柑橘全爪螨PcSOD3的异源表达及其重组酶的抗氧化活性

【目的】笔者课题组前期研究发现,柑橘全爪螨在极端温度、杀螨剂、重金属和紫外线胁迫下,其体内Pc SOD3表达显著上调,暗示Pc SOD3在柑橘全爪螨应对不良环境胁迫的过程中起着至关重要的作用。为了进一步明确Pc SOD3的生理功能,本研究开展了该基因的异源表达及重组酶生化特性的分析工作。【方法】构建p ET28a-Pc SOD3重组表达质粒,并在大肠杆菌中实现异源表达。随后利用镍柱亲和层析法分离纯化Pc SOD3重组酶,采用Western blot对纯化蛋白进行验证。使用WST-1法分析Pc SOD3重组酶的抗氧化活性,测定不同反应体系pH及不同前处理温度下Pc SOD3重组酶的活性。采用氯化铬、叔丁基过氧化氢和过氧化氢异丙苯3种氧化性药剂,诱导大肠杆菌细胞内产生氧化应激反应,并利用Kirby-Bauer纸片琼脂糖扩散法测定上述3种药剂对过表达Pc SOD3大肠杆菌的抑制效果。【结果】在大肠杆菌BL21(DE3)菌株中成功表达Pc SOD3,研究摸索出Pc SOD3重组蛋白最适诱导表达条件:诱导表达温度为18℃,摇床转速为160 r/min,当OD值达0.6时加入IPTG并使其终浓度为0.4mmol·L~(-1),诱导时间为18 h。Western blot验证结果表明所纯化重组蛋白即为Pc SOD3重组酶,重组蛋白大小为25.3k D。WST-1法测得Pc SOD3重组酶活性在p H=7.0的反应体系中最高,约为47.3 U/mg protein,而在前处理温度为25℃时,Pc SOD3重组酶活性最高为40.2 U/mg protein。利用K-B纸片琼脂糖扩散法进行了药敏试验,结果显示氯化铬对过量表达Pc SOD3大肠杆菌产生的抑菌圈均显著小于对空载体对照产生的抑菌圈,通过测量发现抑菌圈较对照缩小近20%;在150 mmol·L~(-1)浓度下叔丁基过氧化氢对过量表达Pc SOD3大肠杆菌产生的抑菌圈与对照相比缩小约25%;而浓度为200 mmol·L-1的过氧化氢异丙苯对过量表达Pc SOD3大肠杆菌产生的抑菌圈与对照相比则缩小约15%。【结论】成功在大肠杆菌中实现了Pc SOD3的功能性表达,并纯化获得了Pc SOD3重组蛋白。明确了Pc SOD3重组酶最适反应p H及最适前处理温度等生化特性,WST-1法证明Pc SOD3重组蛋白在离体条件下具有显著的抗氧化活性,而K-B纸片琼脂糖扩散法则证明过表达Pc SOD3的大肠杆菌抗氧化能力显著增强,说明Pc SOD3具有抗氧化功能。研究结果进一步揭示Pc SOD3在柑橘全爪螨抗氧化损伤过程中具有重要作用。

Heterologous Expression of PcSOD3 from Panonychus citri and the Anti-oxidant Activity of Its Recombinant Enzyme

【Objective】In the author’s previous studies, it was found that PcSOD3 is up-regulated after exposure to thermal stress, acaricide abamectin, heavy metal and UV-B ultraviolet irradiation, which suggested that PcSOD3 played an important role in the response of P. citri to various adverse environmental stress. In order to further elucidate the physiological functions of PcSOD3, the biochemical characteristics of its recombinant protein expressed in Escherichia coli was investigated. 【Method】The pET28a-PcSOD3 expression plasmid for the heterologous expression in E. coli was constructed. The recombinant protein was purified by using a Ni⁺ affinity chromatography column, and confirmed by the Western blot analysis. The enzymatic activity was measured using a SOD total activity assay kit. Moreover, the effects of various pH values of the reaction system and different temperatures for preincubation on the recombinant enzyme were tested using the same kit. Using the Kirby-Bauer disc diffusion assay method, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 were measured by exposing to chromic chloride, t-butylhydroperoxide and cumene hydroperoxide. 【Result】 PcSOD3 was successfully expressed in the BL21 (DE3) strain of E. coli. The best condition for the induction and expression of the recombinant protein was obtained, under which cells were cultured at 18℃ and 160 r/min, IPTG was added to a final concentration of 0.4 mmol·L^-1, the inducing time continued for 18 h. The resulted PcSOD3 recombinant protein was further confirmed by the Western blot analysis, with the molecular weight of 25.3 kD. Moreover, the PcSOD3 recombinant protein was active in the WST-1 activity assay system. Based on the assay system, it was found that PcSOD3 recombinant protein was mostly active (activity was about 47.3 U/mg protein) when the pH of the reaction system was 7.0, while it was mostly active (activity was about 40.2 U/mg protein) when the preincubation temperature was 25℃. As indicated by the Kirby-Bauer test, the diameters of inhibition zones of the E. coli overexpressing PcSOD3 which were exposed to chromic chloride (CdCl₂), were about 20% smaller than the control. Similarly, overexpression of PcSOD3 in E. coli had significantly reduced the inhibition zone (around 25%) caused by exposure to t-butylhydroperoxide at the concentration of 150 mmol·L^-1, compared to the control. Meanwhile, E. coli expressing PcSOD3 showed 15% reduced inhibition when exposed to cumene hydroperoxide.【Conclusion】PcSOD3 was functionally expressed in E. coli. Purified PcSOD3 recombinant protein was obtained. Furthermore, the biochemical characteristics of the recombinant enzyme such as the optimal pH and preincubation temperature was determined. The recombinant enzyme showed a significant antioxidation activity in vitro by WST-1 assay method. The results of Kirby-Bauer disc diffusion assays showed that the E. coli overexpressing PcSOD3 had a significantly elevated tolerance to the oxidative stress, which has proven that PcSOD3 has the antioxidant function. Results of the experiment further revealed that PcSOD3 played a crucial role in the tolerance of P. citri to oxidative stress.