Developing efficient strategies for localizing the enhanced yellow fluorescent protein subcellularly in transgenic Eimeria parasites.

Eimeria species serve as promising eukaryotic vaccine vectors. And that the location of heterologous antigens in the subcellular components of genetically modified Eimeria may determine the magnitude and type of immune responses. Therefore, our study aimed to target a heterologous fluorescent protein to the cell surface or microneme, two locations where are more effective in inducing protective immunity, of Eimeria tenella and E. acervulina sporozoites. We used an enhanced yellow fluorescent protein (EYFP) as a tagging biomarker, fusing variously with some localization or whole sequences of compartmental proteins for targeting. After acquiring stable transgenic Eimeria populations, we observed EYFP expressing in expected locations with certain strategies. That is, EYFP successfully localized to the surface when it was fused between signal peptides and mature products of surface antigen 1 (SAG1). Furthermore, EYFP was efficiently targeted to the apical end, an optimal location for secretory organelle known as the microneme, when fused to the C terminus of microneme protein 2. Unexpectedly, EYFP exhibited dominantly in the apical end with only weak expression on the surface of the transgenic sporozoites when the parasites were transfected with plasmid with EYFP fused between signal peptides and mature products of E. tenella SAG 13. These strategies worked in both E. tenella and E. acervulina, laying a solid foundation for studying E. tenella and E. acervulina-based live vaccines that can be further tailored to the inclusion of cargo immunogens from other pathogens.

Production and Identification of Monoclonal Antibody Against Flumequine and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay / 分析化学

The hapten of Flumequine(FLU) with four carbon atoms spacer arm(FLUABA) was synthesized and coupled to bovine serum albumin(BSA) as immunogen using activated ester method. Balb/c mice were immunized by the artificial immunogen and the splenocytes of immunized mice were fused with Sp2/0 cells to obtain the monoclonal antibody(McAbs). A hybridoma cell line(DB6-E7) secreting anti-flumequine McAbs was obtained by limited dilution method and screened by indirect enzyme-linked immunosorbent assay(ELISA) using heterogenous coating antigen. The results showed that the subtype of the McAb was IgG_1, and the affinity was 8.19×10~8 L/mol. The haptens of FLU, FLUABA and FLUACA, with different space arm, were separately linked to ovalbumin(OVA) for heterologous or homologous coating antigen. The results of indirect ELISA and indirect competitive ELISA(icELISA) indicated that the heterologous coating antigen could improve the sensitivity of ELISA significantly. By heterologous coating antigen(FLU-OVA), the icELISA showed an IC_(50) value of 26.33 μg/L, LOD of 4.0 μg/L, and the workable range of 8-114 μg/L (IC_(20)-IC_(80)). Cross-reactivity studies showed that the McAbs were quiet specific for FLU, no cross-reactivity(<0.1%) was detected between the obtained McAbs and the quinolones compounds or other structural similarity compounds. The developed icELISA for FLU can satisfy the detection criteria of flumequine in animal food-products.