Cloning of claudin-5 Gene from Oreochromis niloticus and Analysis of Its Expression Under Streptococcus agalactiae

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.

Keywords

• oreochromis niloticus
• claudin-5 gene
• expression pattern
• streptococcus agalactiae
• blood-brain barrier
• bacterial meningitis