摘要
目的鉴定恶性疟原虫ApiAP2蛋白家族成员PF3D7_1107800与var基因内含子保守序列(iNPE)存在体内相互结合作用。方法提取恶性疟原虫标准株3D7基因组DNA,PCR扩增PF3D7_1107800基因5′端片段。扩增产物经双酶切后连接入原核表达载体pGex-4T-1,重组质粒转化至大肠埃希菌(E.coli)BL21(DE3),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)分析重组蛋白表达情况,谷胱甘肽亲和层析纯化重组蛋白。20只BALB/c小鼠分为重组蛋白免疫组和PBS对照组,每组10只,免疫组小鼠每鼠背部皮下注射纯化的重组蛋白100μl(约含蛋白20μg),对照组小鼠注射等量PBS,共免疫3次,每次间隔2周,末次免疫后2周处死小鼠,收集血清。重组纯化蛋白G(Protein G)纯化2组小鼠血清,Western blotting鉴定免疫血清抗体情况。染色质免疫沉淀(ChIP)获得恶性疟原虫DNA,qPCR检测PF3D7_1107800蛋白抗体组在C类var基因的内含子区域的富集情况。结果 PCR扩增PF3D7_1107800基因5′端片段结果显示,在约345 bp处有一特异性条带,与理论值相符。重组表达载体pGEX-4T-1-PF3D7_1107800N经测序表明构建成功。重组蛋白经诱导表达和纯化后,SDS-PAGE和Western blotting结果显示,所获的重组蛋白为目的蛋白,其相对分子质量(Mr)约为37 000,与预测值一致。重组蛋白免疫小鼠后,获得抗体血清,Western blotting分析结果显示,血清中抗体可与重组蛋白特异结合,在约Mr200 000处有一目的条带,与预测值一致。ChIP实验产物经qPCR分析显示,在var基因内含子区域的相对富集程度为内参seryl-tRNA合成酶基因区域的2倍以上。结论恶性疟原虫ApiAP2家族成员PF3D7_1107800可在体内与C类var基因内含子区域结合。
Objective To investigate the potential interaction between the ApiAP2 protein family member, PF3D7_l107800, and vat intron of Plasmodium falciparum in vivo. Methods Genomie DNA was extracted from Plas- modium falciparum （3D7 strain）, 5＇ end gene fragment of PF3D7_l107800 was amplified by PCR, and cloned into pGEX-4T-1 vector. The constructed pGEX-4T-1-PF3D7_llO7800N was transformed into E. Coli BL21 （DE3） and followed by expression of the protein induced by IPTG. The recombinant protein was purified through glutathione sepharose. Twenty female BALB/c mice were divided into 2 group. Ten mice in experiment group were immunized with a mixture of the purified protein and Freund＇s adjuvant by subcutaneous injection. Other 10 mice received PBS injection as con- trol. Sera from mice of 2 group were purified with protein G. The effect of the antibody was testified with Western blot- ting. DNA products of ChIP assay was analyzed for enrichment of anti-PF3D7_l107800 group in ups C vat intron by qPCR. Results PCR result of the PF3D7_l107800 gene 5＇ end segment showed that there was a specific band（about 345 bp）, which was consistent with the theoretical value. The constructed vector pGEX-4T-1-PF3D7_llO7800N was con- firmed by gene sequencing. SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was about Mr 37 000. The anti-PF3D7_l107800 serum was obtained after the immunization of mice with the purified protein, and reacted with the recombinant protein, the specific band was about Mr 200 000. qPCR result showed that the foldenrichment of anti-PF3D7_l107800 group in vat intron was two times higher than that of the reference gene region. Conclusion The Plasmodiumfalciparum ApiAP2 family member PF3D7_1107800 binds to ups C vat intron region in vivo.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2014年第1期1-5,共5页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金(No.31271388)
中央高校基本科研业务费专项资金~~
