【摘要】：目的：為了應(yīng)用PCR-DHPLC技術(shù)對萊姆病螺旋體進行準確、快速、高通量的分型檢測；為了后續(xù)制備膠體金試紙，進行現(xiàn)場快速診斷，對致病性伯氏疏螺旋體外膜蛋白A(Outer surface protein,OspA)進行原核表達，為萊姆病螺旋體的快速檢測做準備工作。 方法：以世界范圍內(nèi)常見的3種萊姆病螺旋體致病菌株（阿氏疏螺旋體、伯氏疏螺旋體、伽氏疏螺旋體）為研究對象。應(yīng)用Genbank設(shè)計種屬特異型基因序列OspC通用引物，應(yīng)用PCR方法擴增3種致病菌的特異序列，聯(lián)用DHPLC對擴增片段進行分型檢測。此過程中進行PCR-DHPLC參數(shù)的優(yōu)化，應(yīng)用48株非螺旋體致病性菌株驗證PCR-DHPLC的靈敏度、特異性，并將此方法應(yīng)用到實際樣品檢測中；為了應(yīng)用間接酶聯(lián)免疫法檢測抗體，本研究表達OspA蛋白，為后續(xù)制備膠體金試紙做準備。首先通過查找相關(guān)文獻，選取在伯氏疏螺旋體感染中大量表達的OspA，針對其序列設(shè)計引物,在兩端加入酶切位點NdeI/XhoI，獲取帶有酶切位點的目的基因序列。雙酶切后聯(lián)入帶有His標簽pET-28b載體，將構(gòu)建成功的重組表達載體轉(zhuǎn)入BL21(DE3)表達菌株。通過改變誘導(dǎo)劑濃度、誘導(dǎo)時間、誘導(dǎo)溫度，確定重組融合蛋白的最佳表達條件，分別選取誘導(dǎo)劑IPTG的濃度0.3mM、0.5mM、0.7mM，誘導(dǎo)時間0h、1h、2h、3h、4h、5h，誘導(dǎo)溫度25℃、30℃、37℃，應(yīng)用超聲破碎、6M鹽酸胍溶解、SDS-PAGE鑒定pET-28b-rOspA蛋白表達形式，應(yīng)用Western Blotting鑒定pET-28b-rOspA融合蛋白的正確性和特異性。通過鎳柱純化所獲得融合蛋白，改變洗脫液的濃度以達到最優(yōu)的洗脫目的。 結(jié)果：應(yīng)用特異性通用引物擴增OspC產(chǎn)物片段，長度分別為633bp、630bp、630bp。測序后，通過比對，擴增序列完全正確；將擴增好的片段無需其他處理可直接上樣于DHPLC中，結(jié)果表明，在部分變性條件下55.8℃時，能通過DHPLC將3種螺旋體致病菌進行區(qū)分。從而各自出現(xiàn)了特異性的分離圖譜，可以很明顯地從檢測圖譜上區(qū)分3種萊姆病螺旋體致病菌種；通過PCR-DHPLC分型檢測方法，建立萊姆病致病菌株標準圖譜，PCR-DHPLC方法靈敏度在核酸水平上達0.01pg/μL，具有良好特異性。成功構(gòu)建重組伯氏疏螺旋體pET-28b-rOspA表達載體，經(jīng)測序驗證，轉(zhuǎn)入BL21(DE3)，經(jīng)過IPTG誘導(dǎo)，確定最佳表達條件，即IPTG誘導(dǎo)濃度為0.5mM、溫度為37℃、誘導(dǎo)時間為4h。通過SDS-PAGE確定其為包涵體表達，經(jīng)Western blotting驗證，經(jīng)鎳柱純化，獲得純度較好OspA蛋白。結(jié)論：本研究成功建立3種萊姆病螺旋體致病菌PCR-DHPLC標準圖譜；通過構(gòu)建重組pET-28b-rOspA質(zhì)粒，表達純化后，，獲得純度較好OspA蛋白，為后續(xù)制備萊姆病螺旋體檢測試紙奠定了基礎(chǔ)。
[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

本文編號：2278169