捻转血矛线虫伊维菌素耐药株P-糖蛋白基因多态性的PCR-RFLP分析

Polymorphism analysis of P-glycoprotein gene in Haemonchus contortusof ivermectin resistance strains by PCR-RFLP

为了解P-糖蛋白基因在捻转血矛线虫伊维菌素耐药株和敏感株间的差异,进一步弄清该基因与伊维菌素耐药性产生的关系,用PCR-RFLP方法分析了捻转血矛线虫伊维菌素耐药株P-糖蛋白基因hcpgp-1的多态性,并对所得序列进行测序。结果表明:捻转血矛线虫伊维菌素敏感株虫体的PCR产物不能被内切酶KpnⅠ切开,而伊维菌素耐药株虫体的PCR产物可被内切酶切开;酶切后统计数据显示,10条敏感虫株全部为敏感纯合子(BB);在所检测的20条伊维菌素耐药性虫株中,抗性纯合子(AA)6个(30%),杂合子(AB)13个(65%),敏感性纯合子(BB)1个(5%)。其中,AB基因型频率最大,为优势基因型,其次为AA型,BB型比例最低。抗性等位基因A为优势基因,其频率为62.5%,敏感等位基因B的频率为37.5%。同源性分析发现,具有抗性的糖蛋白基因hcpgp-1与敏感株的同源性较低,在80.2%～89.3%之间。且发现伊维菌素耐药株hcpgp-1基因序列中,有多处碱基发生了突变。本研究结果为进一步了解P-糖蛋白在捻转血矛线虫伊维菌素耐药性产生中的作用,提供了参考数据。

To study the differences of P-glycoprotein gene between ivermectin-resistant（HY） and sus- ceptible strains（HM） of Haemonchus contortus and further clarify the relationship between P-glycoprotein gene and ivermectin resistance in future, the polymorphism of P-glycoprotein gene from ivermectin-resis- rant strain of H. contortus in sheep was analyzed by PCR-restriction fragment length polymorphism. The result showed that the PCR products of HM group were resistive to the restriction enzyme Kpn I , whileHY group were vulnerable to the enzyme. The statistics data showed that 10 nematodes of ivermectin-sus- ceptible strains were all homozygote（BB）;in 20 nematodes of ivermectin-resistance strain, there were 6 ivermectin-resistant homozygotes （AA, 30 %）, 13 heterozygotes （AB, 65% ） and only 1 ivermectin-suscepti- ble homozygote（BB, 5 %）. The heterozygote（AB） was the most dominant genotype, followed by ivermectin- resistant homozygotes（AA）,and ivermectin-susceptible homozygote（BB） was the least. The resistance al- lele A was the most dominant gene,and took 62.5%,while allele B was only 37.5% of the samples. The homologous analysis revealed that the P-glycoprotein gene of ivermectin resistance（hcpgp-1） shared low homology（80. 2% to 89. 3%） with the susceptible strains. Besides, mutant sites on the hcpgp-1 gene of ivermectin-resistant strains were also found. This paper could provide a reference for studying the effects of P-glycoprotein gene in ivermectin resistance. And it also could supply basic information for the mechanism study of resistance to common anthelmintics of H. contortus.