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| Cloning and Prokaryotic Expression of Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Gene from Streptococcus agalactiae in Tilapia |
| LI Guihuan,WANG Bei,LU Yishan,TANG Jufen,HUANG Yucong,WU Zaohe,JIAN Jichang |
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Abstract A pair of specific primers was designed and synthesized based on glyceraldehyde-3-phosphate dehydrogenase(GAPDH) of tilapia Streptococcus agalactiae ZQ0910 published on GenBank .The GAPDH gene was amplified by PCR and then inserted into the pET-32a (+ ) vector to construct the prokaryotic expression plasmid pET-32a-GAPDH . The recombinant GAPDH fusion protein was expressed in Escherichia coli BL21 (DE3 ) cells by the induction of isopropyl-β-D-thiogalactopyranoside (IPTG ) . Sequence analysis revealed that GAPDH gene contained 1011 bp and encoded 336 amino acids and the amino acid sequence of S .agalactiae ZQ0910 showed the highest identity to S .agalactiae 2603 V/R .The SDS-PAGE electrophoresis indicated that the optimal expression was observed under conditions of 0 .06 mmol/L IPTG ,at temperature of 37 ℃ ,and for 5 hours .The recombinant GAPDH fusion protein had 560μg/mL in concentration after purified using HisTrap HP column .The Western blot showed that the target protein had the similar molecular weight (36 .01 ku) to the theoretic molecular weight .The findings prove that a recombinant of prokaryotic expression vector pET-32a (+ )-GAPDH is constructed successfully and are expected to provide a basis for further studies on the immunogenecity of GAPDH and subunit vaccines preparation .
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Received: 25 March 2014
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