微口膜壳绦虫DAZ基因的原核表达、转录水平及组织定位研究  

Prokaryotic expression,transcription level analysis and tissue localization of DAZ gene in Hymenolepis microstoma

作　　者：马静 张艳艳[2] 贾新月 马勋[1] 王正荣[2] 薄新文[1,2] MA Jing;ZHANG Yan-yan;JIA Xin-yue;MA Xun;WANG Zheng-rong;BO Xin-wen(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;State Key Laboratory of sheep Genetic Improvement and Healthy Production/Institute of Animal Husbandry and Veterinary,Xinjiang Academy of Agricultural and Reclamation Sciences,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]新疆农垦科学院畜牧兽医研究所省部共建绵羊遗传改良与健康养殖国家重点实验室,新疆石河子832000

出　　处：《中国预防兽医学报》2023年第2期129-137,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：新疆生产建设兵团国际科技合作(2020BC007、2021BC008);国家自然科学基金(31860701);省部共建绵羊遗传改良与健康养殖国家重点实验室重大专项(2021DZ02)。

摘　　要：为研究微口膜壳绦虫无精子症(DAZ)基因的生物学功能,本研究利用在线软件对WormBase中微口膜壳绦虫DAZ基因(HmN_000202400)及其编码蛋白进行生物信息学分析,并构建DAZ基因的遗传进化树。结果显示,微口膜壳绦虫DAZ基因cDNA全长1494 bp,编码497个氨基酸,存在保守结构域和完整的开放阅读框,无跨膜区和信号肽,且与缩小膜壳绦虫和微小膜壳绦虫亲缘关系较近。本研究经PCR扩增DAZ基因,构建重组质粒pET-22b-DAZ,经双酶切鉴定正确后经IPTG诱导表达,并采用His-Ni柱亲和层析法纯化,经SDS-PAGE检测结果显示,所表达的重组DAZ蛋白(rDAZ)以包涵体形式存在,分子量约为57 ku,且纯化效果较好。利用rDAZ免疫新西兰大白兔,制备兔rDAZ的多克隆抗体,经间接ELISA检测其效价,并利用western blot检测其反应原性。结果显示,该多克隆抗体效价可达1∶128000,且能够与微口膜壳绦虫的天然总蛋白反应,表明获得了反应原性较好的兔rDAZ的多克隆抗体。为研究DAZ的生物学特性,本研究采用荧光定量PCR(qPCR)分别检测DAZ基因在微口膜壳绦虫成虫、似囊尾蚴及虫卵中的相对转录水平;将微口膜壳绦虫制备石蜡切片观察虫体的各组织结构;利用制备的兔rDAZ多克隆抗体采用间接免疫荧光试验(IFA)检测DAZ蛋白在虫体各组织中的定位。qPCR结果显示,DAZ基因在微口膜壳绦虫成虫、似囊尾蚴及虫卵中均不同程度转录,与其虫卵相比,DAZ基因在似囊尾蚴中的转录水平极显著升高(P<0.01),但在成虫中显著降低(P<0.05);石蜡切片观察结果显示,微口膜壳绦虫生殖系统发达,发育成熟的睾丸、卵巢、卵黄腺、子宫、受精囊等主要分布在成节中,而孕节内的其他生殖器官则逐渐退化,唯有子宫扩大并充满虫卵;IFA结果显示,在微口膜壳绦虫的睾丸中可见特异性绿色荧光,而卵巢、卵黄腺、子宫、受精囊等中则无该绿色荧光。上述结果表明,DAZ基�In order to study the biological function of deleted in azoospermia(DAZ)gene of Hymenolepis microstoma,we used online software to analyse the H.microstoma DAZ gene(HmN_00202400)and its encoded protein in WormBase,and constructed the phylogenetic tree of DAZ gene.The results showed that the cDNA of DAZ gene was 1494bp in length,and encoding 497 amino acids,which has conserved domain and complete open reading frame,but no transmembrane region and signal peptide.It was genetically close to H.diminuta and H.nana.In this study,the DAZ gene sequence was amplified by PCR,and the recombinant plasmid pET-22b-DAZ was constructed.The recombinant plasmid pET-22b-DAZ was identified by double enzyme digestion and then induced by IPTG.The recombinant DAZ protein(rDAZ)was purified by His-Ni column affinity chromatography.The SDS-PAGE detection results showed that the rDAZ with molecular weight of about 57ku existed in the form of inclusion body and the purification effect was good.New Zealand white rabbits were immunized with rDAZ to prepare rabbit polyclonal antibody.The titer was determined by indirect ELISA,and its reactivity was detected by western blot.The results showed that the titer of the polyclonal antibody was up to 1∶128000,and it could react to the natural total protein of H.microstoma,indicating that the rabbit polyclonal antibody with good reactivity against rDAZ was obtained.In order to study the biological characteristics of DAZ,fluorescence quantitative PCR(qPCR)was used to detect the relative transcriptional level of DAZ gene in the adult,cysticercoid and egg of H.microstoma.The paraffin sections of H.microporum were prepared and the tissue structures of the worm were observed.The localization of DAZ protein in various tissues of the worm was detected by indirect immunofluorescence assay(IFA)using the rabbit polyclonal antibody against rDAZ.The results of qPCR showed that the DAZ gene was transcribed in different degrees in the adult,cysticercoid and egg of H.microstoma.Compared to the egg,the transcription

关 键 词：微口膜壳绦虫 DAZ基因 原核表达 多克隆抗体 间接免疫荧光试验 

分 类 号：S852.73[农业科学—基础兽医学]