| 摘要: |
| 【目的】制备灰茶尺蠖（Ectropis grisescens Warren）固醇转运蛋白（SCP）的多克隆抗体，并检测灰茶尺蠖SCP2蛋白在灰茶尺蠖不同组织中的表达情况，为进一步研究SCP2对胆固醇的转运和利用机制以及其生物学功能奠定基础。【方法】以灰茶尺蠖5龄1 d幼虫中肠cDNA为模板，应用PCR技术扩增得到EgSCP2片段，将EgSCP2目的片段连入pMD18-T载体后进行测序，利用DNAMAN软件分析该基因序列的准确性及编码蛋白的分子质量。以测序正确的EgSCP2基因片段为模板，扩增EgSCP2的开放阅读框，通过BamHⅠ和Hind Ⅲ限制性内切酶连接到原核表达载体pET32a中，并转化大肠杆菌BL21 (DE3)进行原核表达，离心收集并超声波破碎菌体，取上清液和沉淀分别进行SDS-PAGE检测，在不同温度、不同IPTG终浓度条件下优化EgSCP2重组蛋白表达条件。经Ni-NTA树脂层析柱纯化得到EgSCP2重组蛋白。将该蛋白免疫新西兰大白兔，制备兔抗EgSCP2血清抗体，采用间接ELISA方法测定了该血清抗体的效价，并通过Western blotting检测EgSCPx和EgSCP2蛋白在灰茶尺蠖不同组织中的表达情况。【结果】扩增得到EgSCP2基因开放阅读框（ORF）全长为441 bp，编码146个氨基酸，预测编码蛋白的分子质量为16 ku。优化蛋白诱导条件的试验表明，28 ℃、IPTG浓度为1.0 mmol/L时，EgSCP2重组蛋白表达量最高，该条件下通过大肠杆菌表达的EgSCP2重组蛋白分子质量约为34 ku，其大小与预期结果一致，且其在上清液和沉淀中均有表达。间接ELISA法检测结果表明，制备的兔抗EgSCP2抗体具有较好的灵敏度，效价达到1∶256 000。Western blotting检测结果显示，58 ku EgSCPx在5龄2 d灰茶尺蠖的表皮、脂肪体和中肠均有表达，且在中肠的表达量远高于表皮和脂肪体；16 ku EgSCP2仅在表皮和脂肪体中表达。58 ku EgSCPx蛋白在4龄和5龄灰茶尺蠖的幼虫中肠大量表达，在蛹期少量表达，在成虫期几乎不表达。【结论】纯化获得了EgSCP2重组蛋白，其最优表达条件为温度28 ℃、IPTG终浓度1.0 mmol/L；制备了高效价的灰茶尺蠖EgSCP2多克隆抗体，明确了灰茶尺蠖EgSCPx蛋白在不同组织中的表达规律。 |
| 关键词: 灰茶尺蠖 固醇转运蛋白 原核表达 抗体制备 组织表达 |
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| 基金项目:国家自然科学基金项目(31501902)；河南省科技攻关计划项目(182102110034)；信阳师范学院“南湖学者奖励计划”青年项目（2016058）；信阳师范学院大学生科研基金项目(2017-DXS-141);国家重点研发计划“化学肥料和农药减施增效综合技术研发”试点专项(2016YFD0200900) |
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| Preparation of polyclonal antibody and tissue expression analysis of EgSCP2 from Ectropis grisescens |
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ZHANG Lili,WU Yiqiong,WEI Jiaping,et al
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| Abstract: |
| 【Objective】This study prepared polyclonal antibody of Ectropis grisescens Warren sterol carrier protein (SCP) and investigated the expression of SCP2 protein in different issues of E.grisescens to provide basis for studying function and mechanism of sterol transportation and utilization.【Method】The fragment EgSCP2 was obtained by PCR amplification using cDNA of 1 day old fifth instar larval midgut of E. grisescens.The EgSCP2 was then inserted into pMD18-T for sequencing.The nucleic acid sequence and amino acids encoded were analyzed by DNAMAN software.The open reading frame of EgSCP2 was cloned from the sequenced fragment of EgSCP2 and inserted into the expression vector pET32a by BamH Ⅰ and Hind Ⅲ digestion.The recombinant vector pET32a-SCP2 was then transformed into competent cell Escherichia coli BL21 (DE3).The induced EgSCP2 recombinant protein was collected and crushed by ultrasonic.The supernatant and precipitate were collected,and SDS PAGE was used to analyze the expression of the recombinant protein.The recombinant EgSCP2 protein was expressed at different temperatures and concentrations of IPTG to optimize expression condition.The recombinant protein was purified by Ni NTA resin chromatography column.The purified recombinant protein was used to produce polyclonal antibody via immunizing rabbit.The titer of rabbit anti-SCP2 antiserum was evaluated by indirect ELISA.The expression of SCPx and EgSCP2 protein in different issues from E. grisescens was detected by Western blotting.【Result】The length of the open reading frame of EgSCP2 was 441 bp,encoding 146 amino acids.The predicted molecular weight of EgSCP2 recombinant protein was 16 ku.The EgSCP2 recombinant protein was highly expressed at inducing temperature of 28 ℃ with 1.0 mmol/L IPTG.The molecular weight of EgSCP2 recombinant protein expressed in E. coli was 34 ku,consistent with the predicted molecular weight.The indirect ELISA assay showed that the rabbit anti-SCP2 antiserum had a good sensitivity with the titer of 1∶256 000.Western blotting showed that 58 ku EgSCPx was expressed in epidermis,fat body and midgut of 2 days old fifth instar larvae and it had the highest expression level in the fourth instar and fifth instar larval midgut,as compared to the lower expression level in pupae and trace expression level at adult stage.The 16 ku EgSCP2 was only expressed in epidermis and fat body.【Conclusion】The EgSCP2 recombinant protein was successfully purified and the optimized expression condition was at inducing temperature of 28 ℃ with 1.0 mmol/L IPTG.The polyclonal antibody of EgSCP2 protein with high titer was obtained and the expression of SCPx in different issues from E. grisescens was explicit. |
| Key words: Ectropis grisescens Warren sterol carrier protein prokaryotic expression polyclonal antibody tissue expression |
