TrxA-EstB1融合蛋白的表达、纯化与活性分析
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摘要
农药的大量施用不仅对环境造成严重污染，而且使大量害虫产生抗药性。其中抗性库蚊产生大量酯酶对农药起代谢作用是其产生抗药性的主要原因，抗药性的产生加大了农药治虫的难度，加剧了农药对环境的污染。然而，将抗性蚊虫产生的解毒酶用于农药污染的生物治理是环境污染治理的新思路、新方向，有较好的应用前景。本文旨在将解毒酶酯酶B1基因融合高效表达，为其实际应用创造条件。
本文将从抗性库蚊（Culex pipiens）中分离出的解毒酶基因和硫氧还蛋白融合表达载体pThioHisA用限制性内切酶EcoRI消化，用低熔点胶回收消化片段并纯化，纯化后的载体脱磷，在T4 DNA连接酶作用下将二者进行体外连接，将连接产物转化大肠杆菌DH5α，在100μg/L氨苄青霉素平板上筛选Ampr转化子，将重组质粒分别经BglⅡ、BamHI单酶切，电泳分析获得酯酶基因插入方向正确的融合表达质粒，命名为pThioHisA-B1。
挑取鉴定正确的单菌落，接种于2ml含氨苄青霉素（50μg/ml）的LB培养基中，37℃，180rpm培养过夜，次日按5％接种量转接1次，同样条件下培养至OD550达0.5左右，加入IPTG至浓度1mmol/L，诱导12h。12％ SDS-PAGE电泳检测出工程菌可表达分子量约63KD的TrxA-EstB1融合蛋白，将胶体进行薄层扫描确定诱导7h后目的蛋白表达量最高，占菌体总蛋白的48.7％。
菌体在样品提取液中超声、冻融破壁后，4℃，10000rpm离心20min，分别取离心后的上清液和沉淀进行SDS-PAGE电泳鉴定，发现TrxA-EstB1融合蛋白大部分以包涵体形式存在，少部分以可溶形式存在。为了提高目的蛋白的可溶性表达，本实验在低温下（34℃、32℃、30℃、28℃、25℃）进行目的蛋白表达，最终确定28℃下诱导，可溶性目的蛋白表达量最高，占目的蛋白总数的65％，占菌体总蛋白的30％左右；IPTG浓度在常规用量1mmol/L的基础上降低至0.4mmol/L时，目的蛋白的表达量无显著差异。
以α-NA为底物对所构建工程菌进行酯酶活性测定，结果表明该工程菌能高效降解α-NA,具有较强的酯酶活性，融合伴侣硫氧还蛋白促进了酯
酶B1的正确折叠，形成了天然的蛋白质构象。本研究将工程菌以2.5％海藻酸钠、3％ CaCl2进行包埋固定，以α-NA为底物测定固定化工程菌的酯酶活性，结果表明工程菌固定化后，酶活虽有所降低，但仍有一定的解毒能力。
将最适条件下诱导的工程菌发酵液离心，收集菌体，加样品提取液超声、冻融法将菌体破壁，4℃，10000rpm离心收集上清液，获得粗提蛋白。将粗提蛋白经硫酸铵分级盐析，DEAE-Sepharose CL-6B离子交换层析,Sephadex G-150分子筛层析后，用聚乙二醇包埋浓缩，SDS-PAGE电泳检测提取物纯度,表明TrxA-EstB1融合蛋白已被提纯。对该融合蛋白进行酶活性测定表明它具有酯酶B1的解毒活性，最适反应条件为40℃， pH 7.5。
The use of insecticides does not only bring benefit to human beings but also cause serious environmental pollution. Many insects including Culex pipiens can product detoxificase that can catalyze and degrade many xenobiotic agents besides pesticides, so it becomes harder to kill them. But the use of insect detoxificase provides a novel method in bioremediation of pesticides contamination ,so this thesis is aimed at fusion expressing the detoxificase(esterase B1) at high level. It will pave the way for utilization of esterase B1 in reality.
The DNA fragment of esterase B1 is cloned to MCS of plasmid pThioHisA to construct the thioredoxin(TrxA) and esterase B1 fusion expression vector . The new recombinant plasmid is named pThioHisA-B1. The process is as follows : first, the plasmid pRL-B1 and pThioHisA are digested with restriction endonuclease EcoRI. Then retrieve and purify the 1.33Kb fragment of pRL-B1 and 4.4Kb fragment of pThioHisA through low-melting-point agarose gel electrophoresis. After the purified expression vector fragment is dephosphorylated , link up the two fragments with T4DNA ligase in vitro . After transferring the new recombinant plasmid pThioHisA into the host E.coli DH5αthe positive clones are screened on LB+AMP medium plate with BglⅡand BamHI.
The engineering strain E.coli DH5α/pThioHisA-B1 is cultured in LB +Amp medium to an OD550 of 0.5 at 37℃,180rpm ,then add IPTG to a final concentration of 1mmol/L, 12 hours for induction . Higher levels of expression of TrxA-EstB1 is obtained 7 hours after induction . The expressed fusion protein with molecular weight of about 63KD is about 48.7% of the total bacterial protein as detected by SDS-PAGE and thin-layer gel scanning analysis.
The bacterial pellet is sonicated and centrifuged at 10000rpm for 20 minutes at 4℃ to pellet cell debris and insoluble matter .The SDS-PAGE confirms that the TrxA-EstB1 fusion protein exists in both inclusion body and soluble protein in the cells. The amount of the soluble target protein increases along with decrease of induction temperature and amounts to more than 30% of the total bacterial proteins at 28℃.Under shake incubator the IPTG
concentration is studied ,The result shows:0.4mmol/L IPTG can induce the E.coli express fusion protein and has no obvious difference when which is 1.0mmol/L.
The results of detoxifing experiment indicate that the TrxA-ExtB1 fusion in engineering bacterial of E.coli DH5α/pThioHisA-B1 exhibit a high detoxificase activity in degrading the specific substrate such as α-naphthyl acetate(α-NA).This result shows that the achieved fusion protein keeps the enzyme activity of esterase B1 because the fusion chaperon of TrxA can promote the correct fold of esterase B1 and its soluble expression . We have immobilized the bacterials in 2.5% algmate and 3% CaCl2 in order to make more practical use of the engineering bacterial .It is found that the immobilized cell can work well in degradingα-NA also .
The fermented broth is centrifugated at 10000rpm to collect cell pellet, then the pellet is sonicated in lysate buffer and centrifugated to separate the crude fusion protein. The crude fusion protein is purified with ammonium sulphate precipitation,DEAE-sepharose CL-6B anion-exchange and SephadexG-150 gel filtration chromatographies. The purified TrxA-EstB1 protein is demonstrated to be one single protein band by SDS-PAGE. With respect to optimal temperature and optimal pH,substrate specificity,the purified fusion esteraseB1 exhibited properties similar to those of the wild -type esteraseB1. The optimal reaction temperature and pH are 40℃ and 7.5 ,respectively.
本文将从抗性库蚊（Culex pipiens）中分离出的解毒酶基因和硫氧还蛋白融合表达载体pThioHisA用限制性内切酶EcoRI消化，用低熔点胶回收消化片段并纯化，纯化后的载体脱磷，在T4 DNA连接酶作用下将二者进行体外连接，将连接产物转化大肠杆菌DH5α，在100μg/L氨苄青霉素平板上筛选Ampr转化子，将重组质粒分别经BglⅡ、BamHI单酶切，电泳分析获得酯酶基因插入方向正确的融合表达质粒，命名为pThioHisA-B1。
挑取鉴定正确的单菌落，接种于2ml含氨苄青霉素（50μg/ml）的LB培养基中，37℃，180rpm培养过夜，次日按5％接种量转接1次，同样条件下培养至OD550达0.5左右，加入IPTG至浓度1mmol/L，诱导12h。12％ SDS-PAGE电泳检测出工程菌可表达分子量约63KD的TrxA-EstB1融合蛋白，将胶体进行薄层扫描确定诱导7h后目的蛋白表达量最高，占菌体总蛋白的48.7％。
菌体在样品提取液中超声、冻融破壁后，4℃，10000rpm离心20min，分别取离心后的上清液和沉淀进行SDS-PAGE电泳鉴定，发现TrxA-EstB1融合蛋白大部分以包涵体形式存在，少部分以可溶形式存在。为了提高目的蛋白的可溶性表达，本实验在低温下（34℃、32℃、30℃、28℃、25℃）进行目的蛋白表达，最终确定28℃下诱导，可溶性目的蛋白表达量最高，占目的蛋白总数的65％，占菌体总蛋白的30％左右；IPTG浓度在常规用量1mmol/L的基础上降低至0.4mmol/L时，目的蛋白的表达量无显著差异。
以α-NA为底物对所构建工程菌进行酯酶活性测定，结果表明该工程菌能高效降解α-NA,具有较强的酯酶活性，融合伴侣硫氧还蛋白促进了酯
酶B1的正确折叠，形成了天然的蛋白质构象。本研究将工程菌以2.5％海藻酸钠、3％ CaCl2进行包埋固定，以α-NA为底物测定固定化工程菌的酯酶活性，结果表明工程菌固定化后，酶活虽有所降低，但仍有一定的解毒能力。
将最适条件下诱导的工程菌发酵液离心，收集菌体，加样品提取液超声、冻融法将菌体破壁，4℃，10000rpm离心收集上清液，获得粗提蛋白。将粗提蛋白经硫酸铵分级盐析，DEAE-Sepharose CL-6B离子交换层析,Sephadex G-150分子筛层析后，用聚乙二醇包埋浓缩，SDS-PAGE电泳检测提取物纯度,表明TrxA-EstB1融合蛋白已被提纯。对该融合蛋白进行酶活性测定表明它具有酯酶B1的解毒活性，最适反应条件为40℃， pH 7.5。
The use of insecticides does not only bring benefit to human beings but also cause serious environmental pollution. Many insects including Culex pipiens can product detoxificase that can catalyze and degrade many xenobiotic agents besides pesticides, so it becomes harder to kill them. But the use of insect detoxificase provides a novel method in bioremediation of pesticides contamination ,so this thesis is aimed at fusion expressing the detoxificase(esterase B1) at high level. It will pave the way for utilization of esterase B1 in reality.
The DNA fragment of esterase B1 is cloned to MCS of plasmid pThioHisA to construct the thioredoxin(TrxA) and esterase B1 fusion expression vector . The new recombinant plasmid is named pThioHisA-B1. The process is as follows : first, the plasmid pRL-B1 and pThioHisA are digested with restriction endonuclease EcoRI. Then retrieve and purify the 1.33Kb fragment of pRL-B1 and 4.4Kb fragment of pThioHisA through low-melting-point agarose gel electrophoresis. After the purified expression vector fragment is dephosphorylated , link up the two fragments with T4DNA ligase in vitro . After transferring the new recombinant plasmid pThioHisA into the host E.coli DH5αthe positive clones are screened on LB+AMP medium plate with BglⅡand BamHI.
The engineering strain E.coli DH5α/pThioHisA-B1 is cultured in LB +Amp medium to an OD550 of 0.5 at 37℃,180rpm ,then add IPTG to a final concentration of 1mmol/L, 12 hours for induction . Higher levels of expression of TrxA-EstB1 is obtained 7 hours after induction . The expressed fusion protein with molecular weight of about 63KD is about 48.7% of the total bacterial protein as detected by SDS-PAGE and thin-layer gel scanning analysis.
The bacterial pellet is sonicated and centrifuged at 10000rpm for 20 minutes at 4℃ to pellet cell debris and insoluble matter .The SDS-PAGE confirms that the TrxA-EstB1 fusion protein exists in both inclusion body and soluble protein in the cells. The amount of the soluble target protein increases along with decrease of induction temperature and amounts to more than 30% of the total bacterial proteins at 28℃.Under shake incubator the IPTG
concentration is studied ,The result shows:0.4mmol/L IPTG can induce the E.coli express fusion protein and has no obvious difference when which is 1.0mmol/L.
The results of detoxifing experiment indicate that the TrxA-ExtB1 fusion in engineering bacterial of E.coli DH5α/pThioHisA-B1 exhibit a high detoxificase activity in degrading the specific substrate such as α-naphthyl acetate(α-NA).This result shows that the achieved fusion protein keeps the enzyme activity of esterase B1 because the fusion chaperon of TrxA can promote the correct fold of esterase B1 and its soluble expression . We have immobilized the bacterials in 2.5% algmate and 3% CaCl2 in order to make more practical use of the engineering bacterial .It is found that the immobilized cell can work well in degradingα-NA also .
The fermented broth is centrifugated at 10000rpm to collect cell pellet, then the pellet is sonicated in lysate buffer and centrifugated to separate the crude fusion protein. The crude fusion protein is purified with ammonium sulphate precipitation,DEAE-sepharose CL-6B anion-exchange and SephadexG-150 gel filtration chromatographies. The purified TrxA-EstB1 protein is demonstrated to be one single protein band by SDS-PAGE. With respect to optimal temperature and optimal pH,substrate specificity,the purified fusion esteraseB1 exhibited properties similar to those of the wild -type esteraseB1. The optimal reaction temperature and pH are 40℃ and 7.5 ,respectively.
引文
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