| 陈彦,史宝,王成刚,薄万军,晏科文,陶美君,赵新宇,马晓东.星康吉鳗LH基因的克隆、序列特征分析及原核表达.渔业科学进展,2025,46(3):40-52 |
| 星康吉鳗LH基因的克隆、序列特征分析及原核表达 |
| LH gene cloning, sequence feature analysis and prokaryotic expression in Conger myriaster |
| 投稿时间：2024-04-09 修订日期：2024-05-07 |
| DOI：10.19663/j.issn2095-9869.20240409002 |
| 中文关键词: 星康吉鳗 促黄体生成素基因 基因克隆 序列特征分析 原核表达 |
| 英文关键词: Conger myriaster Luteinizing hormone gene Gene cloning Sequence feature analysis Prokaryotic expression |
| 基金项目:中国水产科学研究院黄海水产研究所基本科研业务费(20603022023023; 2023TD51)、山东省重点研发计划(2021LZGC028)、国家重点研发计划(2023YFD2400405)和财政部和农业农村部：国家现代农业产业技术体系(CARS-47)共同资助 |
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| 中文摘要: |
| 促黄体生成素(luteinizing hormone, LH)在鱼类配子成熟、排卵和类固醇激素合成方面发挥重要作用。通过同源克隆获得星康吉鳗(Conger myriaster) LH基因CDS区序列，利用生物信息学软件分析该基因编码蛋白的结构与特征，并成功构建原核表达重组质粒对LH蛋白进行表达。克隆获得LH基因CDS区为423 bp，编码140个氨基酸。分析LH蛋白理化性质可知，其相对分子质量为15.56 kDa，理论等电点为5.85；经亲疏水性分析发现，LH蛋白为亲水性蛋白；对信号肽和跨膜区分析结果显示，其前1~22个氨基酸为信号肽，无跨膜区；经结构域分析，其存在保守性较强的GHB结构域(由27~132位的105个氨基酸组成)；亚细胞定位分析表明，LH蛋白主要定位于细胞核；对糖基化位点及磷酸化位点分析发现，其存在1个N-糖基化位点和3个O-糖基化位点，共含有17个潜在的磷酸化位点；LH蛋白二级结构主要含有α-螺旋(13.6%)、β-折叠(25%)和无规则卷曲(61.4%)；通过三级结构分析发现，其与日本鳗鲡(Anguilla japonica) LH蛋白三级结构较为相似，与人(Homo sapiens) LH蛋白的三级结构存在一定差异。多序列比对及系统发育树分析结果显示，LH与欧洲鳗鲡(A. anguilla)、花鳗鲡(A. marmorata)和日本鳗鲡进化关系较近，同源性分别为92.14%、91.43%和90.71%。构建的重组质粒pET-28a-LH在Rosetta (DE3)感受态细胞中成功表达，其表达的可溶性LH重组融合蛋白条带在SDS-PAGE电泳检测及Western Blot结果中约为26.5 kDa，与预期表达的分子量相符。该研究为揭示星康吉鳗LH基因的分子特征及蛋白功能奠定了基础，为后续星康吉鳗人工繁育技术的优化提供了理论参考。 |
| 英文摘要: |
| Conger myriaster is an economic valuable fishery resources in the seas surrounding China, Korea, and Japan. C. myriaster presents an important fishing target in Chinese fishery. However, due to intense human fishing activities, C. myriaster populations have been declining steadily. Consequently, safeguarding this species has become imperative. The luteinizing hormone (LH) gene plays an important role in gamete maturation, ovulation, and steroid hormone synthesis in fish. However, the expression of reproductive axis genes and the regulation of reproductive-related hormones, specifically in C. myriaster, are not well understood. In this study, the coding sequence (CDS) of the LH gene was cloned using PCR techniques. The LH gene structure and characteristics were bioinformatically analyzed. The CDS of the LH gene consisted of 423 bp and encoded 140 amino acids. By analyzing the physical and chemical properties of the protein, the LH protein molecular weight was 15.56 kDa. The aliphatic index was 73.71 and the theoretical pI was 5.85. Based on an instability index value of 66.49, the LH protein exhibits a state of instability. Using functional domain analysis, we found that the LH protein possessed a highly conserved GHB characteristic domain (composed of 105 amino acids at 27–132). Hydrophilicity and hydrophobicity analysis of the protein revealed that the LH protein is hydrophilic. The 16th amino acid (Cys) exhibited the strongest hydrophobicity (2.233), while the 65th amino acid (Ser) exhibited the strongest hydrophilicity (–2.333). Through the signal peptide analysis and transmembrane domain analysis of LH protein, the first 1–22 amino acids function as signal peptides without containing any transmembrane domain. According to the analysis of protein glycosylation sites, the LH protein contain 1 N-glycosylation site and 3 O-glycosylation sites. The LH protein contain 17 potential phosphorylation sites. Specifically, there are 9 Serine (Ser), 6 Threonine (Thr), and 2 Tyrosine (Tyr) phosphorylation sites. Subcellular localization analysis revealed that the LH protein was mainly localized in the nucleus. The secondary structure of the LH protein mainly consists of α-helix (13.6%), β-sheet (25%), and random coil (61.4%). Upon comparing the tertiary structure of the LH protein with that of Anguilla japonica and Homo sapiens, the tertiary structure of the LH protein was similar to that of A. japanica. However, certain differences were found compared to the tertiary structure of H. sapiens. Based on the sequence alignment and phylogenetic analysis, LH in C. myriaster exhibited a closer evolutionary relationship with A. anguilla, A. marmorata, and A. japonica. Compared with A. anguilla, A. marmorata, and A. japonica, the C. myriaster LH amino acid sequence revealed the similarities of 92.14%, 91.43% and 90.71%, respectively. In addition, the LH amino acid sequence of C. myriaster compared with H. sapiens, Mus musculus and Bos taurus revealed the similarities of 43.34%, 41.73% and 41.73%, respectively. The LH of C. myriaster exhibits high homology with other teleosts, but displays low homology with mammals, which indicates that LH has evolutionary differences during evolution. In this study, we present the successful production of recombinant C. myriaster LH protein using the pET-28a(+) expression system. The restriction site NdeⅠ/XhoⅠ was inserted at both ends of LH fragment. The 439 bp fragment of LH was amplified using RT-PCR. Subsequently, the fragment was cloned into the pEASY-T1 vector to obtain recombined plasmids, which was designated as pEASY-T1-LH. The recombinant plasmid pEASY-T1-LH and the pET-28a(+) vector were double-digested using NdeⅠ/XhoⅠ enzymes. After double-digested, the fragment was successfully cloned into the pET-28a(+) vector to obtain recombined plasmids, which was designated as pET-28a-LH. In order to investigate the biological activities and physiological significance of pET-28a-LH, we used the pET protein fusion and purification system to produce pET-28a-LH in Trans1-T1 competent cell. Recombinant plasmids (pET-28a-LH) were transferred into Rosetta (DE3) cells, which were cultured under different IPTG induction conditions (0.01, 0.1, 0.5 and 1 mmol/L) at a temperature of 16 ℃ for 16 h. LH could be efficiently expressed across various IPTG concentrations and under the aforementioned induction conditions. Consequently, the subsequent experiments were conducted using the following optimized parameters: an IPTG concentration of 0.5 mmol/L, incubation at 16 ℃ for a duration of 16 h. The precipitate (insoluble fraction) was resuspended in lysis buffer. The supernatant and precipitate were analyzed by SDS-PAGE following induction of expression. SDS-PAGE analysis revealed the presence of distinct 26.5 kDa bands for the LH protein, which were soluble and present in the clear supernatant. Western blot analysis revealed that histidine tagged LH protein reacted with anti-His antibody, LH protein yielded clear bands of 26.5 kDa were detected. The observed size is in accordance with the anticipated molecular weight expression, indicating successful expression of the fusion protein carrying 6×His tag. The protein was purified using a His-tagged nickel affinity chromatography column. This result establishes a solid foundation for future utilization of recombinant LH protein in inducing sexual maturity in cultured C. myriaster, while also providing valuable insights into unraveling the regulatory mechanisms underlying LH gene-mediated steroidogenesis in C. myriaster. |
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