生长素诱导的蛋白敲减系统在刚地弓形虫Chinese 1虫株中的应用及验证

Application and verification of auxin-inducible degron system in Toxoplasma gondii Chinese type 1 strain

目的应用生长素诱导的蛋白敲减(AID)系统调控刚地弓形虫Chinese 1虫株中荧光蛋白的表达。方法人工合成3′端融合有标签FLAG的植物生长素受体--运输抑制剂响应蛋白1(TIR1)的核苷酸序列TIR1-FLAG,将其连接至pTub-e GFP-CAT载体中,构建pTub-TIR1-FLAG-CAT表达质粒;表达质粒经电穿孔转染至弓形虫Chinese 1虫株TgCtwh3,经氯霉素筛选和极限稀释后筛选单克隆虫株。PCR扩增及蛋白质免疫印迹(Western blotting)检测单克隆虫株中TIR1基因的插入和相应蛋白的表达,将验证正确的阳性克隆命名为TIR1虫株。构建表达荧光蛋白mCherry-Ty-AID报告基因的表达质粒pTub-mCherry-Ty-AID-DHFR,并通过电穿孔转染将其整合至TIR1虫株基因组中,乙胺嘧啶筛选后极限稀释筛选获得稳定表达mCherry-AID的虫株,命名为TIR1:m Cherry-AID虫株。将TIR1:mCherry-AID虫株的虫体分为吲哚乙酸(IAA)调控组和对照组,IAA调控组加IAA至终浓度500μmol/L,对照组加等量乙醇,培养3 h后,分别以抗TgGAP45和抗Ty-1抗体为一抗,间接免疫荧光试验(IFA)和Western blotting分析IAA调控下mCherry荧光蛋白的表达情况。结果PCR扩增结果显示,在1816 bp处有一条特异性扩增条带。Western blotting分析结果显示,抗FLAG-Tag抗体可识别TIR1虫株中相对分子质量(Mr)约70000的蛋白;Ty-1抗体可在TIR1:mCherry-AID虫株的对照组中检测到约Mr58000的特异性条带,与理论上C端融合了AID的mCherry-Ty蛋白大小相同;在IAA调控组中未检测到特异性条带。IFA结果显示,TIR1:mCherry-AID虫株对照组可见红色荧光蛋白,IAA调控组未检测到mCherry蛋白。结论应用AID系统可成功调控弓形虫Chinses 1虫株中mCherry荧光蛋白的表达。

Objective To regulate the expression of fluorescent proteins in Toxoplasma gondii Chinese type 1 strain using the auxin-inducible degron(AID)system.Methods The nucleotide sequence of plant auxin receptortransport inhibitor response protein 1(TIR1)fused with a FLAG tag at the 3′end(TIR1-FLAG)was synthesized,and ligated into the pTub-eGFP-CAT vector to obtain the pTub-TIR1-FLAG-CAT expression plasmid.The plasmid was transfected into the Toxoplasma gondii Chinese type 1 strain by electroporation.After chloramphenicol screening and limiting dilution,positive clones were selected,in which the inserted TIR1 gene and its expression was examined by PCR and Western blotting.The verified strain clone with correct insertion was designated as the TIR1 strain.The plasmid pTub-mCherry-Ty-AID-DHFR expressing the mCherry-Ty-AID reporter gene was constructed and integrated into the genome of the TIR1 strain by electroporation,from which a clone capable of stable expression of m Cherry-AID protein was obtained by pyrimethamine screening and ultimate limiting dilution,and designated as TIRI:m Cherry-AID strain.The TIR1:mCherry-AID parasites were divided into the regulating group and control group,and cultured with an auxin,indole-3-acetic acid(IAA)solution(500μmol/L)or an equal volume of ethanol for 3 h accordingly.Then the expression of mCherry fluorescent protein was detected by indirect immunofluorescence assay(IFA)and Western blotting using anti-Tg GAP45 and anti-Ty-1 antibodies.Results The PCR amplification showed a specific amplification band at 1816 bp.Western blotting showed that the anti-FLAG-Tag antibody can recognize a protein band with a relative molecular mass(Mr)of 70000 in the TIR1 strain,and anti-Ty-1 antibody can detect a specific band with Mrof about 58000 in the TIR1:mCherry-AID strain,which equales to the theoretical size of m Cherry-Ty protein fused with AID at the C terminus;however,no specific band was detected in the auxin/IAA group.IFA assay indicated that red fluorescent protein was seen in the TIR1:mCherry-AID in the control group,but no mCherry protein was detected in the auxin/IAA group.Conclusion The AID system can regulate mCherry fluorescent protein expression in T.gondii Chinses 1 strain.