Recombination expression and immunoprotective effects of the P6 outer membrane protein of nontypeable Haemophilus influenzae
CHEN Xiang-peng, WANG Jian, LI Li-li, CHEN Jing, ZHANG Zhen-long
Published 2010-04-30
Cite as Chin J Microbiol Immunol, 2010,30(04): 349-354. DOI: 10.3760/cma.j.issn.0254-5101.2010.04.015
Abstract
Objective To clone and express recombinant outer membrane protein P6, determine its optimal expression conditions and to investigate the immunoprotective effects of the P6 protein on mice. Methods The P6 gene of nontypeable Haemophilus influertzae(NTHi) was amplified by PCR from the NTHi genome and cloned into expression vector pET-32a (+) to generate the pET-32a-P6 recombinants. They were confirmed by nuclease digestion and sequence analysis. The verified recombinant was transformed into E. coli BL21 (DE3). Its optimal expression conditions were determined such as engineering strains, the concentration of IPTG, inducing temperature, inducing time, different medium etc. The recombinant protein was purified by Q Sepharose~(TM) XL ion exchange and gel filtration chromatography. The protein was analyzed by SDS-PAGE, Western blot and sequencing. BALB/c mice were immunized with recombinant protein be-fore challenged by NTHi through intraperitoneal injection. Then the mortality rate of different group was com-pared. Results The recombinant P6 of NTHi was successfully constructed and expressed in E. coli at a rel-atively high level. The purity was up to 95% after purification. The relative molecular mass of the protein is 14 145. 848. The recombinant protein was confirmed to show specific reaction on the antiserum through Western blot. The animal experiments showed the mortality rates of immunization groups were significantly lower than that of the control group (P < 0.05). Conclusion The successful expression of the recombinant P6 will be very helpful for the further study on development of vaccine, its purified, immunological activity and antibody preparation.
Key words:
Nontypeable Haemophilns influenzae; Outer membrane protein; P6; Cloning and expression; Immunoprotective
Contributor Information
CHEN Xiang-peng
Semi-works Production Division 1, National Vaccine and Serum Institute, Beijing 100024, China
WANG Jian
Semi-works Production Division 1, National Vaccine and Serum Institute, Beijing 100024, China
LI Li-li
Semi-works Production Division 1, National Vaccine and Serum Institute, Beijing 100024, China
CHEN Jing
Semi-works Production Division 1, National Vaccine and Serum Institute, Beijing 100024, China
ZHANG Zhen-long
Semi-works Production Division 1, National Vaccine and Serum Institute, Beijing 100024, China