title: An efficient and rapid influenza gene cloning strategy for reverse genetics system.
abstract: Influenza reverse genetics plays vital roles in understanding influenza molecular characteristics and vaccine development. However, current influenza reverse genetics heavily depends on restriction enzyme and ligation for gene cloning. The traditional cloning process of influenza eight fragments for virus rescuing generally requires considerable work. To simplify and increase the pace of gene cloning for influenza reverse genetics system, we developed a rapid restriction enzyme-free ExnaseTM II-based in vitro recombination approach for influenza gene cloning. We used this strategy rapidly and successfully to clone influenza eight genes both from viruses PR8 and H9N2 for virus rescuing. Our data demonstrate that the strategy developed here can accelerate the process of influenza gene cloning into reverse genetics system, and shows high potential for applications in both influenza basic and applied research.
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Shao
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China.
authors.forename: Zhonglei
authors.initials: Z
authors.lastname: Fan
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Wan
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Tian
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Hongjun
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Veterinary Medicine, University of Maryland, and Virginia-Maryland Regional College of Veterinary Medicine, 8075 Greenmead Drive, College Park, MD 20742, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Perez
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China.
authors.forename: Aijian
authors.initials: A
authors.lastname: Qin
authors.affiliation: Ministry of Education Key Lab for Avian Preventive Medicine, and Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, 12 Wenhui Road, Yangzhou 225009, Jiangsu, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, Jiangsu, PR China. Electronic address: jqye@yzu.edu.cn.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Ye
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D009975:Orthomyxoviridae / Q000235:genetics*; D011995:Recombination, Genetic; D059386:Reverse Genetics / Q000379:methods*; D013997:Time Factors; D014773:Virology / Q000379:methods*
keywords: In vitro recombination; Influenza reverse genetics; Rapid gene cloning