Creation And Research Of Noval Silk Material With HEGF Bioactivity

Silk as a textile material enjoy good reputation in the garment and textile industries due to its outstansding properties such as soft gloss, good hygroscopicity, easy processing and good comforts. In addition, it can get bright colors by dyeing. In recent years, with the discovery of its good mechanical properties, environmental stability, biocompatibility, biodegradability and other physical and chemical properties, the silk cause the increasingly attention as an ideal biological material in the field of medical and biological tissue engineering. However, natural silk still include a few defects i.e. weakness of color fastness, mechanical properties is less than the spider silk fiber, good medical material properties can’t meet the medical tissue engineering research needs.In order to overcome the defects in the natural silk, many researchers try to use piggyBac-mediated transgenic technology to create novel functional silk materials. Utilizing the silkworm silk gland to express fluorescent protein to obtain transgenic color silk which can exhibit green, red or orange respective colors in natural light. Fusion expression of spider silk protein in the silkworm silk gland to obtain transgenic silk which mechanical properties has improved. The expression of different medical purposes foreign proteins and peptides to obtain transgenic medical materials which cell adhesion, biological compatibility and calcium ion adsorption has improved. However, transgenic color silk would appear mechanical properties decline, the mechanical properties of transgenic silk isn’t increase obvious, the target protein in the transgenic medical silk material has no biological activity.Human epidermal growth factor (hEGF) is a kind of polypeptide substance which widespread in mammals. It can strongly promote the growth of cells and for burn, scald, ulcer and corneal injury treatment in medicine. In order to explore the feasibility of acquisition the silk material with hEGF biological activity. We use the fibroin heavy chain expression vector R3 to express hEGF in this study. The main results are as following:1. The design and construction of two different hEGF-heavy chain fusion protein transgenic expression vectorsThe fibroin heavy chain expression vector R3, mainly composed of fibroin heavy chain gene N terminal, C terminal sequence and intermediate fusion expression of enhanced green fluorescent protein gene. We replaced the EGFP with hEGF gene to constructed transgenic expression vector pBac{Fib-H P3-hEGF-LBS; 3×P3-DsRed};We truncated signal peptide cutting sites downstream sequence of the heavy chain N-terminal and cysteine which involved in secretion upstream sequence of the C-terminal to constructe transgenic expression vector pBac{Fib-H P3(S)-hEGF-LBS(s); 3×P3-DsRed}.2. The production and molecular identification of the hEGF transgenic linesThe transgenic expression vectors and pHA3PIG microinjected into non-diapause 871 strain eggs and screened experssion of red fluorescent protein in the eyes of G1 silkworm. The positive lines which injected vector pBac{Fib-H P3-hEGF-LBS; 3×P3-DsRed} and pBac{Fib-H P3(s)-hEGF-LBS(s); 3×P3-DsRed} was named TSL-P and TSL-P(s), respectively; The positive rate of TSL-P and TSL-P(s) was 11/60 (18.3%) and 7/31 (22.6%), respectively. The result of Souhern blotting and Inverse PCR about all 18 transgenic lines showed that TSL-P-2 and TSL-P (s)-7 were both single copy and inserted in the silkworm chromosomes 9 and 8, respectively.3. The molecular identification of the hEGF-heavy chain fusion protein in the transgenic silkworm cocoonUsing 0.6 g mL-1 LiSCN to dissolved cocoons, centrifuged to obtain the supernatant silk protein. The result of SDS-PAGE and Western blotting show that:1、The hEGF-heavy chain fusion proteins of TSL-P and TSL-P(s) are expressed in the posterior silk gland and secrecte into cocoon, the average value content of hEGF in TSL-P is 4.9% which is far more than TSL-P(s)(2.1%). TSL-P-2 (16.7%) and TSL-P(s)-7 (3.0%) was the highest pure hEGF content in TSL-P and TSL-P(S), respectively; 2、The actually protein molecular mass of TSL-P fusion protein larger than predicted protein molecular mass, while the actually protein molecular mass of TSL-P(s) fusion protein was the same with the predicted, the results show that hEGF-heavy chain fusion protein has post-translational modifications on the C-terminal of FibH-chain.4. The identification of biological activity of hEGF-heavy chain fusion protein in the degummed silk and cocoon sliceThe silk and cocoon slice were degummed three times in alkaline solution at 55℃ for 30 min to remove the sericin. To detected the biological activity of hEGF-heavy chain fusion protein in degummed silk and cocoon slice. Brdu Immunofluorescence analysis showed that the cell culture medium immersing TSL-P(s) can significantly improve the division rate of human epidermal fibroblast cell; MTT cell proliferation assay results indicate that, degummed cocoon slice of TSL-P(s) can significantly promote the proliferation of human epidermal fibroblast cell. Two experimental results consistently indicate that hEGF-heavy chain fusion protein in the cocoon produced by TSL-P(s) has hEGF biological activity which can promote the division and proliferation of human epidermal fibroblast cells, while and hEGF-heavy fusion protein in TSL-P has almost no biological activity.In this study, we successfully expressed the hEGF-H chain fusion protein in the silk by utilizing the fibroin heavy chain expression vector. We got one transgenic silk material with hEGF biological activity. This would provide an important theory and application value to the application of transgenic technology on the field of silk modifacation and to the blueprint of transgenic silk as novel biomaterials.