日本血吸虫雄虫免疫血清对成虫cDNA文库的筛选及克隆分析

目的　筛选和分析日本血吸虫 (Schistosomajaponicum ,Sj)新基因 ,为血吸虫疫苗研究提供新的候选分子。 方法　以日本血吸虫雄性成虫抗原免疫兔血清为探针 ,筛选Sj成虫cDNA文库 ,对阳性克隆的插入片段进行PCR鉴定及测序分析。通过互联网对测序获得的核苷酸序列进行同源性分析 ,并预测新基因编码蛋白的结构与功能。结果　筛选获 11个阳性克隆 ,其插入SjcDNA片段大小在 0 7～ 2 3kb之间。对部分阳性克隆进行测序分析 ,获两个Sj新基因 ,即Sj-MA及Sj -Cp8(登录号分别为AF5 1980 8和AF5 2 4 896 ) ,分别编码 2 4 9和 71个氨基酸的核内蛋白和胞浆蛋白。Sj-MA蛋白含有 9个蛋白激酶C磷酸化位点、3个酪蛋白激酶Ⅱ磷酸化位点、2个酪氨酸激酶磷酸化位点和 1个N -肉豆蔻酸化位点。Sj-Cp8蛋白含一个跨膜区、3个酪蛋白激酶Ⅱ磷酸化位点、2个N -肉豆蔻酸化位点。结论　筛选Sj成虫cDNA文库所获得两个新基因 ,其编码的蛋白可能为存在于胞浆和胞核内的日本血吸虫的重要信息传递分子 ,有望成为新的血吸虫病疫苗候选分子。

Molecular cloning and characterization of two novel genes of Schistosoma japonicum immunoscreened from adult worm cDNA library with male worm immunized rabbit sera

Aim To clone and characterize new genes encoding vaccine candidate molecules of Schistosoma japonicum (Sj)Method Sj adult worm cDNA library was screened by sera from male worm immunized rabbits Positive clones were identified firstly by PCR and further by sequencing and data analysis through internet Nucleotide BLAST software of NCBI and Expert Protein Analysis System of Swiss Institute of Bioinformatics Result Eleven positive clones were obtained and their PCR product sizes ranged from 0 7～2 3kb Two novel genes named as Sj MA and Sj Cp8 respectively were confirmed by GenBank(GenBank accession number are AF519808 and AF524896 ) The deduced protein of Sj MA localized in nucleus and consisted of 249 amino acids,while Sj Cp8 in cytoplasm and of 71 amino acids Sj MA protein predicted containing nine protein kinase C phosphorylation site,three casein kinaseⅡ phosphorylation site,two tyrosine kinase phosphorylation site and one N myristoylation site Sj Cp8 had one possible transmembrane helix,three casein kinaseⅡ phosphorylation site and two N myristoylation site Conclusion The two novel genes from immunoscrrening of Sj adult worm cDNA predictably encodes two possibly important nuclear and cytoplasmic signal transduction molecules that might be new vaccine candidates against schistosomiasis