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Relationships between Hagfishes, Lampreys, Gnathostomata and Testing Activity of Recombinant Proteins

Author: YuShuiYan
Tutor: LiQingWei
School: Liaoning Normal University
Course: Marine biology
Keywords: phylogenetic relationship Hagfish Lamprey Gnathostome outgroups tree-making method T lymphocyte Cell proliferation Autoimmune disease Kv1.3 potassium channel
CLC: Q819
Type: Master's thesis
Year: 2008
Downloads: 36
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Abstract

It has become clear that the extant vertebrates are divided into three major groups, that is, hagfishes, lampreys, and jawed vertebrates. Morphological and molecular studies, however, have resulted in conflicting views with regard to their interrelationships. To clarify the phylogenetic relationships between them, 48 orthologous protein-coding gene families were analyzed. Even as the analysis of 34 nuclear gene families supported the monophyly of cyclostomes, the analysis of 14 mitochondrial gene families suggested a closer relationship between lampreys and gnathostomes compared to hagfishes. Lampreys were sister group of gnathostomes. The results of this study supported the cyclostomes. Choice of outgroup, tree-making methods, and software may affect the phylogenetic prediction, which may have caused much debate over the subject. Development of new methods for tackling such problems is still necessary. To identify the functional gene fragment, a cDNA library was constructed from the oral gland of Lampetra japonica. After random selection of clones for sequencing and BlastX and BlastN programs used for sequence similarity alignment, a selective blocking of Kv1.3 like protein named L251 was found. L251 belongs to CRISP family which contains three structural domain: Pathoegenesis-related group 1 domain (PR-1domain), a link domain and a cysteine-rich domain (CRD). L251 may share identical conservative sequences and the same functional site with CRD by analyzing of mass interrelated proteins, function and construction.Sequence specific primers were designed to obtain the full-length cDNA encoding the L251 protein. Then the cDNA was directionally cloned into the pET23b and transformed into E. coli Rosetta, and expression was induced by IPTG. After the identification by SDS-PAGE, the recombinant proteins were purified by a specific His-Tag binding step in which the target protein strongly bind to a chromatography column bearing Ni2+ . After denature and renature by dialyzation,the activity of the inhibit proliferation of T lymphocyte was tested in which T lymphocyte were purified from human peripheral blood by Dynal T Cell Negative Isolation Kit Ver II. L251 was tested to inhibit the proliferation of T lymphocyte by the method of MTT and taking counts of cells towards a concentration gradient in 0.1ug/mL, 0.3 ug/mL , 0.5 ug/mL , 0.7 ug/mL. Meantime, the cell cycle was also tested by flow cytometry that showed the dividing cells were blocked at G1/S point towards a concentration gradient . CFSE staining, flow cytometry, F1channel and related software were used to detect the fluorescence intensity and analysis the inhibition for T lymphocyte proliferating.There are elevated Kv1.3 potassium channel expressing on T lymphocyte with autoimmune diseases but low level in normal. The high selective of Kv1.3 blockers make the new therapeutical way about immune inhibitor for autoimmune disease therapy doing no harm to immune system realized. L251 are hopeful to act as the Kv1.3 blockers and as a new immune inhibitor will be turned into a more attractive therapeutic target than classical inhibitor because of more high selective and less harm.

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