【作者】 陈帅印；

【导师】 段广才；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2010， 硕士

【摘要】 幽门螺杆菌(Helicobacter pylori,H. pylori)感染是胃炎、胃癌和消化道溃疡的重要病因。目前临床主要用抗生素和抑酸剂联合疗法,虽然取得了一定疗效,但抗生素经济负担高、耐药株出现和药物的副作用等使得抗生素治疗受限。在此情况下,疫苗就成了最有效的方法,具有很好的应用前景。尿素酶(Urease,Ure)是H. pylori的主要致病因子,UreB亚基已经通过基因工程在大肠杆菌中表达,并在粘膜免疫佐剂配合下,能引起免疫应答。所有的经口免疫模型必须用霍乱毒(Cholera toxin,CT)或大肠杆菌不耐热肠毒素(E.coli labile toxin,LT)作为免疫佐剂。但是CT和LT都有一定的毒性而不能用于人体,加上目前疫苗大多采用鼠伤寒减毒沙门氏菌为抗原提呈载体,对免疫力低下的人群危害大,限制了疫苗的使用。但用乳酸乳球菌(Lactococcus lactis,L. lactis)做为活菌疫苗的宿主菌,就可以避免上述危害。但传统的基因改良菌往往以抗生素抗性基因作为筛选标记,这就存在潜在的因生物转基因污染而引起的抗生素耐药的危害。因此完全的食品级表达系统的研发一直是该领域的热点。材料与方法1.从重组质粒pMD19-T-ureB双酶切得到ureB基因；双酶切胶回收乳酸菌载体pNZ8149(该载体以lacF基因为筛选标记),连接后电转化入lacF基因缺失的乳酸乳球菌NZ3900。挑阳性克隆进行酶切、PCR和测序鉴定。2.用正交试验,对影响目的蛋白诱导产量的因素,如诱导剂浓度、诱导时机和诱导持续时间进行实验,用Brandford法测定目的蛋白浓度,通过方差分析得出最适宜表达条件。3.纯化重组大肠杆菌TB1/pMAL-C2X-UreB的诱导产物MBP-UreB,混合弗氏免疫佐剂免疫小鼠,制备抗UreB免疫血清。用Western-blot免疫印迹法研究重组乳球菌NZ3900/pNZ8149-ureB表达的UreB蛋白的免疫反应性。结果1.PCR扩增、酶切鉴定,食品级表达载体pNZ8149-ureB构建成功,转化得到重组工程菌NZ3900/pNZ8149-ureB。重组菌用乳联菌肤(Nisin)诱导后,SDS-PAGE电泳可见64.1KD的目的蛋白UreB。2.正交实验表明,各因素对实验结果影响大小次序是：诱导时机、诱导剂浓度、诱导时间,只有诱导时机对实验结果的影响有统计学意义;表达条件的最优组合：在菌体培养到OD600为0.4时加入终浓度为25ng/mL的Nisin,诱导表达5h。3.制备的小鼠抗UreB血清,经ELISA鉴定效价为1：800。将乳酸菌诱导表达的目的蛋白UreB转移到硝酸纤维膜上,用制备的小鼠血清进行Western blot分析,可在相应位置显示杂交条带,说明乳酸菌诱导表达的UreB具有良好的免疫反应原性。结论1.成功构建了表达UreB的不含任何抗生素抗性筛选标记的乳酸乳球菌食品级原核表达系统。2.利用正交实验获得了重组工程菌NZ3900/pNZ8149-ureB的最优表达条件。3.乳酸乳球菌食品级表达系统诱导表达的UreB蛋白有良好的免疫原性,为研制H. pylori的直接口服疫苗奠定基础。更多还原

【Abstract】 Helicobacter pylori (H. pylori) is the principal cause of chronic gastritis, pepti ulcers and stomach cancer. Current therapies are mainly based on antibiotics together with antiacid. Although the antibiotic-based triple treatments have recently acquired good therapeutic effect, it is not practical for global control for the high cost of antibiotic, problems with patient’s compliance and the increasing drug resistant strains. So, under these circumstances vaccination against H. pylori infection has been considered the best way to control H. pylori infection all over the word.Urease is acid stable, appears to be essential for stomach colonization by H. pylori. The subunit B(UreB) which has been cloned in E. coli.UreB combination with immunogenic adjuvant can stimulate the mouse to producing immunoresponse against challenge of H. pylori. In all the oral immunization experiments, without immunogenic adjuvant, UreB can not protect the animals gainst challenge of H. pylori. The Cholera toxin (CT) and E. coli heat-labile toxin (LT) used to mucosal adjuvant. Because of the toxicity in volunteers, CT or LT can not be given to people.By contrast, using Lactococcus lactis (L.lactis) to deliver protective antigens to the mucosal surface may be a way to solve these problems. L. lactis is nonpathogenic food-grade bacterium that is generally recognized as safe and widely used in food industry. But these traditional genetically modified organism usually used antibiotics resistance gene screening method. So it has potential risk of the antibiotic-resistant. A number of reaserch groups have reported that L. lactis can be widespread used for the expression of heterogeneous genes.Method1.ureB gene was obtained from the recombinant vector of pMD19-T-ureB by NcoⅠand XbaⅠenzyme digestion. The ureB gene was inserted into pNZ8149 food-grade expression vector which lacF is the food-grade selection.Then the recombinant plasmid pNZ8149-ureB was electrotransformed into L. lactis NZ3900.Due to the lacF deletion, strain NZ3900 is unable to grow on lactose.2. L25 (53) orthogonal design was used to optimization the expression conditions of UreB.Different dosage of inducer, the OD6oo of culture, incubating time after iuduced were chosen. Five levels had set for each factor. Then Brandford method was used to work out the expression quantity of the target protein. ANOVA method was used to analyze the results.3.This study acquired fusion protein MBP-UreB from E. coli TB1/pMAL-C2X-UreB. The MBP-UreB fusion protein immunized mice to produce anti-UreB immune serum. Western-blot was performed to check the immunological activity of UreB expressed by food-gread gene expression system.Results1.The food-grade expression vector was successfully constructed through PCR and enzyme digestion.The recombinant plasmid was electrotransformed into L. lactis NZ3900.2.Nisin 25ng/mL was added to the medium when the recombinant grew to OD600≈0.4 then incubate 5h.As a result, the maximum yield of UreB was estimated to be 7% of total soluble cellular proteins.3.The titre of the antiserum was determined to be 1:800 by enzyme lined immunosorbent assay (ELISA).Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity.Conclusions1.Because all the fragments used to construct the expression vector system L. lactis NZ3900/pNZ8149-ureB were obtained from food-grade bacteriat, the expression vector system can be regarded as a food-grade expression vector system.2.The optimal expression conditions for targeted protein expressed by NZ3900/pNZ8149-ureB have been obtained by orthogonal design. 3.The recombinant protein UreB expressed by L. lactis was detected by Western-blot. All the results make an appealing case for construction of the food-grade vaccine for H. pylori.更多还原