Abstract:

Objective This study aims to express the merozoite surface protein 1 N-terminal proteins of Plasmodium ovale curtisi and P. ovale wallikeri, and analyze their antigenicity and immunogenicity, revealing the potential for being as vaccine candidates of P. ovale. Methods The N-terminal of the Pomsp1 gene was amplified from the genomic DNA of P. ovale. The purified N-terminal sequences of Pocmsp1 and Powmsp1 were separately connected to the plasmid pET30a and pET32a and then transfected into DH5α competent cells. After verification with BamHⅠ and XhoⅠ enzyme digestion and DNA sequencing, the gene sequences were transferred into Escherichia coli BL21 (DE3) pLysS. After induction with 0.1 mmol/L IPTG, the expressed proteins were collected, purified, and verified with SDS-PAGE and Western blotting. Fifteen BALB/c mice were randomly assigned into three groups of 5 each: the rPocMSP1 N-terminal protein group, rPowMSP1 N-terminal protein group, and a negative control group. The mice of the three groups were injected intraperitoneally with the recombinant protein of rPocMSP1 N-terminal and rPowMSP1 N-terminal protein (50 μg of recombinant protein emulsified with Freund’s complete adjuvant) respectively, while the control group mice were injected with PBS. On 21d and 42d after primary immunization, the mice were boosted with the corresponding antigens emulsified with Freund’s incomplete adjuvant. Furthermore, on days 0, 7, 28, and 49 after immunization, tail vein blood samples were collected for serum preparation. ELISA and Western blotting were used to determine the serum specific IgG, analyze antibody titer and antibody affinity index, thereby to evaluate the immunogenicity of rPocMSP1 N-terminal and rPowMSP1 N-terminal proteins. Western blotting was used to determine the cross-reaction of the two recombinant proteins, and protein array was used to analyze their antigenicity in the serum of examinees infected with P. ovale. Results PCR demonstrated that the sequence length of PocMSP1 N-terminal and PowMSP1 N-terminal fragment was 1 068 bp, showing concordant with the expected size. Produced by PCR cloning, expression and purification, the concentration of rPocMSP1 N-terminal and rPowMSP1 N-terminal recombinant protein was 0.5 mg/ml and 1.0 mg/ml respectively. SDS-PAGE showed that the relative molecular mass of the purified rPocMSP1 N-terminal and rPowMSP1 N-terminal protein was 46 000 and 59 000, respectively, and Western blotting indicated the recombinant proteins were successfully expressed. The sera of immunized mice could recognize the corresponding antigens. ELISA detected specific IgG antibodies on 7 d after immunization, revealing specific reaction between the purified recombinant proteins and IgG, which was significantly different compared with that in the negative control group (rPocMSP1 N-terminal: t = 5.824, P < 0.01; rPowMSP1 N-terminal: t = 25.98, P < 0.01). On 28 d after immunization, the mean A₄₅₀ values for serum IgG in groups rPocMSP1 N-terminal and rPowMSP1 N-terminal were 1.043 ± 0.390, and 1.923 ± 1.373, and when the IgG levels continued to rise up to 49 d after immunization the mean A₄₅₀ values for serum IgG in rPocMSP1 N-terminal was 1.217 ± 0.365, and rPowMSP1 N-terminal was 2.463 ± 0.983. rPoMSP1 N-terminal protein induced considerable immune response in mice, with the antibody titer of 1 ∶ 640 000 and 1 ∶ 1 280 000, respectively.ELISA revealed that rPocMSP1 N-terminal and rPowMSP1 N-terminal proteins induced IgG antibody with high affinities in immunized mice, showing the affinity 97.11% by rPocMSP1 N-terminal protein, and 75.72% by rPowMSP1 N-terminal protein. Western blotting showed that the serum IgG antibody in mice immunized with rPocMSP1 N-terminal could recognize rPowMSP1 N-terminal antigen, and vice versa, the antibody in mice immunized with rPowMSP1 N-terminal antigen could recognize rPocMSP1 N-terminal antigen, demonstrating the two antigens have cross reaction property. Analysis with protein array showed that tested with rPocMSP1 N-terminal protein using P. ovale, the sensitivity and specificity was 83.33% and 57.14%, respectively; while rPowMSP1 N-terminal protein presented 97.62% of sensitivity and 54.76% of specificity; in comparison with the reaction using the sera from normal persons, the difference was statistically significant(t = 5.896, 10.42, P < 0.01). Conclusion rPoMSP1 N-terminal protein showed good immunogenicity, inducing humoral immune response in mice, and displaying significant antigenisity in reacting with the serum of people infected with P. ovale.

Key words: Plasmodium ovale, Merozoite surface protein 1, Immunogenicity, Antigenicity