【摘要】 用MA10 4细胞培养增殖了轮状病毒地方分离株JL94。利用一对根据Genebank中已发表的轮状病毒VP7基因cDNA序列而设计并合成的引物 ,通过逆转录—聚合酶链反应 (RT_PCR)从JL94毒株扩增出VP7基因全长cDNA。将其插入pMD18_T载体中 ,构建了重组质粒pMD18_T_JL94 /VP7,并对其进行了HindⅢ ,HindⅢ和BamHⅠ的单、双酶切初步鉴定及其核苷酸序列测定 ,证明pMD18_T_JL94 /VP7中的插入基因为轮状病毒的VP7基因。重新设计一个带有Kozak序列的上游引物 ,通过PCR扩增出带有Kozak序列的vp7基因并将其插入pMD18_T载体中构建了重组质粒pMD18_T_VP7,酶切鉴定其大小和方向后 ,与真核表达载体pcDNA 3.1(+)分别用HindⅢ和BamHⅠ双酶切 ,胶回收纯化后连接。经酶切和核苷酸序列检测鉴定 ,成功地构建了真核表达质粒pcDNA_VP7更多还原

【Abstract】 The native JL94 strain of porcine rotavirus was propagated and harvested on MA104 cell. Using a pair of primer designed according to the published sequence of RV’s VP7 gene cDNA, the full length gene encoding outer capsid protein VP7 was amplified from JL94 strain by reverse transcription_polymerase chain reaction (RT_PCR). The cDNA of VP7gene was inserted into pMD18_T vector and was identified by sequencing, single and double digestion using HindⅢ, HindⅢ and BamHⅠ.Using a new upper primer designed according to the sequence of JL94/VP7 gene cDNA,the gene of vp7 containing Kozak’s sequence was amplified from pMD18_T_JL94/VP7 by PCR. The cDNA of vp7 gene was inserted into pMD18_T vector and was identified by digestion, then it was recombinated into eukaryotic expression plasmid pcDNA3.1(+) and was identified by sequencing and digestion.更多还原