Cloning of claudin-5 Gene from Oreochromis niloticus and Analysis of Its Expression Under Streptococcus agalactiae
[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.
万方数据
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