Vaccinology
Preparation of a fusion protein with pneumococcal surface protein A of two different clades of family Ⅰ and analysis of immunogenicity of the recombinant protein
Haiying Lin, Yonghui Peng, Shuangling Zhang, Chunhua Luo, Meiyun Zheng, Wei Lyu, Duqing Zhan
Published 2015-05-31
Cite as Chin J Microbiol Immunol, 2015, 35(5): 377-381. DOI: 10.3760/cma.j.issn.0254-5101.2015.05.010
Abstract
ObjectiveTo construct a recombinant fusion protein with pneumococcal surface protein A (PspA) of Stretococcus pneumonia (SPN) family Ⅰ clade 1 and 2, and to analyze the immunogenicity of the fusion protein.
MethodsThe gene fragments encoding the α-helix of PspA of the two clades were amplified by PCR and then inserted into the expression vector pET-27b(+ ) to construct the recombinant expression plasmid. The transformed Escherichia coli BL21 strains carrying expression plasmid were induced by IPTG to express the recombinant protein. The titers and affinity of antibodies against PspA protein were measured by ELISA. An opsonophagocytic assay and an animal experiment were performed to evaluate the immunogenicity of the recombinant protein.
ResultsDouble enzyme cutting and gene sequencing confirmed the two purpose gene fragments were correctly expressed in the expression vector pET-27b(+ ). The titers of anti-PspA antibody in the serum of Kunming (KM) mice immunized with the fusion protein were 1×104. The affinity of anti-PspA antibody reached to 2×105. The rates of recombinant PspA6B-PspA05 protein mediated phagocytosis for SPN6B, SPN05 and SPN01 strains were 20%, 15% and 8.8%, respectively. No SPN23F strain was engulfed by macrophages upon the stimulation with PspA6B-PspA05 protein. The survival rates of mice injected with SPN05, SPN6B, SPN01 and SPN23F strains were respectively 75%, 92%, 75% and 33% upon the immunization of PspA6B-PspA05 protein.
ConclusionThe recombinant fusion protein PspA6B-PspA05, constructed with the PspA proteins of Stretococcus pneumonia family Ⅰclade 1 and 2, was successfully expressed in the E. coli prokaryotic system with the advantage of high immunogenicity. High titers of anti-PspA antibodies with high specificity were induced in KM mice upon the stimulation with PspA6B-PspA05 protein. Moreover, a cross-protective immunity was induced in KM mice upon the immunization with PspA6B-PspA05 protein.
Key words:
Stretococcus pneumonia; Pneumococcal surface protein A; Recombinant protein; Immunogenicity
Contributor Information
Haiying Lin
College of Biological Science and Engineering, Institute of Phamaceutical Biotechnology and Bioengineering, Fuzhou University, Fuzhou 350100, China
Yonghui Peng
Shuangling Zhang
Chunhua Luo
Meiyun Zheng
Wei Lyu
Duqing Zhan