Study on Expression of hIGF-Ⅰ in Silkworm Silk Gland Based on the Derived piggyBac Transposon
|Course||Biochemistry and Molecular Biology|
|Keywords||transgenic silkworm IGF-I secretary expression DM mice|
Human insulin-like growth factor-I (hIGF-I) is a class of peptide of cell differentiation and proliferation, besides insulin-like function. The insufficient supply has become a major bottleneck with the increase extentive application. Silk gland of silkworm is a highly specialized organ that has the tremendous ability to synthesize and secrete silk protein. As a good bioreactor, it has extensive prospects for applications of genetic engineering pharmaceutical industry.hIGF-I expressed by silk glands transgenic silkworm could be secreted, which simplifies the extraction process and enhances the expression. Therefore, transgenic vector pigA3GFP-serHS-hIGF-I-neo, based on the piggyBac transposon, containing hIGF-I gene and the promoter ser-HS of sericin-1 gene (ser-1) with the signal peptide sequence of fib-H was structured. The transgenic silkworm was then obtained by sperm-mediated. The molecular idention performed that the expression box of hIGF-I gene and signal peptide sequence of fib-H was intergrated into the genome of silkworm, in which the expressed hIGF-I was partly sectreted into silk gland cavity, and then reached sericin layer of silk, eventually. The hIGF-I expression is approximately 43.8 ng per gram fresh middle silk gland, and 46.7 ng per gram cocoon in G1 generation. These results demonstrated signal peptide sequence of fib-H could lead sectretary expression in middle silk gland, and the secretory expression level was elevated.Besides, we bred hIGF-I-transgenic silkworms (controled by Fhx/p25 promoter) through eight generations by continuously selecting with green fluorescence, and G418. The G8 transgenic silkworms were confirmed by PCR and dot blotting, further more, their posterior silk glands were removed from the fifth instar larvae to make freeze-dried powders so as to measure the hIGF-I. ELISA results showed that the expression level of hIGF-I in the posterior silk glands of G8 transgenic silkworm was approximately 493 ng/g in freeze-dried powder. When the diabetes mellitus (DM) mice were administrated by gavage with freeze-dried powder, the blood glucose in DM mice significantly decreased (P<0.05) in time and dose dependent manners compared with that of DM mice orally administrated with distilled water and normal freeze-dried powders made of untreated silk glands. These results demonstrated that hIGF-I expressed in posterior silk glands of transgenic silkworms could reduce blood glucose by oral administration, and the posterior silk glands made of the hIGF-I-transgenic silkworms may be directly used as perorally administered medicine.