第1类整合子中aadA2基因翻译起始位点的确定  

Determining the start codons of aadA2 gene in class 1 integron

作　　者：魏取好[1,2] 蒋晓飞[2] 李敏[2] 胡庆丰[1] 吕火[2] 祥周永[1] 列吕元[1] 

机构地区：[1]浙江省人民医院检验中心,杭州310014 [2]复旦大学附属华山医院检验医学科,上海200040

出　　处：《中华微生物学和免疫学杂志》2011年第12期1063-1067,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（81101291）;浙江省自然科学基金资助项目（Y2110743）

摘　　要：目的 确定第1类整合子中aadA2基因能否从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白.方法 定点突变含有不同翻译起始密码子的aadA2基因盒,并连同上游的可变区启动子分别克隆入质粒pACYC184中,转化大肠埃希菌JM109,免疫印迹检测含有不同翻译起始密码子的aadA2基因的翻译产物,用微量肉汤稀释法检测链霉素对含有不同翻译起始密码子aadA2基因的大肠埃希菌JM109的最小抑菌浓度.结果 aadA2基因可从上游无核糖体结合位点的密码子ATG及上游具有核糖体结合位点的密码子GTG起始翻译,同时在GTG密码子下游还存在着起始翻译密码子,其翻译产物在免疫印迹中均可与抗氨基糖苷-3″-腺苷酰基转移酶多克隆抗体产生特异性杂交条带,并赋予宿主细菌对链霉素不同水平的耐药.结论 当位于第1类整合子第1位基因盒中,aadA2基因可从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白,这一结构特点使得整合入第1类整合子的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,从而有利于第1类整合子表达从外界环境中捕获的基因.Objective To determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.Methods Site-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.Results aadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3＂-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD（3＂） proteins that can be detected by anti- aminoacyl -3＂-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.Conclusion When inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.

关 键 词：整合子 基因盒 耐药性 核糖体结合位点 

分 类 号：R392[医药卫生—免疫学]