TaMAPK5蛋白的抗体制备及其在小麦与叶锈菌互作过程中的表达特征

doi:10.7668/hbnxb.20194872

摘要/Abstract

摘要：

为了深入探究TaMAPK5在小麦与叶锈菌互作过程中发挥的功能,克隆了TaMAPK5基因的CDS区,构建了原核表达载体pET28a-TaMAPK5,转化到大肠杆菌表达菌株BL21(DE3)中,对目的蛋白诱导表达的异丙基硫代半乳糖苷(IPTG)最佳浓度、诱导时间和诱导温度进行了探索,并通过Ni-NTA亲和层析对目的蛋白进行了纯化,将纯化后的重组蛋白免疫新西兰兔制备TaMAPK5多克隆抗体。结果表明,TaMAPK5的CDS区全长为1 110 bp,TaMAPK5重组蛋白在16 ℃经终浓度为0.050 mmol/L的IPTG诱导48 h效果最好。将该重组蛋白作为抗原免疫新西兰兔,成功制备了可以特异性识别TaMAPK5的多克隆抗体,抗体效价为1∶51 200。Western Blot结果表明,在小麦与叶锈菌不亲和组合中TaMAPK5受叶锈菌侵染诱导表达。成功表达、纯化了TaMAPK5重组蛋白并制备了其多克隆抗体,揭示了TaMAPK5蛋白可能正调控小麦抵抗叶锈菌侵染反应。

关键词: 小麦, TaMAPK5, 原核表达, 蛋白纯化, 多克隆抗体

Abstract:

In order to further explore the function of TaMAPK5 in the interaction between wheat and Puccinia triticina,the CDS region of TaMAPK5 gene was cloned,and the prokaryotic expression vector pET28a-TaMAPK5 was constructed and transformed into E.coli BL21(DE3).The optimal concentration,induction time and induction temperature of isopropyl β-D-thiogalactoside (IPTG) induced expression of the target protein were explored,and the target protein was purified by Ni-NTA affinity chromatography.The purified recombinant protein was used to immunize New Zealand rabbits to prepare TaMAPK5 polyclonal antibody.The results showed that the full length of the CDS region of TaMAPK5 was 1 110 bp,and the TaMAPK5 recombinant protein was induced with IPTG at a final concentration of 0.050 mmol/L and incubated at 16 ℃ for 48 h.The recombinant protein was used as an antigen to immunize New Zealand rabbits,and a polyclonal antibody capable of specifically recognizing TaMAPK5 was successfully prepared.The antibody titer was 1∶51 200.The results of Western Blot showed that TaMAPK5 was induced by P.triticina infection in wheat and P.triticina incompatible combinations.The recombinant protein TaMAPK5 was successfully expressed and purified,and its polyclonal antibody was prepared.It was revealed that TaMAPK5 protein may positively regulate wheat resistance to leaf rust infection.

Key words: Wheat, TaMAPK5, Prokaryotic expression, Protein purification, Polyclonal antibody

邵文先, 王冬梅. TaMAPK5蛋白的抗体制备及其在小麦与叶锈菌互作过程中的表达特征[J]. 华北农学报, 2024, 39(5): 186-192. doi: 10.7668/hbnxb.20194872.

SHAO Wenxian, WANG Dongmei. Antibody Preparation of TaMAPK5 Protein and Its Expression Characteristics in the Interaction Between Wheat and Puccinia triticina[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(5): 186-192. doi: 10.7668/hbnxb.20194872.

http://www.hbnxb.net/CN/Y2024/V39/I5/186

图/表 7

图1 TaMAPK5原核表达载体构建 A.TaMAPK5基因CDS区的扩增:M.DL2000 DNA Marker,1.TaMAPK5原核表达片段;B.双酶切阳性质粒检测:M.DL15000 DNA Marker,1.pET28a-TaMAPK5载体质粒,2.pET28a-TaMAPK5质粒酶切。红色箭头.目的片段。

Fig.1 TaMAPK5 prokaryotic expression vector construction A.Amplification of CDS region of TaMAPK5 gene:M.DL2000 DNA Marker,1.TaMAPK5 prokaryotic expression fragment;B.The detection of digesting positive plasmid:M.DL15000 DNA Marker,1.pET28a-TaMAPK5 vector plasmid,2.pET28a-TaMAPK5 plasmid digest.The red arrow.The target fragment.

图2 TaMAPK5重组蛋白可溶性表达条件筛选 A.IPTG诱导浓度的筛选:M.蛋白质Marker,1.TaMAPK5未经IPTG诱导的总蛋白,2-10.TaMAPK5经0.001,0.005,0.010,0.025,0.050,0.075,0.100,0.125,0.150 mmol/L的IPTG诱导的总蛋白;B.IPTG诱导温度的筛选,M.蛋白质Marker;1-4.未诱导、16,28,37 ℃诱导的上清,5-8.未诱导、16,28,37 ℃诱导的沉淀。红色箭头.目的蛋白。

Fig.2 Screening of conditions for soluble expression of TaMAPK5 recombinant protein A.Screening of IPTG induction concentration:M.Protein Marker,1.Total protein of TaMAPK5 without IPTG induction,2-10.Total protein of TaMAPK5 induced by 0.001,0.005,0.010,0.025,0.050,0.075,0.100,0.125,0.150 mmol/L IPTG;B.Screening of IPTG induction temperature:M.Protein Marker,1-4.Supernatant of uninduced,16,28,37 ℃-induced,5-8.Precipitate of uninduced,16,28,37 ℃-induced.The red arrow.The target protein.

图3 TaMAPK5纯化后的电泳分析及Western Blot检验 A.咪唑洗脱浓度的筛选:M.蛋白质Marker,1.上清液,2.流出液,3-8.0,20,50,100,250,500 mmol/L的咪唑洗脱;B.Western Blot检验:M.蛋白质Marker,1.BSA,2.TaMAPK5;红色箭头.TaMAPK5重组蛋白。

Fig.3 Protein electrophoresis analysis and Western Blot test of purified TaMAPK5 A.Screening of imidazole elution concentration:M.Protein Marker,1.Clear supernatant,2.Effluent,3-8.0,20,50,100,250,500 mmol/L imidazole elution;B.Western Blot test:M.Protein Marker,1.BSA,2.TaMAPK5;The red arrow.TaMAPK5 recombinant protein.

图4 SDS-PAGE检测TaMAPK5抗体纯度 M. 蛋白质Marker;1. Anti-TaMAPK5。

Fig.4 SDS-PAGE for TaMAPK5 antibody purity M. Protein Marker;1. Anti-TaMAPK5.

图5 ELISA检测TaMAPK5多克隆抗体效价

Fig.5 ELISA for TaMAPK5 polyclonal antibody potency

图6 TaMAPK5多克隆抗体特异性检测 A.TaMAPK5重组蛋白中Western Blot检测:M.蛋白质Marker,1.TaMAPK5,2.BSA;B.接菌后48 h的TcLr26总蛋白中Western Blot检测:M.蛋白质Marker,1.TcLr26,2.BSA。

Fig.6 Specificity detection of TaMAPK5 polyclonal antibody A.Western Blot detection of TaMAPK5 recombinant protein:M.Protein Marker,1.TaMAPK5,2.BSA;B.Western Blot detection in total protein of TcLr26 at 48 h after inoculation:M.Protein Marker;1.TcLr26,2.BSA.

图7 Western Blot检测接种后不同时间点TaMAPK5蛋白水平的表达

Fig.7 Western Blot was used to detect the expression of TaMAPK5 protein at different time points after inoculation

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