CAJ | 학술논문

目的从日本血吸虫成虫cDNA中扩增出溶血磷脂酶编码基因(Si1539),并亚克隆至真核表达载体pcDNA3.1(+),旨在提取重组抗原进行免疫原性分析,并用于免疫学诊断.方法提取日本血吸虫成虫总RNA,反转录生成cDNA,PCR法扩增出Sj1539基因,通过克隆载体pGEM-T连接,亚克隆至真核表达载体pcDNA3.1(+),然后转染至人宫颈癌细胞株(HeLa细胞)中表达,表达产物用Western印迹法进行鉴定.结果 Sj1539基因PCR扩增产物片段长度为684 bp.重组质粒pcDNA3.1(+)-Sj1539经BamH I、Xho I双酶切和PCR扩增鉴定,证实Sj1539基因已成功插入.重组质粒pcDNA3.1(+)-Sj1539在HeLa细胞中的表达产物经Western印迹法鉴定能够与日本血吸虫感染的兔血清反应,其相对分子质量(Mr)约为25×103.结论成功构建了日本血吸虫Si1539基因真核表达载体,并获得了表达产物.
목적 종일본혈흡충성충cDNA중확증출용혈린지매편마기인(Si1539),병아극륭지진핵표체재체pcDNA3.1(+),지재제취중조항원진행면역원성분석,병용우면역학진단.방법 제취일본혈흡충성충총RNA,반전록생성cDNA,PCR법확증출Sj1539기인,통과극륭재체pGEM-T련접,아극륭지진핵표체재체pcDNA3.1(+),연후전염지인궁경암세포주(HeLa세포)중표체,표체산물용Western인적법진행감정.결과 Sj1539기인PCR확증산물편단장도위684 bp.중조질립pcDNA3.1(+)-Sj1539경BamH I、Xho I쌍매절화PCR확증감정,증실Sj1539기인이성공삽입.중조질립pcDNA3.1(+)-Sj1539재HeLa세포중적표체산물경Western인적법감정능구여일본혈흡충감염적토혈청반응,기상대분자질량(Mr)약위25×103.결론 성공구건료일본혈흡충Si1539기인진핵표체재체,병획득료표체산물.
Objective Schistasoma japonicum(S.japonicum)lysophospholipase gene(Sjl539)from cDNA of S japonicum adult worms was amplified and subcloned into eukaryotic expression vector pcDNA3.1(+)for expression of recombinant antigen and immunogenicity analysis.Methods Total RNA of S.japonicum was extracted to generato cDNA by RT-PCR.The Sj1539 gent was amplified.The DNA fragment was subcloned into eukaryofic expression vector pcDNA3.1(+)following insertion and amplification in pGEM-T.The recombinant plasmid was transfected into human cervical carcinoma cell strain(Hela cells)and expression products were identified by Western blotting.Results The size of PCR product was approximately 684 bp.It was confirmed that Sj1539 gene had been inserted successfully by the recombinant plasmid digested with two enzymes and PCR.It was verified that the expression product could react with S.japonicum-infected rabbit serum by Western blotting and the molecular weight was approximately 25×103.Conclusions The eukaryotie expression vector carrying Sj1539 gene has been established and the expression product has been obtained.