Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of GhENODL6 from Upland Cotton

doi:10.7668/hbnxb.20196027

Abstract

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

CLC Number:

S562

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.

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Figures/Tables 7

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

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.

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.

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.

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

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

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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