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1、幽门螺杆菌培养方法及其尿素酶纯化的研究硕士研究生：刘建东导师：周曾芬教授单位：昆明医学院第一附属医院消化内科中文论文摘要目的：探讨大量培养幽门螺杆菌（H.pylori）的方法，从H.pylori标准菌株NCTC11639中提取与纯化尿素酶（Urease）。方法：采用单因子试验统计分析技术对H.pylori固体和液体培养技术进行优化筛选。以哥伦比亚培养基（CAB）为基础培养基进行固体培养，在复苏和传代培养中，分别观察不同添加剂（10%冻融羊血（SB）、10%绵羊血清（SBS）和0.1%环糊精（-CD）、不同室温下空气中暴露时间（1h和3h）和不同抽气负压（-0.075Mpa、-0.070Mpa和
2、-0.065Mpa）对H.pylori培养质量（产量和形态）的影响；以BHIB为基础培养基进行液体培养，观察四种不同添加剂（5%小牛血清（CBS）、10%CBS、10%SBS和0.1%-CD）对H.pylori液体培养产量的影响。比较不同固、液培养体系的效费关系。用旋涡振荡和冻融法粗提尿素酶，再用Q Sepharose FF阴离子交换层析、Phenyl Sepharose HP疏水相互作用层析和Source 30S Sepharose阳离子交换层析进行纯化，测定纯化出的尿素酶的活性和纯度，计算蛋白浓度和总蛋白回收率。用SDS-PAGE和Western blotting进行鉴定。结果：H.pyl
3、ori固体培养复苏4天后，添加10%SB、10%SBS和0.1%-CD的CAB平板每块分别可获得0.0618g、0.0576g和0.0335g菌量，传代培养3天后，每块分别可获得0.0802g、0.0922g和0.0447g菌量。无论复苏或传代，H.pylori产量在10%SB和10%SBS培养基之间均无明显统计学差异（P 0.05），但均优于0.1%-CD培养基（P 0.05）。固体培养时，以0.1%-CD作添加剂的平板上，更易观察到较典型的H.pylori形态。液体培养3天后，添加5%CBS、10%CBS、10%SBS和0.1%-CD的四种液体培养基的OD650值分别为0.2532、0.3
4、705、0.3173和0.2336，每100ml H.pylori产量分别为0.101g、0.148g、0.127g和0.093g，10%CBS与10%SBS组 H.pylori产量均高于0.1%-CD组（P 0.05），10%CBS组H.pylori产量高于5%CBS组（P 0.05）。每产1g H.pylori，10%SB、10%SBS和0.1%-CD三种固体培养的费用分别为26.28元、24.23元和38.37元，需要的相对培养批次数分别为1.14次、1.00次、2.04次；5%CBS、10%CBS、10%SBS和0.1%-CD的四种液体培养费用分别为100.40元、89.27元、86.
5、85元和70.11元，需要的相对培养批次数分别为1.50次、1.00次、1.15次和1.67次。最后纯化得到的尿素酶酶活性为19.7 urea/mg/min，纯度在95%以上，总的蛋白回收率为2.58%，SDS-PAGE显示出尿素酶亚单位的分子量66kDa（UreB）和30kDa（UreA），Western blotting显示其可以与兔抗H.pylori全菌血清发生反应。结论：添加10%SB和10%SBS的CAB培养基可获得较高的H.pylori产量，最高可获0.092g/平板，可用于H.pylori的大量培养；采用旋涡振荡和冻融法粗提H.pylori尿素酶得率高，使用离子交换层析（阳离子和
6、阴离子）和疏水相互作用层析的三步纯化法，可大量快速地纯化尿素酶。本研究在云南地区首次建立了H.pylori液体培养体系，这为在云南地区进行H.pylori大量培养及H.pylori相关基础与临床研究奠定了基础。关键词幽门螺杆菌尿素酶培养纯化方法英文论文摘要Culture and Urease Purification of Helicobacter pyloriMaster Candidate: Jiandong LiuSupervisor: Prof. Zengfen ZhouUnit: Gastroenterology Department of the First Affiliated
7、Hospital of Kunming Medical CollegeDetailed AbstractObjective To explore a large-scale culture method for Helicobacter pylori (H.pylori) and purify urease from H.pylori standard strain NCTC11639. Methods Using analytical technique of single factor test statistics, to select a suitable medium and cul
8、ture condition for H.pylori. Columbia Agar base (CAB) as plating media was used, in the process of recovery culture and subculture, to observe the growth of H.pylori when different nutrient supplements (10% sheep blood (SB), 10% sheep blood serum (SBS) and 0.1%-cyclodextrin (-CD) were added, or when
9、 plates were exposed in the atmospere for different periods of time (1h and 3h), or when the different negative pressures (-0.075Mpa, -0.070Mpa and -0.065Mpa) were attained while pumping out the excess oxygen from the jar. Brain Heart Infusion broth (BHIB) was used as the liquid media to observe the
10、 growth of H.pylori when different nutrient supplements (5%CBS, 10%CBS, 10%SBS and 0.1%-CD) were added. Calculations and comparison of the cost and the time consumed for 1g of H.pylori in different cuture medias were performed. The urease was extracted from the bacterium using a spin-surge and alter
11、nate freezing and thawing mothod and purified by Q Sepharose FF anion-exchange chromatography, Phenyl Sepharose HP Hydrophobic interaction and Source 30S Sepharose cation-exchange chromatography. Enzymatic activity of the purified urease was detected with a spectrophotometic assay based on phenol re
12、d. The purity of the purified urease was analysed by the software totallab version 1.0. The protein concentrations were detected based on Bradford method. The overall protein recovery was calculated. The identification of the purified urease was performed by sodium dodecyl sulfate polyacrylamide gel
13、 electrophoresis (SDS-PAGE) and Western blotting. Results From solid plate medias added with 10%SB, 10%SBS and 0.1%-CD, 0.0618g, 0.0576g and 0.0335g of H.pylori was attained respectively after 4 days growth of recovery and 0.0802g、0.0922g and 0.0447g of H.pylori was attained respectively after 3 day
14、s growth of subculture. Whether it was during the recovery or the subculture, theres no statistic significance (P 0.05) of H.pylori production between the 10%SB group and the 10% SBS group, but both were superior to the 0.1% -CD group(P 0.05) in H.pylori production. In the process of solid culture,
15、H.pylori was more frequently observed in a typical curved rod shape when 0.1% -CD was added. The OD650 values of 0.2532, 0.3705, 0.3173 and 0.2336 corresponding to 0.101g, 0.148g, 0.127g and 0.093g of H.pylori were attained from the 100 ml of liquid medias added with 5%CBS, 10%CBS, 10%SBS and 0.1%-CD respectively after
