孔雀石綠（malachite green，MG）是一種易溶于水和乙醇等的基礎染料，可在多種行業(yè)應用，并曾用于水生動物多種疾病如真菌病、寄生蟲病、細菌性疾病等的治療。但因其具有致癌等毒副作用，目前已被禁用。然而市場上仍有在水產品違法使用MG的現(xiàn)象，因此需要加強對該藥品殘留的檢測。本研究選擇羊駝免疫庫篩選抗MG納米抗體，通過噬菌體ELISA篩選高親和力和特異性的納米抗體。通過輔助噬菌體M13的救援，使得納米抗體基因（VHH）與噬菌體外殼蛋白基因融合表達，從而使得VHH蛋白展示在噬菌體表面，通過有限稀釋篩選和多次洗脫的辦法，獲得含有目的基因的單個噬菌克??；將重組質粒PMES4-VHH轉化到表達細胞中，進行體外表達。最后采用NI-NTA柱進行His標簽純化，得到抗MG納米抗體，并進行SDS-PAGE電泳鑒定。結果顯示：噬菌體文庫成功構建，庫容為3.2×108 cfu/mL；通過ELISA篩選，得到16個具有抑制效果的噬菌體克隆，經對噬菌體基因測序并翻譯，得到2種氨基酸序列；將重組質粒轉化至表達菌株表達、純化，最后得到大小約為15 kDa的抗MG納米抗體，從而為MG的下一步檢測奠定了基礎。
Preparation of Alpaca Immune Repertoire and Selection of Nano-antibody against Malachite Green
Malachite green（MG）is a kind of primary dye that is soluble in water and ethanol，and has been applied in many industries，especially to fight against many diseases of aquatic animals，such as fungal diseases，parasitic diseases and bacterial diseases. But it has been banned due to its adverse reaction such as carcinogenicity. However，it is necessary to strengthen the detection of drug residues from MG that is still illegally used in aquatic products in markets. In this study，nano-antibody with high affinity and specificity against MG was selected from alpaca immune repertoire through the phage by ELISA，then was used to support the rescue of phage M13，so that nano-antibody gene（VHH）could be fused and expressed with the coat protein gene of the phage，and VHH protein could be shown on the surface of the phage to obtain the single phagocytic colonies containing the targeting gene by means of limited dilution screening and multiple elution；the recombinant plasmid PMES4-VHH were transformed into expression cells and expressed in vitro，then His-tag purification was carried out by the NI-NTA column to obtain the nano-antibody against MG that was identified by SDS-PAGE electrophoresis. It was concluded that the immune repertoire was successfully constructed with a capacity of 3.2×108 cfu/mL；16 colonies with inhibitory effect were screened by ELISA，two kinds of amino acid sequences were obtained by sequencing and translating the phage genes；PMES4-VHH was transformed into the expression strain for expression and purification，and the nano-antibody of about 15 kDa against MG was produced，which paved the way to future detection of MG.
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