【作者】 李莹；

【导师】 贾晓强；

【作者基本信息】 天津大学 ， 生物化工， 2020， 硕士

【摘要】 近年来,细胞表面展示技术已成为重金属修复领域的研究重点。在此背景下,本研究选取冰晶核蛋白的N端序列（INPN）为细胞外膜的锚定蛋白,首先在大肠杆菌中构建了一系列绿色荧光蛋白表面展示验证载体,考察了INPN的转运和锚定能力,并初步探究了融合模式与蛋白诱导温度对蛋白表达的影响。结果表明,INPN能够在大肠杆菌中发挥其运转载体的功效,且工程菌荧光表达情况受融合方式的影响较大,不合适的连接肽可能导致融合蛋白无法正常折叠而失活;对诱导温度的研究发现,工程菌在22℃诱导下的荧光值相对较高且更稳定。基于上述工作,通过相同的融合蛋白构建方式,将来自耐金属贪铜菌（Cupriavidus metallidurans CH34）中与铅离子调控有关的两个转录调节因子PbrR和PbrR691以及铅封存蛋白PbrD锚定在大肠杆菌上,并通过细胞分级分离、蛋白印迹分析和全细胞免疫荧光印迹分析证实了融合蛋白在细胞外膜的具体定位。基于构建的若干株铅结合蛋白表面展示菌株,考察了融合方式、诱导剂浓度控制下的蛋白表达水平,以及铅离子浓度和混合金属浓度对工程菌铅离子吸附性能的影响。发现最有利于融合蛋白Pb²⁺吸附的最佳融合方式为柔性连接肽（Flexible linker）,最佳的IPTG诱导浓度为0.5 m M,且菌株吸附能力随Pb²⁺浓度的增加表现出先提高后降低的态势,在500-1000μM时吸附能力达到最大化。特异性实验表明,PbrR、PbrR691和PbrD表面展示工程菌对Pb²⁺的吸附性能达到Cd²⁺的5倍以上和Hg²⁺的12倍以上;对Pb²⁺的最大吸附量分别达到942.1、754.3和864.8μmol/g CDW,这也是迄今报道的三种蛋白对铅的最高吸附量。为了检测本研究构建的工程菌的实际应用效果,建立了一种基于本氏烟草植物的生物检定法。本研究中构建的PbrR,PbrR691和PbrD表面展示工程菌处理下的烟草种子萌芽率分别为对照组的2.5,2.7和2.4倍,平均芽长分别达到对照组的8.5,7.9和9倍;土壤解毒试验中,三种工程菌处理下的植物总生物量分别达到对照组的7.45,13.35和7.3倍,说明工程菌解毒效果明显。总的来说,本研究设计并构建的PbrR,PbrR691和PbrD表面展示工程菌对Pb²⁺具有较高的吸附能力和良好的选择特异性,具有很好的应用价值,为重金属污染环境的原位修复提供了基础和依据。更多还原

【Abstract】 Recently,the application of cell surface display has become the research focus in the field of heavy metal repair.In this study,the N-terminal sequence of ice-nucleation protein（INPN）was selected as the anchor protein of the outer membrane of the cell.First,a series of enhanced green fluorescent protein surface display expression system was constructed in E.coli to investigate the transport and anchoring function of INPN.And the effect of fusion pattern between the anchor protein and the target protein and the culture temperature on the fluorescence expression of engineered bacteria in this system was studied.The results show that INPN can be used as a carrier protein in E.coli cells,and the fluorescence expression of engineered bacteria is greatly affected by the fusion pattern.Inappropriate linker peptide may cause the target protein to misfold.Studies on the induction temperature found that 22℃is the most favorable temperature for protein expression.Based on the above work,through the same fusion pattern,the two transcription regulators PbrR and PbrR691 and the lead storage protein PbrD from the Cupriavidus metallidurans strain CH34 were displayed on the extracellular membrane of Escherichia coli cells,with only the N-domain of ice-nucleation protein as an anchor protein to achieve specific adsorption of lead ions(Pb²⁺)and bioremediation of lead pollution;the specific localization of fusion proteins on the outer membrane was confirmed by cell fractionation,western bloting analysis,and whole-cell immunofluorescence.We also investigated the effects of fusion pattern,the expression level of fusion protein controlled by the concentration of inducer directly,heavy metal concentration,and heavy metal type on the adsorption of Pb²⁺by these engineered bacteria,and the optimal linker peptide（flexible linker）and inducer concentration（0.5 m M）were obtained.The engineered bacteria showed specific selectivity and strong adsorption capacity for Pb²⁺.Under the interference of other heavy metal ions,the adsorption capacity of engineered bacteria for lead has reached 5 times higher than cadmium and12 times higher than mercury;and the maximum Pb²⁺adsorption amounts of the three proteins（PbrR,PbrR691,and PbrD）were 942.1,754.3,and 864.9μmol/g cell dry weight,respectively,which are the highest lead adsorption amounts of these three proteins reported to date.To confirm the actual detoxification effect of the engineered bacteria constructed in this study,a bioassay method based on the Nicotiana benthamiana plant was established.The engineered E.coli bacteria were applied to seed germination and growth of Nicotiana benthamiana in a lead-contaminated environment,and clear detoxification effects were observed.The seed germination rates treated with PbrR,PbrR691 and PbrD surface displaying strains were 2.5,2.7,and 2.4 times higher than that of the control group,and the average bud length reached 8.5,7.9,and 9 times than that of the control group respectively;in the soil detoxification test,the total plant biomass under the treatment of the three engineered bacteria reached 7.45,13.35,and 7.3 times of the wild type strain BL21,indicating that the detoxification effects of the engineered bacteria are obvious and can be used as cell adsorbents that specifically binds lead ions.In summary,the PbrR,PbrR691,and PbrD surface displaying bacteria designed and constructed in this study have high adsorption capacity,selective specificity,and good application value for Pb²⁺,which may provide a convincing basis for in situ remediation of lead-contaminated water or soil.更多还原