白念珠菌H3K4甲基转移酶N末端肽段Set1-208p的原核细胞表达及鉴定  

Prokaryotic expression and identification of recombinant N-terminal region(Set1-208p) of Candida albicans histone 3 lysine 4 methyltransferase

作　　者：孔倩倩[1,2] 廖红[1,2] 李芳秋[2] 马春芳[2] 史利宁[2] 胡毓安[2] 

机构地区：[1]南京师范大学生命科学学院,南京210097 [2]南京军区南京总医院临床中心实验科,南京210002

出　　处：《临床检验杂志》2012年第11期919-921,930,共4页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省科技支撑计划-社会发展项目(BE2009673);南京军区医学科技创新重点课题(10Z027)

摘　　要：目的原核细胞表达白念珠菌H3K4甲基转移酶N末端肽段Set1-208p,鉴定重组蛋白质的抗原性。方法用比对软件对白念珠菌H3K4甲基转移酶与其他物种甲基转移酶进行比对。选择与人类无同源性的抗原特异性片段,以白念珠菌C1标准株基因组DNA为模板,PCR扩增Set1-208p的编码基因,以pET28a(+)为载体,构建重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导重组蛋白质表达。用抗His标签的单克隆抗体鉴定融合蛋白,并用确诊侵袭性白念珠菌感染(IC)患者血清进行抗原性鉴定。结果成功克隆了白念珠菌抗原特异性H3K4甲基转移酶片段Set1-208p基因并诱导表达,获得了纯化的重组肽段,所得蛋白质分子量(Mr)约为29 000,带有His标签,经western blot鉴定,诱导的重组蛋白可与IC患者血清特异性反应。结论成功诱导表达了具有良好抗原反应性的Set1-208p重组蛋白质,为建立相应的抗原、抗体ELISA检测方法提供基础。Objective To express N-terminal region （Setl-208p） of Candida albicans histone 3 lysine 4 methyltransferase （Setlp） by prokaryotic expression system, and identify the antigenieity of the recombinant fragment. Methods The amino acid sequence of C. al- bicans Setlp was compared with that of other species by multiple-alignment program. N-terminal region （Setl-208p） of C. albicans Setl p, the antigen-specific fragment without significant homology to other human proteins, was selected for prokaryotic expression. The genomie DNA of C. albicanswas C1 strain was used as the template and the coding sequence of Setl-208p was amplified by PCR. The PCR products were cloned into prokaryotic expression vector pET-28a（ ＋ ） to constructed recombinant plasmid which was transferred into E. coli BL21 （DE3） to express the recombinant protein with IPTG induction. The recombinant His6-tagged Setl-208p was further identified by western blot with the monoelonal antibody against His6-tag and its antigenicity was confirmed with the sera from the patients with systemic candidiasis. Results The recombinant His6-tagged Setl-208p with relative molecular weight approximately 29 000 was successfully expressed and purified. Western blot showed that the recombinant protein was able to specifically react with the sera from the patients with systemic candidiasis. Conclusion The successful expression of recombinant Setl-208p with specific immunogenicity may be helpful to establish an ELISA method to detect the antibody against Setl p.

关 键 词：白念珠菌 H3K4甲基转移酶 基因克隆 原核细胞表达 

分 类 号：Q784[生物学—分子生物学]