title: [Cloning Hap gene from non-typeable Haemophilus influenzae and expression of Hap protein in prokaryotic cell].
abstract: 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.
authors: Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
authors: Wanyi
authors: W
authors: Li
authors: Yu
authors: Y
authors: Kuang
authors: Feng
authors: F
authors: Yao
authors: Yuan
authors: Y
authors: Yang
authors: Changchun
authors: C
authors: Chen
authors: Zhonghua
authors: Z
authors: Jiang
authors: Mingyuan
authors: M
authors: Li
mesh_terms: 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
chemical_list: D001425:Bacterial Outer Membrane Proteins; C091552:Hap protein, Hemophilus influenzae; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases