The Construction And Application Of Transgenic Secretory Expression Vectors Expressing HIGF-Ⅰ In Silkworm Gland Bioreactor

The mulberry silkworm Bombyx mori belongs to Lepidoptera Bombycidae. Based on the character of strong promoter of the gene and high level secretion of fibroin of Bombyx mori,,It was feasible and hopeful for Bombyx mori silk gland bioreactor to express foreign medicinal protein safely and efficiently.Recently the technical system has been gradually constructed to express foreign proteins using transgenic silkworms. Human insulin-like growth factor- I(hIGF- I)possesses the multi-function in biology,This reseach expected to express the hIGF- I in silk gland of silkworm,with the help of transgenic technique,which might develop a new way in hIGF- I expression, as well as improve the applicational value of bombyx mori.We amplified the promoter of heavy chain gene ( fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+)to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal I\Kpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS- DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS- DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function.The secretory expression pigA3GFP-FibHS-IGF-PolyA-IE-Neo transposon vector was composed of several cloned elements: human insulin-like growth factor- I driven by FibHS, the polyadenylation acid signal of fibroin light-chain gene, and the neo gene driven by IE- 1promoter.Then the mixture of transgenic secretory expression vectors pigA3GFP-FibHS-IGF-PolyA-IE-Neo and the helper plasmid helper-PigA3 in the ratio of 1:1 were transferred into BmN cells,we screened the transgenic cells by antibiotics G418 at final concentration 800μg/mL continuously for about three months.,finally we obtained stable transformed cells。The analysis of transgenic BmN cells and culture fluid by ELISA showed the amount of hIGF- I expression in transgenic BmN was about 29.5ng/105cells.Then the mixture of transgenic secretory expression vectors pigA3GFP-FibHS-IGF-PolyA-IE-Neo and the helper plasmid helper-PigA3 in the ratio of 1:1 were transferred into silkworm by sperm-mediated gene transfer and particle bombardment gene transfer. In G0 genneration,after screening by the antibiotic G418 and GFP,we got three transgenic silkworms emitting the green flourescent protein obviously.we successfully amplified the GFP gene using the genome of three transgenic bombyx mori as template repectively and utilizing the spcial primers.The consequence illustrated that the silkworams we obtained were transgenic animals.the outcome of reverse PCR experiment showed that we failed to find the speical insert sites developed by piggyBac transposon ;The result of ELISA described that,in G0 generation, the amount of expressive hIGF- I in cocoon was approximate 756ng/g.