摘要
参考 Genbank收录的 TGEV- Miller株的基因序列 ,自行设计合成 1对引物 (TGEVP5 /P6 ) ,对不同代次的 TGEV疫苗弱毒 STC3及种毒 、种毒 进行了 RT- PCR扩增 ,产物经琼脂糖凝胶电泳分析 ,均出现 1条大约 12 6 2 bp的目的条带 ,经 Eco R 酶切 ,都产生了 871bp和391bp左右的两个片段 ,与预期大小相符。将种毒 RT- PCR扩增目的条带回收纯化后克隆入PMD18- T载体中 ,转化宿主菌 DH5 α,挑选阳性克隆 (命名为 PTs) ,提取重组质粒 ,用 Hpa 、Eco R 对重组质粒进行酶切鉴定以及 PCR扩增 ,然后进行序列测定 ,并进行了序列分析 ,证实与国外标准毒株 Miller、Fs772 / 70、Purdue。
A pair of primers was designed according to the S gene sequence of TGEV Miller strain in Genebank. Different generation TGEV vaccine virus STC3 strain and seed virus1, seed virus2 were RT PCR amplified. All PCR products come to be 1 262 bp band with agarose gel electrophoresis. with EcoRⅡenzyme digestion a 871 bp and a 391bp band were got according with the expectation. Recycled PCR product of seed viru2 was inserted into the PMD18 T vector. The vector was transformed to host bacteria DH5a.The positive clone was named PT3. The recombinant plasmid was identified by restriction enzyme HapⅠ and EcoRⅠ. After sequencing and sequence analysising, gene cloned here was found to be very high homologues with standard strains Miller, Fs772/70, Purdue and T014.
出处
《动物医学进展》
CSCD
2003年第3期93-97,共5页
Progress In Veterinary Medicine
