CAJ | 학술논문

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(single nuclear polymorphism,SNP).同时将序列导入Mega 4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-P2-10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 Sj-TSP2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-TSP2等位基因.
목적 분석일본혈흡충Sj-TSP2서렬다태성.방법 추제단조일본혈흡충성충적총RNA,역전록합성cDNA,PCR확증목적편단.장목적편단극륭도재체pBlue2KSM중전화대장간균,도취10개양성극륭측서.장획득적Sj-TSP2서렬도입DnaSP서렬분석연건진행분석,계산등위기인수목,감정단핵감산다태성위점(single nuclear polymorphism,SNP).동시장서렬도입Mega 4서렬분석연건,계산착의돌변솔여동의돌변솔적비치(dN/dS),진행진화분석,구건진화수.결과 공추제료60조일본혈흡충성충적총RNA,기중46조통과RT-PCR획득료목적기인Sj-TSP2；서렬분석공감정료20개등위기인,주요시유점돌변인기,Sj-P2-10시우세등위기인.공감정료85개SNP위점,대응우63개밀마자적변이,기중36개시동의돌변,27개시착의돌변,유22개착의돌변발생재막외대배결구역중.결론 Sj-TSP2기인고도변이,대다수돌변존재막외대배구,공감정료20개Sj-TSP2등위기인.
Objective To identify the polymorphic sites of Schistosoma japonicum tegument protein tetraspanin-2 (Sj-TSP2).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 Neighborjoining tree and calculate the ratio of non-synonymous to synonymous substitutions (dN/dS).Results 46 fulllength ORFs of Sj-TSP2 were successfully amplified by RT-PCR from 60 individual worms.In total,92 fulllength 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.