融合蛋白FliC-Pfs25在trxB和gor基因双突变大肠埃希菌中的表达

Expression of FliC-Pfs25 fusion protein in a host strain of Escherichia coli with double mutations in trxB and gor genes

目的检测恶性疟原虫表面蛋白25(Plasmodium falciparum surface protein 25,Pfs25)与鼠伤寒沙门菌1相鞭毛蛋白(phase 1 flagellin of Salmonella enterica serovar Typhimurium,FliC)的融合蛋白FliC-Pfs25,在硫氧还蛋白还原酶基因(thioredoxin reductase gene,trxB)和谷胱甘肽还原酶基因(glutathione reductase gene,gor)双突变大肠埃希菌Origami2(DE3)中的表达。方法将含有Pfs25和FliC基因的重组质粒pET28a(+)-fliC-pfs25转化Origami2(DE3),IPTG诱导表达融合蛋白FliC-Pfs25,破菌上清经Ni-Sepharose纯化,纯化产物进行SDS-PAGE及Western blot鉴定。将纯化的融合蛋白配制成不同的疫苗制剂免疫BALB/c小鼠,ELISA法检测小鼠免疫血清中抗Pfs25抗体水平。结果融合蛋白FliC-Pfs25在Origami2(DE3)中以可溶性形式表达;纯化的融合蛋白可被两个抗Pfs25的单抗mAb 1A6和mAb 4D10识别;融合蛋白与铝佐剂结合在小鼠中激发出抗Pfs25的抗体应答,但Origami2(DE3)表达的FliCPfs25与抗Pfs25单抗的反应以及在小鼠中激发抗Pfs25抗体的能力,均弱于大肠埃希菌BL21(DE3)表达的FliCPfs25。结论融合蛋白FliC-Pfs25在大肠埃希菌Origami2(DE3)中成功获得表达,但trxB和gor两个基因的突变未显示出能够提高该融合蛋白在大肠埃希菌中的表达量。

Objective To investigate the expressions of FliC-Pfs25, a fusion protein of Plasmodium falciparum surface protein 25（Pfs25）and Salmonella enterica serovar Typhimurium phase 1 flagellin（FliC）, in E.coli Origami2（DE3）with double mutations in thioredoxin reductase（trxB）and glutathione reductase（gor）genes. Methods Recombinant plasmid pET28a（＋）-fliC-pfs25 was transformed into E.coli Origami2（DE3）and induced with IPTG. The fusion protein of FliCPfs25 was purified from the sonicated cell supernatant by Ni-Sepharose affinity chromatography, and identified by SDSPAGE and Western blot. The purified fusion protein was prepared into various vaccine preparations of FliC-Pfs25 and used to immunize BALB/c mice, and the Pfs25-specific antibody in mouse serum was determined by ELISA. Results The fusion protein of FliC-Pfs25 was expressed in a soluble form in E.coli Origami2（DE3）. The purified fusion protein was recognized by two anti-Pfs25 monoclonal antibodies, mAb 1A6 and mAb 4D10. The fusion protein in combination with aluminum adjuvant induced Pfs25-specific antibody response in mice. However, compared with the same protein expressed in E.coli BL21（DE3）, the fusion protein showed a weak reaction with anti-Pfs25 monoclonal antibody and a low ability in inducing anti-Pfs25 antibody response in mice. Conclusion The fusion protein of FliC-Pfs25 was successfully expressed in E.coli Origami2（DE3）. However, the mutations of trxB and gor genes showed no promoting effect on the quality of the expressed fusion protein.