Construction and Prokaryotic Expression Sip-GAPDH Fusion Gene of Streptococcus agalactiae from GIFT Strain of Nile Tilapia（Oreochromis niloticus）

Abstract

Abstract: The DNA fragment encoding the Sip and GAPDH gene of Streptococcus agalactiae strain ZQ0910 isolated from GIFT Strain of Nile Tilapia（Oreochromis niloticus）were obtained by PCR. The Sip-GAPDH gene was constructed in a Sip-linker-GAPDH format with the standard 15-amino acid linker（Gly4Ser）3 by SOEing PCR technique, and the final full length product was cloned into the pET-32a（+）vector for protein expression in Escherichia coli strain BL21（DE3）. The result showed that the expression fusion protein Sip-GAPHD were about 102.01 kD and Western blotting analysis confirmed that the 102.01 kD protein was the fusion protein, because it was specifically recognized by mouse anti-His monoclonal antibody. Inducing the cells at 37℃ in 0.1 mmol/L of IPTG for 5 hours was the optimal conditions for expression of the recombinant Sip-GAPDH fusion protein.

Key words: Streptococcus agalactiae, Sip-GAPDH fusion gene, SOEing PCR, Prokaryotic expression

Wang Bei , Li Guihuan, Lu Yishan , Tang Jufen , Huang Yucong , Wu Zaohe , Jian Jichang. Construction and Prokaryotic Expression Sip-GAPDH Fusion Gene of Streptococcus agalactiae from GIFT Strain of Nile Tilapia（Oreochromis niloticus）[J]. Biotechnology Bulletin, 2014, 0(7): 137-142.

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