捻转血矛线虫锌金属蛋白酶NAS-31的表达特性及功能初步研究  

Primary study on the expression pattern and function of zinc metalloproteinase NAS-31 in Haemonchus contortus

作　　者：黄艳 张惠 童丹妮 周静茹 吴飞 陈学秋[1] 杨怡 马光旭 杜爱芳[1] HUANG Yan;ZHANG Hui;TONG Danni;ZHOU Jingru;WU Fei;CHEN Xueqiu;YANG Yi;MA Guangxu;DU Aifang(Institute of Preventive Veterinary Medicine,College of Animal Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学动物科学学院动物预防医学研究所,杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2021年第6期805-814,共10页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家重点研发计划(2017YFD0501203);国家自然科学基金(32002304);浙江省基础公益计划(LGN18C180002);国家重点基础研究发展计划(2015CB150300)。

摘　　要：捻转血矛线虫病由捻转血矛线虫(Haemonchus contortus)寄生于反刍动物皱胃之中引起。为探究捻转血矛线虫锌金属蛋白酶NAS-31基因(Hc-nas-31)在虫体自由生活时期(特别是3期幼虫)中发挥的功能,本研究通过实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)检测目的基因在捻转血矛线虫各发育阶段的转录水平,利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)、基因步移及融合PCR方法扩增并分析其基因结构;构建原核表达载体后转化至BL21感受态细胞中进行诱导表达;将纯化后的蛋白免疫新西兰大白兔以制备多克隆抗体;通过虫体间接免疫荧光分析Hc-NAS-31在捻转血矛线虫体内的表达特性并预测其功能。结果表明:Hc-nas-31在各时期虫体中均有转录,其中在3期幼虫中的转录水平最高;蛋白质印迹结果显示,制备的抗体能特异性结合Hc-NAS-31天然蛋白;间接免疫荧光分析发现,Hc-NAS-31蛋白主要表达于3期幼虫的上皮细胞合胞体内,在成虫中则分布于肠道、生殖腺(性腺)、肌肉组织及早期虫卵中。本实验明确了NAS-31在捻转血矛线虫中的表达特性,为进一步研究Hc-nas-31的生物学功能奠定了基础。Haemonchosis is caused by Haemonchus contortus parasitic in the ruminant abomasum.In order to study the function of zinc metalloproteinase NAS-31 gene(Hc-nas-31)in the free-living period(especially L3)of this kind of worm,real-time quantitative polymerase chain reaction(qRT-PCR)was conducted to detect the transcription level of the target gene in different developmental stages of H.contortus.Rapid amplification of cDNA ends(RACE),genome walking and fusion PCR methods were used to amplify Hc-nas-31 gene and the gene structure was analyzed after that.Then,prokaryotic expression plasmid was constructed and transformed into BL21 to express the recombinant protein.A New Zealand white rabbit was immunized with the purified recombinant Hc-NAS-31(rHc-NAS-31)to prepare polyclonal antibodies,which were used to analyze the expression pattern of Hc-NAS-31 in H.contortus by indirect immunofluorescence assay.The results showed that Hc-nas-31 was transcribed in all stages of the worms,while the transcription level of L3 was the highest.Polyclonal antibody was made successfully and its specific binding with Hc-NAS-31 natural protein was confirmed by Western blotting.Indirect immunofluorescence analysis showed that Hc-NAS-31 protein was mainly expressed in the epithelial syncytia of L3,while in adults,it was distributed in the intestines,gonads,muscle tissues and early eggs.In summary,we confirmed the expression pattern of NAS-31 in H.contortus.This experiment lays the foundation for further research on the biological functions of Hc-nas-31.

关 键 词：捻转血矛线虫 捻转血矛线虫锌金属蛋白酶NAS-31基因 转录水平 表达特性 

分 类 号：S855.9[农业科学—临床兽医学]