抗黄曲霉毒素B_(1)多价纳米抗体融合蛋白的构建及活性分析  

Construction and Bioactivity Analysis of Fusion Protein of Polyvalent Anti-aflatoxin B_(1) Nanobody

作　　者：帅文苑 何庆华[1,4] 钟引凤 黄云祥 张航 刘传勇 张乐平 涂追 SHUAI Wen-yuan;ZHONG Yin-feng;HUANG Yun-xiang;ZHANG Hang;LIU Chuan-yong;ZHANG Le-ping(State Key Laboratory of Food Science and Technology,Nanchang University,Nanchang 330047,China;School of Food Sciences,Nanchang University,Nanchang 330047,China;School of Life Sciences,Nanchang University,Nanchang 330031,China,4.Jiangxi Province Key Laboratory of Modern Analytical Sciences,Nanchang University,Nanchang 330031,China)

机构地区：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047 [2]南昌大学食品学院,江西南昌330047 [3]南昌大学生命科学学院,江西南昌330031 [4]江西省现代分析科学重点实验室,江西南昌330031

出　　处：《分析测试学报》2022年第2期213-219,共7页Journal of Instrumental Analysis

基　　金：国家自然科学基金项目(31860260);江西省科技创新平台资助项目(20192BCD40001)。

摘　　要：纳米抗体来源于天然缺失轻链的重链抗体可变区,是已知最小抗原结合单元。该研究构建了抗黄曲霉毒素B_(1)(AFB_(1))纳米抗体的单价及多价串联体,分别与绿色荧光蛋白(GFP)编码片段融合并克隆至原核表达载体p ET30。以大肠杆菌BL21(DE3)作为表达宿主,通过异丙基-β-D硫代吡喃半乳糖苷诱导,亲和层析技术分别纯化单、双及三价抗AFB_(1)纳米抗体与GFP的融合蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明三种融合蛋白均为可溶性表达,表达量分别为6.0、7.5、4.0 mg/L。采用酶联免疫吸附法和荧光扫描法对重组融合蛋白的抗原识别活性及荧光特性进行测定。结果显示,未融合蛋白、单价及双价串联重组蛋白的半抑制浓度(IC;)分别为12.10、14.10、2.19 ng/m L,表明单价重组蛋白的IC;值与未融合蛋白相似,而双价重组蛋白的IC;值与之相比提高了5.5倍。当浓度为0.7 mg/m L时,两种重组蛋白的荧光强度均可达3000以上,荧光强度与GFP相当,且二价重组蛋白表现出更高的荧光强度;三价串联重组蛋白表现出与抗原的结合活性,但AFB_(1)的阻断活性较差,且荧光强度仅为1700左右。该研究为后续建立基于荧光信号的免疫学检测方法奠定了基础,同时也为优化免疫学检测中纳米抗体亲和活性提供了思路和借鉴。Nanobody,derived from the variable domain of heavy chain only antibodies(VHHs),is the smallest antigen recognition unit.It is an ideal candidate for the next generation of immunoassays due to its high structural stability and high expression yields compared with the conventional antibody.This study aimed to investigate effects of the multivalency and gene fusion of anti-aflatoxin B_(1)(anti-AFB_(1))nanobody on the bioactivities of recombinant proteins.The anti-AFB_(1)nanobody,namely G8,was screened from an immune VHH phage display library in a previous work.The coding sequence of G8 was cloned into the vector pET30 between the restriction enzyme sites NdeⅠand SfiⅠ,generating vector pET30-G8.The overlap PCR was performed to assemble the tandem structures(DiG8 and TriG8)with flexible linker(GGSGG).The overlap PCR products encoding DiG8 and TriG8 were purified,and cloned into the vector pET30 at the same site as the G8,generating vector pET30-DiG8 and pET30-TriG8,respectively.To construct expression vectors for the recombinant fusion proteins,the green fluorescent protein(GFP)encoded gene was firstly amplified by PCR with specific primers F0 and R0,and then subcloned into the restriction sites between SfiⅠand NotⅠof vectors pET30-G8,pET30-DiG8,and p ET30-TriG8,respectively.The resulting vectors(pET30-G8-GFP,pET30-DiG8-GFP,and pET30-TriG8-GFP),which fused GFPs at the C terminals of monomeric,dimer and trimer anti-AFB_(1)nanobody,were confirmed via colony PCR,restrictive digestion,and sequencing.The vectors were transformed to Escherichia coli BL21(DE3),respectively.To induce the expression of the target proteins,isopropylβ-D-1-thiogalactopyranoside(IPTG)was added to the culture.Fusion proteins each contain six histidine tag at their C-terminals which were purified by affinity chromatography,respectively.SDS-PAGE analysis showed that the three fusion proteins(G8-GFP,DiG8-GFP and TriG8-GFP)were solubly expressed in E.coli yielding at least 6.0,7.5,4.0 mg/L,respectively.Enzyme-linked immunosorbent assay(ELISA)a

关 键 词：黄曲霉毒素B_(1) 纳米抗体 串联重组 原核表达 酶联免疫吸附法 

分 类 号：O657.3[理学—分析化学] O629.73[理学—化学]