科研动态

孔雀石绿（malachite green，MG）是一种易溶于水和乙醇等的基础染料，可在多种行业应用，并曾用于水生动物多种疾病如真菌病、寄生虫病、细菌性疾病等的治疗。但因其具有致癌等毒副作用，目前已被禁用。然而市场上仍有在水产品违法使用MG的现象，因此需要加强对该药品残留的检测。本研究选择羊驼免疫库筛选抗MG纳米抗体，通过噬菌体ELISA筛选高亲和力和特异性的纳米抗体。通过辅助噬菌体M13的救援，使得纳米抗体基因（VHH）与噬菌体外壳蛋白基因融合表达，从而使得VHH蛋白展示在噬菌体表面，通过有限稀释筛选和多次洗脱的办法，获得含有目的基因的单个噬菌克隆；将重组质粒PMES4-VHH转化到表达细胞中，进行体外表达。最后采用NI-NTA柱进行His标签纯化，得到抗MG纳米抗体，并进行SDS-PAGE电泳鉴定。结果显示：噬菌体文库成功构建，库容为3.2×108 cfu/mL；通过ELISA筛选，得到16个具有抑制效果的噬菌体克隆，经对噬菌体基因测序并翻译，得到2种氨基酸序列；将重组质粒转化至表达菌株表达、纯化，最后得到大小约为15 kDa的抗MG纳米抗体，从而为MG的下一步检测奠定了基础。

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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