[Prokaryotic expression and antigenic activity analysis on the matrix protein genes of two strains of human metapneumovirus recently identified in Beijing].
Human metapneumovirus (hMPV) is a recently identified respiratory virus more like human respiratory syncytial virus in clinical symptoms. Matrix protein (M) is one of the most important structural proteins. For further studying of hMPV, the full length of M genes from the recombinant plasmid pUCm-M1816 and pUCmM1817 were cloned by PCR and sub-cloned into the pET30a(+) vector, which is a prokaryotic expression vector, after dual-enzyme digestion with Bam HI and Xho I. The positive recombinated plasmids were transformed into E. coli BL21 (DE3) and expressed under the inducing of IPTG. Target proteins were characterized by SDS-PAGE and Western blotting. In this article, we' ve successfully constructed the recombinated plasmids pET30a-M1816 and pET30a-M1817 which have correct open reading frames confirmed by dual-enzyme digestion analysis and sequencing. The fusion proteins with 6 x His-N were highly produced after inducing by 1mmol/ L IPTG at 37 degrees C. A unique protein band with approximate 27.6 kD was characterized by SDS-PAGE. Most of the target protein existed in inclusion body. Western blot analysis showed that the target protein has specific binding reaction to rabbit antiserum against polypeptides of the matrix protein of hMPV. So the M genes were highly expressed in the prokaryotic system and the expressed M proteins have specific antigenic activities. It can be used for further studying of hMPV infections in Beijing.
Cao
Shou-Chun
SC
Laboratory of Virology, Beijing Municipal Laboratory of Infection and Immunity, Capital Institute of Pediatrics, Beijing 100020, China.
Qian
Yuan
Y
Li
Guo-Hua
GH
Zhu
Ru-Nan
RN
Zhao
Lin-Qing
LQ
Ding
Ya-Xin
YX
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002681:China; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D029121:Metapneumovirus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011817:Rabbits; D013045:Species Specificity; D015678:Viral Structural Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000956:Antigens, Viral; D007106:Immune Sera; D015678:Viral Structural Proteins