Cloning And Expression Of Chicken IL-2 And GM-CSF

Eimeria tenella is an apicomplexan protozoan parasite that replicates within intestinal epithelial cells of the cecal causing the economically important disease coccidiosis.So how to control the disease is a big problem we are facing.In this thesis,we focus on the research of IL-2 and GM-CSF gene.Chicken IL-2 and GM-CSF primers were designed and synthesized by the sequence in GenBank,T wo cDNA fragments of 391bp and 435bp were amplified by RT-PCR from cecal tonsil of chicken which were artificially vaccinated with E.tenella sporulated oocysts.and cloned to pGEM-T vector respectively,They were identified to be interleukine-2 and Granulocyte-macrophage colony stimulating factor cDNA with the methods of restriction enzyme digestion,PCR and sequencing.They are identical to the nucleotide sequences reported in GenBank,Compared with the sequences of chickens repoted in GenBank,there were mutation nucleotides of one to two,IL-2 shared 54.5% homology with Coturnix japonica. and shared 29.9%~34.5% homology with mammalian's.GM-CSF shared 35.2%~45.4% homology with mammalian's.Chickenβ-actin as inner-reference,the expression profiling of ChIL-2 and ChGM-CSF were detected by semi-quantitative RT-PCR at various time-points during E.tenella vaccinated。The results indicated that the expression level of ChIL-2 in experimental group at day 9 post-primary and 7 post-secondary infection significantly increased.The expression of ChGM-CSF in experimental group at day 1 post-primary(P<0.05)and 7 post-secondary(P<0.01)vaccinated significantly increased,decreased significantly(P<0.01)at day 9 post-primary and at day 3 post-secondary vaccinatedAnother two pairs of primers with restriction enzyme for prokaryotic expression was designed respectively according to two genes'CDs complete sequence.Firstly,two genes were amplified by RT-PCR,then cloned into pGEM-T vector and transformed into the JM109 competent cells,the plasmid of white spot was identified by PCR and sequenced. The positive plasmid was cutted by two restriction enzymes. The objective gene fragments were retrieved and cloned into expressing vector pGEX-6p-1 and transformed into BL21 competent cells.The plasmid of white spot was identified by PCR and restriction enzyme of endonucleases.Two fusion proteins were expressed by different inducing condition of IPTG.refolded the inclusionbody of the fusion protein,purified by Glutathione Sepharose 4B.The specific band was identified GST antibody through Western blot analysis.their biological activity were asssyed by lymphoproliferation test.