摘要
目的构建肠道病毒71型(EV71)P1和3CD基因双顺反子重组腺病毒,并检测3CD蛋白酶的切割作用和表达产物的抗原性。方法采用RT-PCR法从阜阳分离的EV71中扩增P1和3CD基因,构建穿梭质粒pShuttle-CMV-P1-3CD、pShuttle-CMV-P1和pShuttle-CMV-3CD;经同源重组获得重组腺病毒rAd-P1-3CD、rAd-P1和rAd-3CD,分别转染293细胞,RT-PCR法检测目的基因的转录,免疫荧光法检测目的蛋白的表达。结果3种重组腺病毒经PCR和酶切鉴定表明构建正确;RT-PCR检测表明,3个重组腺病毒均已转录目的基因mRNA;免疫荧光检测仅观察到rAd-P1-3CD表达产物具有特异性,而rAd-P1、rAd-3CD未能检测到特异性表达产物。结论已成功构建EV71P1和3CD基因双顺反子重组腺病毒,表达产物具有EV71特异抗原性,提示P1产物只有在被3CD蛋白酶切割后才体现抗原性。
Objective To construct a bicistronic recombinant adenovirus with P1 and 3CD genes of enterovirus 71（EV71） and determine the cleavage effect of 3CD protease as well as the antigenicity of expressed product. Methods P1 and 3CD genes were amplified from EV71 strain isolated from Fuyang City, Anhui Province, China for construction of shuttle plasmids pShuttle-CMVP1-3CD, pShuttle-CMV-P1 and pShuttle-CMV-3CD, based on which recombinant adenoviruses rAd-P1-3CD, rAd-P1 and rAd-3CD were obtained by homologous recombination and transfected to 293 cells respectively. The transcription of target gene was determined by RT-PCR, and the expression of target protein by IFA. Results Both PCR and restriction analysis proved that recombinant adenoviruses rAd-P1-3CD, rAd-P1 and rAd-3CD were constructed correctly. RT-PCR showed transcription of target mRNA in the 293 cells transfected with the 3 kinds of recombinant adenoviruses. Only specific expressed product of rAd-P1-3CD was observed by IFA, while no specific expressed product of rAd-P1 or rAd-3CD. Conclusion The bicistronic recombinant adenovirus with P1 and 3CD genes of EV71 was successfully constructed, and the expressed product showed EV71-specific antigenicity, indicating that the P1 protein showed antigenicity only after being cleaved with 3CD protease.
出处
《中国生物制品学杂志》
CAS
CSCD
2010年第4期341-345,共5页
Chinese Journal of Biologicals
