Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig

Study on the Expression and Bioactivities of Porcine Interleukin-2/6 Fusion Protein in Vivo and in Vitro

Author MaWenTao
Tutor WuZhiMing
School Henan Agricultural University
Course Preventive Veterinary Medicine
Keywords porcine interleukin-2 porcine interleukin-6 chimeric gene fusion expression bioactivity
CLC S858.28
Type Master's thesis
Year 2011
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Vaccine inoculation is the important method to control the porcine infectious diseases.immunization failure and low protective rate often occurred due to vaccine defects and immunerepress disease.so it’s of importance to inhance immunity and protective efficiency in controlling porcine infectious diseases.the cytokine can inhance the immune effect and immunity. Therefore the research on the cytokine as the immunoenhancer comes to be the focus.Interleukin-2 and interleukin-6 belongs to the Th1 and Th2 cytokine respectively,mediating cellular immune and humoral immune.in order to develop a high-efficient immunoenhancer balancing Th1 and Th2 responses,The porcine inteleukin-6 mature peptide gene was cloned from the total cellular RNA of porcine cell induced by bacterial lipopolysaccharides (LPS) by SOE-PCR (splicing by overlap extension-PCR) method,and subsequently sub-cloned into prokaryotic expression vector pQE30. The recombinant protein was expressed in E.coli. JM109 and purified under the innovated reconbinant fusion protein purification method of denaturing by 8M urea, refolding by a self-innovative renaturation buffer and dialyzing by PBS buffer etc.the method of the porcine IL-6 ELISA assay was used to detect the specificial immunoactivity of the rPoIL-6 protein.the cell proliferation activity induced by rPoIL-6 protein was assessed by MTS assay. The result show that the PoIL-6 mature peptide gene with a length of 573bp was successfully cloned;the expressed protein molecular weight was 20KD and reached a purity of more than 95% after purification. rPoIL-6 protein was specifically reacted with McAb to PoIL-6;the rPoIL-6 protein activation was 1.23x106UI/ mg and had a significant proliferation activity on porcine kidney cells.In order to explore a high efficient porcine genetic engineering immune enhancement agent for vaccine adjuvant,the recombinant chimeric gene of PoIL-2-linker-PoIL-6 constructed by porcine interleukin-2 (PoIL-2) gene linked interleukin-6 (PoIL-6) gene via a flexible linker by splicing overlap extension PCR (SOE-PCR) method was cloned into prokaryotic expression vector pQE30. The recombinant PoIL-2-linker-PoIL-6 fusion protein (rPoIL-2-linker-PoIL-6) was expressed in a pQE30/E.coli JM109 system and purified with the innovated recombinant fusion protein purification method. The activities of rPoIL-2-linker-PoIL-6 protein were estimated by detection its specific immune response to monoclonal antibody (MAb) against PoIL-2 and PoIL-6 by ELISA assay, and the abilities of promoting the proliferation of porcine peripheral blood T lymphocyte (PBLC) and spleen lymphoblast cells. The results indicated chimeric gene of PoIL-2-linker-PoIL-6 was successfully constructed and abundantly fusion expressed in E.coli. and the fusion rPoIL-2-linker-PoIL-6 protein was successfully purified with the molecular mass of about 28 kD and more than 95% pure on SDS-PAGE. The rPoIL-2-linker-PoIL-6 protein displayed the specific immune response to monoclonal antibody (MAb) against PoIL-2 and PoIL-6, as well as the bioactivities of significant promoting the proliferation of porcine PBLC and spleen lymphoblast cells in vivo. This study primarily proved that rPoIL-2-linker-PoIL-6 protein had the duplex bioactivity of PoIL-2 and PoIL-6 protein in vitro, which laid a foundation for further widely use of rPoIL-2 and PoIL-6 protein in vivo.

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