弓形虫二氢叶酸还原酶一胸背酸合成酶ROP17真核表达载体的构建及ROP17参与自噬的研究

Construction of a eukaryotic expression vector of Toxoplasma DHFR-TS-ROP17 and study of the role of ROP17 in autophagy

目的构建刚地弓形虫二氢叶酸还原酶一胸苷酸合成酶(dihydrofolate reductase-thymidylate synthase,DHFR-TS)-棒状体蛋白17真核表达载体pIRES/TgDHFR-TS-TgROP17,将其转染人胚肾细胞(HEK 293T)后观察ROP17对细胞自噬的作用。方法以弓形虫速殖子总RNA为模板.逆转录PCR(RT-PCR)扩增DHFR-TS基因片段,克隆至真核表达载体pIRES的多克隆位点B,构建重组质粒pIRES/TgDHFR-TS.经菌液PCR、双酶切和测序鉴定正确后.用脂质体法转染人胚肾细胞(HEK 293T),RT-PCR法和Western blot检测DHFR-TS的表达。以弓形虫速殖子ecDNA为模板,PCR获得ROP17.将其引人重组质粒pIRES/TgDHFR TS的多克隆位点A.构建重组载体pIRES/TgDHFR-TS-TgROP17。重组载体经菌液PCR、双酶切和测序鉴定后转染人胚肾细胞(HEK 293T),对转染细胞血清饥饿诱导,通过.Western blot检测自噬标志蛋白LC3-II、Beclin-1和P62的表达。结果RT-PCR检测显示,从RH株弓形虫速殖子中扩增出约1809 bp的片段。菌落PCR、双酶切及测序显示工具载体pIRES/TgDHFR-TS构建成功;转染293T细胞后RT-PCR和Western blot转染pIRES/TgDHFR TS的细胞中有TgDHFR TS的表达,基因大小为1809 bp.蛋白相对分子质量约为68X103。菌液PCR、双酶切和测序显示重组载体pIRES/TgDHFR-TS-TgROP17构建成功,转染293T细胞后RT-PCR和Western blot检测转染pIRES/TgDHFR TS TgROP17的细胞中有TgROP17的表达.基因大小及蛋白分子质量与预期相符,而转染pIRES/TgDHFR-TS质粒组未见相应基因及蛋白表达。经血清饥饿诱导后,Western blot.检测显示随着去血清时间的延长,LC3-I向LC3-II的转化、Beelin-1蛋白逐渐增加、P62蛋白逐渐减少。结论成功构建了工具载体pIRES/TgDHFR-TS和重组载体pIRES/TgDHFR-TS-TgROP17,且能在真核细胞中表达,表达的弓形虫棒状体蛋白17(ROP17)能促进血清饥饿诱导的细胞自噬。

Objective To construct the recombinant eukaryotic expression plasmid pIRES/TgDHFR TS TgROP17 and study the role of ROP17 in cell autophagy.Methods Total RNA of T.gondii was extracted from RH strain.tachyzoites.The DHFR-TS and ROP17 genes were amplified with reverse transcription-PCR(RT-PCR).First,TgDH-FR-TS was cloned into the multiple cloning site B of the eukaryotic expression vector pIRES to construct the recombinant plasmid plRES/TgDHFR-TS,The recombinant pIRES/TgDHFR-TS was identified using colony-PCR,double enzyme digestion,and sequencing.Then the pIRES/TgDHFR TS recombinant plasmid was transfected into 293T cells,and the expression of DHFR TS was detected using RT-PCR and Western blotting.Second,TgROP17 was introduced into the multiple cloning site A of the recombinant plasmid pIRES/TgDHFR TS to construet the recombinant pIRES/TgDHFR-TS TgROP17 plasmid.The recombinant plRES/TgDHFR-TS TgROP17 was identified using colony-PCR,double en-xyme digestion,and sequencing.The pIRES/TgDHFR TS-TgROP17 plasmid was transfected into 293T cells,and the expression of ROP17 was detected using RT-PCR and Western blotting.Third,the transfected cells were serum:starved,and the autophagy marker proteins LC3-II.Beclin-1.and P62 were detected with Western blotting.Results RT-PCR results indicated that a fragment of about 1.809 bp was amplified from RH strain T.gondii tachyzoites.Colony PCR and double enzyme digestion and sequencing indicated that the recombinant pIRES/TgDHFR TS plasmid was successfully constructed.RT-PCR and Western blotting indicated expression of TgDHFR-TS in pIRES/TgDHFR TS transfected 293T cells with a gene 1.809 bp in length and had a relative molecular mass(Mr)of about 68 KDa.Asa result of RT-PCR,a fragment of about 1,860 bp was amplified from RH strain T.gondii tachyzoites.Colony PCR and double enzyme digestion and sequencing indicated that the recombinant plasmid pIRES TgDHFR TS TgROP17 was sessfully con-structed.RT-PCR and Western blotting indicated that TgROP17 was expressed in pIRES/TgDHFR-TS TgROP17-transfected 293T cells.The gene was 1.860 bp in length and had a relative molecular mass(Mr)of about 70 KDa.After serum starvation.Western bloting indicated that the LC3-I1 and Beclin-l protein gradully increased while the P62 pro-tein gradually decreased in a time dependent manner.C onclusion The recombinant plasmid plRES/TgDHFR TS and the recombinant plasmid pIRES/TgDHFR-TS TgROP17 were sucessfully constructed.Morover,TgROP17 promotes serum starvation-induced cell autophagy.