Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion, migration and proliferation. Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly, repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall, it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of "head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a, and then the B121 (DE3) pLysS. The fragments were in agreement with the desired through digestion, agarose gel electrophoresis respectively. By registration into the GenBank data-base, the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102 kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore, a larger quantity of synthetical proteins with high density fermentation were searched after, and a suit of high efficient purification methods for 32 multimers protein were established.
Animals ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Fibroins ; biosynthesis ; genetics ; Molecular Sequence Data ; Oligopeptides ; biosynthesis ; chemistry ; genetics ; Polymers ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Transformation, Bacterial

OBJECTIVE: To observe the effect of acupuncture at METHODS: Sixty patients with type-2 diabetic peripheral neuropathy were randomly divided into an observation group and a control group, 30 cases in each one. Both groups were treated with basic treatment, and the observation group was additionally treated with acupuncture at Neiting (ST 44), Xiangu (ST 43), Dadu (SP 2), Taibai (SP 3), Zusanli (ST 36), etc. once every other day, 3 times a week for 4 weeks. The changes of TCM symptom score, Toronto clinical assessment (TCSS) score, visual analogue scale (VAS) score of pain and serum tumor necrosis factor α(TNF-α) level were observed before and after treatment in the two groups, and the clinical effects of the two groups were evaluated. RESULTS: Compared before treatment, the TCM syndrome score and the TCSS score in the two groups were reduced after treatment ( CONCLUSION Acupuncture at
Acupuncture Points ; Acupuncture Therapy ; Diabetes Mellitus, Type 2/therapy* ; Diabetic Neuropathies/therapy* ; Humans ; Rivers ; Treatment Outcome