产肠毒素大肠杆菌STa突变体、LTB和STb融合蛋白在乳酸菌中组成型分泌表达及其免疫原性分析

发布时间：2018-05-23 19:28

本文选题：产肠毒素大肠杆菌 + mSTa-LTB-STb融合蛋白　；参考：《中国预防兽医学报》2017年12期

【摘要】：为获得产肠毒素大肠杆菌(ETEC)抗原STa突变体(mSTa)、LTb和STb融合蛋白在乳酸菌表达系统中组成型分泌表达,本研究将mSTa、LTb、STb三价抗原融合基因,克隆于p23启动子-USP45分泌信号肽之后,插入到乳酸乳球菌表达载体pTX8048中,构建了重组表达载体pTX-sls,将其电转化到宿主菌乳酸乳球菌L.lactisNZ9000中进行表达,应用westernblot、ELISA方法鉴定蛋白表达情况。将重组乳酸乳球菌pTX-sls/NZ9000口服免疫BALB/c小鼠,分别测定了免疫后不同时间血清中特异性IgG、粪便中特异性的sIgA水平以及血清的中和活性;采用MTT法检测免疫小鼠脾淋巴细胞增殖情况,流式细胞术检测Th细胞免疫类型。结果显示,目的蛋白mSTa-LTB-STb以分泌形式组成型表达,可被阳性血清所识别。在免疫小鼠血清和粪便中均可检测到特异性IgG、sIgA,血清抗体具有一定的中和毒素作用。结果表明,该重组乳酸乳球菌具有作为口服疫苗的潜在应用价值,本研究为研制ETEC乳酸菌活载体口服疫苗奠定了基础。
[Abstract]:In order to obtain the constitutive secretory expression of STa mutants of enterotoxigenic Escherichia coli (STa) and STb fusion protein in lactic acid bacteria expression system, the fusion gene was cloned into the signal peptide secreted by p23 promoter -USP45. The recombinant expression vector pTX-slswas constructed by inserting into the expression vector pTX8048 of Lactococcus lactis. The recombinant expression vector pTX-slswas transformed into the host strain of Lactococcus lactis L.lactisNZ9000 for expression. Western blottELISA was used to identify the expression of pTX-sls. BALB/c mice were immunized orally with recombinant Lactococcus lactis pTX-sls/NZ9000. The specific sIgA levels in serum, the specific sIgA level in feces and the neutralization activity of serum were determined at different time after immunization, and the proliferation of spleen lymphocytes in immunized mice was detected by MTT method. Th cell immune type was detected by flow cytometry. The results showed that the target protein mSTa-LTB-STb was expressed in secretory form and could be recognized by positive serum. Specific IgG sIgA can be detected in sera and feces of immunized mice, and serum antibodies can neutralize toxins to some extent. The results showed that the recombinant Lactococcus lactis had potential application value as oral vaccine. This study laid a foundation for the development of oral vaccine of ETEC lactobacillus live vector.
【作者单位】：东北农业大学动物医学院;
【基金】：国家自然科学基金(31101845)
【分类号】：S852.61

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