The Construction And Application Of Transgenic Vectors Expressing HGM-CSF In Silkworm Silk Gland Bioreactor

The mulberry silkworm Bombyx mori(Lepidoptera:Bombycidae) is the tamed member of the millions of insects and reared economically in large scale.The larva of silkworm has a pair of highly developed silk glands with the ability to synthesize fibroin.Recently,the technical system has been gradually constructed to express foreign proteins using transgenic silkworms.However,there are some drawbacks in current technical systems such as lower expression level of foreign proteins and heavy selecting jobs.In the present research,a transgenic vector based on piggyBac transposon for silkworm was constructed by using fibroin promoters.The promoters of fibroin heavy chain(fib-H) and fibroin light chain(fib-L) were cloned and their motifs were analyzed through bioinformatics.Transient expression vectors using DsRed as reporter gene under the control of fib-H and fib-L promoters were constructed and assayed in the body of silkworm and Bran cells.Transgenic vectors used for expressing foreign proteins under the control of fib-H and fib-L promoters were constructed respectively and used in the transgenic research of BmN cells and silkworms.Promoters of fib-H(EF540776,489 bp),fib-L(EF540777,606 bp),polyA signal sequence(EF216676, 289 bp),enhancer(DQ679478,375 bp) from intron-1 of fib-L gene,and neomycin resistance gene(neo^r) were cloned for the construction of transgenic vectors.The second structures of fib-H and fib-L were analyzed. Transient expression showed that both of the promoters fib-H,fib-L had specific inductive activities in the posterior silk gland,and also a few of activities in BmN cells.Two transgenic vectors were constructed by respectively combining fib-H and fib-L promoters with hGM-CSF,the polyA signal sequence,enhancer,neo^r and the GFP reporter gene already included in the original vector.Bran cells were transfected by piggyBac transposon with neo^r and GFP double selection elements.Through screen by G418 at the final concentration of 800μg/mL for 3 months,a stable transformation cell strain was obtained.Approximate 75%of the BmN cells were observed emitting green fluorescence.Foreign genes were proved existing in the genome of the cells by PCR.The two constructed transgenic vectors were used to conduct exploration of transgenic experiment on silkworm.The green fluorescence spots were observed in 3 transgenic larvae at 3 day 2^nd instar silkworm,and 3 pupa.with green fluorescence was also observed.The two transgenic vectors constructed in this research may lead deep research on the expression of foreign proteins by transgenic silkworm bioreactor.