禽流感病毒M2蛋白跨膜区基因的缺失

根据禽流感病毒 (AIV ) A/ Chicken/ Korea/ MS96 / 96 (H9N2 )株的核苷酸序列 ,设计并合成引物 ,通过 RT- PCR,从AIV H9N1株感染的 MDCK细胞总 RNA中扩增出 2 94 bp的 AIV全长的 M2基因。通过软件分析其序列中的跨膜区后 ,将其与p GEM- T easy载体连接产物 p GEM- T/ M2为模板 ,通过 PCR扩增出约 90 bp左右 M2的膜外区编码序列和约 16 0 bp左右 M2的胞内区编码序列。将两个扩增产物同时作为模板 ,以 OE- PCR(overlap extension- PCR)扩增出约 2 5 0 bp的缺失跨膜区的 M2基因M2 d。测序结果表明 ,M2 d的序列除在跨膜区以 4个甘氨酸序列替代外 ,其余部分与 M2完全一致 ,由此说明 OE- PCR扩增法成功地将禽流感病毒 M2基因跨膜区缺失

Deletion of coding sequence of transmembrane domain of M2 protein of AI virus

Primers were designed according to the nucleotide sequence of avian influenza virus. Full- length M2 protein gene of avian influenza virus H9N2 was amplified from total RNA of infected MDCK cells by using RT- PCR. The transmembrane domain was analyzed by computer. Then using p GEM- T/ M2 ,the ligation product of M2 and p GEM- T easy as template,The 90 bp coding sequences of outer membrane and 16 0 bp coding sequence of inner membrane were amplified by using PCR. U sing the two amplified products as template,the remaining sequences about 2 5 0 bp which delete the transmembrane domain of M2 were ampli- fied by using overlap extension- PCR. Sequence anylasis showed that the remaining sequences were identical to the M2 coding sequences except the transmembrane protein domain which was substitute by 4 glycines. It demonstrated that the overlap extension PCR successfully deleted the coding sequence of transmembrane do- main of M2 .