猪A组轮状病毒Vp4全基因的克隆与序列分析

Cloning and Sequencing of Full Vp4 Gene of Group A Porcine Rotavirus

应用RT-PCR技术,从猪A组轮状病毒总RNA中扩增了2 331 bp的Vp4全基因,通过T-A克隆技术,将PCR产物克隆至克隆载体pGEM-T中,转化至受体菌株JM109中,通过质粒提取、限制性内切酶酶切、PCR、序列测定等方法鉴定,构建出重组阳性克隆质粒pGEM-T-Vp4。经DNASTAR软件分析核苷酸和氨基酸序列,结果显示该实验株与黑龙江株和美国株的同源性最高,均达到99%以上,并具有轮状病毒Vp4全基因的抗原表位区。

The antigenic determinants of Vp4 gene of porcine group A Rotavirus were amplified from cell infected with rotavims by the reverse transcription-polymerase chain reaction （RT- PCR）. The length of full Vp4 gene was 2 331 bp. The products of RT - PCR were ligated with plasmid pGEM - Teasy and transformed to JM109. By the analysis of restriction endonuclease cutting, PCR and sequencing, the recombinant plasmids pGEM - T - Vp4 was constructed. The results of nucleotide and amino acid sequences analysis showed that the experimental rotavims Vp4 gene has over 99% similarity with the rotavirus Vp4 genes of America strain and Heilongjiang strain, which has a positive open reading frame.