单增李斯特菌iap、actA、plcB基因的原核表达、产物的免疫原性分析以及重组p60蛋白多抗的制备

发布时间：2019-05-23 15:46

【摘要】：目的：单增李斯特菌(Listeria monocytogenes，LM)是李斯特菌属最重要的人和动物致病菌，由于其具有低温生长的特性，因此是冷藏食品和即食食品最重要的致病菌之一。本文通过原核表达单增李斯特菌特异性膜表面蛋白的基因iap，actA，plcB，并分析其产物作为制备LM特异性膜表面蛋白单克隆抗体的可行性，为今后制备这些蛋白的单克隆抗体并建立单增李斯特菌的免疫学磁珠分离快速检测方法的建立奠定基础。方法：分别设计actA，plcB，iap基因引物，并以提取的单增李斯特菌DNA为模板进行PCR扩增，产物回收后，连接入pMD18-T克隆载体，转化E.coliDH5α，提取质粒并进行双酶切鉴定及测序鉴定。鉴定正确的的重组质粒与表达载体PET28a(+)/PET32a（+）构建重组表达质粒，产物转化E.coli BL21，经含卡那霉素/氨苄青霉素的LB培养基筛选，挑取阳性菌落并进行测序。将鉴定正确的菌液ITPG诱导表达，超声破碎菌体，SDS-PAGE对表达产物进行可溶性表达分析，利用亲和层析分离纯化目的蛋白，运用westernblotting和ELISA分析重组蛋白的免疫原性，将纯化的p60蛋白免疫小鼠，制备鼠多克隆抗体，ELISA检测效价以及交叉反应性。结果：双酶切及测序鉴定显示，重组克隆载体及表达载体均构建成功。分析诱导表达产物，ActA蛋白和p60蛋白为可溶性表达，plcB为包涵体表达。镍离子亲和层析纯化，得到了纯度较高的PlcB蛋白和p60蛋白，但是ActA没有纯化成功。免疫原性分析表明：p60蛋白和ActA蛋白与兔和鼠免疫血清均具有反应性，plcB与兔免疫血清反应良好。以重组表达的p60蛋白作为免疫原制备鼠多克隆抗体，3次免疫后效价达到1:8000，并且与属内威尔斯、英诺克菌存在微弱交叉反应。结论：原核表达单增李斯特菌毒力因子基因actA，plcB，iap，，分析了所表达蛋白的免疫原性，其中重组p60蛋白对于家兔和小鼠均有较好的免疫原性，免疫小鼠后所得多抗与LM菌体特异性结合，这表明以其为靶点，制备LM特异性的膜表面单抗是可行的。
[Abstract]:Objective: Listeria monocytogenes (Listeria monocytogenes,LM) is the most important human and animal pathogen of listeria. Because of its low temperature growth characteristics, Listeria monocytogenes is one of the most important pathogenic bacteria in refrigerated food and ready-to-eat food. In this paper, the gene iap,actA,plcB, of Listeria monocytogenes specific membrane surface protein was expressed in prokaryotic cells and the feasibility of using its product as monoclonal antibody to LM specific membrane surface protein was analyzed. It lays a foundation for the preparation of monoclonal antibodies against these proteins and the establishment of a rapid method for the separation and detection of Listeria monocytogenes by immunological magnetic beads. Methods: actA,plcB,iap gene primers were designed, and the extracted Listeria monocytogenes DNA was used as template for PCR amplification. after the product was recovered, the product was ligated into pMD18-T clone vector and transformed into E.coliDH5 伪. The plasmid was extracted and identified by double enzyme digestion and sequencing. The correct recombinant plasmid and expression vector PET28a () / PET32a () were identified to construct the recombinant expression plasmid. The product was transformed into E.coli BL21, and screened by LB medium containing kanamycin / ampicillin. The positive colonies were selected and sequenced. The correct ITPG induced expression was identified, the expressed product was analyzed by SDS-PAGE, the target protein was isolated and purified by affinity chromatography, and the immunogenicity of the recombinant protein was analyzed by westernblotting and ELISA. Mouse polyclonal antibody was prepared by immunizing mice with purified p60 protein. The titer and cross-reaction of mouse polyclonal antibody were detected by ELISA. Results: double enzyme digestion and sequencing showed that the recombinant clone vector and expression vector were constructed successfully. The induced expression products were analyzed. ActA protein and p60 protein were soluble and plcB was expressed as inclusion body. High purity PlcB protein and p60 protein were obtained by nickel ion affinity chromatography, but ActA was not purified successfully. Immunogenicity analysis showed that p60 protein and ActA protein were reactive to rabbit and mouse immune serum, and plcB reacted well with rabbit immune serum. Mouse polyclonal antibody was prepared by using recombinant p60 protein as immunogen. The titer of mouse polyclonal antibody reached 1 鈮

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