广西HIV-1重组流行株基因变异的研究和快速基因分型方法的建立
发布时间：2018-08-02 07:41
【摘要】：目的:(1)研究广西 HIV-1 重组流行株亚型 env 基因 V3-V4 区及其临近区域的基因变异和其与生物表型之间的关系。(2)建立一种快速基因分型方法,对广西 HIV-1 重组流行株进行亚型鉴定。方法:(1)应用 nest-PCR 对 50 份来自广西主要流行区的 HIV-1 毒株 env 和 gag 基因区进行扩增后,使用 ABI 310型测序仪测序,然后应用 CLUSTAL、MEGA 和 dnatools 等生物学软件对 env基因区 V3-V4 区及其临近区域进行系统树和氨基酸分析。(2)从 50 份 HIV阳性样品中提取核酸,使用 HIV-1 M 组通用引物对 env 和 gag 基因区进行第一轮扩增,第二轮使用分别检测 C、CRF01-AE 亚型的二套特异性引物放入同一反应管中进行扩增,根据不同亚型扩增的目的条带位置不同来判断亚型;另外对 gag 基因区设计了一套引物,专用于检测 B′/C;同时使用通用的内侧引物进行扩增,以验证特异性引物是否正确,扩增出的所有样品均进行基因测序和系统树分析以验证结果。结果:(1)从 50 份毒株中获得 46 份基因序列,3 份为 CRF08-BC(6.52%),43 份为 CRF01-AE(93.48%);系统树分析显示,3 份 CRF08-BC 中 2 份与 CRF08-BC.98CN006 和 CRF08-BC.97CNGX6f靠近,还有 1 份样品则接近 C.95IN21068;43 份 CRF01-AE 重组毒株中 41 份与分离于广西地区的 CRF01-AE.97CNGX2f 和泰国代表毒株 THCM240 接近,另外 2 份与 90CF402 聚成一簇;24 份毒株 env V3-V4 区核苷酸序列分析显示,广西的样本与分离于该地区的代表毒株基因距离较小,CRF08-BC 和CRF01-AE 重组毒株的氨基酸替换主要发生在 C3 和 V4 区,而 V3 区氨基酸序列相对保守;46 份毒株 V3 区及临近基因区氨基酸序列分析显示,CRF08-BC毒株 V3 顶端四肽为 GPGQ,而 CRF01-AE 毒株则存在着 7 种类型: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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
本文编号：2158681
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346
【引证文献】
相关硕士学位论文 前1条
1 谭建新;HIV-1B'亚型毒株全基因组分子克隆[D];河北大学;2007年
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