CAJ | 학술논문

目的：优化艰难梭菌毒素B受体结合区CDB3融合蛋白诱导表达条件，在获得高表达融合蛋白后对其进行纯化，为艰难梭菌毒素B受体结合区CDB3单克隆抗体的制备奠定基础。方法以诱导前菌液600 nm处的光密度值（OD600）、诱导温度、诱导剂异丙基-β-D巯基半乳糖苷（IPTG）浓度和诱导时间为单变量，分别诱导融合蛋白的表达，在此基础上采用正交试验对融合蛋白的诱导表达条件进行优化，通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳半定量分析融合蛋白的表达量及表达形式，以Western blot对融合蛋白进行鉴定，以GST 亲和层析柱对融合蛋白进行纯化。结果融合蛋白诱导表达的最佳条件为诱导前菌液OD6000．8、IPTG浓度0．6 mmol/L、诱导时间12 h、诱导温度30℃，融合蛋白以可溶性蛋白表达形式为主，经纯化后，融合蛋白纯度超过90％。结论本研究成功优化了艰难梭菌毒素B受体结合区CDB3融合蛋白的诱导表达条件，获得大量表达，并成功对其进行了纯化，为后续单克隆抗体的制备奠定了基础。
목적：우화간난사균독소B수체결합구CDB3융합단백유도표체조건，재획득고표체융합단백후대기진행순화，위간난사균독소B수체결합구CDB3단극륭항체적제비전정기출。방법이유도전균액600 nm처적광밀도치（OD600）、유도온도、유도제이병기-β-D구기반유당감（IPTG）농도화유도시간위단변량，분별유도융합단백적표체，재차기출상채용정교시험대융합단백적유도표체조건진행우화，통과십이완기광산납-취병희선알응효전영반정량분석융합단백적표체량급표체형식，이Western blot대융합단백진행감정，이GST 친화층석주대융합단백진행순화。결과융합단백유도표체적최가조건위유도전균액OD6000．8、IPTG농도0．6 mmol/L、유도시간12 h、유도온도30℃，융합단백이가용성단백표체형식위주，경순화후，융합단백순도초과90％。결론본연구성공우화료간난사균독소B수체결합구CDB3융합단백적유도표체조건，획득대량표체，병성공대기진행료순화，위후속단극륭항체적제비전정료기출。
Objective To investigate the best condition for the induced expression of CDB3 receptor binding region fusion protein of Clostridium difficile toxin B ,to analyze the forms of its expression ,and to lay the foundation for the preparation of monoclonal antibodies .Methods The fusion protein were induced to expression at different conditions ,including the optical density at 600 nm(OD600) of bacterial before induction ,the induction temperature , the concentration of inducer isopropy-β-D-thiogalactoside(IPTG) and the induction time .Then ,the orthogonal test was used to determine the optimal conditions for the expression of fusion protein ,and the expression level and the form of its expression were analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis and identified by Western blot .Results The optimal conditions for the induced expression of CDB3 receptor binding region fusion pro-tein of Clostridium difficile toxin B were the OD600 of bacterial before induction with 0 .8 ,the concentration of IPTG of 0 .6 mmol/L ,the induction time of 12 h ,and the induction temperature of 30 ℃ .The main form of expression was soluble protein and the purity could be more than 90% .Conclusion This study successfully induced the expression of CDB3 receptor binding region fusion protein of Clostridium difficile toxin B and the fusion protein was expressed a-bundantly .The main form of expression was soluble protein and was successfully purified ,which could lay the foun-dation for the preparation of monoclonal antibodies .