摘要
目的获取日本血吸虫(Sj)精氨酸酶(ARG)新基因的DNA编码序列及其启动子序列,实验验证ARG基因编码序列的完整性。方法以日本血吸虫成虫DNA做模板,PCR扩增ARG基因编码区的DNA序列并测序;根据SjARG基因已扩增的DNA序列设计2条巢式引物,用TaKaRaLATaqPCRCloninginvitroKit,扩增SjARG基因的启动子序列;对已扩增得到的序列进行TATA盒的寻找以分析启动子序列的位置,实验验证我们曾获取的SjARG新基因编码序列的完整性。结果扩增得到长约1000bpARG基因DNA序列,SjARG基因DNA序列内没有内含子,与cDNA序列完全一致。对启动子序列扩增后,得到一略大于250bp的序列,测序后分析发现其中有36bp与ARG基因DNA序列的5'端重叠。因此,将精氨酸酶基因的DNA序列又向前延伸了221bp,与前面得到的序列拼接后得到一条1486bp的总序列,将该序列在NCBI上进行ORF的寻找发现其最长的ORF达1164bp,起始密码子在第156位,终止密码子在第1319位,该起始密码子前32位为TATA盒的位置。因此,确定该SjARG基因全长cDNA编码序列长应为1164bp。结论成功扩增获得了日本血吸虫精氨酸酶基因编码区的DNA序列,其不含有内含子;并扩增得到了该基因的启动子序列,从而获得了SjARG基因真正的全长cDNA序列,为进一步的功能鉴定奠定了基础。
Objective To obtain DNA sequence of Schistosoma japonicum (Sj) arginase (ARG) novel gene and its promoter and to identify the completeness of their CDS. Methods Prepare DNA from Schistosoma japonicum adult worms, then amplify the corresponding sequences of DNA by using the same primers for amplifying CDS. Compare DNA with cDNA sequences by ClustalW in EBI to find out if there were introns among the DNA sequences. Amplify the promoter sequence of ARG gene by using TaKaRa LA Taq PCR Cloning in Vitro Kit, then analyze the position of TATA box. Results DNA sequences of Sj ARG gene was amplified from DNA of Sj adult worms. Comparing the cDNA sequences with the DNA sequences of Sj ARG showed that the two sequences of Sj ARG had no difference. It was extended for 69bp from amplifying promoter sequence by TaKaRa LA Taq PCR Cloning in Vitro Kit. There existed a sequence of 32bp before initiation codon ATG named, TATA box. The real ORF length of ARG gene should be 1 164bp. Conclusion The DNA sequence encoding Sj arginase was firstly sequenced and proved to have no intron. As the ARG gene's promoter sequence was amplified in this study, the full cDNA length of the arginase was obtained for further study.
出处
《热带医学杂志》
CAS
2005年第1期1-4,40,共5页
Journal of Tropical Medicine
关键词
日本血吸虫
大陆株
精氨酸酶
启动子序列
序列分析
Schistosoma japonicum
Chinese strain
arginase
promoter sequence
sequence analysis
