[Construction and identification of recombinant adenovirus containing the polyproteins coding regions of O type foot-and-mouth disease virus by homologous recombination in Escherichia coli].
The gene coding for the polyprotein (PP) of foot-and-mouth disease virus (FMDV)was obtained by PCR from recombinant plasmid rpMD18-T/PP. The PCR product was digested with Xba I and Not I and inserted into the cloning site of the adenovirus shuttle vector pAdTrack-CMV, previously digested with the same enzymes. This recombinant shuttle plasmid was designated rpAd-CMV/PP. The recombinant adenovirus vector rpAd/PP was obtained by homologous recombination of plasmid rpAd-CMV/PP and adenovirus skeletal vector pAdeasy-1 in E. coli. Plasmid rpAd/PP was linearized by Pme I and transformed into 293 competent cells to pack the adenovirus using liposome mediated gene transfer method and, as a result, the recombinant adenovirus rAd/PP that contained the polyprotein coding gene was obtained. Obvious CPE could be observed under an inverted microscope, the green fluorescence protein expression can be detected under fluorescence microscope and the empty capsid of FMDV was observed under electron microscope. These results indicated that the recombinant adenovirus rAd/PP expressed the PP protein and that this protein could be assembled into the empty capsid of FMDV. The recombinant adenovirus obtained in this study can be used for further research for making FMDV recombinant adenovirus vaccine.
Zhang
Xing-Wang
XW
Key Laboratory of Animal Virology of Ministry of Agriculture, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China. ZXW2002@xinhuanet.com
Wang
Qin
Q
Liu
Ji-Xing
JX
Yin
Xiang-Ping
XP
Li
Zhi-Yong
ZY
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005536:Foot-and-Mouth Disease / Q000821:virology; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000502:physiology; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020815:Polyproteins / Q000235:genetics* / Q000378:metabolism; D016865:Reassortant Viruses / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D019065:Virus Assembly
D020815:Polyproteins; D014764:Viral Proteins