In vitro assembly of an infectious cDNA clone of infectious bronchitis virus and its application as a gene transfer vector /

Bibliographic Details
Main Author:	Youn, Soonjeon, 1971-
Other Authors:	Collisson, Ellen W. (Thesis advisor)
Format:	Thesis eBook
Language:	English
Published:	[College Station, Tex.] : [Texas A&M University], [2005]
Subjects:	Major veterinary microbiology. infectious cDNA clone IBV in vitro assembly coronavirus
Online Access:	Link to OAK Trust copy

Description
Abstract:	An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.
Item Description:	"Major Subject: Veterinary Microbiology" Title from author supplied metadata (automated record created on Feb. 17, 2005.) Vita. Abstract. Electronic resource.
Format:	Mode of access: World Wide Web. System requirements: World Wide Web access and Adobe Acrobat Reader.
Bibliography:	Includes bibliographical references.