从噬菌体展示的抗黄曲霉毒素B1(AFB1)纳米抗体文库中,通过四轮亲和淘选,得到抗AFB1纳米抗体G8。将编码纳米抗体G8的基因与碱性磷酸酶(AP)基因融合,构建了重组融合表达载体pET25b(+)-G8-AP,将其转化大肠杆菌BL21(Rosetta,DE3)感受态细胞,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导融合基因表达。SDS-PAGE结果表明,融合蛋白G8-AP为可溶性表达,Ni2+-NTA亲和层析柱纯化融合蛋白,对硝基苯磷酸二钠(pNPP)法测得纯化后的G8-AP的碱性磷酸酶比活力为(364.5±8.3) U/mg。ELISA检测结果表明,G8-AP具有特异识别AFB1的活性,与其它真菌毒素(AFB2、AFG1、AFG2、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮、伏马菌素B1)无交叉反应。基于G8-AP建立了检测AFB1的一步ELISA法。在优化的条件下,即甲醇浓度20%、盐离子浓度20 mmol/L、pH 7.4条件下,方法的半数抑制浓度(IC50)为19.8 ng/mL,线性范围为4.3-92 ng/mL,检出限为2.6 ng/mL。对玉米和小麦样品加标回收实验结果表明,基质对本方法的干扰不明显,可用于实际样品检测。
A phage clone (designated as the anti-aflatoxin B1 nanobody, G8) specific binding to aflatoxin B1 (AFB1) was screened from an immunized nanobody phage library. The DNA fragment encoding G8 was subcloned into the vector pET25b(+)-alkaline phosphatase (AP), and fused to the N terminal of AP. The fusion protein G8-AP was expressed in E. coli BL21 (Rosetta, DE3) as soluble protein, and purified by immobilized metal affinity chromatography. The purified G8-AP exhibited AP activity of 364.5±8.3 U/mg. Enzyme-linked immunosorbent assay (ELISA) showed that G8-AP was also capable of recognizing AFB1, with negligible cross-reactivity toward other six common mycotoxins. A one-step ELISA was established to detect AFB1 using G8-AP. The effects of methanol concentration, ionic strength, and pH on the detection sensitivity of the G8-AP-ELISA were evaluated and optimized. Under the optimized conditions (methanol 20%, NaCl 20 mmol/L, pH 7.4), the half inhibitory concentration (IC50) of the G8-AP based ELISA was 19.8 ng/mL, with the linear range of 4.3-92 ng/mL and the detection limit of 2.6 ng/mL. The recoveries of AFB1 for corn and wheat samples were 90.4%-101.5%.
参考文献
| [1] | .Abrar M, Anjum F M, Butt M S, Pasha I, Randhawa M A, Saeed F, Waqas K.. Crit. Rev. Food Sci. Nutr., 2013,53:862-874 |
| [2] | GB2761-2011, Maximum Levels of Mycotoxins in Foods.. National Standards of the People's Republic of China 食品中真菌毒素的限量.中华人民共和国国家标准. GB2761-2011 |
| [3] | GB13078-2001, Hygienical Standards for Feeds.. National Standards of the People's Republic of China. 饲料卫生标准.中华人民共和国国家标准. GB13078-2001 |
| [4] | .Iqbal J, Asghar M A, Ahmed A, Khan M A, Jamil K.. Toxicol. Mech. Methods, 2014,24:544-551 |
| [5] | .Gazioglu I, Kolak U.. J. AOAC Int., 2015,98:939-945 |
| [6] | .Lupo A, Roebuck C, Dutcher M, Kennedy J, Abouzied M.. J. AOAC Int., 2010,93:587-599 |
| [7] | .Ji Y, Ren M, Li Y, Huang Z, Shu M, Yang H, Xiong Y, Xu Y.. Talanta, 2015,142:206-212 |
| [8] | .Wang D, Hu W, Xiong Y, Xu Y, Li C M.. Biosens. Bioelectron., 2015,63:185-189 |
| [9] | .Yu F Y, Gribas A V, Vdovenko M M, Sakharov I Y.. Talanta, 2013,107:25-29 |
| [10] | .Zhang B, Du D L, Yin Y M.. Food Anal. Method, 2014,7(9):1798-1803 |
| [11] | .Simons B, Kaplan H, Hefford M A.. J. Immunol. Methods, 2006,315:88-98 |
| [12] | .Hu Z Q, Li H P, Zhang J B, Huang T, Liu J L, Xue S, Wu A B, Liao Y C.. Anal. Chim. Acta, 2013,764:84-92 |
| [13] | .Xue S, Li H P, Zhang J B, Liu J L, Hu Z Q, Gong A D, Huang T, LiaoY C.. Anal. Chem., 2013,85(22):10992-10999 |
| [14] | .Yang S L, Shang Y J, Yin S H, Wang D, Cai J P, Gong Z L, Serge M, Liu X T.. J. Virol. Methods, 2015,213:84-92 |
| [15] | .Farajnia S, Ahmadzadeh V, Tanomand A, Veisi K, Khosroshahi S A, Rahbarnia L.. Immunopharm. Immunot., 2014,36(5):297-308 |
| [16] | .Ahmad Z A, Yeap S K, Ali A M, Ho W Y, Alitheen N B, Hamid M.. Clin. Dev. Immunol., 2012, 2012:980250 |
| [17] | .XU Fang, YU Guang-Hui, WANG Pu.. J. Zhengjiang University Tech., 2014,42(1):45-49 .徐 放, 遇广辉, 王普..浙江工业大学学报,2014,42(1):45-49 |
| [18] | .Muyldermans S, Baral T N, Retarnozzo V C, De B P, De G E, Kinne J, Leonhardt H, Magez S, Nguyen V K, Revets H.. Vet. Immunol. Immunop., 2009,128(1-3):178-183 |
| [19] | .Kaczmarek J Z, Skottrup P D.. Mol. Immunol., 2015,65(2):384-390 |
| [20] | .Liu X, Xu Y, Wan D B, Xiong Y H, He Z Y, Wang X X, Gee S J, Ryu D J, Hammock B D.. Anal. Chem., 2015,87:1387-1394 |
| [21] | .Liu X, Xu Y, Xiong Y H, Tu Z, Li Y P, He Z Y, Qiu Y L, Fu J H, Gee S J, Hammock B D.. Anal. Chem., 2014,86(15):7471-7477 |
| [22] | .LIU Xia, XU Yang, TU Zhui, HE Qing-Hua.. J.Food Sci.Biotech., 2011,30(6):950-955 .刘 夏, 许杨, 涂追, 何庆华..食品与生物技术学报,2011,30(6):950-955 |
| [23] | .Xu Y, Xiong L, Li Y P, Xiong Y H, Tu Z, Fu J H, Tang X.. Food Chem., 2015,177:97-101 |
| [24] | .XU Hui-Fang, ZHANG Xian-En, ZHANG Zhi-Ping, ZHANG Yong-Mei.. Prog. Biochem. Biophys., 2003,30(1):89-94 .徐卉芳, 张先恩, 张治平, 张用梅..生物化学与生物物理进展,2003,30(1):89-94 |
| [25] | .Liu X, Xu Y, He Q H, He Z Y, Xiong Z P.. J. Agr. Food Chem., 2013,61(20):4765-4770 |
| [26] | .WANG Lü, ZOU Xu-Qiang, CHEN Chao-Chao, SHAO Hui-Feng, CHEN Xue-Lan.. Chinese J. Anal. Chem., 2015,43(6):856-861 .王 吕, 熊斯诚, 邹旭强, 陈超超, 邵辉锋, 陈雪岚..分析化学,2015,43(6):856-861 |
| [27] | .XIE Hui, ZHANG Xian, WANG Xin, FAN Peng-Cheng, SHI Yu-Fei, FANG Wei-Huan.. Microbiology China, 2015,42(10):2033-2040 .谢 珲, 章先, 王歆, 凡鹏程, 时玉菲, 方维焕..微生物学通报,2015,42(10):2033-2040 |
| [28] | .YANG Dan-Yan, LIN Chen-Shui.. J. Zhengjiang University Tech., 2009,37(4):372-375 .杨丹燕, 林陈水..浙江工业大学学报,2009,37(4):372-375 |
| [29] | .Kim S H, Cha S H, Karyn B, Park S W, Son S W, Kang H G.. Toxicol. Res., 2011,27(2):125-131 |
- 下载量()
- 访问量()
- 您的评分：
-
10%
-
20%
-
30%
-
40%
-
50%