日本血吸虫8k CaBP基因启动子区的克隆与分析

Cloning and analysis of the promoter region of Schistosoma japonicum 8k CaBP gene

目的克隆日本血吸虫8kCaBP(Sj8CaBP)基因启动子区并对该序列进行分析。方法提取日本血吸虫基因组DNA,并按ClontechUniversalGenomeWalkerTMKit的操作手册说明,建立日本血吸虫基因组DNA文库。根据手册要求及Sj8CaBP基因的cDNA序列,设计合成该基因染色体步移的特异性引物与试剂盒提供的端子引物进行巢式PCR。将得到的PCR产物克隆测序,测序结果用启动子区的分析软件及与其cDNA序列比对进行分析。结果得到的Sj8CaBP基因长度为1079bp(GenBank登录号AY262018),该基因包含Sj8CaBP基因完整的开放阅读框(ORF),包括1个内含子及2个外显子,在其5'端UTR区具有明显的启动子区,包括TATA盒及CAAT盒,没有发现GC盒及一些血吸虫基因启动子区所常见的AP-1(Activeprotein1)结合位点。结论从日本血吸虫基因组文库中获得一个长1079bp的Sj8CaBP基因片段(GenBank登录号为AY262018),经分析证明,该片段包含一个启动子区,具有TATA盒及CAAT盒的结构及1个内含子2个外显子.

Objective To clone and analyze the promoter sequence of Schistosoma japonicum 8k Calcium binding protein(CaBP) (Sj8CaBP) gene. Methods The genomic DNA was extracted from S. japonicum adult worms. The DNA library was constructed according to the user's manual for Clontech Universal Genome WalkerTM Kit , and the gene-specific primer 1 and 2 (GSP1 and GSP2) of Sj8CaBP gene were designed and synthesized. GSP1 and GSP2 were coupled with the adaptor primer 1 and 2 (AP1 and AP2), then nested PCR was performed. After proper purification, the PCR product was linked to pMD-18 T vector, and the recombinant T-vector was sequenced, analyzed with the promoter prediction software and compared with the cDNA sequence of Sj8CaBP. Results The cloned Sj8CaBP gene was about 1 079 bp in length ( GenBank accession No.AY262018), which contained a full open-reading frame with 1 intron and 2 exons. The 5'UTR has a putative promoter region including a TATA-box and a CCAAT-box. The GC-box binding region was not found in this gene, nor was that for active protein 1 (AP-1) often seen in other schistosome genes. Conclusion A 1 079 bp Sj8CaBP gene fragment (GenBank accession No.AY262018) was obtained from a S. japonicum genomic DNA library, containing a promoter region with TATA-box, CCAAT-box, 1 intron and 2 extrons.