닭의 장내에 기생하는 Eimerian Protozoa의 표면 항원에 결합하는 재조합 닭 항체 단백질 8C3 ScFv의 클로닝, 정제, 및 특성화
Cloning, purification, and characterization of recombinant chicken 8C3 ScFv antibody fragment specific to surface antigens of intestinal parasite, Eimerian Protozoa
닭 장 기생 Eimerian Protozoa 표면 항원 항체 단백질 8C3 ScFv 클로닝 정제;
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Avian coccidiosis is a very important disease of poultry industry. Coccidiosis, caused by intestinal parasites belonging to genus Eimeria, is an obligate protozoan disease of chickens, resulting in a significant economic loss in the poultry industry. In recent years, many researchers studied of avian coccidiosis. Coccidial therapeutic drug have been used at some poultry farms in many countries. However, despite increasing interest in developing protection strategies, the use of whole parasites or chemotherapy has several problems. The application of coccidial therapeutic drug is hindered by high costs and development of drug resistance. To bypass these problems, we applied the antibody engineering to develop an immunotherapeutic antibody molecule using recombinant DNA technology. In general, antibodies have several merits over small molecule drug, because it is easier and faster in development, as proper immunization of the target proteins usually raises good antibody response. Therefore, in this study, recombinant chicken single-chain variable domain fragment(8C3 ScFv) has been produced in Escherichia coli, using cDNA derived from hybridoma cells by RT-PCR. Expressed recombinant 8C3 ScFv showed antigen binding activity specific to Eimerian Protozoa such as Eimeria acervulina and Eimeria tenella. Unlike mammals such as mice and humans, the immunoglobulin gene diversification in chickens is mainly constructed by gene conversion. Gene conversion in chickens make it possible to amplify variable region genes using a single pair of primers per heave(VH) and light(VL) chain. The sequencing analysis of cloned 8C3 VH and VL with germline chicken VH and VL. And other VH and VL gene, and confirmed the gene conversion for the immunoglobulin gene diversification in chicken. For the expression of a soluble ScFv, orientation and linker design are important factors. Therefore, this study has examined the effects of four different linker(LK, 20LK, EK and 12EK) and orientation(VH-VL/VH-VL) to determine the most suitable linker and orientation for the production of soluble 8C3 ScFv. Eight different forms of 8C3 ScFv(HL-LK, LH-LK, HL-20LK, LH-20LK, HL-EK, LH-EK, HL-12EK and LH-12EK) were constructed, expressed and characterized. I found that LH-12EK, LH-LK and LH-EK 8C3 ScFv were secreted as a soluble form and showed their antigen binding specificity. In addition, I expanded the experiments to study the possibility of phage antibody production. To do this, the 5 chicken ScFv including 8C3 LH-12EK were cloned into phagemid vector(pHEN) and were produced as a soluble recombinant phage antibody. Phage antibodies showed antigen binding specificity in ELISA, suggesting phage antibody can be applied to select antigen specific chicken ScFv antibody molecules derived from synthetic ScFv antibody library constructed by combinatorial phage-displayed technique.