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

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。

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

To study the differences of P-glycoprotein gene between ivermectin-resistant(HY) and susceptible 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-tant 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 KpnⅠ,while HY group were vulnerable to the enzyme.The statistics data showed that 10 nematodes of ivermectin-susceptible 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-susceptible 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 allele 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.