Full-length cDNA cloning and prokaryotic expression of immunoglobulin M heavy chain in Weever (Lateolabrax japonicus)
LIU Yue, ZHANG Qi-zhong*, CUI Miao
Institute of Hydrobiology, Jinan University, Engineering Research Center of Tropical and Subtropical Aquatic Ecological En-gineering Ministry of Education, Key Laboratory of Aquatic Eutrophication and Control of Harmful Algal Blooms of Guang-dong Higher Education Institutes, Guangzhou 510632, China
In the present study, the complete cDNA sequence of the heavy chain of immunoglobulin M (IgM) in Lateolabrax japonicus was cloned using the method of homologous cloning and RACE technique. The full-length cDNA of IgM was 1,901 bp, and encoded a polypeptide of 582 amino acids. A phylogenetic tree was constructed based on the alignment of amino acid sequences from the constant regions in IgM heavy chain of L. japonicas and other related teleost fishes. The results showed that the IgM, IgZ, and IgD from various fishes clustered together, respectively, and formed three major branches, while the branches of IgM and IgZ generated from a single root suggested a closer evolutionary relationship between IgM and IgZ. RT-PCR results showed the highest IgM expression level in head-kidney, and an almost silence expression in heart, muscle and brain. The recombinant expression plasmid (pQE30-IgM) was constructed with encoding fragment of L. japonicas IgM heavy chain cDNA and the prokaryotic expression vector (pQE30), and transformed into Escherichia coli M15. The recombinant protein was identified by SDS-PAGE and Western blot using anti- 6×His monoclonal antibody which was specially binding with 6×His of the fusion protein.
刘悦, 张其中*, 崔淼. 鲈鱼IgM重链基因cDNA的克隆及其原核表达[J]. , 2013, 32(2): 218-223.
LIU Yue, ZHANG Qi-zhong*, CUI Miao. Full-length cDNA cloning and prokaryotic expression of immunoglobulin M heavy chain in Weever (Lateolabrax japonicus). , 2013, 32(2): 218-223.
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