Prokaryotic expression and polyclonal antibody preparation of Nesfatin-1 protein in Micropterus salmoides
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摘要: Nesfatin-1蛋白可以通过调节基因表达及信号通路途径影响鱼类的脂肪生成和代谢。为探究大口黑鲈 (Micropterus salmoides) Nesfatin-1蛋白的生理功能，构建其原核表达质粒并进行蛋白表达纯化，制备了该蛋白的鼠源多克隆抗体。通过扩增Nesfatin-1蛋白的基因序列，构建原核表达载体获得pET32a-Nesfatin-1重组质粒。经异丙基-β-D-硫代半乳糖苷 (IPTG) 诱导蛋白表达，镍离子亲和层析法纯化融合蛋白，并免疫Balb/c小鼠，获得针对Nesfatin-1的多克隆抗体。结果显示：大口黑鲈Nesfatin-1蛋白的基因序列为246 bp；Nesfatin-1融合蛋白在菌液上清液中的质量浓度为1.05 mg·mL−1，相对分子质量约为30 kD；Western blot结果显示多克隆抗体能与重组蛋白特异性结合；间接ELISA检测该抗体效价达到1∶204 800以上；免疫组织荧光结果显示该抗体能特异性识别大口黑鲈肝胰腺中的蛋白，弥散性分布于肝胰腺中且血管周围呈强阳性反应。研究成功表达纯化出大口黑鲈Nesfatin-1融合蛋白，并制备了特异性鼠源多克隆抗体，为后续深入研究Nesfatin-1生物学作用提供了功能性蛋白与特异性抗体。
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关键词:
- 大口黑鲈 /
- Nesfatin-1蛋白 /
- 原核表达 /
- 多克隆抗体
Abstract: Nesfatin-1 protein can affect fish adipogenesis and metabolism by regulating gene expression and signaling pathways. To investigate its physiological functions in largemouth bass (Micropterus salmoides), we constructed and purified a prokaryotic expression plasmid, and prepared a murine polyclonal antibody to this protein. The pET32a-Nesfatin-1 recombinant plasmid was obtained by amplifying the gene sequence of Nesfatin-1 protein and constructing a prokaryotic expression vector. The protein expression was induced by isopropyl-β-D-thiogalactosid (IPTG), and the fusion protein was purified by nickel ion affinity chromatography and immunized in Balb/c mice to obtain a polyclonal antibody against Nesfatin-1. The results show that the Nesfatin-1 protein had a gene sequence length of 246 bp; the concentration of the Nesfatin-1 fusion protein in the supernatant of the bacterial broth was 1.05 mg·mL−1, and its relative molecular mass was 30 kD. Western blot analysis shows that the polyclonal antibody could specifically bind to the recombinant protein; the potency of the antibody reached over 1: 204 800 by indirect ELISA. The results of immunofluorescence detection indicates that the antibody specifically recognized the protein in the hepatopancreas of largemouth bass and was diffusely distributed in the hepatopancreas, showing strong positive reaction around the blood vessels. In this study, the Nesfatin-1 fusion protein of largemouth bass was successfully expressed and purified, and a specific murine polyclonal antibody was prepared, which can provide the functional protein and specific antibody for further study of the biological role.-
Key words:
- Micropterus salmoides /
- Nesfatin-1 protein /
- Prokaryotic expression /
- Polyclonal antibody
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图 1 Nesfatin-1 蛋白的基因扩增产物电泳图
Figure 1. Electrophoresis of gene amplification product of Nesfatin-1 protein
图 2 蛋白诱导表达产物的 SDS-PAGE 分析
a. IPTG浓度对pET-32a-Nesfatin-1蛋白表达的影响；b. pET-32a-Nesfatin-1融合蛋白的表达形式。
Figure 2. SDS-PAGE analysis of prokaryotic expression products
a. pET-32a-Nesfatin-1 protein expression in BL21 under different IPTG concentration conditions; b. Type of pET-32a-Nesfatin-1 fusion protein.
图 4 Western blot 检测 Nesfatin-1 蛋白多克隆抗体的特异性
Figure 4. Identification of specificity of anti-Nesfatin-1 polyclonal antibody by Western blot
图 5 Nesfatin-1 蛋白在大口黑鲈肝胰腺组织中的定位
Figure 5. Localization of Nesfatin-1 protein in hepatopancreas of M. salmoides
表 1 ELISA 检测多克隆抗体效价
Table 1. Titer of polyclonal antibody detected by ELISA
稀释
Dilution多克隆抗体①
P阴性对照②
N比值
P/N空白对照
PBS1∶100 2.51 0.25 9.97 0.09 1∶200 2.46 0.16 15.38 0.07 1∶400 2.37 0.11 21.55 0.08 1∶800 2.17 0.08 27.13 0.06 1∶1 600 1.95 0.07 27.86 0.06 1∶3 200 1.87 0.06 31.17 0.06 1∶6 400 1.53 0.07 21.86 0.05 1∶12 800 1.23 0.06 20.50 0.07 1∶25 600 0.94 0.06 15.67 0.06 1∶51 200 0.73 0.06 12.17 0.06 1∶102 400 0.54 0.06 9.00 0.05 1∶204 800 0.36 0.06 6.00 0.06 注：① 表示两组待测多抗检测结果的平均值；② 表示两组阴性对照检测结果的平均值。 Note: ① Means of two groups of polyclonal antibody results; ② Means of two groups of negative control results. 
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