大肠杆菌重组LTKA63突变体和LTB黏膜免疫佐剂活性的研究

Determination of fbe mucosal immunoadjuvantivity of recombinant EschericMa cott LTKA63 mutant and LTB

目的构建大肠杆菌不耐热肠毒素A亚单位(heat-labile enterotoxin subunit A,LTA)突变体 LTKA63及B亚单位(heat-labile entemtoxin subunit B,LTB)基因原核表达系统,确定rLTKA63和rLTB黏膜免疫佐剂活性。方法采用高保真PCR从大肠杆菌44815株基因组DNA中扩增LTA和LTB基因,采用定位突变技术构建LTKA63突变体,T-A克隆后测序。构建LTKA63和LTB原核表达载体,采用SDS-PAGE鉴定表达产物。采用三色流式细胞术确定rLTKA63激活T细胞途径,以GM1-ELISA检测rLTB结合牛GM1的能力。建立幽门螺杆菌SS1株小鼠感染模型,分别以幽门螺杆菌(Hp)重组尿素酶B亚单位(rUreB)和黏附素(rHpaA)为抗原,分别检测rLTKA63和rLTB对rUreB和rHpaA的免疫保护增强作用。结果从大肠杆菌44815株DNA模板中扩增获得预期大小的LTA和LTB基因条带。LTA基因定位突变后获得序列正确的LTKA63突变体。rLTKA63和rLTB表达量分别占细菌总蛋白的30％和20％左右。LTKA63作用后T_H1 和T_H2细胞较阴性对照组分别增加19．9％和42．3％,GM1-ELISA结果证实rLTB能与牛GM1结合。 33．3％(4／12)rUreB和41．7％(5／12)rHpaA免疫小鼠胃黏膜标本中分离出幽门螺杆菌,且rUreB和rHpaA特异性S-IgA检测结果均为阴性。rUreB或rHpaA加用rLTKA63或rLTB免疫后,小鼠胃黏膜标本中幽门螺杆菌分离阳性率下降为16．7％～25．0％(P>0．05),rUreB和rHpaA特异性S-Iga阳性率可达41．6％- 58．3％(P<0．01)。结论成功地构建了LTKA63和LTB原核表达系统,所表达的rLTKA63和rLTB均具有较强的黏膜免疫佐剂活性。

Objective To construct the prokaryotic expression systems of detoxified mutant LTKA63 of Es- cherichia coli heat-labile enterotoxin subunit A （LTA） gene and E. coli heat-labile enterotoxin subunit B （LTB） gene, and to determine the mucosal immunoadjuvantivity of the rLTKA63 and rLTB. Methods The LTA and LTB genes from E. col/strain 44815 genome DNA were amplified by high fidelity PCR. Site mutation technique was applied to construct the mutant LTKA63. All the cloned genes were sequenced after T-A cloning, Then the prokaryotic expression systems of LTKA63 and LTB genes were constructed and their expressed products were identified using SDS-PAGE. The pathways activating T lymphocyes of rLTKA63 were determined using the three colors flow cytometry. And GM1-ELIZA was used to demonstrate the ability binding bovine GM1 of rLTB. By using recombinant urease subunit B （rUreB） and adhesin A （ rHpaA ） as the antigens, the effects of rLTKA63 and rLTB on increasing the immunoprotection of rUreB and rHpaA in Helicobacter pylori strain SS1 infected mice were determined respectively. Results The amplification fragments of LTA and LTB genes with expected size were obtained from E. coli strain 44815 genome DNA templates. The mutant LTKA63 with correct sequence was obtained through the site mutation of the cloned LTA gene. The expression outputs of rLTKA63 and rLTB were approximate 30% and 20% of the total bacterial proteins, respectively. After co-incubation with LTKA63, TH 1 and TH2 lympbocytes were increased by 19.9% and 42.3% respectively. The result of GMI-ELISA indicated that rLTB could combine bovine GM1. 33.3% （4/12） of the gastric biopsy samples in the mice immunized with rUreB alone and 41.7% （5112） of the gastric biopsy samples in the mice immunized with rHpaA alone were positive for H. pylori isolation, and the rUreB or rHpaA specific S-IgAs in all the animals were negative. When rUreB or rHpaA co-administrated with rLTKA63 or rLTB, the positive H. pylori isolation rates were as lower as 16.7%-25.0% （ P 〉0.05） and the rUreB and rHpaA specific S-IgAs positive rates reached 41.6%-58.3% （P〈0.01）. Conclusion The prokaryotic expression systems of LTKA63 and LTB genes were successfully established in this study. Both the expressed rLTKA63 and rLTB have strong mucosal adjuvant activities.