本文選題：黃鱔 + 醛酮還原酶��； 參考：《水產(chǎn)科學》2017年06期

【摘要】：為實現(xiàn)原核表達黃鱔醛酮還原酶基因并制備其多克隆抗體,筆者利用基因特異性引物自黃鱔肝臟cDNA中擴增黃鱔醛酮還原酶全長序列,將之克隆至原核表達載體pET-28a(+)中,構建了重組表達載體;經(jīng)測序驗證后的重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)后進行IPTG誘導。利用Ni離子親和柱純化了重組蛋白,通過多次免疫新西蘭兔制備了多克隆抗體;采用Western blot和免疫組化法鑒定兔抗醛酮還原酶多克隆抗體的特異性,用間接ELISA技術檢測多抗的效價。結果發(fā)現(xiàn)成功構建pET/Eakr原核表達載體,并實現(xiàn)了重組蛋白的融合表達和純化;Western blot檢測表明制備的兔多克隆抗體不僅能特異性地識別來源于黃鱔4種組織的醛酮還原酶蛋白而且還可識別來源于黃顙魚、團頭魴、烏鱧、鯽魚和草魚的同源蛋白。免疫組化結果提示該多抗可特異性識別黃鱔小腸、肝胰組織和脾臟的醛酮還原酶蛋白。制備的多抗效價達1∶6400。
[Abstract]:In order to express aldosterone reductase gene in prokaryotic expression and prepare polyclonal antibody, the full-length aldosterone reductase was amplified from the liver cDNA of Monopterus Albus by gene specific primers and cloned into the prokaryotic expression vector pET-28a (). The recombinant expression vector was constructed and transformed into E. coli BL21DE3 by sequencing. The recombinant protein was purified by Ni ion affinity column, polyclonal antibodies were prepared by immunizing New Zealand rabbits, and the specificity of polyclonal antibodies against aldehyde-ketone reductase was identified by Western blot and immunohistochemistry. The titers of polyclonal antibodies were detected by indirect Elisa. The results showed that pET-Eakr prokaryotic expression vector was successfully constructed. The fusion expression of the recombinant protein and the purification of the recombinant protein by Western blot showed that the rabbit polyclonal antibody could recognize not only aldehydes and ketone reductase proteins from four tissues of Monopterus Albus, but also from Pelteobagrus fulvidraco, bream, snakehead Argus. The homologous proteins of crucian carp and grass carp. The results of immunohistochemistry indicated that the polyclonal antibody could specifically identify aldehydes and ketone reductase proteins in small intestine, liver, pancreas and spleen of Monopterus Albus. The titer of the prepared polyclonal antibody is 1: 6400.
【作者單位】： 長江大學生命科學學院;武漢大學生命科學學院;
【基金】：國家大學生創(chuàng)新創(chuàng)業(yè)訓練計劃項目(201510489007) 濕地生態(tài)與農(nóng)業(yè)利用教育部工程研究中心開放課題項目(KF2015016)
【分類號】：S917.4

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本文編號：2033922