基因重组人脑钠素十聚体发酵工艺及纯化工艺研究-生物化工专业论文.docxVIP

摘要本文在摇瓶实验中，采用正交设计法，优化适合基因重组人脑钠素十聚体 摘要 本文在摇瓶实验中，采用正交设计法，优化适合基因重组人脑钠素十聚体 (10hBNP)表达的最佳培养基组成为(g／L)：葡萄糖2、胰蛋白胨5、酵母粉5、 NaCI 1、MgS040．1、KH2P04 0．5。在上述培养基中，接种量2％，37C，转速170r／rain 下培养至菌体密度OD600为1．0时，加入诱导剂IPTG使其在发酵液中浓度为 0．5mmol／L进行诱导培养4h，诱导后培养温度为34。C，控制pH为7．0，溶氧DO 为50％，得到10hBNP最大表达量为29．77％，菌体密度为3．28。对用乳糖取代 IPTG作为诱导剂进行了初步研究，表明乳糖有利于菌体生长，但诱导效果低于 1PTG。 此外，还系统研究了lOhBNP包含体的洗涤和溶解工艺，确定最佳洗涤工艺． ／为：20倍于包含体质量(mL／g)的含3mol／L尿素与o．5％Triton X-100的缓冲液 洗涤3~4次；最佳溶解工艺为：pH8．0的50mmol／LTris．HCI缓冲体系，溶有8mol／L 尿素、10mmol／L B．ME、150mm01／L NaCl和1rnnaol,rL EDTA，包含体浓度为 10mg／mL于37。C下溶解3h。)7。 在对10hBNP纯化实验过程中，采用固定化金属螯合亲和层析的方法，结果 表明，采用咪唑梯度法，并在杂蛋白洗脱缓冲液中加入16mmol／L眯唑和20％乙 醇，可进一步纯化产物的纯度，使之达到94．31％，收率约为38．9％。 关键词：重组人脑钠素十聚体发酵洗涤溶解固定化金属螯合亲和层析 、／ ×，K X ∥ 钦辞工艺，姬钇圪 ∥ AbstractIn Abstract In this thesis，recombinant Escherichia coli HMSl74 strain harbouring plasmid pQE．3—10hBNP,which encodes human brain natriuretie peptide(IOhBNP)，is used in fermentations to improve the levels of 10hBNP．The medium is optimized through the orthogonal experiment，and the result is as follows(g／L)：glucose 5，tryptone 5，yeast 5，NaCl l，MgS04 0．1，KH2P04 O．5．Under the condition of inoculum 2％，37。C， 1 70r／min，and when the biomass density OD600 is 1．0 the inducer,IPTG of the concentration 0．5 mmol／L is added for the next 4hr induction culture．The culture temperature is altered from 37C to 34C，pH maintained at 7．0，DO at 50％after the induction，and the highest expression of 10hBNP Can be obtained as to 29．77％，and the biomass density is 3．28．Lactose is used as all inducer instead of IPTG in the experiment，and the result shows that lactose Can benefit the biomass growth，but its induction effect is inferior to nlat ofIPTG． In addition，the technological condition for washing and solution of 10hBNP inclusion body are also studied．The optimal washing condition is obtained：the inclusion body is washed with 50mmol／L rids-HCI(pH8．01 containing 3mmol／L urea and 0．5％Triton X一100 at a concentration of 50mg／mL for 3--4 times．The optimal solubilization condition is：the