沙眼衣原体pORF5质粒蛋白免疫优势片段的筛选与鉴定  

Screen and identification of immunodominant fragment of pORF5 plasmid protein from Chlamydia trachomatis

作　　者：何战胜[1] 邹燕[1] 粟胜梅 雷文波[1] 李忠玉[1] 

机构地区：[1]南华大学医学院病原生物学研究所/特殊病原体防控湖南省重点实验室,衡阳421001

出　　处：《中国免疫学杂志》2016年第1期59-64,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.81102230,No.31470277);湖南省高校创新平台开放基金(13K081);湖南省重点研发计划(2015JC3087);湖南省高层次卫生人才“225”工程培养项目(2013-13);湖南省普通高校带头人培养项目(2014-186);特殊病原体防控湖南省重点实验室(2014-5);湖南省高等学校“分子靶标新药研究”协同创新中心(2014-405)资助项目

摘　　要：目的:鉴定沙眼衣原体pORF5质粒蛋白的免疫原性,并进一步筛选和确定pORF5质粒蛋白免疫优势片段。方法:以沙眼衣原体D血清型DNA为模板,设计pORF5基因全长和9个不同片段特异引物进行PCR扩增,PCR产物经Bam HⅠ、NotⅠ双酶切后插入经同样双酶切的原核表达载体pGEX-6p中,构建pORF5质粒蛋白不同长度片段的原核表达重组体,重组体经PCR和测序鉴定后,转化XL1 Blue大肠杆菌表达10种不同长度的GST融合蛋白;ELISA方法检测pORF5质粒蛋白的免疫原性,Western blot鉴定pORF5质粒蛋白的免疫反应性;ELISA测定10个不同片段与沙眼衣原体生殖道感染患者血清、鼠免疫血清以及抗pORF5单克隆抗体的免疫反应性,鉴定pORF5质粒蛋白免疫优势片段。结果:pORF5质粒蛋白免疫原性强,能刺激机体产生高效价抗体;破坏pORF5质粒蛋白天然空间结构,其免疫反应性基本消失;在ELISA反应中,N端缺失66氨基酸的F6片段的免疫反应强度与pORF5全长基本相似,F2与F3出现较弱的免疫反应,其余片段免疫反应消失。结论:pORF5质粒蛋白为构象依赖性免疫优势抗原,其免疫优势表位和构象表位位于C端,本研究为进一步探讨pORF5质粒蛋白的生物学功能和疫苗的研制提供实验依据。Objective： To investigate the immunogenicity of pORF5 plasmid protein, and further to screen and identify its im- munodominant domian. Methods： 10 different fragments of pORF5 gene including full length were amplified from the DNA of Chlamydia trachomatis serovar D by PCR and cloned into appropriate site of pGEX-6p vector to construct recombinant vectors after digested with BamH I and Not I restriction endonucleases. After identification by PCR and sequencing, the recombinant plasmids were transformed into XL1 Blue E. coli to express the GST fusion proteins. ELISA and Western blot were carried out to identify the immunogenicity and immunoreaction of pORF5 plasmid protein. 10 different fragments were reacted with sera from patients urogenitally infected with Chlamydia trachomatis, mouse polyclonal antibodies and mouse monoclonal antibodies of pORF5 plasmid protein with ELISA method. Results： pORF5 plasmid protein displayed strong immunogenicity and could induce a strong antibody response in human. The reactivity of human antibodies almost completely disappeared,when the native structure of pORF5 plasmid protein was de- stroyed. F6 that only lacked the N-terminal 66 amino acids was recognized by antibodies in ELISA as strongly as the whole pORF5 plasmid protein was. However, no other fragments were significantly recognized although there was a minimal reactivity of F2 and F3 with antibodies. Conclusion： pORF5 plasmid protein was an immunodominant antigen containing conformation-dependent epitope, and the C-terminal three quarters of pORF5 amino acid sequence was required for maintaining its immune dominance and conformation. The significance of the above findings lay a foundation for the further study on pORF5 protein function and vaccine development.

关 键 词：沙眼衣原体 pORF5质粒蛋白 免疫原性 免疫优势表位 

分 类 号：R374.1[医药卫生—病原生物学]