摘要
鸡柔嫩艾美耳球虫属于具有顶复合器的原生动物,主要寄生于鸡的盲肠中,常常引起大批鸡只的死亡,如何更好的控制球虫病是兽医工作者面临的一个课题,本文对细胞因子IL-2和GM-CSF展开研究。
根据GenBank上发表的鸡的IL-2和GM-CSF序列,设计引物。逆转录多聚酶链式反应(RT-PCR)方法,从人工感染柔嫩艾美耳球虫的雏鸡盲肠扁桃体中扩增到了391bp和435bp的DNA片段,将两片段与pGEM-T载体连接后转化JM109感受态细胞,经蓝白斑筛选、PCR鉴定后,将阳性质粒进行测序。结果表明,扩增片断为鸡IL-2和GM-CSF。IL-2和GM-CSF基因与GenBank中报道的序列仅有1-2个碱基的差异,IL-2与鹌鹑的同源性为54.5%,而与哺乳动物IL-2的同源性在29.9%~34.5%之间。GM-CSF与哺乳动物的同源性在35.2%~45.4%之间。
以鸡β-Actin为内参进行半定量RT-PCR检测柔嫩艾美耳球虫孢子化卵囊免疫前后鸡盲肠扁桃体IL-2和GM-CSF基因表达的动态变化。结果表明,ChIL-2在第一次免疫后的第九天和第二次免疫后的第七天ChIL-2的表达明显升高;ChGM-CSF在第一次免疫后的第一天(P<0.05)和第二次免疫后的第七天(P<0.01),ChGM-CSF的表达量显著升高,在第一次免疫后的第九天和第二次免疫后的第三天ChGM-CSF的表达量显著降低(P<0.01)。
按照pGEX-6p-1表达载体的多克隆位点,重新设计IL-2和GM-CSF带有相应限制性内切酶酶切位点的引物,通过RT-PCR克隆获得两目的基因,与pGEM-T载体连接后转化JM109感受态细胞,经蓝白斑筛选、测序正确后,进行双酶切,胶回收酶切后的目的基因片段,将其与pGEX-6p-1表达载体连接,转化入BL21感受态细胞中。经双酶切和PCR鉴定为阳性的菌株,测序鉴定正确后,用IPTG最佳诱导条件表达IL-2和GM-CSF蛋白。通过菌体裂解,包涵体的洗涤、溶解、复性后,过Sepharose 4B柱纯化GST融合蛋白。Western Blot检测IL-2和GM-CSF融合蛋白,通过淋巴细胞增殖试验证明纯化的IL-2和GM-CSF融合蛋白具有一定的生物学活性。
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 vaccinated
Another 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.
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