| Spider silk which light and tenacity,is one of the best natural protein fibers with excellent comprehensive properties.It have important application in industry and military such as parachutes ropes,military protective clothing and space using composite materials because of it unique mechanical feature of high strength elasticity and high energy to break. At the same time, because of its good biocompatibility and degradation,silk fibers hold great potential application in the biomedical field used for wound dressing,artificial tendons, drug carriers,tissue scaffolds and so on.The problem is that nature spider silk production quantity is little and spiders can’t be scale breeding, through genetic engineering to obtaine large amount of spider silks for their applications that is a effective way to solve the problem.In genetic engineering the microorganism expression system with advantage of growing fast,yield high,short production cycle,cultivation condition simple, genetic background clearly and also has variety of expression vectors,strains and purification system,the native or synthetic spider silk protein expressed in the Escherichia coli and yeast expression system at early stage.There has developed many spider silk protein expression system,but none of them can make silk fiber which has excellent mechanical feature from expressed silk proteins due to study on the spider silk protein assembly in vivo,spinning mechanism after protein expressed and other related research is lagging. The silkworm is the one of economic insects which are domesticated.The ability of it’s silk gland to synthesis and secretion protein is huge,and the silk rich in Gly and Ala just like spider silk. The ideal way to obtain silk fibers with outstanding mechanical properties,one can use transgenic silkworm as a candidate host for the mass production of spider silk protein,through its spinning device to obtain recombinant spider silk.In this paper,the part of dragline silk protein gene from Nephila clavata genome DNA was amplified by PCR and sequenced.Analysis the sequences and forecasting its protein,showed that typical mould (A) n, (GGX) n of dragline silk protein existing in the cloning sequence,the spiral structure of amino acid residues absolute advantage and there has major ampullate spidroinl and 2 domain at the 3’-terminal.The cloning sequence was ligated into pET-28a(+) and construct prokaryotic expression vector pET28-NcMaSp, NcMaSp was expression by the BL21 (DE3) strains.SDS-PAGE analysis shows that molecular weight of products expressed is 27kD just as expection,dragline silk protein expressed by Escherichia coli both soluble and unsoluble.On that basis, the expression of dragline silk protein was purified by His-tag affinity chromatography.In order to construct silkworm transposon expression vector as a tool in expression dragline silk protein,the sericinl gene promoter(named serl) and 3’terminal non- repetitive sequence from Bombyx mori genome DNA was amplified by PCR and sequenced. Analysis serl gene promoter sequence shows the sequence of TATA box was TATAAAA at position -24～-30,the CAAT box at position -112～-115.Predicte the site of signal peptide of fusion protein shows the signal peptide at position 20-21 of amno acid sequence. Construction recombinant plasmid pFFa [MCS-ser-signal-NcMaSpl-ser3’] based on their sequences analysis.The serl-signal-NcMaSpl-ser3’ fragment of the recombined plasmid was fusd with piggyBac[3xp3-DsRed] plasmid consist,and the special expression vector piggyBac[serl-signal-NcMaSpl-ser3’,3xp3-DsRed] in the silkworm gland was constructed completely. Through microinjection, the expression plasmids of transposon piggyBac and an help plasmid hsp were introduced into Bombyx mori eggs.The foundation was established that improve silk protein structure and mechanism properties of transgenic silkworms and develop high performance silk fibers through above basic work. |
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