Molecular cloning and expression of OspA peptide from a Chinese Borrelia garinii strain PD91 and preliminary study on its immunoprotectivity

Abstract

Objective To clone and express the 126-274 aa OspA peptide (OspA-pep) of Chinese Borrelia garinii (B.garinii) strain PD91 and to preliminarily study its immune protectivity. Methods The gene encoding the 126-274 aa OspA-pep of B. garinii PD91 was amplified by polymerase chain reaction (PCR) and then cloned into the prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-OspA-pep. Escherichia coli BL21 (DE3) competent cells transfected with the recombinant plasmid were induced by IPTG to express the target protein. The recombinant OspA-pep (rOspA-pep) was purified with Ni-IDA resin chromatography and its immunogenicity was analyzed by Western blot. New Zealand white rabbits were immunized with different doses of rOspA-pep (20, 30, 40, 50, 60, 80 and 100 μg). The titers of specific IgG antibodies in rabbit serum samples before and after immunization were detected by indirect immunofluorescence assay (IFA). The optimal immune dose was determined according to the antibody titer after immunization. In vitro neutralization test was performed to detect the immune protection of rOspA-pep using serum samples of the optimal immunization group. The optimal dose of rOspA-pep was used to immunize New Zealand white rabbits to observe the changes in antibody titer. Results The recombinant plasmid pET-30a-OspA-pep was successfully constructed and highly expressed in host bacteria. Western blot showed that rOspA-pep had obvious antigen-antibody reaction with polyclonal antibody against B. garinii PD91 strain. IFA results showed the titers of IgG antibody in serum samples of rabbits immunized with rOspA-pep increased significantly (up to 1∶2 480) and 40 μg was the optimal dose. The neutralization rates of antibodies induced by 40 μg of rOspA-pep were 100% against 106 strain/ml of representative B. garinii PD91 and Borrelia afzelii (B.afzelii) FP1 strains, 100% against 107 strain/ml of FP1 strain, and 60% against 107 strain/ml of PD91 strain. After immunization with 40 μg rOspA-pep on 1 d and 30 d, the titers of specific IgG antibody in rabbit serum samples reached the peak within two months, and maintained at that level for about 3-4 months before a gradual decline. Conclusions The 126-274 aa OspA peptide fragment of Chinese B. garinii PD91 strain possessed good immunogenicity and induced antibodies with better in vitro neutralizing activity, which suggested that it could be used as a candidate component of the second generation subunit vaccine in China. Key words: Borrelia garinii; OspA peptide; Cloning and expression; Immunoprotectivity