香蕉果实MaNPC1基因原核表达及多克隆抗体制备

【目的】对香蕉果实非特异性磷脂酶C基因（MaNPC1）开放阅读框（ORF）进行原核表达，并制备其多克隆抗体，为深入探究MaNPC1在香蕉果实抵御炭疽病中的作用机制提供理论依据。【方法】克隆MaNPC1基因ORF序列，对其进行生物信息学分析及抗原性预测，并通过双酶切法构建其原核表达载体，利用热激法转入大肠杆菌Rosetta 2（DE3）感受态细胞中进行诱导表达，重组蛋白经Ni-NTA树脂层析柱纯化后免疫新西兰兔，以制备多克隆抗体。同时，运用Western blotting和实时荧光定量PCR分别检测香蕉果实贮藏过程中在炭疽病侵染胁迫下MaNPC1蛋白表达水平及MaNPC1基因相对表达量。【结果】重组质粒pGEX-6p-3-MaNPC1经双酶切后得到1650 bp的特异性条带，说明原核表达载体构建成功，将其转入大肠杆菌Rosetta 2（DE3）感受态细胞后成功表达。MaNPC1蛋白分子式为C₂₇₄₇H₄₂₄₉N₇₆₉O₇₉₃S₁₀，分子量为61.06 k D，理论等电点（pI）为8.96；丙氨酸、缬氨酸、亮氨酸和丝氨酸含量较高，分别占氨基酸总数的8.7%、8.2%、7.8%和7.7%；不稳定指数为47.14，表明其为不稳定蛋白；蛋白抗原区段丰富且亲水性氨基酸数目明显高于疏水性氨基酸，有利于后续抗体的制备。制备的多克隆抗体效价较高，为1∶2048000。Western blotting检测发现，香蕉果实贮藏过程中MaNPC1蛋白表达水平呈上升—下降—上升的变化趋势；炭疽病侵染组MaNPC1蛋白除贮藏15 d时的表达水平比对照组低外，其他贮藏时间均略高于对照组。实时荧光定量PCR检测结果表明，MaNPC1基因相对表达量在香蕉果实贮藏过程中呈逐渐上升趋势；贮藏3~15 d，炭疽病侵染组MaNPC1基因相对表达量均显著高于对照组（P<0.05）。【结论】炭疽病侵染能提高MaNPC1蛋白表达水平，故推测MaNPC1蛋白参与香蕉果实抵抗炭疽病侵染。

Prokaryotic expression of banana MaNPC1 gene and preparation of its polyclonal antibody

【Objective】 Prokaryotic expression of the open reading frame(ORF) of banana fruit non-specific phospholipase C gene(MaNPC1) and preparation of its polyclonal antibody were conducted, to provide theoretical basis for indepth exploration of the mechanism of MaNPC1 in banana fruit resistance to anthracnose.【Method】 The ORF sequence of MaNPC1 gene was cloned, bioinformatics analysis and antigenicity prediction were carried out, and its prokaryotic expression vector was constructed by double enzyme digestion method, and the heat shock method was used to transfer it into Escherichia coli Rosetta 2(DE3) competent cell for induction expression, and the recombinant protein was purified by Ni-NTA resin chromatography column to immunize New Zealand rabbits to prepare polyclonal antibodies. At the same time, Western blotting and real-time fluorescent quantitative PCR were used to detect the MaNPC1 protein expression level and the relative expression level of MaNPC1 gene under the stress of anthracnose infection during banana fruit storage.【Result】 The pGEX-6 p-3-MaNPC1 recombinant plasmid was double-enzyme digested to obtain a specific band of 1650 bp, indicating that the prokaryotic expression vector was successfully constructed and transferred to E. coli Rosetta 2(DE3) competent cell for successful expression. The molecular formula of MaNPC1 protein was C₂₇₄₇H₄₂₄₉N₇₆₉O₇₉₃S₁₀, the molecular weight was 61.06 k D, and the theoretical isoelectric point(pI) was 8.96;the contents of alanine, valine, leucine and serine were high, accounting for 8.7%, 8.2%, 7.8% and 7.7% of the total number of amino acids, respectively;the instability index was 47.14, indicating that it was an unstable protein. The protein antigen segment was rich and the number of hydrophilic amino acids was higher than that of hydrophobic amino acids, which was easy for subsequent antibody preparation. The prepared polyclonal antibody had a higher titer of 1:2048000. Western blotting analysis showed that the MaNPC1 protein expression level of banana fruits showed an upward-decreasing-increasing trend during storage. The MaNPC1 protein expression level of the anthracnose infection group was lower than that of the control group when stored for 15 d, and the other storage time was slightly higher than the control group. Real-time fluorescent quantitative PCR detection showed that the relative expression of MaNPC1 gene showed a gradual upward trend during storage of banana fruits;after storage for 3 to 15 d, the relative expression of MaNPC1 gene in the anthracnose infection group was significantly higher than that in the control group(P<0.05). 【Conclusion】 Anthracnose infection can increase the expression level of MaNPC1 protein, indicating that MaNPC1 protein participates in banana fruit resistance to anthracnose infection.