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| 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 |
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| Abstract: |
| 【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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