日本脑炎病毒WHe株全长cDNA的长链RT-PCR法的扩增

Amplification of full-length cDNA of Japanese encephalitis virus WHe strain by long RT-PCR

为深入探索日本脑炎病毒(Japanese encephalitis virus,JEV)WHe株的致病机理,并为新型疫苗研发提供技术支持,采用长链RT-PCR技术对其基因组全长cDNA进行了扩增鉴定,并对含复杂二级结构的3′末端和5′末端非编码区扩增片段进行了测序分析。扩增结果表明,扩增出的JEV WHe株基因组全长cDNA分子近11 kb大小;非编码区测序分析表明,扩增产物为WHe株所特有。提示长链RT-PCR法可用于JEV WHe株基因组全长cD-NA的扩增。

In order to elucidate the pathogenesis of Japanese encephalitis virus （JEV） Wile strain and develope novel vaccine,the full-length cDNA was amplified by long reverse transcription-polymerase chain reaction（long RT-PCR）. The full-length cDNA was identified by PCR and the fragments which contain the 3rand 5runtranslation regions were cloned into pGEM-T vector, and the sequences of inserted fragments were determined by Invitrogen Corporation. The results showed that the approximate 11 kb full-length cD- NAs of WHe strain were amplified by long RT-PCR and its correctness was proved by partial nucleotide sequence analysis. The genomic cDNAs of WHe strain were successfully amplified.