LH Gene Cloning, Sequence Feature Analysis and Prokaryotic Expression in Conger myriaster
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, saf...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Science Press, PR China
2025-06-01
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| Series: | Progress in Fishery Sciences |
| Subjects: | |
| Online Access: | http://journal.yykxjz.cn/yykxjz/ch/reader/view_abstract.aspx?file_no=20240409002 |
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| Summary: | 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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| ISSN: | 2095-9869 |