蚊抗药性相关新基因NYD-Ch和NYD-Tr的初步功能研究
发布时间：2018-12-18 06:48
【摘要】：【目的】研究抗药性相关糜蛋白酶基因(NYD-Ch)和胰蛋白酶基因(NYD-Tr)与抗药性之间的关系。 【材料和方法】采用PCR方法,以淡色库蚊抗性品系cDNA文库为模板,扩增出抗性品系中高表达的NYD-Ch和NYD-Tr,经T/A克隆测序鉴定后,扩增纯化质粒,经酶切后纯化目的片段亚克隆到真核表达载体pIE1-3中,用PCR结合酶切鉴定,核酸测序验证。将上述重组表达载体分别单独转入和共转入蚊细胞,经筛选获得稳定转染的细胞系,RT-PCR和Western blotting鉴定目的基因的稳定表达,并用MTT和流式检测稳定表达的细胞对溴氰菊酯杀虫剂的敏感性。 【结果】成功构建昆虫细胞表达载体并建立稳定转染细胞系,使目的基因在其中稳定表达。MTT结果显示:pIE1-3/NYD-Ch单独转染组,细胞存活率与对照组无显著性差异(p0.05);pIE1-3/NYD-Tr单独转染组,120μM溴氰菊酯浓度时,其细胞存活率较空载体组和未转染组分别增加38.20%(p0.05)和9.66%(p0.05);pIE1-3/NYD-Ch和pIE1-3/NYD-Tr共转染组,120μM溴氰菊酯浓度时,其细胞存活率较空载体组和未转染组分别增加67.37%(p0.01)和38.83%(p0.001);160μM药物浓度时,其细胞存活率较空载体组和未转染组分别增加48.83%(p0.05)和24.26%(p0.05)。 【结论】NYD-Ch和NYD-Tr在细胞对抗溴氰菊酯的毒性作用中有一定的保护力,提示NYD-Ch和NYD-Tr与抗药性相关。
[Abstract]:[objective] to study the relationship between drug resistance associated chymotrypsin gene (NYD-Ch) and trypsin gene (NYD-Tr) and drug resistance. [materials and methods] using cDNA library of Culex pipiens pallens resistance strain as template, the highly expressed NYD-Ch and NYD-Tr, in the resistant lines were amplified by PCR method, and the purified plasmids were amplified by T / A cloning and sequencing. The target fragment was subcloned into eukaryotic expression vector pIE1-3 after digested by enzyme, identified by PCR binding enzyme digestion and verified by nucleic acid sequencing. The recombinant expression vectors were transferred into mosquito cells separately and cotransferred into mosquito cells. Stable transfected cell lines were obtained by screening. The stable expression of the target gene was identified by RT-PCR and Western blotting. The sensitivity of stably expressed cells to deltamethrin was detected by MTT and flow cytometry. [results] insect cell expression vector was successfully constructed and stable transfection cell line was established, in which the target gene was stably expressed. MTT results showed that pIE1-3/NYD-Ch was transfected alone. There was no significant difference between the cell survival rate and the control group (p0.05). The cell survival rate of pIE1-3/NYD-Tr alone transfected with 120 渭 M deltamethrin increased by 38.20% (p0.05) and 9.66% (p0.05) respectively compared with the empty vector group and the untransfected group. The cell survival rate of pIE1-3/NYD-Ch and pIE1-3/NYD-Tr co-transfected group increased 67.37% (p0.01) and 38.83% (p0.001) respectively compared with empty vector group and untransfected group at 120 渭 M deltamethrin concentration. The cell survival rate was increased by 48.83% (p0.05) and 24.26% (p0.05) respectively at 160 渭 M concentration compared with empty vector group and untransfected group. [conclusion] NYD-Ch and NYD-Tr have protective effect on the cytotoxicity of deltamethrin, suggesting that NYD-Ch and NYD-Tr are related to drug resistance.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R384.1
本文编号：2385514
[Abstract]:[objective] to study the relationship between drug resistance associated chymotrypsin gene (NYD-Ch) and trypsin gene (NYD-Tr) and drug resistance. [materials and methods] using cDNA library of Culex pipiens pallens resistance strain as template, the highly expressed NYD-Ch and NYD-Tr, in the resistant lines were amplified by PCR method, and the purified plasmids were amplified by T / A cloning and sequencing. The target fragment was subcloned into eukaryotic expression vector pIE1-3 after digested by enzyme, identified by PCR binding enzyme digestion and verified by nucleic acid sequencing. The recombinant expression vectors were transferred into mosquito cells separately and cotransferred into mosquito cells. Stable transfected cell lines were obtained by screening. The stable expression of the target gene was identified by RT-PCR and Western blotting. The sensitivity of stably expressed cells to deltamethrin was detected by MTT and flow cytometry. [results] insect cell expression vector was successfully constructed and stable transfection cell line was established, in which the target gene was stably expressed. MTT results showed that pIE1-3/NYD-Ch was transfected alone. There was no significant difference between the cell survival rate and the control group (p0.05). The cell survival rate of pIE1-3/NYD-Tr alone transfected with 120 渭 M deltamethrin increased by 38.20% (p0.05) and 9.66% (p0.05) respectively compared with the empty vector group and the untransfected group. The cell survival rate of pIE1-3/NYD-Ch and pIE1-3/NYD-Tr co-transfected group increased 67.37% (p0.01) and 38.83% (p0.001) respectively compared with empty vector group and untransfected group at 120 渭 M deltamethrin concentration. The cell survival rate was increased by 48.83% (p0.05) and 24.26% (p0.05) respectively at 160 渭 M concentration compared with empty vector group and untransfected group. [conclusion] NYD-Ch and NYD-Tr have protective effect on the cytotoxicity of deltamethrin, suggesting that NYD-Ch and NYD-Tr are related to drug resistance.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R384.1
【引证文献】
相关博士学位论文 前1条
1 胡小邦;视蛋白基因的克隆及其与抗药性/耐药性关系的研究[D];南京医科大学;2007年
相关硕士学位论文 前1条
1 段晓雷;PR-XP1基因的克隆及在C6/36耐药细胞株中抗性的初步研究[D];西北农林科技大学;2011年
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