罗非鱼源无乳链球菌甘油醛-3-磷酸脱氢酶基因的克隆与原核表达

摘要

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 .%为对罗非鱼源无乳链球菌ZQ0910株甘油醛-3-磷酸脱氢酶基因进行克隆及表达进行研究，根据GenBank上已登录的相关基因设计引物，采用 PCR方法扩增该株细菌的甘油醛-3-磷酸脱氢酶基因，然后将其定向克隆至原核表达载体pET-32a（＋）中，在大肠杆菌BL21（DE3）中进行异丙基-β-D-硫代半乳糖苷诱导表达。结果显示，甘油醛-3-磷酸脱氢酶基因有1011个碱基，编码336个氨基酸；同源基因序列比对显示，无乳链球菌ZQ0910株与无乳链球菌2603V/R的甘油醛-3-磷酸脱氢酶基因的同源性最高；经异丙基-β-D-硫代半乳糖苷诱导表达后，SDS-PAGE电泳显示甘油醛-3-磷酸脱氢酶蛋白表达的最优条件为：异丙基-β-D-硫代半乳糖苷浓度为0．06 mmol/L、温度37℃、诱导5 h ，蛋白表达量最大；HisTrap HP柱纯化后融合蛋白的质量浓度为560μg/mL ；Western Blot验证甘油醛-3-磷酸脱氢酶的分子量为36．01 ku。研究结果表明，成功克隆与表达了甘油醛-3-磷酸脱氢酶基因，这将为进一步研究甘油醛-3-磷酸脱氢酶的免疫原性及亚单位疫苗提供理论依据。