| 陈杨慧，张素莹，黄永雄，张子瑞，王 蓓.尼罗罗非鱼 claudin-5 基因克隆及其在无乳链球菌胁迫下的表达分析[J].广东农业科学,2024,51(7):25-34 |
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尼罗罗非鱼 claudin-5 基因克隆及其在无乳链球菌胁迫下的表达分析 |
| Cloning of claudin-5 Gene from Oreochromis niloticus and Analysis of Its Expression Under Streptococcus agalactiae |
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| DOI：10.16768/j.issn.1004-874X.2024.07.003 |
| 中文关键词: 尼罗罗非鱼 claudin-5 基因 表达模式 无乳链球菌 血脑屏障 细菌性脑膜炎 |
| 英文关键词: Oreochromis niloticus claudin-5 gene expression pattern Streptococcus agalactiae blood-brain barrier bacterial meningitis |
| 基金项目:国家自然科学基金（32073006）；国家科技部“一带一路”创新人才交流外国专家项目（DL2023030009） |
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| 中文摘要: |
| 【目的】克隆尼罗罗非鱼（Oreochromis niloticus）紧密连接蛋白 5（claudin-5）基因，分析其在无乳
链球菌（Streptococcus agalactiae）刺激下的表达模式，为进一步了解该基因功能提供依据。【方法】根据 NCBI 已
公布的尼罗罗非鱼基因组预测序列（GenBank 登录号：XM_005473513.4），选取其 claudin-5 基因的 CDS 区，设计
claudin-5 基因的克隆及实时荧光定量 PCR 引物。利用生物信息学对克隆得到的 claudin-5 进行理化性质和进化分析，
采用 qRT-PCR 分析尼罗罗非鱼 claudin-5 在各组织中及无乳链球菌刺激下的表达模式。【结果】通过 PCR 克隆技
术获得尼罗罗非鱼 claudin-5 基因全长序列，共 651 bp，编码 216 个氨基酸；蛋白分子量约 23 kD，理论等电点（pI）
为 8.53；具有 4 个跨膜结构域和 3 个蛋白激酶 C（PKC）磷酸化位点。尼罗罗非鱼与奥利亚罗非鱼（O. aurea）的
claudin-5 氨基酸序列相似性高达 97.55%；系统发育进化树结果也显示，二者 claudin-5 氨基酸序列聚为一支，表明
二者具有较近的亲缘关系。尼罗罗非鱼 claudin-5 在脑部的表达量极高，是血细胞等组织的 300 倍左右，具有明显
的组织特异性。经无乳链球菌刺激后，尼罗罗非鱼脑和头肾的 claudin-5 表达量在 12 h 时达到最高值，在 24 h 时有
所下调，其中在脑部的表达量于 48 h 表达量最低，但仍高于 0 h，而在头肾的表达量在 48~96 h 时出现明显上调后
下降；肠中 claudin-5 表达量在无乳链球菌刺激后 6~12 h 时上升缓慢，在 48 h 时表达量大幅度上调，并达到最高值，
之后在 48~96 h 时出现下调，但低于 0 h；肝脏中 claudin-5 表达量在无乳链球菌刺激后出现明显下调，在 24 h 略有
回升然后继续下调。【结论】尼罗罗非鱼 claudin-5 基因在脑组织的表达量极高，具有明显的组织特异性，其编码
蛋白在宿主抵抗无乳链球菌入侵的过程中发挥重要作用，尤其是通过血脑屏障抵抗无乳链球菌，保护中枢神经系统。 |
| 英文摘要: |
| 【Objective】In the study, claudin-5 gene from Oreochromis niloticus was cloned and the expression pattern
of claudin-5 gene stimulated by Streptococcus agalactiae was analyzed, with an aim to provide basis for understanding the
function of claudin-5 in O. niloticus.【Method】According to the genome prediction sequence (GenBank accession No.:
XM_005473513.4) of O. niloticus released by NCBI, the CDS region of claudin-5 was selected to design the cloning primer and
qRT-PCR primer of claudin-5 gene. The physicochemical properties of claudin-5 and their evolutionary characteristics were
analyzed by bioinformatics methods. The expression of claudin-5 in various tissues of O. niloticus and the expression pattern
stimulated by S. agalactiae were analyzed by qRT-PCR.【Result】The full-length of claudin-5 gene of O. niloticus obtained
by PCR cloning technology was 651 bp, encoding 216 amino acids. The molecular weight of the protein encoded by claudin-5
was about 23 kD, and the theoretical isoelectric point (pI) was 8.53, with 4 transmembrane domains and 3 PKC phosphorylation
sites. The homology analysis showed that the amino acid sequence of O. niloticus claudin-5 was 97.55% similar to that of O.
aurea. Phylogenetic tree results also showed that the claudin-5 amino acid sequences of the two were clustered into one branch,
and they were closely related. The expression level of claudin-5 in the brain of O. niloticus was extremely high, about 300 times
that of blood cells. Therefore, it had obvious tissue specificity of expression of claudin-5. After stimulation by S. agalactiae, the
expression levels of claudin-5 in brain and head kidney reached the highest at 12 h and then began to decrease. Among them,
the expression level in brain was the lowest at 48 h, but it was still higher than that at 0 h, while the expression level in head
kidney decreased after obvious up-regulation at 48-96 h. The expression of claudin-5 in intestine increased slowly at 6-12
h and increased significantly at 48 h and reached the highest value. Afterwards, it was downregulated at 48-96 h, but lower
than that at 0 h. The expression of claudin-5 in liver significantly decreased, then rose slightly at 24 h and then continued to
decrease.【Conclusion】The expression of claudin-5 in brain tissue was extremely high, with obvious tissue specificity. After
stimulated by S. agalactiae, claudin-5 of O. niloticus played an important role in the host’s resistance to S. agalactiae invasion,
especially in protecting the central nervous system through the blood-brain barrier. |
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