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昆虫几丁质酶是昆虫几丁质代谢不可或缺的关键酶类。本研究旨在构建美洲大蠊几丁质酶Pa Cht1基因原核表达载体,纯化并鉴定Pa Cht1重组蛋白,为后续酶活等测定奠定基础。定向克隆Pa Cht1基因成熟肽序列至原核表达载体pET32a(+)上,将构建成功的pET32a-Cht1重组表达质粒导入大肠埃希菌Rosetta中,分别对诱导剂IPTG浓度和诱导时间进行优化,确定最佳诱导浓度和诱导时间,并对表达产物进行可溶性分析。表达产物经镍离子柱纯化后通过Western Blot鉴定。结果表明,美洲大蠊几丁质酶Pa Cht1成熟肽基因编码区长1 083 bp,编码360个氨基酸。最佳诱导浓度和诱导时间分别为0.2 mmol/L和4 h。所得重组蛋白大小约60 k D,与预期结果大小一致,重组蛋白主要以包涵体的形式存在。Western Blot鉴定重组蛋白可与6×His-tag单克隆抗体特异性结合。说明成功构建了原核表达载体pET32a-Cht1,并诱导表达获得了重组几丁质酶蛋白。
Insect chitinase is an essential enzyme for chitin metabolism in insects. The aim of this study was to construct a prokaryotic expression vector for Pa Cht1 gene of chitinase from American cockroach, and to purify and identify Pa Cht1 recombinant protein, which laid the foundation for subsequent determination of enzyme activity. The mature peptide sequence of Pa Cht1 gene was cloned into the prokaryotic expression vector pET32a (+). The recombinant plasmid pET32a-Cht1 was successfully introduced into Escherichia coli Rosetta to optimize the IPTG concentration and induction time. Good induction concentration and induction time, and the expression product was analyzed for solubility. The expressed product was purified by nickel ion column and identified by Western Blot. The results showed that the gene coding Pa Cht1 mature peptide of cockroach Periplaneta americana was 1,083 bp in length and encoded 360 amino acids. The best induction concentration and induction time were 0.2 mmol / L and 4 h respectively. The resulting recombinant protein was about 60 kD in size, consistent with the expected size of the recombinant protein mainly in the form of inclusion bodies. Western Blot identification of recombinant proteins with 6 × His-tag monoclonal antibody specific binding. The successful construction of prokaryotic expression vector pET32a-Cht1, and induced expression of recombinant chitinase protein.