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目的研究乳酸脱氢酶(LDH)与蚊溴氰菊酯抗性之间的关系。方法应用RT-PCR及cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术扩增白纹伊蚊LDH全长基因;用实时荧光定量PCR检测敏感和抗性蚊C6/36细胞中LDH基因的表达水平;用乳酸脱氢酶活性检测试剂盒检测敏感和抗性蚊细胞中LDH酶活性。结果 LDH基因全长共1831 bp,其中ORF为996 bp,共编码331个氨基酸。该基因与埃及伊蚊、黑腹果蝇、小家鼠和人的乳酸脱氢酶的同源性分别为89%、72%、66%和65%。实时荧光定量PCR结果表明,LDH基因在抗性蚊细胞中高表达,为敏感蚊细胞的1.87倍(P<0.05)。LDH酶活性检测结果显示,LDH在敏感蚊细胞中的酶活性范围为1～95 U/gprot,均值为40 U/gprot;在抗性蚊细胞中的酶活性范围为20～150 U/gprot,均值为75 U/gprot(n=15,P<0.01)。结论抗性蚊细胞中的LDH表达量和酶活性均高于敏感蚊细胞,提示LDH可能与蚊溴氰菊酯抗性相关。
Objective To study the relationship between lactate dehydrogenase (LDH) and deltamethrin resistance. Methods The LDH full-length gene of Aedes albopictus was amplified by RT-PCR and rapid amplification of cDNA ends (RACE). The LDH gene in sensitive and resistant mosquito C6 / 36 cells was detected by real-time fluorescence quantitative PCR The expression level of LDH in sensitive and resistant mosquito cells was detected by lactate dehydrogenase activity assay kit. Results The total length of LDH gene was 1831 bp, of which the ORF was 996 bp and encoded a total of 331 amino acids. The gene shares 89%, 72%, 66% and 65% identity with Aedes aegypti, Drosophila melanogaster, Mus musculus and human lactate dehydrogenase, respectively. Real-time PCR results showed that LDH gene was highly expressed in resistant mosquito cells, which was 1.87 times of that of sensitive mosquito cells (P <0.05). LDH enzyme activity test results showed that LDH enzyme activity in sensitive mosquito cells ranged from 1 to 95 U / gprot, with an average value of 40 U / gprot; and the enzyme activity range of resistant mosquito cells was 20 to 150 U / gprot, The mean was 75 U / gprot (n = 15, P <0.01). Conclusion LDH expression and enzyme activity in resistant mosquito cells were higher than those in sensitive mosquito cells, suggesting that LDH may be related to deltamethrin resistance.