【摘要】 【目的】本文在前期获得绿盲蝽Apolygus lucorum磷脂酶C基因(Al PLC)的基础上,明晰Al PLC基因在绿盲蝽不同日龄若虫中的表达特征,阐明Al PLC重组蛋白的酶学性质。【方法】qRTPCR法分析1-13日龄绿盲蝽若虫Al PLC的表达量;构建含有Al PLC基因的原核表达载体p Czn1-Al PLC;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pNPPC(P-硝基苯基磷酸胆碱)为底物测定不同温度和不同p H下的重组Al PLC蛋白酶活性,最终确定了其酶活性的最佳p H和最适温度。【结果】Al PLC 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Al PLC可在大肠杆菌Escherichia coli中表达一个约79 k D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/m L条件下,该酶最适温度为57℃(179.54±3.96 nmol/μg·min),最适p H为8.5(374.99±2.84 nmol/μg·min)。【结论】结果表明Al PLC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Al PLC的功能奠定基础。更多还原

【Abstract】 【Aim】Based on our previous work of cloning the phospholipase C gene Al PLC from Apolygus lucorum,this study aims to determine the expression profiles of Al PLC 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 theexpression pattern of Al PLC 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 p H were measured by p-nitrophenylphosphorylcholine( p-NPPC). Finally,the optimum temperature and p H for the enzyme activity of PLC were analyzed. 【Results 】Al PLC 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 p Czn1-Al PLC expressed the target recombinant protein of 79 k D 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 Al PLC 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/m L,the most suitable temperature for its reaction was 57℃( 179. 54 ± 3. 96 nmol/μg · min) and the optimal p H was 8. 5( 374. 99 ± 2. 84 nmol/μg · min). 【Conclusion】The expression of Al PLC shows the developmental stage-specificity. Higher enzyme activity of Al PLC can be achieved under higher temperature and alkaline environment. The results provide the basis for analyzing the Al PLC function at the protein level.更多还原