【作者】 何正祥；

【导师】 张部昌； 马清钧；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 在大肠杆菌中分泌型表达重组人生长激素既有利于消除其在临床应用上的抗原性又有利于提取纯化，因此在重组人生长激素的大规模发酵生产中越来越受到青睐。本研究应用T7启动子与ompA₃引导序列构建了高效分泌型表达重组人生长激素工程菌pET-ompA₃-hGH／BL21（DE3），在摇瓶水平上，采用多因素正交优选法初步筛选培养基成分，进而考察葡萄糖对重组人生长激素表达量的影响。在此培养基基础上优化诱导表达条件，分别考察了诱导起始菌密度、诱导温度、诱导剂浓度和诱导表达时间对重组人生长激素表达量的影响。在摇瓶水平优化后进一步考察了工程菌在发酵罐水平目的蛋白的表达情况。在5L发酵罐中将工程菌pET-ompA₃-hGH／BL21（DE3）进行补料式发酵，诱导表达分泌型重组人生长激素。通过对发酵培养基成分、补料成分、补料体积、补料时机和滴加流速的选择，分泌表达重组人生长激素的量可达到250mg／L，占周质蛋白的44％～54％，发酵周期缩短为8小时。用rhGH纯蛋白免疫动物制备的多克隆抗体制成的抗体亲和层析柱，一步纯化大肠杆菌周质中分泌表达的重组人生长激素，产物纯度达到96％以上，纯化产物在分子量大小、免疫活性和二硫键的形成上均与天然生长激素相一致。更多还原

【Abstract】 Periplasmic expression of rhGH in Escherichia coli was propitious to avoid antigenicity in its clinic application and convenient for purification. Therefore, in modern industry secretary expression of rhGH was being in favor. The engineering bacteria expressing rhGH in secretion type was constructed by using T7 promoter and ompA₃ leader sequence. The effect of phosphate, yeast extract and lactose on expression level of rhGH was determined by orthogonal test, and the influence of different starting glucose concentration on rhGH expression level was also studied to optimize culture medium compositions. Induction stage, induction temperature, IPTG concentration and expression time were studied to optimize condition in secretory expression of rhGH in engineering bacteria. rhGH expression conditions and level of engineering bacteria in fermentation were based on results obtained in shaking flasks. Engineered pET-ompA3-hGH/BL21（DE3） expressed secretion rhGH by inducing and supplying nutrimental components in 5 L fermentor. Expression level of rhGH could reach 250 mg/L culture solution by means of selecting components of original culture media, components of supplemental media, volume of supplemental media, the time of supplying supplemental media and supplying speed of supplemental media, secretion rhGH was found to represent 44%-54% of the total proteins in periplasm, and the fermentation time was reduced to 8 hrs. Secretion rhGH was purified in one-step with affinity chromatography prepared by the polyclonal antibodies from the rabbit immunized with rhGH, and homogeneous rhGH was obtained, with the purity above 96% identified with reduced and nonreduced SDS-PAGE and reversed-phase HPLC. The molecular mass size, immunity activity and formation of disulphide bond were identical to those of native hGH.更多还原