFP) promoted by 3xp3 was used as reporter.We got the target fragment from the digest production that using AscI digest The vector,which confirmed that human interferon-ωgene has been successfully cloned into the expression vector.3.Screening of transgenic silkworm individuals which express Human IFN-ωprotein.In the study,we used the silkworm Daizo(P50) saved by Department of silkworm genetic resources in our school as the injection of genetically modified material.Then the admixture of high-purity plasmid of expression and auxiliary vector was micro-injected into the 1-3h early embryos of Daizo(P50)(recorded as G0 generation).Screening positive individuals under fluorescence microscope from birth eggs of G0-generation moth,then recorded as G1 generation.4.Molecular detection of transgenic individualsAfter the self-crossed progeny of G1 generation was born,we extracted genome from five of the mating individuals,detected the copies of huIFN-ωgene in the chromosome of offsprings by southern blot.It's found that there were 2copies in all of the five chosed individuals,that is huIFN-ωgene transports twice in the genome.The result of RT-PCR revealed that huIFN-ωgene has expression of high levels of transcription,in silk gland5.Activity detection of recombinant protein.We extracted and puried recombinant protein expressed in the mid-silkgland of offsprings on day six of fifth stage,the result of western blot demonstrated protein of huIFN-ωspecific expressed in mid-silkgland.For further identification of activity of the recombinant protein, hepG2 cells from human lung cancer was used to detect anti proliferatice effect of the recombinant protein by MTT assay,and it showed that proliferation of hepG2 cells was blocked.