[Construction of recombinant yellow fever virus 17D containing 2A fragment as a vaccine vector].
The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.
Xiaowu
Pang
P
Beijing Oriental Tengen Tech Development Co., Ltd., Beijing 100078, China.
Fu
Wen-Chuan
WC
Guo
Yin-Han
YH
Zhang
Li-Shu
LS
Xie
Tian-Pei
TP
Xinbin
Gu
G
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D000939:Epitopes / Q000276:immunology; D005536:Foot-and-Mouth Disease / Q000517:prevention & control; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000276:immunology; D005818:Genetic Engineering; D005822:Genetic Vectors; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014613:Vaccines, Attenuated; D014765:Viral Vaccines / Q000235:genetics* / Q000276:immunology; D015005:Yellow fever virus / Q000235:genetics / Q000276:immunology
D000939:Epitopes; D014613:Vaccines, Attenuated; D014765:Viral Vaccines