腺病毒抗原表位嵌合蛋白的原核表达及其免疫原性  

Prokaryotic expression and immunogenicity of adenovirus antigenic epitope chimeric protein

作　　者：王新国[1,2] 齐永[1] 潘英[1] 李素芹[1] 李佳萌[1] 陈红霞[1] 李素梅[1,3] 陈晨[1,2] 徐艺菲[1,2] 张玉彬[2] 李越希[1,2] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]中国药科大学生命科学与技术学院,江苏南京210009 [3]南京医科大学基础医学院,江苏南京210029

出　　处：《中国生物制品学杂志》2017年第7期685-689,共5页Chinese Journal of Biologicals

基　　金：国家重大新药创制课题(2015ZX09J15105-001-003)

摘　　要：目的设计含有人腺病毒(human adenovirus,HAd V)3、7、11、14及55型抗原表位的嵌合蛋白,利用大肠埃希菌进行原核表达,并检测其抗原性及免疫原性。方法对5种型别HAd V的六邻体蛋白氨基酸序列进行分析,筛选出其中具有强抗原性的片段,片段之间用2个甘氨酸和1个丝氨酸进行连接,形成1个多抗原片段串联的嵌合蛋白。优化嵌合蛋白的基因序列并化学合成全长基因,克隆至原核表达载体p ET28a(+)中,转化大肠埃希菌BL21(DE3),筛选高表达嵌合蛋白的工程菌。用High Affinity Ni-NTA resin纯化该嵌合蛋白,并将其作为免疫原免疫BALB/c小鼠,制备抗血清,间接ELISA法检测嵌合蛋白的抗原性及免疫原性。结果成功构建了高表达嵌合蛋白的工程菌,表达的嵌合蛋白相对分子质量约55 000,表达量达95%;破菌上清中嵌合蛋白的纯度较高,纯化后纯度在90%以上。嵌合蛋白免疫小鼠4次后,小鼠抗血清效价达1∶320 000。制备的腺病毒抗原表位嵌合蛋白能有效检测血清中腺病毒3、7、11、14及55型抗体,且能有效刺激机体产生针对各型别腺病毒抗原表位的抗体。结论表达制备的包含5种型别HAd V抗原表位的嵌合蛋白具有较好的抗原性及免疫原性,为进一步研制腺病毒疫苗、单克隆抗体及诊断试剂奠定了基础。Objective To construct a chimeric protein of antigenic epitopes from human adenovirus（HAd V） of types 3,7, 11, 14 and 55, express in E. coli and determine its antigenicity and immunogenicity. Methods The amino acid sequences of HAd V hexons of five types were analyzed by using the software ANTHEWIN, from which the epitopes with strong antigenicity were screened and linked together with Gly-Gly-Ser for construction of a chimeric protein. The gene sequence of the chimeric protein was optimized, synthesized chemically and cloned into prokaryotic expression vector p ET-28a（＋）. The constructed recombinant plasmid was transformed to E. coli BL21（DE3）, and the recombinant strain for high expression of chimeric protein was screened. The expressed chimeric protein was purified by High Affinity Ni-NTA resin chromatography and used as an antigen to immunize BALB/c mice for preparation of antiserum. The antigenicity and immunogenicity of chimeric protein were determined by ELISA. Results The recombinant E. coli for high expression of chimeric protein was constructed. The expressed chimeric protein, with a relative molecular mass of 55 000, contained95% of total somatic protein. The purity of chimeric protein in bacterial supernatant reached more than 90% after purification. The titer of antiserum of mice immunized with the chimeric protein for 4 times reached to 1 ∶ 320 000. The prepared chimeric protein was effective in determination of antibodies against HAd V types 3, 7, 11, 14 and 55, and stimulated the production of antibodies against antigenic epitopes of various types. Conclusion The expressed chimeric protein of antigenic epitopes of HAd V of five types showed good antigenicity and immunogenicity, which laid a foundation of developing HAd V vaccine, monoclonal antibodies and diagnostic reagents.

关 键 词：人腺病毒 抗原表位 嵌合蛋白 抗原性 免疫原性 

分 类 号：R373.9[医药卫生—病原生物学] R392-33[医药卫生—基础医学]