Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (2): 116-123.

• Papers • Previous Articles     Next Articles

Cloning and Prokaryotic Expression of Secretory Form of Immunoglobulin M（sIgM）Heavy Chain Gene in GIFT Strain of Nile Tilapia（Oreochromis niloticus）Induced by Streptococcus agalactiae

Wang Pei1，2，3, Lu Yishan1，2，3, Wang Bei1，2，3, Tang Jufen1，2，3, Cai Jia1，2，3, Wu Zaohe2，3，4, Jian Jichang1，2，3,   

  1. （1. Fisheries College，Guangdong Ocean University，Zhanjiang 524088；2. Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals，Zhanjiang 524088；3. Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals，Zhanjiang 524088；4 .Zhongkai University of Agriculture and Engineering，GuangZhou 510225）
  • Online:2014-02-27 Published:2014-02-27

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Abstract: GIFT strain of Nile tilapia（Oreochromis niloticus）, induced by inactivated Streptococcus agalactiae, was used to construct the head kidney cDNA library. The full-length sIgM cDNA of Nile Tilapia was cloned using homological cloning and rapid amplification of cDNA ends（RACE）methods. Results showed the full-length of sIgM cDNA was 1 921 bp, containing a 5' untranslated region（5'-UTR）of 41 bp, a 3'-UTR of 140 bp and an open reading frame of 1 740 bp encoding 579 amino acids with an estimated molecular weight of 64.26 kD and an estimated isoelectric point of 5.36. In the N-terminal of sIgM exists a signal peptide structure. The phylogenetic trees constructed showed that sIgM of Nile tilapia shared the closest relationship with the corresponding proteins of Paralichthys olivaceus and Rachycentron canadum. The sIgM had four CH domains. The constant segment of sIgM gene was amplified and inserted into the pET-28a（+）vector to construct the prokaryotic expression plasmid pET28a-sIgM. The optimal expression condition was set as 0.05 mmol/ L IPTG, induced temperature 37℃, and induced time 4 hours.The recombinant sIgM fusion proteins was purified by His TrapTM HP column, and then identified by western blot using His-Tag Mouse mAb.