一株新的致倦库蚊细胞系的建立与鉴定

摘要

[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％ heatinactivated 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 frozen in liquid nitrogen,Cxq-1 was successfully resuscitated for several times.DNA amplification fingerprinting polymerase chain reaction clearly distinguished Cxq-1 from two other insect cell lines.Sequencing of ribosome DNA internal transcribed spacer of Cxq-1 indicated that its rDNA-ITS1 and rDNA-ITS2 sequences has higher than 99％ identity with those of C.pipens quinquefasciatus registered in the GenBank,confirming the origin of Cxq-1.[Conclusion] The newly established cell line Cxq-1 from newly hatched larvae of C.pipens quinquefasciatus has been confirmed to be a cell line of this mosquito.This cell line provides an important platform for studying the molecular mechanisms of insecticide resistance,mosquito-borne viruses and parasites in the future.%[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rDNA-ITS1和rDNA-ITS2序列与GenBank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.