CAJ | 학술논문

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。
위대라비어원무유련구균ZQ0910주감유철-3-린산탈경매기인진행극륭급표체진행연구，근거GenBank상이등록적상관기인설계인물，채용 PCR방법확증해주세균적감유철-3-린산탈경매기인，연후장기정향극륭지원핵표체재체pET-32a（＋）중，재대장간균BL21（DE3）중진행이병기-β-D-류대반유당감유도표체。결과현시，감유철-3-린산탈경매기인유1011개감기，편마336개안기산；동원기인서렬비대현시，무유련구균ZQ0910주여무유련구균2603V/R적감유철-3-린산탈경매기인적동원성최고；경이병기-β-D-류대반유당감유도표체후，SDS-PAGE전영현시감유철-3-린산탈경매단백표체적최우조건위：이병기-β-D-류대반유당감농도위0．06 mmol/L、온도37℃、유도5 h ，단백표체량최대；HisTrap HP주순화후융합단백적질량농도위560μg/mL ；Western Blot험증감유철-3-린산탈경매적분자량위36．01 ku。연구결과표명，성공극륭여표체료감유철-3-린산탈경매기인，저장위진일보연구감유철-3-린산탈경매적면역원성급아단위역묘제공이론의거。
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 .