[Preliminary application of a mosquito densovirus-mediated artificial intron in vitro and in vive of mosquito].
An artificial intron consisting of the 5'-donor site (from the first intron of the human beta-globin gene) and the 3'-acceptor site (from the intron of an immunoglobulin gene heavy chain variable region) was obtained with a splice overlap extension PCR and was then inserted in frame into the coding sequence of nostructural protein NS1 gene fused to GFP gene in a recombinant mosquito densovirus plasmid p7NS1-GFP. The constructed plasmid was named as p7NS1-Intron-GFP. The plasmid p7NS1-Intron-GFP was co transfected with the helper plasmid pUCA into C6/36 cells, then the packaged recombinant and wild type viruses were purified and recovered. The second-instars of Aedes albopictus larvae were exposed to recombinant and wild type virus mixed stock. The high level GFP expression in C6/36 cells and larvae was observed under fluorescence microscope, indicating that the inserted artificial intron exerted its normal function in self-splicing both in vitro and in vivo. This study laid a foundation for application of an artificial intron in insect cells and development of new strategy for genetic engineering technology of mosqtuito and its pathogens.
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China. wangyanhai79@126.com
Yan-hai
YH
Wang
Zhi-fa
ZF
Lai
Jin-bao
JB
Gu
D000818:Animals; D002460:Cell Line; D009033:Culicidae / Q000235:genetics* / Q000821:virology; D017990:Densovirus / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D016133:Polymerase Chain Reaction
D049452:Green Fluorescent Proteins