摘要
目的构建一种嵌合过敏原,同时保有草和豚草过敏原的特征,而其抗原性得到改变。方法以业经克隆到草和豚草过敏原基因片段的混合物为模板,利用桥式PCR和重叠引物法,扩增得到嵌合基因,利用测序、蛋白表达及抗原性分析软件对所得嵌合基因编码蛋白与MHCⅡ类分子的结合表位进行评价。结果通过以上方法获得嵌合基因3例,序列分析表明,3例嵌合基因AL03-1、AL03-2及AD03-4分别与原始克隆LCM9、LCS13(LCM20)及D03具有较高的同源性,而其抗原性分别比相似的原始克隆略强、略强及明显增强,融合表达带型与原始克隆的也基本一致。结论采用桥式PCR及重叠引物法,嵌合基因及其表达载体得到成功构建,所得的嵌合基因将满足不同体质或不同免疫力的人群、不同免疫治疗阶段的脱敏治疗的临床需要。
Objective To create a chimeric allergens that bear the characteristics of the allergens from Humulus scandens and short ragweed. Methods Bridging PER（BPCR） and partially overlapping primer-based PCR（POP-PCR） were employed to obtain the chimeric genes by splicing two sorts of allergen gene fragments. The PCR products were cloned and their sequences were subsequently analyzed and compared with those from the original clones. The antigenicity of chimeric genes was predicted by the software SYFPEITHI to investigate the binding epitope to classⅡ MHC moleculars. Results Three chimeric genes（AL03-1, AL03-2, AD03-4） were obtained. All sequences of which were homologous to the original clones but with some difference, i.e. AL03-1 was similar to LEM9, AL03-2 was similar to LCS13 （LEM20）, and AD03-4 was similar to D03. Epitope prediction by software SYFPEITHI demonstrated that the binding ability with class Ⅱ MHC molecules of the three chimeric genes was remodeled. The band profiles of all the chimeric proteins on SDS-PAGE were similar to those of the original clones. Conclusion The construction of three chimeric genes and their expression vector construction were facilitated by BPCR and POP-PER methods. The proteins encoded by the chimeric genes would meet the different clinical needs by different atopic patients with diverse constitutions and in different immunotherapeutic stages.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第1期78-84,共7页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助(No.30470996)
广东省自然科学基金资助(No.034617)
关键词
过敏原
嵌合基因
抗原性
Allergens
Chimeric genes
Antigenicity
