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目的构建大肠杆菌不耐热肠毒素B亚单位(LTB)和幽门螺杆菌(Hp)尿素酶B亚单位(UreB)、幽门螺杆菌粘附素A(HpaA)的融合基因ltBureBhpaA及其原核表达系统,鉴定重组表达产物rLTBUreBHpaA的免疫原性、佐剂活性及对Hp感染小鼠的保护作用。方法采用连接引物PCR构建ltBureBhpaA融合基因,TA克隆后测序。采用pET42a质粒及其宿主菌E.coliBL21DE3亚克隆构建原核表达系统pET42altBureBhpaAE.coliBL21DE3,并用不同浓度IPTG诱导表达。SDSPAGE用于检测rLTBUreBHpaA的表达及其产量,免疫双扩散试验及Western印迹法检测rLTBUreBHpaA抗原性和免疫反应性。建立牛GM1的ELISA(GM1ELISA)检测重组蛋白中rLTB的佐剂活性。采用HpSS1株BaLb/C小鼠感染模型,检测rLTBUreBHpaA的免疫保护作用。结果ltBureBhpaA核苷酸序列与各原始基因序列完全相同。不同浓度的IPTG均可诱导pET42altBureBhpaAE.coliBL21DE3表达rLTBUreBHpaA,其产量约为细菌总蛋白的15%。rLTBUreBHpaA家兔抗血清的双扩效价为1∶8。商品化兔抗Hp全菌抗体、兔抗UreB或HpaA均能识别rLTBUreBHpaA并与之结合。GM1ELISA结果证实rLTBUreBHpaA仍具有结合牛GM1的活性。rLTBUreBHpaA(200μg/每只小鼠)免疫后,可使小鼠100%免于HpSS1株的感染。10μgrLTB与rUreB或rHpa共同免疫小鼠,可使保护率分别从66.7%提高至81.8%和83.3%。结论本研究成功地构建了ltBureBhpaA融合基因及其原核表达系统。目的表达产物rLTBUreBHpaA有良好的免疫原性、佐剂活性及免疫保护效果,具有作为Hp基因工程疫苗的应用前景。
Objective To construct the fusion gene ltBureBhpaA of Escherichia coli heat-labile enterotoxin B subunit (LTB) and Hp urease B subunit (UreB), Helicobacter pylori adhesin A (HpaA) and its prokaryotic expression system The immunogenicity, adjuvant activity and protective effect of rLTBUreBHpaA on Hp-infected mice were identified. Methods The fusion gene of ltBureBhpaA was constructed by ligating primer PCR and sequenced after TA cloning. The prokaryotic expression system pET42altBureBhpaAE.coliBL21DE3 was constructed by subcloning pET42a plasmid and its host strain E. coli BL21DE3 and induced with different concentrations of IPTG. SDSPAGE was used to detect the expression of rLTBUreBHpaA and its production, immunodouble diffusion assay and Western blotting to detect the antigenicity and immunoreactivity of rLTBUreBHpaA. Establishment of a bovine GM1 ELISA (GM1 ELISA) for the adjuvant activity of rLTB in recombinant proteins. HpSS1 strain BaLb / C mouse infection model was used to test the protective effect of rLTBUreBHpaA. As a result, the ltBureBhpaA nucleotide sequence is exactly the same as each original gene sequence. Different concentrations of IPTG induced pET42altBureBhpaAE.coliBL21DE3 expressed rLTBUreBHpaA, which produced about 15% of total bacterial protein. The double-titers of rLTBUreBHpaA rabbit antisera were 1: 8. Commercial rabbit anti-Hp whole antibodies, rabbit anti-UreB or HpaA, recognize and bind to rLTBUreBHpaA. GM1 ELISA results confirmed that rLTBUreBHpaA still has the activity of binding to bovine GM1. After immunization with rLTBUreBHpaA (200 μg / mouse) mice were 100% protected from infection with the HpSS1 strain. Co-immunization of mice with 10 μg rLTB and rUreB or rHpa increased the protection rates from 66.7% to 81.8% and 83.3%, respectively. Conclusion This study successfully constructed ltBureBhpaA fusion gene and prokaryotic expression system. The target product rLTBUreBHpaA has good immunogenicity, adjuvant activity and immune protection effect, and has the potential application as an Hp gene engineering vaccine.