一株新的致倦库蚊细胞系的建立与鉴定

【目的】以致倦库蚊Culex pipiens quinquefasciatus初孵幼虫组织为材料建立细胞系并对其进行分子鉴定。【方法】将实验室筛选的对硫磷抗性的致倦库蚊深桂品系的初孵幼虫剪碎后放入添加20%胎牛血清的改良M199培养基原代培养,并对原代细胞用胰蛋白酶传代培养成细胞系;通过显微镜观察其生物学特性以及DNA扩增指纹图谱和内转录间隔区序列鉴定其组织来源。【结果】该细胞系Cxq-1在M199完全培养基中生长良好,已成功传代培养12个月,并于液氮中冻存,并多次成功复苏。通过DNA扩增指纹图谱鉴定,Cxq-1与实验室培养的其他昆虫细胞DNA指纹图谱的条带各异,表明确为同源昆虫致倦库蚊细胞系...

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Bibliographic Details
Published in昆虫学报 Vol. 61; no. 1; pp. 79 - 85
Main Author 罗岚;崔峰
Format Journal Article
LanguageChinese
Published 2018
Subjects
致倦库蚊;细胞系;DNA扩增指纹图谱;内转录间隔区;分子鉴定
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Summary:【目的】以致倦库蚊Culex pipiens quinquefasciatus初孵幼虫组织为材料建立细胞系并对其进行分子鉴定。【方法】将实验室筛选的对硫磷抗性的致倦库蚊深桂品系的初孵幼虫剪碎后放入添加20%胎牛血清的改良M199培养基原代培养,并对原代细胞用胰蛋白酶传代培养成细胞系;通过显微镜观察其生物学特性以及DNA扩增指纹图谱和内转录间隔区序列鉴定其组织来源。【结果】该细胞系Cxq-1在M199完全培养基中生长良好,已成功传代培养12个月,并于液氮中冻存,并多次成功复苏。通过DNA扩增指纹图谱鉴定,Cxq-1与实验室培养的其他昆虫细胞DNA指纹图谱的条带各异,表明确为同源昆虫致倦库蚊细胞系。通过内转录间隔区序列测定技术鉴定,Cxq-1的r DNA-ITS1和r DNA-ITS2序列与Gen Bank中致倦库蚊相应核酸序列的一致性大于99%,表明其来源组织确为致倦库蚊。【结论】新建立的蚊初孵幼虫细胞系Cxq-1确为致倦库蚊细胞系。该细胞系为实验室今后开展杀虫剂抗性分子机制、蚊媒病毒、寄生虫等研究提供了重要平台。
Bibliography:11-1832/Q
Culex pipiens quinquefasciatus ; cell line ; DNA amplification fingerprinting ; internal transcribed spacer; molecular identification
Aim】 This study aims to establish and molecularly identify a cell line based on tissues from the newly hatched larvae of the mosquito Culex pipiens quinquefasciatus （Diptera： Culicidae）. 【Methods】 The shredded tissues of the newly hatched larvae of the parathionresistant strain Shengui of C. pipens quinquefasciatus were cultured in M199 complete medium supplemented with 20% heatinactivated fetal bovine serum, and the primary cells were obtained. The primary cells were treated with trypsin and subcultured into a cell line. The tissue origin of the cell line was identified by observing its biological characteristics under a microscope and comparing DNA amplification fingerprinting and sequencing the ribosome DNA internal transcribed spacer. 【Results】 The cell line Cxq-1 was well maintained in M199 complete medium and subcultured continually for 12 months. After being fr
ISSN:0454-6296