关键词: 异麦芽糖酶, 原核表达, &alpha, -葡萄糖苷酶, 大肠杆菌

Abstract:

Objective: To construct the prokaryotic expression vector pET-28a(+)-hISO and to explore the expression of hISO fusion protein in E.coli,and to provide a foundation for follow-up experiment research.Methods: The 1 770 bp fragment of hISO gene was amplified from the total RNA of Caco-2 cells by RT-PCR and inserted into pET-28a(+)to construct the recombinant plasmid pET-28a(+)-hISO.The recombinant plasmids were transformed into E.coli BL21(DE3)to induce the protein expression with IPTG after PCR,digestion and DNA sequencing.The recombinant plasmid was analyzed and identified by SDS-PAGE and Western blotting method.Results: The recombinant plasmid pET-28a(+)-hISO was constructed successfully after identified by PCR,digestion and sequencing. The fusion protein was 68 860 when the soluble fusion protein was purified,and using elution buffer containing 40 mmol·L^-1 and 60 mmol·L^-1 imidazole could get high concentration and purity protein.Conclusion: The prokaryotic expression vector pET-28a(+)-hISO is constructed successfully and the recombinant protein is expressed in the E.coli BL21(DE3).

Key words: isomaltase, prokaryotic expression, α-glucosidase, Escherichia.coli