卫氏并殖吸虫成虫λZAP Ⅱ cDNA文库的构建及诊断候选抗原的筛选

Construction of a Paragonimus westermani ZAP Ⅱ cDNA library and screening of potential diagnostic antigens

目的构建卫氏并殖吸虫成虫λZAPⅡcDNA文库,使用卫氏并殖吸虫病患者混合血清筛选诊断候选抗原。方法用卫氏并殖吸虫囊蚴感染实验犬,约70 d后取犬肺,收集卫氏并殖吸虫成虫,提取虫体总RNA,经mRNA纯化试剂盒纯化后构建卫氏并殖吸虫成虫的λZAPⅡcDNA文库。用卫氏并殖吸虫病患者混合血清筛选λZAPⅡcDNA文库,获得阳性克隆,对插入片段测序并进行同源性分析。结果构建的卫氏并殖吸虫成虫λZAPⅡcDNA文库的滴度为5.2×105 pfu/ml,重组率为98.0%,文库插入片段平均长度为1.1 kb。经免疫筛选获得PwⅠ、PwⅡ、PwⅢ和PwⅣ等4类阳性克隆。其代表克隆Pw1、Pw13、Pw18和Pw21的插入片段长度依次约为720、1263、1052、717 bp,均包含开放阅读框,顺序编码213、394、324和209个氨基酸,分别与卫氏并殖吸虫卵黄铁蛋白(yolk ferritin)基因、虫卵抗原(egg antigen)基因、前原组织蛋白酶L(pre-procathepsin L)基因和华支睾吸虫硫氧还蛋白过氧化物酶(thioredoxin peroxidase)基因同源。结论成功构建了卫氏并殖吸虫成虫λZAPⅡcDNA文库,筛选获得4类被卫氏并殖吸虫病患者血清特异性识别的阳性克隆,为进一步寻找卫氏并殖吸虫免疫诊断抗原奠定了基础。

Objective To construct aλZAP II cDNA library of Paragonimus westermani and immunoscreen potential antigens for immuno-diagnosis of a P.westermani infection.Methods P.westermani samples were collected from dogs infected with metacercariae 70 days post-infection.Total RNA of P.westermani was extracted and mRNA obtained using a purification kit was used to construct aλZAP II cDNA-library.The library was immunoscreened with pooled sera from 10 patients infected with P.westermani to obtain positive clones.The inserted fragments of positive clones were identified via amplification with PCR,and the obtained genes were sequenced and analyzed for their homology.Results The titer of the library was 5.2×10~5 pfu/ml.The average length of the inserted fragment in the library was 1.1 kb with a recombination efficiency of 98.0%.Four positive clones,Pw_I,Pw_II,Pw_IIIⅢ,and Pw_IVⅣwere identified,and theirs respective clones Pw1,Pw13,Pw18,and Pw21 had an inserted fragment length of about 720,1,263,1,052,and 717 bp,respectively.Sequencing revealed that all 4 clones contained open reading frames.The deduced amino acid sequences of the 4 clones contained 213,394,324,and 209 amino acid residues.They were homologous with the P.westermani yolk ferritin gene,P.westermani egg antigen gene,P.westermani pre-procathepsin L,and C.sinensis thioredoxin peroxidase gene,respectively.Conclusion AλZAP II cDNA library of P.westermani has been constructed and 4 positive clones have been identified.These clones are recognized by mixed sera from people infected with P.westermani.This study has provided preliminary information for further identification of highly reactive antigens in order to develop immunodiagnostics for a P.westermani infection.