国际医学寄生虫病杂志
國際醫學寄生蟲病雜誌
국제의학기생충병잡지
INTERNATIONAL JOURNAL OF MEDICAL PARASITIC DISEASES
2013年
3期
130-134
,共5页
孙嘉慧%徐斌%鞠川%陈军虎%卢艳%陈韶红%韩建平%胡薇
孫嘉慧%徐斌%鞠川%陳軍虎%盧豔%陳韶紅%韓建平%鬍薇
손가혜%서빈%국천%진군호%로염%진소홍%한건평%호미
田鼠巴贝虫%cDNA文库%免疫筛选
田鼠巴貝蟲%cDNA文庫%免疫篩選
전서파패충%cDNA문고%면역사선
Babesia microti%cDNA library%Immunoscreening
目的 构建田鼠巴贝虫cDNA文库,从中筛选免疫诊断候选抗原. 方法 用田鼠巴贝虫感染BALB/c小鼠,约7d后待虫血密度达到70％时取血,提取虫体总RNA,经mRNA纯化试剂盒纯化后,构建田鼠巴贝虫的cDNA文库.用田鼠巴贝虫感染阳性小鼠血清筛选cDNA文库,获得阳性克隆,PCR鉴定阳性克隆插入片段大小,测序并进行同源性分析. 结果 所构建的田鼠巴贝虫cDNA文库的滴度为5.2×105噬菌斑形成单位(plaque forming unit,pfu)/ml,重组率为91.0％,文库插入片段平均长度为1.1 kb.从cDNA文库中筛选获得Bm2、Bm4、Bm6、Bm7、Bm9和Bm15等6个阳性克隆,插入片段依次约为633、614、1 073、890、1 001和1 032 bp,6个片段均包含开放阅读框,依次编码159、100、254、217、60和244个氨基酸,相对分子质量(Mr)依次约为17 900、11 400、28 600、24 000、6 800和28 900. 结论 构建了田鼠巴贝虫cDNA文库,并筛选获得6个能被感染阳性小鼠血清识别的田鼠巴贝虫阳性克隆,为进一步筛选田鼠巴贝虫免疫诊断抗原奠定了基础.
目的 構建田鼠巴貝蟲cDNA文庫,從中篩選免疫診斷候選抗原. 方法 用田鼠巴貝蟲感染BALB/c小鼠,約7d後待蟲血密度達到70％時取血,提取蟲體總RNA,經mRNA純化試劑盒純化後,構建田鼠巴貝蟲的cDNA文庫.用田鼠巴貝蟲感染暘性小鼠血清篩選cDNA文庫,穫得暘性剋隆,PCR鑒定暘性剋隆插入片段大小,測序併進行同源性分析. 結果 所構建的田鼠巴貝蟲cDNA文庫的滴度為5.2×105噬菌斑形成單位(plaque forming unit,pfu)/ml,重組率為91.0％,文庫插入片段平均長度為1.1 kb.從cDNA文庫中篩選穫得Bm2、Bm4、Bm6、Bm7、Bm9和Bm15等6箇暘性剋隆,插入片段依次約為633、614、1 073、890、1 001和1 032 bp,6箇片段均包含開放閱讀框,依次編碼159、100、254、217、60和244箇氨基痠,相對分子質量(Mr)依次約為17 900、11 400、28 600、24 000、6 800和28 900. 結論 構建瞭田鼠巴貝蟲cDNA文庫,併篩選穫得6箇能被感染暘性小鼠血清識彆的田鼠巴貝蟲暘性剋隆,為進一步篩選田鼠巴貝蟲免疫診斷抗原奠定瞭基礎.
목적 구건전서파패충cDNA문고,종중사선면역진단후선항원. 방법 용전서파패충감염BALB/c소서,약7d후대충혈밀도체도70％시취혈,제취충체총RNA,경mRNA순화시제합순화후,구건전서파패충적cDNA문고.용전서파패충감염양성소서혈청사선cDNA문고,획득양성극륭,PCR감정양성극륭삽입편단대소,측서병진행동원성분석. 결과 소구건적전서파패충cDNA문고적적도위5.2×105서균반형성단위(plaque forming unit,pfu)/ml,중조솔위91.0％,문고삽입편단평균장도위1.1 kb.종cDNA문고중사선획득Bm2、Bm4、Bm6、Bm7、Bm9화Bm15등6개양성극륭,삽입편단의차약위633、614、1 073、890、1 001화1 032 bp,6개편단균포함개방열독광,의차편마159、100、254、217、60화244개안기산,상대분자질량(Mr)의차약위17 900、11 400、28 600、24 000、6 800화28 900. 결론 구건료전서파패충cDNA문고,병사선획득6개능피감염양성소서혈청식별적전서파패충양성극륭,위진일보사선전서파패충면역진단항원전정료기출.
Objective To construct a cDNA library of Babesia microti and immunoscreen candidate antigens for immuno-diagnosis of Babesia infection.Methods The blood samples were collected from the mice infected with B.microti 7 days post infection when the parasitemia density reached 70％.Total RNA of B.microti was extracted and the mRNA obtained by a purifying kit was used to construct the cDNA-library.The library was immunoscreened with pooled sera from B.microti infected mice to obtain positive clones.The inserted fragments of positive clones were identified by PCR amplification,and the obtained genes were sequenced and analyzed for their homology.Results The titer of the library was 5.2 × 101 plaque forming unit (pfu)/ml.The inserted fragment length of the library ranged from 500 to 3 000 bp with a recombination efficiency being 91.0％.Six positive clones,Bm2,Bm4,Bm6,Bm7,Bm9 and Bm15 were found and their inserted fragment length was about 633,614,1 073,890,1 001 and 1 032 bp,respectively.Sequence analysis revealed that all the 6 clones contained open reading frames.The deduced amino acid sequences of the 6 clones contained 159,100,254,217,60 and 244 amino acid residues,with Mr of 17 900,11 400,28 600,24 000,6 800 and 28 900,respectively.Conclusion A cDNA library of B.microti has been constructed and 6 positive clones identified,which can be recognized by the sera of mice infected with B.microti.This study provided preliminary information for further identification of highly reactive antigens for development of immunodiagnostics of B.microti infection.
