本文關(guān)鍵詞： 卡介苗 分型 聚合酶鏈反應(yīng) 胞嘧啶-鳥嘌呤　出處：《華中科技大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:對目前國內(nèi)卡介苗(BCG)生產(chǎn)用株進行基因分型,檢測BCG DNA中胞嘧啶-鳥嘌呤(CpG)的含量。 方法:收集菌體分別經(jīng)機械破碎或溶菌處理,用溴化十六烷基三甲基銨(CTAB)進行沉淀,再經(jīng)酚:氯仿:異戊醇抽提,對所得DNA進行紫外掃描、凝膠電泳檢測。主要采用PCR技術(shù)對11種BCG菌株及對比株提取的DNA進行體外擴增,引物針對BCG菌株某些特異性缺失及擴增染色體片段而設(shè)計,通過瓊脂糖凝膠電泳檢測擴增情況,并對所得產(chǎn)物進行測序鑒定。以CpG特異的甲基化酶(CpG Methylase)M.SssI對BCG DNA進行修飾,將CpG中的胞嘧啶(C)轉(zhuǎn)化為5-甲基胞嘧啶(MC),利用限制性內(nèi)切酶HpaⅡ進行消化以檢測甲基化程度,甲基化完全的DNA經(jīng)相關(guān)酶水解后應(yīng)用反相高效液相色譜(RP-HPLC)方法檢測樣品中CpG的質(zhì)量百分含量。 結(jié)果:機械破碎抽提BCG DNA產(chǎn)率高、純度高,溶菌處理抽提DNA完整性好。在BCG相關(guān)片段的PCR擴增中,國內(nèi)BCG生產(chǎn)用株均顯示相同PCR結(jié)果,SenX3-regX3擴增片段為353 bp,RD Denmark/Glaxo擴增產(chǎn)物分別為281 bp(AL/AR)、1637 bp(BL/BR),nRD18擴增產(chǎn)物分別為655 bp(AL/AR)、406 bp(L/R/wtR),IS6110 copyⅡ擴增產(chǎn)物417 bp,RD2及nRD18(AL/CR)擴增無產(chǎn)物。BCG上海新菌種DNA樣品的CpG質(zhì)量百分含量為23.61%,巴斯德株抽提DNA樣品的CpG質(zhì)量百分含量為23.53%。 結(jié)論:CTAB沉淀是一種比較簡便易行的BCG DNA提取方法。國內(nèi)BCG生產(chǎn)用株均顯示相同基因型,特異性缺失RD2,nRD18和RD Denmark/Glaxo均存在,IS6110為單拷貝,senX3-regX3基因間區(qū)域的77-bp分枝桿菌散在分布重復(fù)單位(MIRUs)為三重拷貝。在目前具有代表性的國際公認菌株中未能找到與國內(nèi)株基因型匹配者。采用甲基化特異酶修飾、酶水解、RP-HPLC的方法,能檢測出DNA樣品中CpG的質(zhì)量百分含量,而且這種方法的準確性得到了較好的驗證。
[Abstract]:Aim: to detect the content of cytosine guanine guanine (CpG) in BCG DNA by genotyping of domestic BCG production strains. Methods: the bacteria were collected by mechanical crushing or bacteriolysis, precipitated by cetyltrimethylammonium bromide (CTAB), then extracted by phenol: chloroform: isoamyl alcohol. The DNA was scanned by UV. PCR technique was used to amplify DNA extracted from 11 BCG strains and control strains in vitro. Primers were designed for the specific deletion and amplification of chromosomal fragments of BCG strains. The amplification was detected by agarose gel electrophoresis. The BCG DNA was modified with CpG specific methylase CpG Methylase)M.SssI, and the cytosine cytosine in CpG was transformed into 5-methylcytosine, which was digested by restriction endonuclease Hpa 鈪，

本文編號：1510854