CAJ | 학술논문

目的构建细粒棘球蚴原头节的λZAPⅡcDNA文库,并对构建的文库进行免疫筛选.方法采集感染细粒棘球蚴的绵羊育囊中的原头节,用Trizol试剂抽提总RNA,用mRNA纯化试剂盒纯化mRNA作为模板合成cDNA,并对其进行末端平端化、EcoR Ⅰ接头连接和磷酸化及Xho Ⅰ酶切,然后应用Sepharose CL-2B分离柱对其进行分级分离,收集大于500 bp的cDNA片段,使用GigapackⅢGold试剂盒将合成的cDNA连接到λZAP-Ⅱ载体,并将重组载体包装到λZAP-Ⅱ噬菌体中,构建细粒棘球蚴cDNA文库.用囊型棘球蚴病患者混合血清对所构建的文库进行免疫筛选,并用NCBI中的Blast模块及其它生物信息软件对筛选出的阳性克隆进行生物信息学分析.结果成功构建了细粒棘球蚴原头节λZAPⅡcDNA文库,文库的滴度为1.8 ×105噬菌斑形成单位(plaque forming unit,pfu)/ml,插入片段长度范围在1.0～4.0 kb间,平均插入片段长度为2.6 kb,插入效率为93.8％.对所构建的文库进行免疫筛选,共筛选到5个阳性克隆,其中有2个为细粒棘球蚴新发现基因,新发现基因存在众多B细胞表位.结论成功构建了细粒棘球蚴原头节λZAPⅡcDNA文库,初步免疫筛选得到2个新发现基因,生物信息学分析预测它们具有潜在的诊断价值.
목적 구건세립극구유원두절적λZAPⅡcDNA문고,병대구건적문고진행면역사선.방법 채집감염세립극구유적면양육낭중적원두절,용Trizol시제추제총RNA,용mRNA순화시제합순화mRNA작위모판합성cDNA,병대기진행말단평단화、EcoR Ⅰ접두련접화린산화급Xho Ⅰ매절,연후응용Sepharose CL-2B분리주대기진행분급분리,수집대우500 bp적cDNA편단,사용GigapackⅢGold시제합장합성적cDNA련접도λZAP-Ⅱ재체,병장중조재체포장도λZAP-Ⅱ서균체중,구건세립극구유cDNA문고.용낭형극구유병환자혼합혈청대소구건적문고진행면역사선,병용NCBI중적Blast모괴급기타생물신식연건대사선출적양성극륭진행생물신식학분석.결과 성공구건료세립극구유원두절λZAPⅡcDNA문고,문고적적도위1.8 ×105서균반형성단위(plaque forming unit,pfu)/ml,삽입편단장도범위재1.0～4.0 kb간,평균삽입편단장도위2.6 kb,삽입효솔위93.8％.대소구건적문고진행면역사선,공사선도5개양성극륭,기중유2개위세립극구유신발현기인,신발현기인존재음다B세포표위.결론 성공구건료세립극구유원두절λZAPⅡcDNA문고,초보면역사선득도2개신발현기인,생물신식학분석예측타문구유잠재적진단개치.
Objective To construct the λ ZAP Ⅱ cDNA library of Echinococcus granulosus for screening of immunodiagnostic candidate molecules.Methods The protoscoleces of Echinococcus granulosus (E.granulosus) were isolated from the fertile cysts of E.granulosus infected sheep,and total RNA of the protoscoleces was extracted using Trizol reagent,then mRNA was purified from the total RNA using mRNA purification kit.cDNA was synthesized using cDNA synthesis kit with the purified mRNA as template,then blunt-ended,linked with EcoR Ⅰ adapter,phosphorylated,and cut by Xho Ⅰ enzyme using the λ ZAP Ⅱ EcoR I/CIAP-kit.The cDNA obtained was then separated by Sepharose CL-2B colum,and cDNA longer than 500 bp was collected and cloned into λ ZAP Ⅱ vector.The recombinant vectors were packaged into λ ZAP Ⅱ phage thus to constitute cDNA library.The cDNA library was immunoscreened by mixed patient sera of cystic echinococcosis and the positive clones obtained were sequenced and analysed by Blast module in NCBI and other informatics sofiwares.Results The λ ZAP Ⅱ cDNA library was constructed with the titer of 18 × 105plaque forming unit(pfu) /ml,the length range of the inserts 1.0 ～4.0 kb,the average length of 2.6 kb,and the insert efficiency of 93.8％. Five positive clones were obtained through immunoscreening of the cDNA library,and 2 of them are new genes of E.granulosus with multiple B cell epitopes.Conclusion The λ ZAP Ⅱ cDNA library of E. granulosus was successfully constructed and positive clones were obtained through immunoscreening,among them 2 clones are new genes which could become candidate molecules for diagnosing cystic echinococcosis.