关键词: 苜蓿夜蛾, 丝氨酸蛋白酶, cDNA克隆, 原核表达, 重组蛋白, 酶活性

Abstract: 【Aim】 This study aims to obtain the full length cDNA sequence of serine protease (SP) from the midgut of Heliothis viriplaca and determine the activity of SP expressed in prokaryotic expression system after purification and renaturation process. 【Methods】The full length cDNA sequence of SP was amplified from the midgut of larval H. viriplaca by RT-PCR and RACE technique, and then the cDNA sequence was expressed in Escherichia coli expression system. After being purified, the recombinant protein was renatured using gradient dialysis technique. The activity of renatured protein was determined with BApNA as the substrate. 【Results】 The cloned SP cDNA sequence from the midgut of H. viriplaca was named as HvSP (GenBank accession no.：JX866720). The full length cDNA sequence of HvSP is 880 bp in length with an open reading frame of 762 bp, encoding 254 amino acid residues with the predicted molecular weight of 26.9 kDa and pI of 9.49. Multiple sequence alignment indicated that HvSP shares 52%-95% amino acid sequence identities with SPs from other lepidopteran insects, and shares the highest amino acid sequence identity (95%) with SP from Helicoverpa armigera (GenBank accession no.: CAA72962). The recombinant vector pET21b-HvSP was expressed in prokaryotic expression system, and the recombinant protein was determined as the target protein using Western-blot analysis. Protein solubility analysis indicated that the recombinant protein was inclusion body protein. The renatured recombinant protein had the highest activity (35.74 U/mL) when the pH was 10.0 in Glycine-NaOH buffer. 【Conclusion】 A novel serine protease cDNA sequence was obtained from H. viriplaca, and recombinant protein expressed in E. coli expression system was active after denaturation, purification and renaturation processes. The results provide a foundation for further research of physiological function of serine protease in lepidopteran insects.

Key words:  Heliothis viriplaca, serine protease, cDNA cloning, prokaryotic expression, recombinant protein, enzyme activity