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首页> 中文期刊> 《中国农业科学》 >家蚕碱性磷酸酶原核表达纯化、结构与活性分析

家蚕碱性磷酸酶原核表达纯化、结构与活性分析

         
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摘要

[目的]碱性磷酸酶(alkaline phosphatase,ALP)是生物体内磷酸代谢的关键调控酶.不同物种ALP的性质与其生理功能密切相关.研究家蚕(Bombyxmori)ALP(BmALP)的性质和结构,为揭示ALP在昆虫体内的生理功能和调控机制提供依据.[方法]取5龄3d家蚕中肠组织,利用Trizol法提取总RNA,然后以其为模板反转录合成cDNA.以家蚕中肠cDNA为模板,利用Primer Premier 6.0软件分别设计上下游引物,通过PCR克隆BmALP.将BmALP与不同的表达载体分别进行双酶切,然后连接并转化表达菌株,利用大肠杆菌表达重组蛋白.比较不同表达载体在上清中的表达情况,选择可溶性重组蛋白表达最好的载体,利用Origami B(DE3)细胞大量表达重组蛋白,借助Ni-NTA亲和层析纯化重组的His-Trx-BmALP蛋白,然后加入Prescission蛋白酶在4℃酶切20h,再次利用Ni-NTA亲和层析去除融合标签His-Trx.利用凝胶过滤层析分析BmALP分子量及其在溶液中的状态,利用圆二色光谱研究BmALP的二级结构及其随温度的变化.通过酶活分析研究BmALP的最适pn、最适温度、Km、结构稳定性及金属离子对其酶学活性的影响.[结果]从家蚕中肠组织提取了总RNA,反转录合成了cDNA,并以该cDNA为模板成功克隆了BmALPo分别构建了BmALP的pSKB2、ppSUMO和pET32M.3C 3种表达载体.表达分析发现,pET32M.3C载体有助于重组的融合蛋白His-Trx-BmALP以可溶性蛋白的形式在细胞裂解后的上清液中表达,然后利用pET32M.3C载体大量表达重组蛋白,并通过Ni-NTA亲和层析纯化获得了可溶性的His-Trx-BmALP.加入Prescission蛋白酶酶切,再次通过Ni-NTA亲和层析去除了融合标签His-Trx.凝胶过滤分析显示BmALP在溶液中形成稳定的二聚体结构.圆二色光谱研究表明BmALP包含有α一螺旋结构,其含量随温度的升高逐渐减少.酶活分析揭示BmALP的最适pH和最适温度分别为11.0和45℃.测定BmALP的Km值为1.40 mmol·L-1.在10℃处理2h后,BmALP的残余活性最高;35℃处理2h后,其活性完全丧失.Mg2+和Zn2+促进了BmALP催化反应的进行,其最适浓度分别为40和5 mmol·L-1.在20mmol·L-1浓度范围内,Cu2+激活了BmALP活性,其中10 mmol·L-1时激活效果最好,浓度超过20 mmol·L-1时,Cu2+则抑制了BmALP活性.[结论]克隆了家蚕碱性磷酸酶基因BmALP,表达纯化了BmALP蛋白并分析了其结构和性质,为深入研究其结构和功能打下了基础.%[Objective] Alkaline phosphatase (ALP) is the key enzyme in the metabolism of phosphoric acid in vivo.The properties of ALP in different species are closely related to their physiological functions.The characterization of the property and structure of Bombyx mori ALP (BmALP) will facilitate to reveal the physiological function and regulation mechanism of ALP in insects.[Method] The total RNA was extracted by Trizol method from the midgut ofB.mori larvae on day 3 of the 5th instar,and then cDNA was synthesized with the extracted total RNA as the template by reverse transcription.The upstream and downstream primers were designed by Primer Premier 6.0 software,and BmALP was cloned with the synthesized cDNA as the template by PCR.BmALP and different expression vectors were double digested,respectively,then ligated and transformed into the expression strain.The recombinant protein was expressed by Escherichia coli.The expressions of different expression vectors in the supematant were compared,and the vector with the best expression of soluble recombinant protein was chosen.The recombinant protein was expressed in large scale using Origami B (DE3) cells,and digested with Prescission protease at 4℃ for 20 h followed by the purification via Ni-NTA affinity chromatography.Then the fusion His-Trx tag was removed using Ni-NTA affinity column again.The molecular weight and the state of BmALP in solution were analyzed by gel filtration chromatography.The secondary structure of BmALP and the effects of temperature on its structure were studied by circular dichroism spectroscopy.The optimum pH,optimum temperature,Km,structural stability and the effect of metal ions on the activity of BmALP were studied by the activity assay.[Result] The total RNA was extracted from the midgut of B.mori and cDNA was synthesized by reverse transcription.BmALP was successfully cloned with the cDNA as the template.The expression vectors of BmALP with pSKB2,ppSUMO and pET32M.3C were constructed,respectively.The expression analysis showed that the pET32M.3C vector facilitated the expression of the recombinant fusion protein His-Trx-BmALP in the form of a soluble protein in the supernatant of cell lysate.Then the recombinant BmALP was expressed in large scale with the pET32M.3C vector.The soluble recombinant His-Trx-BmALP was purified via Ni-NTA affinity chromatography.After the digestion of His-Trx-BmALP by Prescission protease,the fusion His-Trx tag was removed by Ni-NTA affinity column.Gel filtration analysis showed that BmALP formed a stable dimer in solution.Circular dichroism spectroscopy showed BmALP contained α-helical structure,and its content decreased with increasing temperature.Enzymatic activity analysis revealed that the optimum pH and temperature of BmALP were 11.0 and 45 ℃,respectively.The Km of BmALP was measured to be 1.40 mmol·L-1.After 2 h incubation at 10℃,BmALP had the highest residual activity,and the residual activity was completely lost after incubation at 35℃ for 2 h.Mg2+ and Zn2+ promoted the catalytic reaction of BmALP with the optimal concentration of 40 and 5 mmol·L-1,respectively.Cu2+ activated BmALP activity within 20 mmol·L-1,and the optimal concentration was 10 mmol·L-1,however,Cu2+ inhibited the activity of BmALP while its concentration was higher than 20 mmol·L-1.[Conclusion] BmALP was cloned.BmALP protein was expressed and purified and its structure and properties were analyzed.The results of this study provided a basis for further study of its structure and function.

著录项

  • 来源
    《中国农业科学》 |2017年第14期|2837-2850|共14页
  • 作者

    何华伟; 王叶菁; 侯丽; 李瑜; 位曙光; 赵朋; 蒋文超; 赵萍;

  • 作者单位

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

    西南大学生物技术学院;

    重庆400715;

    西南大学重庆市蚕丝纤维新材料工程技术研究中心;

    重庆400715;

    西南大学生物技术学院;

    重庆400715;

    西南大学生物技术学院;

    重庆400715;

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

    西南大学家蚕基因组生物学国家重点实验室;

    重庆400715;

  • 原文格式 PDF
  • 正文语种 chi
  • 中图分类
  • 关键词

    家蚕; 碱性磷酸酶; 表达纯化; 结构; 性质;

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