禽流感病毒H5N1亚型HA基因表达及其产物的应用.pdfVIP

第 21 卷 6 期 中 国 病 毒 学 21 （6 ）：560-564 2006 年 11 月 VIROLOGICA SINICA November 2006 禽流感病毒H5N1 亚型HA 基因表达及其产物的应用 郑其升 1 ，毕志香 2 ，曹瑞兵 1 ，周 斌 1 ，李 鹏 1 ，陈溥言 1** (1.南京农业大学农业部动物疫病诊断与免疫重点开放实验室, 江苏南京 210095, 山东畜牧兽医职业技术学院, 山东潍坊 262100) Correcting a Mutant HA Gene of the Avian Influenza Virus (AIV) H5N1 Subtype by SOE and the High Expression of the Corrected Gene in E.coli and It’s Application 1 2 1 1 1 1** ZHENG Qi-sheng , BI Zhi-xiang , CAO Rui-bing , ZHOU Bin , LI Peng , CHEN Pu-yan (1.Key Laboratory of Animal Disease Diagnosis and Immunology, Nanjing Agricultural University, Nanjing 210095, China; 2. Shandong Animal Science and Veterinary Vocational College, Weifang 262100, China) Abstract ：Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank (Accession number ：DQ023145), the HA gene of AIV H5N1 subtype with the signal peptide sequence was obtained by PCR from the recombinant plasmid pUC-HA, which contains the full length of HA gene. Sequence analysis showed that a single base, an A, was replaced by aT to generate a stop codon (TAA) at position 967 of HA gene. Splicing by overlap extension ( SOE) method was applied to correct this mutation with two overlapped mutation primers P3 and P4, and then the correct HA gene was inserted into the expression vector pET-32a (+) to geerate the recombinant plasmid pET-HA-2. The target gene was successfully expressed in the host cell E.coli BL21(DE3) in inclusion bodies when induc