^(125)Ⅰ标记rLTB-UreB及口服BALB/c小鼠示踪实验研究

目的 观察^(125)Ⅰ标记基因重组融合蛋白LTB-UreB灌喂BALB/c小鼠后的体内分布。方法 采用氯胺T氧化法标记rLTB-UreB,Sephadex-G25凝胶过滤纯化,纸层析鉴定放射化学纯度及比活度。将BALB/c小鼠随机分为PBS口服空白对照组、Na^(125) Ⅰ口服对照组和^(125)Ⅰ-rLTB-UreB实验组,于不同时间采集标本,γ放射计数器检测CPM(每分钟计数值),SPSS分析结果。结果 纯化后的标记蛋白纯度>90％。实验组PP结和肠系膜淋巴结CPM升高,与对照组比较差异有非常显著意义。结论 ^(125)Ⅰ标记融合蛋白口服后可在肠道粘膜下淋巴组织沉积,为融合蛋白作为幽门螺杆菌口服疫苗候选抗原提供重要实验依据。

ltB-ureB融合基因原核表达系统的构建及其产物免疫性和佐剂活性的鉴定

构建lt B- ure B融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T- A克隆法从幽门螺杆菌(H elicobacter pylori,Hp)临床菌株Y0 6和大肠杆菌4 4 85 1株DNA中获得了ure B和lt B全长基因扩增片段及其克隆,并构建了lt B- ure B融合基因及其原核表达系统p ET32 a- lt B- ure B- E.coli BL2 1DE3.在E.coli BL2 1DE3宿主菌中用不同浓度的IPTG诱导表达,并用Hp全菌抗体的Western blot、EL ISA以及GM1 -EL ISA分别证实了目的重组蛋白(r L TB- Ure B)的免疫性和佐剂活性.与报道的相关序列比较,所克隆的ure B和lt B核苷酸序列同源性分别为96 .88%～97.82 %和99.12 %～99.71% ,氨基酸序列同源性为99.6 5 %～99.82 %和97.5 8%～99.19% . 0 .1～1.0 mm ol/ L 的IPTG均能有效地诱导目的重组蛋白r L TB- Ure B的表达,该蛋白主要以包涵体形式存在,其产量约为细菌总蛋白的35 % .Western blot结果证实r L TB- U re B不仅能与商品化的Hp全菌抗体结合,免疫家兔后也能产生特异性抗体,表明r L TB- U re B有良好的免疫反应性及抗原性.GM1 - EL ISA结果显示r L TB- Ure B能与牛GM1 结合,表明r L TB- Ure B有佐剂活性.以兔抗r L TB- U re B为一抗,发现所检测的10 9株Hp临床分离菌株均表达Ure B;以r L TB- U re B为包被抗原,发现所检测的12 5例Hp感染者血清中均存在U re B抗体;表明U re B广泛存在于不同的Hp菌株中,并有很强的抗原性,也提示r L TB- Ure B确有自然表达U re B的抗原特异性.本文成功地构建了L TB- Ure B融合基因原核高效表达系统,所表达的L TB- U re B融合蛋白有良好的免疫性和佐剂活性,为Hp基因工程疫苗的产业化奠定了坚实的基础.

重组LTB-UreB融合蛋白的结构研究

重组LTB-Ure B融合蛋白可有效的预防幽门螺杆菌的感染,但对其蛋白结构研究至今并无相关报道。由于蛋白质的一级结构对其空间结构、性质及功能都是十分重要的,因此对重组LTB-Ure B融合蛋白的一级结构进行了研究,为进一步研究其功能及作用机制奠定基础。文章利用不同方法对重组LTB-Ure B融合蛋白的纯度、分子质量、N-端、C-端及肽段覆盖率进行了分析,结果显示,重组LTB-Ure B融合蛋白一级结构的各项分析数据与理论结果均相符。该研究建立了重组LTB-Ure B融合蛋白一级结构研究方法,获得了蛋白结构相关信息,可为重组LTB-Ure B融合蛋白的功能研究奠定基础。

Construction of prokaryotic expression system of ItB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

AIM: To construct ItB-ureBfusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein.METHODS: The ureB gene from a clinical Helicobacter pylori (H pylon) strain Y06 and the ItB gene from Escherichia coli (E. coli) strain 44851 were linked into ItB-ureB fusion gene by PCR. The fusion gene sequence was analyzed after T-A cloning. A prokaryotic recombinant expression vector pET32a inserted with ltB-ureB fusion gene (pET32a-ltB-ureB) was constructed. Expression of the recombinant LTB-UreB protein (rLTB-UreB) in E. coliBL21DE3 induced by isopropylthio-β-D-galactoside (IPTG) at different concentrations was detected by SDS-PAGE. Western blot assays were used to examine the immunoreaction of rLTB-UreB by a commercial antibody against whole cell of Hpylori and a self-prepared rabbit anti-rUreB serum, respectively,and determine the antigenicity of the recombinant protein on inducing specific antibody in rabbits. GM1-ELISA was used to demonstrate the adjuvanticity of rLTB-UreB.Immunoreaction of rLTB-UreB to the UreB antibody positive sera from 125 gastric patients was determined by using ELISA.RESULTS: In comparison with the corresponding sequences of original genes, the nucleotide sequence homologies of the cloned ltB-ureB fusion gene were 100%. IPTG with different dosages of 0.1-1.0 mmol/L could efficiently induce pET32a-ltB-ureB-E, coli BL21DE3 to express the rLTB-UreB.The output of the target recombinant protein expressed by pET32a-ureB-E.coli BL21DE3 was approximately 35% of the total bacterial proteins, rLTB-UreB mainly presented in the form of inclusion body. Western blotting results demonstrated that rLTB-UreB could combine with the commercial antibody against whole cell of H pylori and anti-rUreB serum as well as induce rabbit to produce specific antibody. The strong ability of rLTB-UreB bindingbovine GM1 indicated the existence of adjuvanticity of the recombinant protein. All the UreB antibody positive sera from the patients (125/125) were positive for rLTB-UreB.CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of Hpylorigenetically engineered vaccine.

小鼠口服重组LTB和HpUreB免疫活性的研究

目的 观察重组大肠杆菌不耐热肠毒素B亚单位 (rLTB)的粘膜佐剂活性以及重组Hp尿素酶B亚单位 (rUreB)的免疫学活性 ,为制备安全有效的Hp疫苗奠定基础。方法 ELISA检测rLTB的GM1(神经节苷脂 )结合活性 ,利用本室基因工程重组表达的rLTB和rUreB口服免疫BALB/c小鼠后 ,在第 4和 /或 5周ELISA方法检测血清IgG ,以及唾液、粪便抽提物、肠粘液sIgA水平。采用3 H掺入实验 ,检测小鼠脾脏淋巴细胞对特异性抗原的增殖反应。结果 ①rLTB具有GM1结合活性。②与不加佐剂的对照比较 ,加rLTB的实验组 ,能够有效地诱发唾液及肠道sIgA的产生。③口服HprUreB抗原能够刺激小鼠产生特异性的IgG和sIgA。④3 HT dR掺入实验提示加佐剂组较不加佐剂组 ,脾脏淋巴细胞对特异性抗原的增殖反应更强。⑤不加佐剂组血清IgG ,免疫第 5周较第 4周高 ;加佐剂则是第 4周比第 5周高。结论 重组LTB具有良好的粘膜佐剂功能 ;HprUreB具备良好的免疫原性 ;小鼠口服rLTB和rUreB后可以产生保护性sIgA ;