本文選題：指數(shù)富集配體系統(tǒng)進(jìn)化 + 金黃色葡萄球菌腸毒素B　； 參考：《福建醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 【目的】 以金黃色葡萄球菌腸毒素B為靶物質(zhì)篩選獲得的富集單鏈DNA文庫(kù)為起始文庫(kù),從中篩選獲得能高親和力和高特異性結(jié)合金黃色葡萄球菌腸毒素B的單個(gè)DNA適配子,并利用適配子建立的夾心法檢測(cè)患者血清標(biāo)本中SEB的含量,輔助診斷金黃色葡萄球菌感染;同時(shí)研究適配子在大鼠體內(nèi)對(duì)SEB超抗原活性的抑制作用。 【方法】 1.將富集文庫(kù)的篩選產(chǎn)物分別進(jìn)行擴(kuò)增、純化、膠回收和TA克隆轉(zhuǎn)化至大腸桿菌DH5α中,LB液體增菌保存。挑取單個(gè)菌落培養(yǎng),作為菌種保存,抽提質(zhì)粒,利用相應(yīng)引物進(jìn)行PCR擴(kuò)增,并以羧基磁珠作為分離介質(zhì)獲得單個(gè)適配子。 2.分別利用地高辛、熒光素和生物素標(biāo)記的適配子直接測(cè)試法,對(duì)適配子的結(jié)合率和特異性等性質(zhì)進(jìn)行鑒定。 3.分別采用抗體一適配子夾心法和文庫(kù)一適配子夾心法,用于SEB的定量檢測(cè);同時(shí)對(duì)兩種檢測(cè)方法進(jìn)行比對(duì)。利用文庫(kù)一適配子夾心法檢測(cè)臨床患者血清中SEB的含量。 4.將大鼠隨機(jī)分成4組,分別進(jìn)行不同的干預(yù)和處理;隨后檢測(cè)各組大鼠血清中IL-4, IL-6, ALT, AST,肌酐和尿酸的含量,并對(duì)各組檢測(cè)結(jié)果進(jìn)行分析。 【結(jié)果】 1.篩選產(chǎn)物進(jìn)行克隆和增菌培養(yǎng)后,選擇其中40個(gè)克隆子進(jìn)行了檢測(cè),共獲得了30個(gè)條帶位置正確的適配子,并從中篩選得到了一條結(jié)合力和特異性均較好的適配子。 2.文庫(kù)一適配子夾心法和抗體一適配子夾心法均可用于SEB的檢測(cè),且兩種檢測(cè)方法的結(jié)果無(wú)統(tǒng)計(jì)學(xué)差異。而健康對(duì)照組與細(xì)菌感染組血清標(biāo)本中SEB的含量具有統(tǒng)計(jì)學(xué)差異。 3.適配子在大鼠體內(nèi)對(duì)SEB超抗原活性具有一定的抑制作用,可有效抑制IL-4和IL-6的分泌,并能降低血清中AST, ALT,尿酸和肌酐的含量。 【結(jié)論】 本實(shí)驗(yàn)成功篩選到了針對(duì)SEB的高親和力和高特異性的ssDNA適配子,建立了文庫(kù)一適配子夾心法可以對(duì)血清標(biāo)本中SEB的含量進(jìn)行檢測(cè)。適配子的作用類似于抗體,能夠與SEB特異性的結(jié)合,它可以作為金黃色葡萄球菌感染的檢測(cè)分子和治療藥物,具有潛在的使用價(jià)值。
[Abstract]:[objective] to select the enriched single strand DNA library from Staphylococcus aureus enterotoxin B as the target material. A single DNA aptamer with high affinity and specificity binding to Staphylococcus aureus enterotoxin B was obtained. The content of SEB in serum samples was detected by the sandwich method established by the aptamer to assist the diagnosis of Staphylococcus aureus infection. At the same time, the inhibitory effect of aptamer on SEB superantigen activity in rats was studied. [methods] 1. The screening products of the enriched library were amplified, purified, reclaimed and transformed into Escherichia coli DH5 偽. A single aptamer was obtained from a single colony culture as a strain preservation, plasmid extraction, PCR amplification with the corresponding primers, and carboxyl magnetic beads as the separation medium. 2. The binding rate and specificity of aptamers were identified by direct assay of aptamers labeled with digoxin, fluorescein and biotin respectively. 3. The antibody-aptamer sandwich method and library-aptamer sandwich method were used for the quantitative detection of SEB, and the two detection methods were compared. The content of SEB in serum of clinical patients was detected by library-aptamer sandwich method. 4. 4. The rats were randomly divided into 4 groups for different intervention and treatment. The serum levels of IL-4, IL-6, alt, AST, creatinine and uric acid in each group were determined. The results of each group were analyzed. [results] 1. After the screening products were cloned and cultured, 40 clones were selected for detection, and 30 aptamers with the correct position were obtained. An aptamer with good binding ability and specificity was obtained. Library-aptamer sandwich method and antibody-aptamer sandwich method can be used to detect SEB, and there is no statistical difference between the two methods. The SEB content in serum samples of healthy control group and bacterial infection group was significantly different from that of bacterial infection group. The aptamer can inhibit the activity of SEB superantigen and inhibit the secretion of IL-4 and IL-6 in rats. It can reduce the content of AST, alt, uric acid and creatinine in serum. [conclusion] the high affinity and specificity of ssDNA adaptor for SEB were successfully screened in this experiment. A library-aptamer sandwich method was established to detect the content of SEB in serum samples. Aptamers have similar effects to antibodies and can bind to SEB specifically. They can be used as detection molecules and therapeutic drugs for Staphylococcus aureus infection.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 江麗;蘭小鵬;曾燕麗;甘龍杰;;用SELEX技術(shù)篩選葡萄球菌腸毒素B特異性適配體[J];臨床檢驗(yàn)雜志;2009年06期

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本文編號(hào)：2019720