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
S943; [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...
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| Published in | 广东农业科学 Vol. 51; no. 7; pp. 25 - 34 |
|---|---|
| Main Authors | , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/广东省水产经济动物病害控制重点实验室,广东 湛江 524088
2024
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1004-874X |
| DOI | 10.16768/j.issn.1004-874X.2024.07.003 |
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| Abstract | S943; [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 12h时达到最高值,在 24h时有所下调,其中在脑部的表达量于 48h表达量最低,但仍高于 0h,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48h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 0h;肝脏中claudin-5表达量在无乳链球菌刺激后出现明显下调,在 24h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. |
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| AbstractList | S943; [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 12h时达到最高值,在 24h时有所下调,其中在脑部的表达量于 48h表达量最低,但仍高于 0h,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48h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 0h;肝脏中claudin-5表达量在无乳链球菌刺激后出现明显下调,在 24h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. |
| Abstract_FL | [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.agalactiaeinvasion,especially in protecting the central nervous system through the blood-brain barrier. |
| Author | 黄永雄 张素莹 陈杨慧 王蓓 张子瑞 |
| AuthorAffiliation | 广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/广东省水产经济动物病害控制重点实验室,广东 湛江 524088 |
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| Author_FL | HUANG Yongxiong CHEN Yanghui ZHANG Suying WANG Bei ZHANG Zirui |
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| DocumentTitle_FL | Cloning of claudin-5 Gene from Oreochromis niloticus and Analysis of Its Expression Under Streptococcus agalactiae |
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| Keywords | blood-brain barrier 血脑屏障 claudin-5 gene Oreochromis niloticus expression pattern claudin-5基因 无乳链球菌 尼罗罗非鱼 bacterial meningitis 细菌性脑膜炎 Streptococcus agalactiae 表达模式 |
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| Title | 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 |
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