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Protein expression and application of recombinant chicken interleukin-2
|關鍵字:||chicken interleukin-2 (chIL2)|
recombinant chIL2 (rchIL2 protein)
|摘要:||本研究目的是表現雞介白素-2 （chicken interleukin-2, chIL2）之重組蛋白、製備其多株抗體、以及開發偵測chIL2 之酵素連結免疫吸附法（enzyme-linked immunosorbent assay, ELISA）試劑。我們以大豆素A （concanavalin A, ConA）刺激雞的周邊血白血球，萃取RNA，進行反轉錄聚合酶鏈反應（reverse transcription-polymerase chain reaction, RT-PCR）選殖chIL2之cDNA基因，構築chIL2原核表現質體pETchIL2，送入大腸桿菌誘導重組chIL2 （recombinant chIL2, rchIL2） 蛋白之表現，純化之重組蛋白經質譜儀分析確認為rchIL2。其次，將rchIL2蛋白分別施打於小鼠及兔子，產生的多株抗體以西方墨漬法分析其特異性。於本研究中小鼠及兔子進行免疫注射時，抗原rchIL2蛋白係融合pET32載體所表現之tag protein與chIL2，所產生的抗體包含anti- tag protein抗體(anti-tag protein antibody) 與anti-chIL2抗體（anti-chIL2 antibody）。為開發chIL2 之 ELISA 套組，必須先製備高特異性的抗chIL2抗體，我們於是將anti-rchIL2抗體通過已結合tag protein之鎳離子管柱，由此去除anti-tag protein 抗體而得到純化的anti-chIL2抗體。將純化的小鼠及兔子anti-chIL2多株抗體組成 ELISA 套組，進行rchIL2的偵測，結果顯示此套組對chIL2具有特異性，可偵測rchIL2蛋白之最低濃度為10 pg/ml。
本研究已開發chIL2重組蛋白、小鼠及兔子anti-chIL2多株抗體、及偵測chIL2 之 ELISA套組，這些試劑可提供於禽病防疫或相關研究。|
Aims of this study are to express recombinant chicken interleukin-2 (rchIL2) protein, to prepare polyclonal antibody specific for chicken interleukin-2 (chIL2), and to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of chIL2. Chicken peripheral leukocytes were stimulated with concanavalin A (ConA), total RNA was extracted, and chIL2 cDNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR). The chIL2 cDNA was cloned into pET32 vector to construct pETchIL2 plasmid. The plasmid was introduced into Escherichia coli and rchIL2 protein was expressed upon induction. Identity of rchIL2 confirmed by mass spectrometry, the six mice and one rabbit were immunized purified with rchIL2 protein and specificity of the elicited polyclonal antibodies were determined by Western blot analysis. Because rchIL2 expressed in this study comprises tag protein encoded by pET32 vector along with chIL2, anti-rchIL2 antibodies produced by mice and rabbit thereby contain both anti-tag protein antibody and anti-chIL2 antibody. To develop ELISA for chIL2 detection, we need to prepare anti-chIL2 antibody with high specificity. Purification of anti-chIL2 antibody was achieved by passing anti-rchIL2 antibodies through a tag protein-bound Ni2+-resin column to remove anti-tag protein antibody. Both mouse and rabbit anti-chIL2 antibodies were purified and used to develop an ELISA kit. The result showed that the ELISA kit has high specificity for chIL2, and the detection limit reached 10 pg/ml of rchIL2 protein. In conclusion, in this study, we have expressed rchIL2 protein, prepared mouse and rabbit anti-chIL2 polyclonal antibodies, and developed an ELISA kit for chIL2 detection. These reagents can be provided for disease control in poultry or related research.
|Appears in Collections:||微生物暨公共衛生學研究所|
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