[Cloning Hap gene from non-typeable Haemophilus influenzae and expression of Hap protein in prokaryotic cell].
This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.
Li
Wanyi
W
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Kuang
Yu
Y
Yao
Feng
F
Yang
Yuan
Y
Chen
Changchun
C
Jiang
Zhonghua
Z
Li
Mingyuan
M
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006193:Haemophilus influenzae / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases