绿盲蝽磷脂酶C(AlPLC)的表达、纯化及酶学性质

doi:10.16380/j.kcxb.2017.11.002

›› 2017, Vol. 60 ›› Issue (11): 1247-1254.doi: 10.16380/j.kcxb.2017.11.002

绿盲蝽磷脂酶C(AlPLC)的表达、纯化及酶学性质

谭永安¹, 赵旭东^2,3, 郝德君^2,3, 肖留斌^1,*, 柏立新¹, 赵静¹, 孙洋¹, 姜义平¹

(1. 江苏省农业科学院植物保护研究所, 南京 210014; 2. 南京林业大学南方现代林业协同创新中心, 南京 210037; 3. 南京林业大学林学院, 南京 210037)

出版日期:2017-11-20 发布日期:2017-11-20

Expression, purification and enzymatic characteristics of phosphodiesterase C (AlPLC) of Apolygus lucorum (Hemiptera: Miridae)

TAN Yong-An¹, ZHAO Xu-Dong^2,3, HAO De-Jun^2,3, XIAO Liu-Bin^1,*, BAI Li-Xin¹, ZHAO Jing¹, SUN Yang¹, JIANG Yi-Ping¹

(1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 3. College of Forestry, Nanjing Forestry University, Nanjing 210037, China)

Online:2017-11-20 Published:2017-11-20

摘要/Abstract

摘要： 【目的】本文在前期获得绿盲蝽Apolygus lucorum磷脂酶C基因(AlPLC)的基础上，明晰AlPLC基因在绿盲蝽不同日龄若虫中的表达特征，阐明AlPLC重组蛋白的酶学性质。【方法】qRT-PCR法分析1-13日龄绿盲蝽若虫AlPLC的表达量；构建含有AlPLC基因的原核表达载体pCzn1-AlPLC；进一步将该表达载体经IPTG诱导表达和蛋白纯化，获得具有磷脂酶活性的重组蛋白；以p-NPPC(P-硝基苯基磷酸胆碱)为底物测定不同温度和不同pH下的重组AlPLC蛋白酶活性，最终确定了其酶活性的最佳pH和最适温度。【结果】AlPLC mRNA在绿盲蝽测定的若虫期持续表达，在4, 8, 12以及13日龄若虫中表达量相对较高。重组蛋白AlPLC可在大肠杆菌Escherichia coli中表达一个约79 kD的蛋白，12% SDS-PAGE显示该蛋白主要以包涵体形式存在；经过变性，复性获得具有磷脂酶活性的纯化蛋白。在蛋白浓度为0.25 mg/mL条件下，该酶最适温度为57℃(179.54±3.96 nmol/μg·min)，最适pH为8.5(374.99±2.84 nmol/μg·min)。【结论】结果表明AlPLC在绿盲蝽若虫中表达具有特异的发育历期特性，获得的重组蛋白在较高温度和碱性环境下有较高磷脂酶活性。本研究结果为后续在蛋白水平上解析AlPLC的功能奠定基础。

关键词: 绿盲蝽, 磷脂酶C, 原核表达, 蛋白纯化, 酶学特性, 酶活性

Abstract: 【Aim】 Based on our previous work of cloning the phospholipase C gene AlPLC from Apolygus lucorum, this study aims to determine the expression profiles of AlPLC gene in different day-old nymphs of A. lucorum, to obtain the recombinant protein which has the phospholipase enzyme activity and to clarify its enzymatic characteristics. 【Methods】 Using the qRT-PCR technique, we determined the expression pattern of AlPLC in one day-old to 13 day-old nymphs of A. lucorum. The recombinant plasmid containing target gene was specifically expressed after induction by IPTG. The recombinant protein was purified by GST agarose affinity chromatography and molecular sieve chromatography. Then the enzymatic characteristics of this recombinant protein under different temperatures and pH were measured by p-nitrophenylphosphorylcholine (p-NPPC). Finally, the optimum temperature and pH for the enzyme activity of PLC were analyzed. 【Results】 AlPLC was found to be continuously expressed throughout the surveyed nymphal stage of A. lucorum, and highly expressed in 4, 8, 12 and 13 day-old nymphs. The recombinant plasmid pCzn1-AlPLC expressed the target recombinant protein of 79 kD after IPTG induction in Escherichia coli. The 12% SDS-PAGE showed that the recombinant protein was mainly present as inclusion bodies. The purified protein of AlPLC with the phosphodiesterase activity was obtained after denaturation and renaturation. This recombinant protein had higher phospholipase activity by using p-NPPC as substrates, and under the protein concentration of 0.25 mg/mL, the most suitable temperature for its reaction was 57℃ (179.54±3.96 nmol/μg·min) and the optimal pH was 8.5 (374.99±2.84 nmol/μg·min). 【Conclusion】 The expression of AlPLC shows the developmental stage-specificity. Higher enzyme activity of AlPLC can be achieved under higher temperature and alkaline environment. The results provide the basis for analyzing the AlPLC function at the protein level.

Key words: Apolygus lucorum, phospholipase C, prokaryotic expression, protein purification, enzymatic characteristics, enzyme activity

谭永安, 赵旭东, 郝德君, 肖留斌, 柏立新, 赵静, 孙洋, 姜义平. 绿盲蝽磷脂酶C(AlPLC)的表达、纯化及酶学性质[J]. , 2017, 60(11): 1247-1254.

TAN Yong-An, ZHAO Xu-Dong, HAO De-Jun, XIAO Liu-Bin, BAI Li-Xin, ZHAO Jing, SUN Yang, JIANG Yi-Ping. Expression, purification and enzymatic characteristics of phosphodiesterase C (AlPLC) of Apolygus lucorum (Hemiptera: Miridae)
[J]. , 2017, 60(11): 1247-1254.

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绿盲蝽磷脂酶C(AlPLC)的表达、纯化及酶学性质

Expression, purification and enzymatic characteristics of phosphodiesterase C (AlPLC) of Apolygus lucorum (Hemiptera: Miridae)

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