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当前位置: 首页 > 文章 > 苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达 昆虫学报 2014,57 (9) 1008-1017
Position: Home > Articles > cDNA cloning,sequence analysis and prokaryotic expression of a serine protease from the midgut of Heliothis viriplaca (Lepidoptera:Noctuidae) Acta Entomologica Sinica 2014,57 (9) 1008-1017

苜蓿夜蛾中肠丝氨酸蛋白酶cDNA的克隆、序列分析及原核表达

作  者：
周晓群;高艳玲;赵奎军;樊东
单  位：
东北农业大学农学院
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
摘  要：
【目的】本研究旨在从苜蓿夜蛾Heliothis viriplaca中肠克隆出丝氨酸蛋白酶(serine protease,SP)基因的cDNA序列,测定原核表达后的蛋白经纯化及复性后的活性。【方法】运用RT-PCR和cDNA末端快速扩增方法(rapid amplification of cDNA ends,RACE)克隆苜蓿夜蛾幼虫中肠丝氨酸蛋白酶cDNA全序列,用大肠杆菌Escherichia coli表达系统进行表达。重组蛋白经纯化后,利用梯度透析法进行复性,以BApNA为底物,进行活性测定。【结果】克隆获得的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为HvSP(GenBank登录号:JX866720),该基因全长880 bp,开放阅读框长762 bp,编码254个氨基酸,推测分子量和pI值分别为26.9 kDa和9.49。由HvSP推导的氨基酸与鳞翅目昆虫SP氨基酸序列的一致性在52%~95%之间,其中与棉铃虫Helicoverpa armigera SP(GenBank登录号:CAA72962)的氨基酸序列一致性最高,达95%。成功构建重组载体pET21b-HvSP进行原核表达,Western-blot鉴定确定为目的蛋白。蛋白可溶性分析发现重组蛋白为包涵体。在Glycine-NaOH缓冲液中,当pH为10.0时,复性的重组蛋白活性达到最高,为35.74 U/mL。【结论】本研究在苜蓿夜蛾体内获得了一个新的丝氨酸蛋白酶基因,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性。该结果为进一步研究丝氨酸蛋白酶在鳞翅目昆虫体内的生理功能奠定了基础。
译  名：
cDNA cloning,sequence analysis and prokaryotic expression of a serine protease from the midgut of Heliothis viriplaca (Lepidoptera:Noctuidae)
作  者：
ZHOU Xiao-Qun;GAO Yan-Ling;ZHAO Kui-Jun;FAN Dong;College of Agronomy,Northeast Agricultural University;
关键词：
Heliothis viriplaca;;serine protease;;cDNA cloning;;prokaryotic expression;;recombinant protein;;enzyme activity
摘  要：
【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 is880 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 GlycineNaOH buffer. 【Conclusion】A novel serine protease cDNA sequence was obtained from H. viriplaca,andrecombinant 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.

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