【摘要】：目的:(1)研究廣西 HIV-1 重組流行株亞型 env 基因 V3-V4 區(qū)及其臨近區(qū)域的基因變異和其與生物表型之間的關(guān)系。(2)建立一種快速基因分型方法,對(duì)廣西 HIV-1 重組流行株進(jìn)行亞型鑒定。方法:(1)應(yīng)用 nest-PCR 對(duì) 50 份來(lái)自廣西主要流行區(qū)的 HIV-1 毒株 env 和 gag 基因區(qū)進(jìn)行擴(kuò)增后,使用 ABI 310型測(cè)序儀測(cè)序,然后應(yīng)用 CLUSTAL、MEGA 和 dnatools 等生物學(xué)軟件對(duì) env基因區(qū) V3-V4 區(qū)及其臨近區(qū)域進(jìn)行系統(tǒng)樹(shù)和氨基酸分析。(2)從 50 份 HIV陽(yáng)性樣品中提取核酸,使用 HIV-1 M 組通用引物對(duì) env 和 gag 基因區(qū)進(jìn)行第一輪擴(kuò)增,第二輪使用分別檢測(cè) C、CRF01-AE 亞型的二套特異性引物放入同一反應(yīng)管中進(jìn)行擴(kuò)增,根據(jù)不同亞型擴(kuò)增的目的條帶位置不同來(lái)判斷亞型;另外對(duì) gag 基因區(qū)設(shè)計(jì)了一套引物,專(zhuān)用于檢測(cè) B′/C;同時(shí)使用通用的內(nèi)側(cè)引物進(jìn)行擴(kuò)增,以驗(yàn)證特異性引物是否正確,擴(kuò)增出的所有樣品均進(jìn)行基因測(cè)序和系統(tǒng)樹(shù)分析以驗(yàn)證結(jié)果。結(jié)果:(1)從 50 份毒株中獲得 46 份基因序列,3 份為 CRF08-BC(6.52%),43 份為 CRF01-AE(93.48%);系統(tǒng)樹(shù)分析顯示,3 份 CRF08-BC 中 2 份與 CRF08-BC.98CN006 和 CRF08-BC.97CNGX6f靠近,還有 1 份樣品則接近 C.95IN21068;43 份 CRF01-AE 重組毒株中 41 份與分離于廣西地區(qū)的 CRF01-AE.97CNGX2f 和泰國(guó)代表毒株 THCM240 接近,另外 2 份與 90CF402 聚成一簇;24 份毒株 env V3-V4 區(qū)核苷酸序列分析顯示,廣西的樣本與分離于該地區(qū)的代表毒株基因距離較小,CRF08-BC 和CRF01-AE 重組毒株的氨基酸替換主要發(fā)生在 C3 和 V4 區(qū),而 V3 區(qū)氨基酸序列相對(duì)保守;46 份毒株 V3 區(qū)及臨近基因區(qū)氨基酸序列分析顯示,CRF08-BC毒株 V3 頂端四肽為 GPGQ,而 CRF01-AE 毒株則存在著 7 種類(lèi)型:GPGQ
[Abstract]:Objective: (1) to study the gene variation of the V3-V4 region of the env gene and its adjacent region of Guangxi HIV-1 recombinant epidemic strain and its relationship with the biological phenotype. (2) to establish a rapid genotyping method to identify the subtype of Guangxi HIV-1 recombinant epidemic strain. Methods: (1) 50 HIV-1 strains from Guangxi were amplified by nest-PCR and sequenced by ABI 310 sequencer. Then the systematic tree and amino acid analysis of the V3-V4 region of the env gene region and its adjacent region were carried out by using CLUSTALMEGA and dnatools. (2) the nucleic acid was extracted from 50 HIV positive samples. HIV-1 M universal primers were used to amplify the gene region of env and gag in the first round, and two sets of specific primers to detect the subtype of CRF01-AE in the same reaction tube were used in the second round. The subtype was judged according to the location of the target bands of different subtypes; a set of primers was designed for detecting BU / C in the gag gene region; and a common inner primer was used to verify whether the specific primers were correct. All the amplified samples were sequenced and phylogenetic tree analysis to verify the results. Results: (1) 46 strains of CRF08-BC were obtained from 50 strains, 3 of them were CRF08-BC (6.52%), 43 of them were CRF01-AE (93.48%), and 2 of them were close to CRF08-BC.98CN006 and CRF08-BC.97CNGX6f by phylogenetic tree analysis. One sample was close to that of C. 95IN21068C 43 CRF01-AE recombinant strains, 41 of which were close to CRF01-AE.97CNGX2f isolated from Guangxi and THCM240 from Thailand, and the other 2 were clustered with 90CF402 to form a cluster of 24 strains of env V3-V4, and the nucleotide sequence analysis showed that The amino acid substitution between the samples of Guangxi and the representative strains of CRF08-BC and CRF01-AE occurred mainly in the C3 and V4 regions. The amino acid sequence analysis of V3 region and its adjacent gene region in 46 strains of V3 region showed that the tetrapeptide at the top of V3 of CRF08-BC strain was GPGQ.There were 7 types of TGQs in CRF01-AE strain.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 譚建新;HIV-1B'亞型毒株全基因組分子克隆[D];河北大學(xué);2007年

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本文編號(hào)：2158681