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Title: Application of silkworm (Bombyx mori) functional genes in fabricating scaffolds for tissue engineering and regenerative medicine
Authors: Li, Zhi
Degree: Ph.D.
Issue Date: 2015
Abstract: The aim of this study was to integrate protein engineering techniques with fiber engineering technology to develop novel bioactive functional scaffolds that can be used as artificial skin, reconstructed tissue, and wound treatments for tissue engineering and regenerative medicine (TERM).Based on the published silkworm database and corresponding references, altogether 41 silkworm AMP genes in seven different families (attacin, cecropin, defensin, enbocin, lebocin, moricin, and gloverin) were identified, and one representative gene from each family were analyzed and cloned, including attacin2, cecropinB1, defensinA, enbocin1, lebocin3, moricinA1, and gloverinB. In order to gain recombinant silkworm AMPs, three common heterologous expression systems, involving yeast, Escherichia coli, and the insect cell system were utilized. Two AMP genes, attacin2 and cecropinB1, were sub-cloned into a vector pET-28a(+) separately and then were transformed into an expression host cell BL21. Through induced expression by IPTG, recombinant protein attacin2 (Bmattacin2) was detected by SDS-PAGE, and then it was confirmed by Western blot. The investigation of solubility showed the recombinant Bmattcin2 was expressed as an inclusion body. Further, the amino acid sequence of this recombinant Bmattacin2 was confirmed by using MALDI- TOF- TOF. The characterization of recombinant Bmattacin2 revealed it was a soluble glycine-rich AMP with positive net charges and it had a high ratio of {220}-helix(25%) in its secondary structure which may relate to its antimicrobial activity. The Radial diffusion assay and Minimal inhibitory concentration assay indicated Bmattacin2's inhibition of both Gram-positive and Gram-negative bacteria.Interestingly, Bmattacin2 exhibited particular activity in regard to killing cancer cells while it had no cytotoxicity in relation to normal cells. The degradation test showed majority of Bmattacin2 degraded after three days' immersion in PBS buffer. All of these characterizations indicated Bmattacin2 is an ideal bioactive material for application in TERM. Although organic solvents were proved to have a negative effect on Bmattacin2's function to some degree, Bmattacin2 still demonstrated antibacterial activity towards E.coli and Siaureus, indicating that Bmattacin2 could be used to make fibrous scaffolds together with synthetic polymers such as PLLA by electrospinning.
In order to prepare massive Bmattacin2 for scaffold preparation, Bmattacin2's expression was optimized. Three methods were applied, including the pilot method, DoE and double-colony selection. The optimization indicated the maximum expression was to induce expression at OD6oo value 0.2 by 0.2 mM IPTG for 1 hour, and this made Bmattacin2's expression percentage reach up to 42.7%, equal to about 3.0mg/L. On the basis of optimized expression conditions, enough pure Bmattacin2 was prepared and then was used to make the nanomembrane together with PLLA by electrospinning. The final constitution of Bmattacin2 in the nanofibrous membrane was 2%. The microstructure and morphology of electrospun PLLA/Bmattacin2 membrane were observed. The degradation of PLLA/Bmattacin2 membrane was conducted. Further, cell experimentation verified that electrospun PLLA/Bmattacin2 membrane had good compatibility with human normal cells and had significant inhibitive activity on E.coli and Siaureus. Moreover, the scaffold demonstrated significant anticancer cell activity.In conclusion, the combination of protein engineering and fiber fabrication in this study has created a set of novel technologies to prepare bioactive scaffolds with characteristics of good compatibility, antimicrobial activity, and anticancer effect, which has a promising potential for biomedical applications such as skin reconstruction, wound treatment, and healing in TERM.
Subjects: Silk -- Therapeutic use.
Tissue scaffolds
Hong Kong Polytechnic University -- Dissertations
Pages: xxii, 224 leaves : illustrations (chiefly color) ; 30 cm
Appears in Collections:Thesis

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