CAJ | 학술논문

目的：高效表达出血型大肠埃希菌O157：H7转位紧密黏附素受体Tir （translocation inti min receptor）的C末端蛋白，并初步鉴定其活性。方法：对EHEC O157：H7标准株EDL933 Tir完整的编码区基因序列和氨基酸序列进行生物信息学分析，选取C末端基因（命名为Tir-C），构建原核表达质粒PET-30a（＋）-Tir-C，转至E．coli BL21/DE3中，IPTG诱导表达，SDS-PAGE检测相对分子质量，Western blot鉴定免疫原性。结果：PCR扩增出675 bp的目的片段，构建的质粒经酶切鉴定及测序与预期一致。目的蛋白在裂解上清中有表达，纯化浓度达500μg/ mL，并有较好的免疫原性。结论：成功表达了有抗原活性的重组Tir-C蛋白，为研究0157：H7疫苗奠定了基础。
목적：고효표체출혈형대장애희균O157：H7전위긴밀점부소수체Tir （translocation inti min receptor）적C말단단백，병초보감정기활성。방법：대EHEC O157：H7표준주EDL933 Tir완정적편마구기인서렬화안기산서렬진행생물신식학분석，선취C말단기인（명명위Tir-C），구건원핵표체질립PET-30a（＋）-Tir-C，전지E．coli BL21/DE3중，IPTG유도표체，SDS-PAGE검측상대분자질량，Western blot감정면역원성。결과：PCR확증출675 bp적목적편단，구건적질립경매절감정급측서여예기일치。목적단백재렬해상청중유표체，순화농도체500μg/ mL，병유교호적면역원성。결론：성공표체료유항원활성적중조Tir-C단백，위연구0157：H7역묘전정료기출。
Objective To identify the immune activity of the recombinant protein Preli minarily after expressing Tir C-ter minal of enterohemorrhagic Escherichia coli(EHEC) in E.coli BL21/DE3 efficiently. Methods Tir C-ter minal (marked as Tir-C)was amplified by PCR considering the result of Bioinformatics analysis of Tir. The recombinant plasmid(designed as PET-30a(+)-Tir-C)was identified by PCR,sequencing and digested by restriction endonucleases. The positive recombinants were transformed into E.coli BL21/DE3 and induced by IPTG to express the Tir-C. The Tir-C protein was detected by SDS-PAGE and identify the antigenic of the recombinant protein Preli minarily by Western blot. Results The 675bp DNA was gained. The plasmid PET-30a (+)-Tir-C was built. Tir-C was expressed mainly in supernatant of lysis and was purified by Ni+affinity chromatograghy. Concentration of the purified protein is about 500 μg/mL and a unique band was detected with the relative molecular mass of approximately 24KDa by Western blot. Conclusion The recombinant Tir-C was expressed successfully and had immunoreactivity to some extent, which deserves the investigations for vaccine against EHEC.