摘要
目的分析日本血吸虫Sj—TSP2序列多态性。方法抽提单条日本血吸虫成虫的总RNA，逆转录合成cDNA，PCR扩增目的片段。将目的片段克隆到载体pBlue2KSM中转化大肠杆菌，挑取10个阳性克隆测序。将获得的Sj—TSP2序列导入DnaSP序列分析软件进行分析，计算等位基因数目，鉴定单核苷酸多态性位点（singlenuclearpolymorphism，SNP）。同时将序列导入Mega4序列分析软件，计算错义突变率与同义突变率的比值（dN／dS），进行进化分析，构建进化树。结果共抽提了60条日本血吸虫成虫的总RNA，其中46条通过RT—PCR获得了目的基因Sj—TSP2；序列分析共鉴定了20个等位基因，主要是由点突变引起，Sj—TSP＇2-10是优势等位基因。共鉴定了85个SNP位点，对应于63个密码子的变异，其中36个是同义突变，27个是错义突变，有22个错义突变发生在膜外大环结构域中。结论引．11SP2基因高度变异，大多数突变存在膜外大环区，共鉴定了20个Sj—TSP＇2等位基因。
Objective To identify the polymorphic sites of Schistosoma japonicum tegument protein tetraspanin-2 （Sj-TSP＇2）. Methods Paired worms were separated in cold PBS with the help of forceps under a microscope. Total RNA of individual worms was extracted by Trizol reagent. First strands cDNAs were synthesized using reverse transcriptase. The open reading frame（ORF） of Sj-TSP2 was amplified by PCR. The resulting PCR products were sequenced after inserted into the pBlu2KSM vector between BamH I and EcoR I sites. DnaSP was used to calculate the alleles and the polymorphic sites. Mega 4 was used to build Neighbor- joining tree and calculate the ratio of non-synonymous to synonymous substitutions （dN/dS）. Results 46 full- length ORFs of Sj-TSP2 were successfully amplified by RT-PCR from 60 individual worms. In total, 92 full- length sequences were obtained from 46 worms, containing 20 unique alleles. 20 alleles were generated by point mutation and Sj-TSP2-10 was the predominant allele. There were a total of 85 polymorphism sites. Of the 63 resulting codon changes, 27 gave rise to amino acid substitution （ non-synonymous changes）. Among them, 22 amino acid substitutions were located in the extracellular loop 2. Conclusion Sj-TSP2 was an extensive polymorphic gene with 20 alleles identified.
出处
《国际医学寄生虫病杂志》
CAS
2011年第5期257-260,共4页
International JOurnal of Medical Parasitic Diseases
基金
基金项目：国家973重点基础研究发展计划（2007CB513100）
