title: [Gene sequence analysis and prokaryotic expression of sigmaB protein of Muscovy duck reovirus YB strain].
abstract: Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.
authors.affiliation: University Key Lab for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, The Animal Science College of Fujian Agricultural and Forestry University, Fuzhou 350002, China. pingping19781015@yahoo.cn
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Wu
authors.forename: Hong-Xing
authors.initials: HX
authors.lastname: Zhang
authors.forename: Yi-Jian
authors.initials: YJ
authors.lastname: Wu
authors.forename: Dian-Lin
authors.initials: DL
authors.lastname: Han
authors.forename: Shao
authors.initials: S
authors.lastname: Wang
authors.forename: Bao-Cheng
authors.initials: BC
authors.lastname: Wu
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D030101:Orthoreovirus, Avian / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D010802:Phylogeny; D011201:Poultry Diseases / Q000821:virology*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012088:Reoviridae Infections / Q000662:veterinary* / Q000821:virology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
chemical_list: D036022:Capsid Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C053179:sigma protein 3, Reovirus