日本血吸虫(中国大陆株)表达基因的分离和EST序列测定

ISOLATION AND IDENTIFICATION OF EXPRESSED SCHISTOSOMA JAPONICUM CHINESE STRAIN GENES FROM A DIRECTIONAL cDNA LIBRARY BY EST STRATEGY

目的 运用表达序列标签技术（ＥＳＴ方法） ，从ＳｊｃＤＮＡ文库中分离、鉴定和测定血吸虫表达基因序列。方法 应用ＥＳＴ方法，从Ｓｊ ｃＤＮＡ 文库中随机挑选出单个重组克隆进行ＰＣＲ直接序列分析，通过互联网将获得的ＥＳＴ 序列送入ＮＣＢＩＧｅｎＢａｎｋ 进行同源性检索，并将发现的未知基因ＥＳＴ序列送入ＮＣＢＩｄｂＥＳＴ 以获得ＧｅｎＢａｎｋ 进入号。结果 分离了１００ 个Ｓｊ重组克隆，并已对２５ 个克隆ｃＤＮＡ 插入片段的ＰＣＲ 产物进行直接序列分析，获得了２１ 个ＥＳＴ 序列。其中２ 个ＥＳＴ 序列为ＧｅｎＢａｎｋ 中已知的血吸虫基因序列，１９ 个为未知基因序列。这１９ 个ＥＳＴ序列已被ＮＣＢＩｄｂＥＳＴ和ＧｅｎＢａｎｋ 接受并获得了新序列进入号。结论 采用ＥＳＴＰＣＲ直接序列分析方法可大量获得血吸虫表达基因ＥＳＴ序列，为进一步开展日本血吸虫（中国大陆株） 表达基因的研究奠定了基础。

Aim To isolate and identify expressed Schistosoma japonicum(Chinese strain) genes from a directional cDNA library by expressed sequence Tags (EST) strategy for searching Schistosoma vaccine candidates and Schistosoma Genome Project Methods the cDNA clones were selected randomly,and the ESTs were obtained by PCR directed sequencing from 5’ end of the sequence,then the ESTs were compared with all sequences in GenBank data bases The novel ESTs were submitted to NCBI dbEST and GenBank for obtaining accession numbers Results 100 recombinant clones were picked up from the cDNA,and the inserts of cDNA were identified by PCR 25 ESTs were generated by PCR directed sequencing Among them,2 ESTs belonged to“identified genes of Schistosoma mansoni”for they showed matched with Schistosome sequences in the databases 19 ESTs were defined as novel genes EST sequences for they showed only partial homology with non Schistosome genes or other organism in the database These novel genes ESTs were submitted into NCBI dbEST and were given the accession numbers of sequence in GenBank Conclusion This study shows that EST strategy,i e picking up recombinant cDNA clones and obtaining partial sequences of cDNAS can been used to discover Schistosoma japonicum expressed genes