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| 星康吉鳗(Conger myriaster)LH基因的克隆、序列特征分析及原核表达 |
| Cloning, sequence feature analysis and prokaryotic expression of Conger myriaster LH gene |
| 投稿时间：2024-04-09 修订日期：2024-05-07 |
| DOI： |
| 中文关键词: 星康吉鳗 促黄体生成素(LH)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(LH) 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资助项目(20603022023023, 2023TD51)、山东省重点研发计划项目(2021LZGC028)、国家重点研发计划项目(2023YFD2400405)、财政部和农业农村部：国家现代农业产业技术体系资助项目(CARS-47) |
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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423bp,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,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(由27~132位的105个氨基酸组成)结构域；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-糖基化位点与25个O-糖基化位点,共含有30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 %)、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。通过多序列比对及系统发育树分析表明,LH与欧洲鳗鲡(A. anguilla)、花鳗鲡(A. marmorata)、日本鳗鲡进化关系较近,同源性分别为92.14 %、91.43 %、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 one of the most economic valuable fishery resources in the seas surrounding China, Korea, and Japan. C. myriaster is also one of the important fishing target in Chinese fishery. However, due to intense human fishing activities, the population of C. myriaster in the wild has been declining steadily. Consequently, safeguarding this species has become imperative. Currently, various artificial breeding methods for C. myriaster have been explored in China. Nevertheless, the primary obstacle hindering the development of its artificial culture lies in achieving sexual maturity under controlled conditions and obtaining glass eel seedlings through artificial breeding technology. Therefore, urgent research for C. myriaster breeding techniques is warranted to address these challenges effectively. Pituitary gonadotropin hormone (GtHs) consist of follicle-stimualting hormone (FSH) and luteinizing hormone (LH), which play crucial roles in the synthesis of sex steroids, responsible for gonadal development and maturation. They exhibit distinct physiological regulatory functions in both gonad development and maturation as well as gamete release. The luteinizing hormone (LH) gene has been shown to plays an important role in gamete maturation, ovulation and the synthesis of steroid hormones in fish. However, there is limited research on the expression of reproductive axis genes and the regulation of reproductive-related hormones specifically in C. myriaster. In this study, the coding sequence (CDS) of LH gene was cloned through the PCR techniques. The structure and characteristics of LH gene encoding protein were analyzed by bioinformatics software. The CDS of LH gene consisted of 423 bp, encoded 140 amino acid. By analyzing the physical and chemical properties of the protein, the LH protein molecular weight is 15.56 kDa. The aliphatic index is 73.71 and the theoretical pI is 5.85. Based on an instability index value of 66.49, it can be classified that this particular protein exhibits a state of instability. Through the functional domain analysis of the protein, the LH protein possessing a highly conserved GHB characteristic domain (composed of 105 amino acids at 27-132). Through the analysis of hydrophilicity and hydrophobicity of the protein revealed that the LH protein is hydrophilic protein. 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 25 O-glycosylation sites. Through the analysis of protein phosphorylation sites, it was found that the LH protein contain 30 potential phosphorylation sites. Specifically, there are 15 Serine (Ser) phosphorylation sites, 10 Threonine (Thr) phosphorylation sites, and 5 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, it was observed that the LH protein tertiary structure is similar to the A. Japanica. And, there was a certain difference with 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. The LH amino acid sequence of C. myriaster compared with A. anguilla, A. marmorata and A. japonica 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. Restriction site NdeI/XhoI was inserted at both ends of LH fragment. The 439bp fragment of LH was amplified by 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 Chemical competent cell. The pET-28a-LH fragment was purified from agarose gels. 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 mM) at a temperature of 16 ℃ for a duration of 16 h. It was observed that 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 mM, incubation at 16 ℃ for a duration of 16 h. The Rosetta(DE3) cells were harvested by centrifugation at 8000 × g for 10 min following induction of expression. Subsequently, the Rosetta(DE3) Chemical competent cells were disrupted using ultrasonic waves. The resulting cell lysate was centrifuged at 12,000 × g for 10 min. The clear supernatant (soluble fraction) was collected. The precipitate (insoluble fraction) was resuspended in lysis buffer. The supernatant and precipitate were analyzed by SDS-PAGE. SDS-PAGE analysis revealed the presence of distinct 26.5 kDa bands for the LH protein, which was found to be 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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