Construction of spider draggling silk protein MaSp1 prokaryotic expression vector and its expression and purification in Escherichia coli / 军事医学

ABSTRACT

Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .