莱姆病螺旋体PCR-DHPLC图谱的建立及伯氏疏螺旋体OspA的表达纯化

发布时间：2018-10-17 22:09

【摘要】：目的：为了应用PCR-DHPLC技术对莱姆病螺旋体进行准确、快速、高通量的分型检测；为了后续制备胶体金试纸，进行现场快速诊断，对致病性伯氏疏螺旋体外膜蛋白A(Outer surface protein,OspA)进行原核表达，为莱姆病螺旋体的快速检测做准备工作。 方法：以世界范围内常见的3种莱姆病螺旋体致病菌株（阿氏疏螺旋体、伯氏疏螺旋体、伽氏疏螺旋体）为研究对象。应用Genbank设计种属特异型基因序列OspC通用引物，应用PCR方法扩增3种致病菌的特异序列，联用DHPLC对扩增片段进行分型检测。此过程中进行PCR-DHPLC参数的优化，应用48株非螺旋体致病性菌株验证PCR-DHPLC的灵敏度、特异性，并将此方法应用到实际样品检测中；为了应用间接酶联免疫法检测抗体，本研究表达OspA蛋白，为后续制备胶体金试纸做准备。首先通过查找相关文献，选取在伯氏疏螺旋体感染中大量表达的OspA，针对其序列设计引物,在两端加入酶切位点NdeI/XhoI，获取带有酶切位点的目的基因序列。双酶切后联入带有His标签pET-28b载体，将构建成功的重组表达载体转入BL21(DE3)表达菌株。通过改变诱导剂浓度、诱导时间、诱导温度，确定重组融合蛋白的最佳表达条件，分别选取诱导剂IPTG的浓度0.3mM、0.5mM、0.7mM，诱导时间0h、1h、2h、3h、4h、5h，诱导温度25℃、30℃、37℃，应用超声破碎、6M盐酸胍溶解、SDS-PAGE鉴定pET-28b-rOspA蛋白表达形式，应用Western Blotting鉴定pET-28b-rOspA融合蛋白的正确性和特异性。通过镍柱纯化所获得融合蛋白，改变洗脱液的浓度以达到最优的洗脱目的。 结果：应用特异性通用引物扩增OspC产物片段，长度分别为633bp、630bp、630bp。测序后，通过比对，扩增序列完全正确；将扩增好的片段无需其他处理可直接上样于DHPLC中，结果表明，在部分变性条件下55.8℃时，能通过DHPLC将3种螺旋体致病菌进行区分。从而各自出现了特异性的分离图谱，可以很明显地从检测图谱上区分3种莱姆病螺旋体致病菌种；通过PCR-DHPLC分型检测方法，建立莱姆病致病菌株标准图谱，PCR-DHPLC方法灵敏度在核酸水平上达0.01pg/μL，具有良好特异性。成功构建重组伯氏疏螺旋体pET-28b-rOspA表达载体，经测序验证，转入BL21(DE3)，经过IPTG诱导，确定最佳表达条件，即IPTG诱导浓度为0.5mM、温度为37℃、诱导时间为4h。通过SDS-PAGE确定其为包涵体表达，经Western blotting验证，经镍柱纯化，获得纯度较好OspA蛋白。结论：本研究成功建立3种莱姆病螺旋体致病菌PCR-DHPLC标准图谱；通过构建重组pET-28b-rOspA质粒，表达纯化后，，获得纯度较好OspA蛋白，为后续制备莱姆病螺旋体检测试纸奠定了基础。
[Abstract]:Objective: to make accurate, rapid and high-throughput typing and detection of Lyme disease spirulina by PCR-DHPLC technique, and to prepare colloidal gold test paper for field rapid diagnosis. The prokaryotic expression of outer membrane protein (A (Outer surface protein,OspA) of Borrelia burgdorferi was carried out to prepare for rapid detection of Lyme disease. Methods: three common pathogenic strains of Lyme disease spirochetes (Treponema arabinoides, Borrelia burgdorferi, Spirulina Galersini) were studied. Genbank was used to design OspC primers for species-specific gene sequence, PCR method was used to amplify the specific sequences of three pathogenic bacteria, and DHPLC was used to detect the amplified fragments. In this process, PCR-DHPLC parameters were optimized, 48 strains of non-spirochetes pathogenicity strains were used to verify the sensitivity and specificity of PCR-DHPLC, and the method was applied to the detection of actual samples, in order to use indirect enzyme-linked immunosorbent assay (Elisa) to detect antibodies, In this study, OspA protein was expressed to prepare colloidal gold test paper. Firstly, the primer was designed for the OspA, expressed in the infection of Borrelia burgdorferi, and the target gene sequence with the enzyme digested site NdeI/XhoI, was obtained by adding the restriction site NdeI/XhoI, at the two ends of the primer to obtain the target gene sequence with the enzyme cleavage site. The recombinant expression vector was transformed into BL21 (DE3) expression strain after double enzyme digestion into pET-28b vector with His tag. The optimal expression conditions of recombinant fusion protein were determined by changing the concentration of inducer, inducing time, inducing temperature, and determining the optimal expression conditions of recombinant fusion protein. The concentration of the inducer IPTG was 0.3mMU 0.5mMN 0.7mMrespectively, the induction time was 0 hh, 1h, 2h, 3h, 4h, 5h, and the induction temperature was 25 鈩

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