视蛋白基因的克隆及其与抗药性/耐药性关系的研究
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摘要
长期以来，大范围和不加选择地使用杀虫剂导致了媒介抗药性的发生和发展。发现和研究新的抗药性相关基因将有助于阐明抗药性的分子基础和寻找新的治理抗药性的靶标。
本研究根据本实验室已经获得的淡色库蚊NYD-OP1～7基因的序列，设计引物，从淡色库蚊抗性品系成蚊cDNA文库中扩增出NYD-OP7基因的全长序列，T／A克隆入pGEM-T Easy载体，经测序、对位拼接获取全长序列。获得的淡色库蚊NYD-OP7基因长1437bp，开放阅读框为1116bp，编码371个氨基酸(GenBank登录号：AY749413)。
用BlastX软件将该基因推导编码的氨基酸序列与蛋白质公共数据库Swissprot分析，用ClustalW软件作序列比对和聚类分析。推导的氨基酸序列与埃及伊蚊(Aedes aegypti)和冈比亚按蚊(Anopheles gambiae)的视蛋白具有80％以上的同源性。
定量PCR的方法研究其在敏感品系和抗性品系淡色库蚊各发育阶段中的表达差异。结果显示，NYD-OP7在淡色库蚊溴氰菊酯敏感和抗性品系卵及1、2、3、4龄幼虫中有微弱表达，在蛹及成蚊中有较高表达，而且该基因在抗性品系蛹和成蚊中的表达较敏感品系显著升高。
将该基因克隆入昆虫细胞表达载体pIE1-3，转染蚊C6／36细胞，G418筛选两周后获得稳定表达的细胞系，[~3H]-TdR检测结果显示：各种浓度的溴氰菊酯处理48小时后，在15μM、20μM和25μM浓度组，转染组细胞的存活率较对照组分别升高了11.08％、13.07％和11.10％(P＜0.05)。
综上，所有结果表明，NYD-OP7基因与淡色库蚊溴氰菊酯抗性相关，为一个新的昆虫抗药性相关基因。
肿瘤细胞对化疗药物产生耐药常常导致化疗失败，研究肿瘤耐药性相关基因将有助于阐明肿瘤耐药性产生的分子机制，也有助于发现新的检测及逆转肿瘤耐药性的方法。
Opsin 3(OPN3)基因，也被称作encephalopsin或panopsin基因，是一个发现时间不长的视网膜外光感受器基因，目前该基因的功能尚不清楚。
本研究采用RT-PCR的方法检测了OPN3基因在人体多种组织以及多种肿瘤细胞系中的表达，结果显示OPN3基因在胸腺、睾丸、肝脏、胰腺和胎盘组织中的表达比较强，在前列腺和肾脏组织中的表达比较弱；同时，OPN3基因在人肺癌紫杉醇耐药株A549_(PTX)细胞中的表达是A549细胞的3.46倍；在人肝癌5-FU耐药株Bel7402_(5-FU)中的表达是Bel7402细胞的0.37倍(P＜0.05)。
随后，本研究构建了OPN3基因的真核细胞表达载体和RNA干涉表达载体，选取A549细胞和A549_(PTX)细胞为研究对象，建立了稳定转染的细胞系，通过[~3H]-TdR的方法研究了分别过表达和低表达OPN3基因后，A549细胞和A549_(PTX)细胞对于紫杉醇敏感性的变化，结果显示：在各种浓度的紫杉醇处理72小时后，与对照组细胞相比，过表达OPN3基因后的A549细胞的存活率均有不同程度的下降，尤其在2，5，25，100，500 nM组，其存活率分别下降了14.26％，24.76％，18.19％，26.79％和27.51％(P＜0.05)；同时，低表达OPN3基因后的A549_(PTX)细胞的存活率均有不同程度的上升，尤其在5nM组，其存活率升高了34.32％(P＜0.05)。
将OPN3基因克隆入pEGFPN2质粒，将重组质粒转染A549细胞、HFLF细胞和293细胞，激光共聚焦显微镜下观察融合蛋白的表达定位，结果显示：OPN3和GFP的融合蛋白在各组细胞的胞膜，胞浆及胞核均有散在表达，对照空载体的GFP蛋白在整个细胞内广泛表达，没有特异性。
综上，本研究首次发现OPN3基因与A549细胞对紫杉醇的耐药性相关，而且OPN3蛋白还具有很特别的细胞定位特征，值得进一步深入研究。
One mosquito opsin gene, NYD-OP7, has been cloned from Culexpipiens pallens. Within the sequence, an open reading frame (ORF) of1116 bp was found to encode a putative 371 amino acids protein whichexhibits high identity with opsins from Aedes and Anopheles mosquitoes.
Transcript expression of NYD-OP7 was determined by real-timePCR in all life stages of deltamethrin-susceptible and -resistant strains ofthe Culex mosquito. The results demonstrated that this gene is expressedat all developmental stages, and it is expressed predominantly at thepupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 isoverexpressed in deltamethrin-resistant strain than in -susceptible strain.Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cellscan confer moderate deltamethrin resistance.
Our study provides the first direct evidence that increased expressionof an opsin gene may play some role in the development of deltamethrinresistance in Cx. pipiens pallens.
Opsin 3 (OPN3), also called encephalopsin or panopsin, is a recentlydiscovered extraretinal photoreceptor, and its function is largely unknownuntil now.
In this study, based on the RT-PCR results, we showed that OPN3was moderately expressed in the thymus, testis, liver, pancreas andplacenta and weakly in the prostate gland and kidney. Meanwhile, theexpression of OPN3 was significantly higher in the paclitaxel -resistantline than in the drug naive parental A549 cell line. Then we confirmed theoverexpression by real-time PCR analysis.
Using cDNA transfection and RNA interference, we successfullyestablished stable transfectants with upregulation and downregulation ofOPN3 expression, respectively. In vitro drug sensitivity assaydemonstrated that overexpression of OPN3 sensitized A549 cells topaclitaxel whereas downregulation of OPN3 conferred A549_(PTX) cellresistance to paclitaxel.
We also investigated cell localization of OPN3 by fusing it to anenhanced green fluorescent protein (EGFP). Green fluorescence signalwas found clearly in the nuclei, body and membrane of cells transfected with OPN3-EGFR In contrast, EGFP (vector control) was distributedmore or less evenly throughout the whole cell.
Our data for the first time suggests that OPN3 gene may play somerole in the development of paclitaxel resistance in human lung cancercells.
本研究根据本实验室已经获得的淡色库蚊NYD-OP1～7基因的序列，设计引物，从淡色库蚊抗性品系成蚊cDNA文库中扩增出NYD-OP7基因的全长序列，T／A克隆入pGEM-T Easy载体，经测序、对位拼接获取全长序列。获得的淡色库蚊NYD-OP7基因长1437bp，开放阅读框为1116bp，编码371个氨基酸(GenBank登录号：AY749413)。
用BlastX软件将该基因推导编码的氨基酸序列与蛋白质公共数据库Swissprot分析，用ClustalW软件作序列比对和聚类分析。推导的氨基酸序列与埃及伊蚊(Aedes aegypti)和冈比亚按蚊(Anopheles gambiae)的视蛋白具有80％以上的同源性。
定量PCR的方法研究其在敏感品系和抗性品系淡色库蚊各发育阶段中的表达差异。结果显示，NYD-OP7在淡色库蚊溴氰菊酯敏感和抗性品系卵及1、2、3、4龄幼虫中有微弱表达，在蛹及成蚊中有较高表达，而且该基因在抗性品系蛹和成蚊中的表达较敏感品系显著升高。
将该基因克隆入昆虫细胞表达载体pIE1-3，转染蚊C6／36细胞，G418筛选两周后获得稳定表达的细胞系，[~3H]-TdR检测结果显示：各种浓度的溴氰菊酯处理48小时后，在15μM、20μM和25μM浓度组，转染组细胞的存活率较对照组分别升高了11.08％、13.07％和11.10％(P＜0.05)。
综上，所有结果表明，NYD-OP7基因与淡色库蚊溴氰菊酯抗性相关，为一个新的昆虫抗药性相关基因。
肿瘤细胞对化疗药物产生耐药常常导致化疗失败，研究肿瘤耐药性相关基因将有助于阐明肿瘤耐药性产生的分子机制，也有助于发现新的检测及逆转肿瘤耐药性的方法。
Opsin 3(OPN3)基因，也被称作encephalopsin或panopsin基因，是一个发现时间不长的视网膜外光感受器基因，目前该基因的功能尚不清楚。
本研究采用RT-PCR的方法检测了OPN3基因在人体多种组织以及多种肿瘤细胞系中的表达，结果显示OPN3基因在胸腺、睾丸、肝脏、胰腺和胎盘组织中的表达比较强，在前列腺和肾脏组织中的表达比较弱；同时，OPN3基因在人肺癌紫杉醇耐药株A549_(PTX)细胞中的表达是A549细胞的3.46倍；在人肝癌5-FU耐药株Bel7402_(5-FU)中的表达是Bel7402细胞的0.37倍(P＜0.05)。
随后，本研究构建了OPN3基因的真核细胞表达载体和RNA干涉表达载体，选取A549细胞和A549_(PTX)细胞为研究对象，建立了稳定转染的细胞系，通过[~3H]-TdR的方法研究了分别过表达和低表达OPN3基因后，A549细胞和A549_(PTX)细胞对于紫杉醇敏感性的变化，结果显示：在各种浓度的紫杉醇处理72小时后，与对照组细胞相比，过表达OPN3基因后的A549细胞的存活率均有不同程度的下降，尤其在2，5，25，100，500 nM组，其存活率分别下降了14.26％，24.76％，18.19％，26.79％和27.51％(P＜0.05)；同时，低表达OPN3基因后的A549_(PTX)细胞的存活率均有不同程度的上升，尤其在5nM组，其存活率升高了34.32％(P＜0.05)。
将OPN3基因克隆入pEGFPN2质粒，将重组质粒转染A549细胞、HFLF细胞和293细胞，激光共聚焦显微镜下观察融合蛋白的表达定位，结果显示：OPN3和GFP的融合蛋白在各组细胞的胞膜，胞浆及胞核均有散在表达，对照空载体的GFP蛋白在整个细胞内广泛表达，没有特异性。
综上，本研究首次发现OPN3基因与A549细胞对紫杉醇的耐药性相关，而且OPN3蛋白还具有很特别的细胞定位特征，值得进一步深入研究。
One mosquito opsin gene, NYD-OP7, has been cloned from Culexpipiens pallens. Within the sequence, an open reading frame (ORF) of1116 bp was found to encode a putative 371 amino acids protein whichexhibits high identity with opsins from Aedes and Anopheles mosquitoes.
Transcript expression of NYD-OP7 was determined by real-timePCR in all life stages of deltamethrin-susceptible and -resistant strains ofthe Culex mosquito. The results demonstrated that this gene is expressedat all developmental stages, and it is expressed predominantly at thepupae and adult stages. Meanwhile, in pupae and adults, NYD-OP7 isoverexpressed in deltamethrin-resistant strain than in -susceptible strain.Importantly, stable expression of NYD-OP7 in the mosquito C6/36 cellscan confer moderate deltamethrin resistance.
Our study provides the first direct evidence that increased expressionof an opsin gene may play some role in the development of deltamethrinresistance in Cx. pipiens pallens.
Opsin 3 (OPN3), also called encephalopsin or panopsin, is a recentlydiscovered extraretinal photoreceptor, and its function is largely unknownuntil now.
In this study, based on the RT-PCR results, we showed that OPN3was moderately expressed in the thymus, testis, liver, pancreas andplacenta and weakly in the prostate gland and kidney. Meanwhile, theexpression of OPN3 was significantly higher in the paclitaxel -resistantline than in the drug naive parental A549 cell line. Then we confirmed theoverexpression by real-time PCR analysis.
Using cDNA transfection and RNA interference, we successfullyestablished stable transfectants with upregulation and downregulation ofOPN3 expression, respectively. In vitro drug sensitivity assaydemonstrated that overexpression of OPN3 sensitized A549 cells topaclitaxel whereas downregulation of OPN3 conferred A549_(PTX) cellresistance to paclitaxel.
We also investigated cell localization of OPN3 by fusing it to anenhanced green fluorescent protein (EGFP). Green fluorescence signalwas found clearly in the nuclei, body and membrane of cells transfected with OPN3-EGFR In contrast, EGFP (vector control) was distributedmore or less evenly throughout the whole cell.
Our data for the first time suggests that OPN3 gene may play somerole in the development of paclitaxel resistance in human lung cancercells.
引文
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