[Sequence analysis and prokaryotic expression of nucleocapsid protein genes of human respiratory syncytial viruses isolated from children in Beijing].
:To characterize nucleocapsid (N) protein genes of human respiratory syncytial viruses isolated from children in Beijing and express the N genes in E. coli,seven HRSV strains (three subtype A and four subtype B) were isolated from clinical samples of infants and children with acute respiratory infections and visited the Children's Hospital affiliated to Capital Institute of Pediatrics in Beijing during the period of Jan. 2006 to Mar. Full length of N genes from seven HRSV strains were amplified by reverse-transcription PCR (RT-PCR). The seven PCR amplicons were sequenced after cloning into pUCm-T and the sequences were compared with the N genes from HRSVs in GenBank. N gene was amplified from recombinant plasmid pUCm-N9968 by PCR and then sub-cloned into the prokaryotic expression vector pET30a(+) after digestion with EcoR I and Xho I . The pET30a-N9968 was transformed into E. coli BL21 (DE3) and expressed by inducing with IPTG. Target protein was characterized by SDS-PAGE electrophoresis and Western blot. The amplified N genes were 1 176 bp in length and the deduced N proteins were 391 amino acids in length. The nucleotide identities of N genes among these seven strains were 85.4%-99.7% and the de-duced amino acid similarities were 95.4%-100%. The recombinant plasmid pET30a-N9968 had correct open reading frame confirmed by dual-enzyme digestion and sequence analysis. The fusion protein 6 x HisN was produced after inducing by 1 mmol/L IPTG at 37 degrees C. A unique protein band with molecular weight 49 kD was characterized by SDS-PAGE and purified by Ni2+ affinity chromatography column. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein had specific binding reaction to specific monoclonal antibody and human sera, indicating that the expressed protein is of specific antigenicity.
Sun
Yu
Y
Laboratory of Virology, Beijing Municipal Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Xing
Jiang-Feng
JF
Zhu
Ru-Nan
RN
Deng
Jie
J
Zhao
Lin-Qing
LQ
Wang
Fang
F
Qian
Yuan
Y
D001483:Base Sequence; D002648:Child; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis; D018113:Respiratory Syncytial Virus, Human / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins