噬菌体展示HIV-1 Tat38-61碱性区51和55位随机突变体文库的构建
Constructing a phage-displayed random mutation library of HIV-1 Tat38-61 at the sites of 51 and 55 amino acids in basic region
投稿时间:2010-12-22  
DOI:  
中文关键词:HIV-1 Tat,碱性区,随机突变,噬菌体展示
英文关键词:HIV-1 Tat, basic region, random mutation, phage display
基金项目:国家自然科学基金 (Nos. 30872246, 30872405, 30972632, 30972799),安徽省自然科学基金 (No. 090413136) 资助,上海市基础研究重点项目 (No. 08Jc1405200) 资助。
作者单位E-mail
葛宜兵 安徽医科大学 病理生理学教研室合肥 230032  
杨旭芳 中国人民解放军第二军医大学 微生物学教研室上海 200433  
杜哲明 武警江西总队医院外一科南昌 330000  
庞强 中国人民解放军第二军医大学 微生物学教研室上海 200433  
曹洁 中国人民解放军第二军医大学 微生物学教研室上海 200433  
陈秋莉 中国人民解放军第二军医大学 微生物学教研室上海 200433  
王锦红 中国人民解放军第二军医大学 微生物学教研室上海 200433  
张华群 中国人民解放军第二军医大学 微生物学教研室上海 200433  
廖文婷 中国人民解放军第二军医大学 微生物学教研室上海 200433  
祁培培 中国人民解放军第二军医大学 微生物学教研室上海 200433  
刘超 中国人民解放军第二军医大学 微生物学教研室上海 200433  
章萍萍 安徽医科大学 病理生理学教研室合肥 230032  
邓松华 安徽医科大学 病理生理学教研室合肥 230032 desoh@126.com 
潘卫 中国人民解放军第二军医大学 微生物学教研室上海 200433 pwpanwei@yahoo.com.cn 
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中文摘要:
      为了构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库,进一步研究HIV-1 Tat38-61表位的分子进化筛选,采用含随机核苷酸序列的引物,通过Overlap PCR的方法获得51和55位氨基酸随机突变的全长Tat编码序列,再以此为模板PCR扩增出两端含有Xba I识别序列的Tat38-61突变体片段HIV-1 Tat38-61(51N/55N),克隆至噬菌体展示载体pCANTAB5S上,转化大肠杆菌TG1,经M13K07辅助噬菌体拯救,构建噬菌体展示Tat38-61(51N/55N) 碱性区突变体文库。结果显示文库的库容量为5.0×106,滴度为2.65×1012 TU/mL,阳性克隆率为56.50%;序列分析显示文库中51、55位核苷酸与氨基酸均呈随机性分布,达到了对文库进行分子进化筛选的要求,为获得可用作疫苗候选物的新型Tat突变体奠定基础。
英文摘要:
      We constructed a phage-displayed random mutation library of Tat38-61(51N/55N), for studying the molecular evolution screening of HIV-1 Tat38-61 epitope. We used primers containing the random nucleotide sequences, and introduced the random mutations at the sites of 51 and 55 amino acids coding sequences into full-length Tat sequences by overlapping PCR. With the randomly mutated full-length Tat as template, the Tat38-61(51N/55N) mutants which contained recognition sequences for the Xba I in both ends were amplified by PCR using the designed primers. The mutants were cloned into Xba I site in the phagemid vector pCANTAB5S, then the recombinants were transformed into E. coli TG1, a phage-displayed the random mutation library of Tat38-61(51N/55N) was constructed by the rescue of help virus M13KO7. The results showed that the library consisted of about 5.0×106 colonies and the phage library titer was 2.65×1012 TU/mL. More than 56.50% colonies in the library were positive for insertion. Sequence analysis showed that the nucleotides encoding amino acids at the sites of 51 and 55 distributed randomly. The constructed mutation library could meet the requirements for the following molecular evolution screening, and might prepare the Tat mutants for the further study of new Tat vaccine candidates.
葛宜兵,杨旭芳,杜哲明,庞强,曹洁,陈秋莉,王锦红,张华群,廖文婷,祁培培,刘超,章萍萍,邓松华,潘卫.噬菌体展示HIV-1 Tat38-61碱性区51和55位随机突变体文库的构建[J].生物工程学报,2011,27(5):755~763
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