陆地棉GhENODL6的原核表达、纯化与多克隆抗体制备

doi:10.7668/hbnxb.20196027

摘要/Abstract

摘要：

为明确早期结瘤蛋白(ENODL)在棉花抗黄萎病中的生物学功能,制备了GhENODL6蛋白的多克隆抗体,为后续机制解析提供技术支撑。以前期鉴定的GhENODL6基因为材料,该基因编码一个由139个氨基酸残基组成的蛋白,包含信号肽、跨膜结构域及ENODL功能域。采用原核表达系统,截除跨膜结构域后构建pET-30a-ENODL6Δ重组表达载体,并系统优化表达条件,确定最佳诱导参数为:0.50 mmol/L IPTG、初始OD₆₀₀为0.8、25 ℃诱导5 h,获得大量包涵体形式的重组蛋白。经Ni-NTA亲和层析结合尿素变性溶解,成功纯化出纯度超过90%的目标蛋白。以此蛋白为抗原免疫新西兰兔,制备兔源多克隆抗体,效价达1∶102 400。Western Blot结果表明,该抗体可特异性识别分子质量约为20.47 ku的GhENODL6蛋白。综上所述,成功建立了GhENODL6蛋白的高效表达与多克隆抗体制备体系,为后续研究其蛋白互作网络、亚细胞定位及在抗病信号通路中的作用机制提供了可靠工具。

关键词: 陆地棉, 黄萎病, 原核表达, 包涵体, 蛋白质印迹

Abstract:

This study aims to generate polyclonal antibodies against GhENODL6,a member of the early nodulin-like (ENODL) family, to elucidate its biological function in cotton defense against V. dahliae and provide a technical tool for subsequent mechanistic analyses.The previously identified GhENODL6 gene was used as the experimental material,encoding a 139-amino-acid protein containing a signal peptide,transmembrane domain,and ENODL functional domain.To enable soluble expression,the transmembrane domain was truncated,and the modified gene was cloned into the pET-30a vector to construct the recombinant plasmid pET-30a-ENODL6Δ.Expression conditions were systematically optimized,and the highest yield of recombinant protein in inclusion bodies was achieved with 0.50 mmol/L IPTG at an initial OD₆₀₀ of 0.8,followed by induction at 25 ℃ for 5 hours.The target protein was purified via Ni-NTA affinity chromatography under denaturing conditions with urea,yielding a purity exceeding 90%.Polyclonal antibodies were raised in rabbits using the purified protein as antigen,achieving a serum titer of 1∶102 400.Western Blot analysis confirmed that the antibody specifically recognizes GhENODL6 with an apparent molecular weight of approximately 20.47 ku.In summary,this study has established a robust prokaryotic expression and purification system for GhENODL6 and generated a high-affinity polyclonal antibody,providing a valuable tool for investigating its protein interactions,subcellular localization,and potential role in disease resistance signaling pathways.

Key words: Gossypium hirsutum, Verticillium wilt, Prokaryotic expression, Inclusion bodies, Western Blotting

中图分类号:

S562

郑鑫鑫, 解美霞, 张冬梅, 吴立强, 张艳, 王省芬, 杨君. 陆地棉GhENODL6的原核表达、纯化与多克隆抗体制备[J]. 华北农学报, 2026, 41(5): 33-40. doi: 10.7668/hbnxb.20196027.

ZHENG Xinxin, XIE Meixia, ZHANG Dongmei, WU Liqiang, ZHANG Yan, WANG Xingfen, YANG Jun. Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of GhENODL6 from Upland Cotton[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(5): 33-40. doi: 10.7668/hbnxb.20196027.

http://www.hbnxb.net/CN/Y2026/V41/I5/33

图/表 7

表1 试验所用引物

Tab.1 Primers used in the experiment

引物名称 Primer name	引物序列(5'-3') Primer sequences(5'-3')	引物用途 Primer uses
NC-L6F	agtggtctctgtccagtcctATGGCTTCAATTGGTTTC	pNC-ET28-ENODL6载体构建
NC-L6R	ggtctcagcagaccacaagtAAAACGTGGCC
PGE-L6F	cggatccATGGCTTCAATTGGTTTCAAT	pGEX-6P-1-ENODL6载体构建
PGE-L6R	cggaattcAAAACGTGGCCAAGGGAC
P30-L6F	cggatccTCTGAAAACTGGCGTTATG	pET-30a-ENODL6Δ载体构建
P30-L6R	cggaattcAAAACGTGGCCAAGGG

图1 GhENODL6原核表达载体构建 A.pNC-ET28-ENODL6载体构建和插入片段检测;B.pGEX-6P-1-ENODL6载体构建和插入片段检测;C.pET-30a-ENODL6Δ载体构建和插入片段检测。M.DNA Marker DL2000;1-3.PCR产物。

Fig.1 Construction of prokaryotic expression vectors of GhENODL6 A.Construction of pNC-ET28-ENODL6 vector and detection of inserted fragments;B.Construction of pGEX-6P-1-ENODL6 vector and detection of inserted fragments;C.Construction of pET-30a-ENODL6Δ vector and detection of inserted fragments.M.DNA Marker DL2000;1-3.PCR products.

图2 利用不同表达载体对GhENODL6进行重组表达检测 A.利用pNC-ET28-ENODL6载体表达重组蛋白:2-4.重组蛋白在整个细胞、细胞裂解沉淀和上清中的表达检测;B.利用pGEX-6P-1-ENODL6载体表达重组蛋白:2-4.重组蛋白在整个细胞、细胞裂解沉淀和上清中的表达检测;C.利用pET-30a-ENODL6Δ载体表达重组蛋白:2-3.重组蛋白在细胞裂解沉淀和上清中的表达检测;M.蛋白质Marker,1.空表达载体的表达检测。

Fig.2 Detection of recombinant expression of GhENODL6 using different expression vectors A.Recombinant protein was expressed using the pNC-ET28-ENODL6 vector:2-4.The expression detection of the recombinant protein in the whole cell,cell lysate precipitate and supernatant,respectively;B.Recombinant protein was expressed using the pGEX-6P-1-ENODL6 vector:2-4.Expression detection of the recombinant protein in the whole cell,cell lysate precipitate and supernatant,respectively;C.Recombinant protein was expressed using the pET-30a-ENODL6Δ vector;2-3.Expression detection of the recombinant protein in the cell lysate precipitate and supernatant,respectively;M.Protein Marker,1.Expression detection of the empty expression vector.

图3 重组蛋白在不同条件下诱导表达的SDS-PAGE分析 A-D.通过IPTG浓度、培养OD₆₀₀值、诱导温度及诱导时间对pNC-ET28-ENODL6的表达条件进行优化;E-H.通过IPTG浓度、培养OD₆₀₀值、诱导温度及诱导时间对pGEX-6P-1-ENODL6的表达条件进行优化;I-L.通过IPTG浓度、培养OD₆₀₀值、诱导温度及诱导时间对pET-30a-ENODL6Δ的表达条件进行优化。M.蛋白质Marker;CK.空表达载体。

Fig.3 SDS-PAGE analysis of recombinant protein expression under different induction conditions A-D.The expression conditions of pNC-ET28-ENODL6 were optimized by IPTG concentration,culture OD₆₀₀ value,induction temperature and induction time;E-H.The expression conditions of pGEX-6P-1-ENODL6 were optimized by IPTG concentration,culture OD₆₀₀ value,induction temperature and induction time;I-L.The expression conditions of pET-30a-ENODL6Δ were optimized by IPTG concentration,culture OD₆₀₀ value,induction temperature and induction time.M.Protein Marker;CK.Empty expression vector.

图4 GhENODL6Δ重组蛋白的纯化检测 M.蛋白质Marker;1-4.纯化的重组蛋白。

Fig.4 Purification detection of GhENODL6Δ recombinant protein M.Protein Marker;1-4.Purified recombinant protein.

图5 多克隆抗体效价检测图中虚线标示阴性对照控制线。

Fig.5 Detection of polyclonal antibody titers The dotted line in the picture indicates the negative control line.

图6 利用多克隆抗体对GhENODL6Δ进行Western Blotting检测 M.蛋白质Marker;1-2.pET-30a-ENODL6Δ经尿素溶解后纯化;3.空载体对照。

Fig.6 Western Blotting detection of GhENODL6Δ using polyclonal antibodies M.Protein Marker;1-2.pET-30a-ENODL6Δ purified after urea dissolution;3.Empty vector control.

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