NUCLEOTIDE SEQUENCE OF UGANDAN HIV ISOLATES

描述：（改编自申请人摘要）研究者建议 采用聚合酶链反应（PCR）、DNA克隆和测序 技术，试图确定编码HIV基因组的多样性， 流行地区约300株代表性分离物中的V3环 乌干达 从艾滋病患者获得的受感染单核细胞的裂解物 在地理上不同的地区将被用作病毒的来源， 分离株 包含核苷酸残基6597-6955的HIV基因组区段 其编码整个V3环将通过PCR使用引物#1扩增 （5 '-CGCTGCAGAAGAAGAGGTAGTAATTAGAT）和引物#2 （5'-CCCTGCAGTAGAAAAATTCCCCTCTACAA）。 扩增的DNA将 从5%-10%聚丙烯酰胺梯度凝胶上电洗脱，然后亚克隆 插入pGEM-蓝色质粒的pst I位点。 插件将在 用双脱氧核苷酸链法进行测序 终止 从乌干达分离株获得的序列数据将在 并与美国标准菌株进行比较 目前用于疫苗设计和开发。

DESCRIPTION: (Adapted from applicant's abstract) The investigator proposes to employ polymerase chain reaction (PCR), DNA cloning and sequencing techniques to attempt to determine diversity in the HIV genome encoding the V3 loop in about 300 representative isolates from endemic localities in Uganda. Lysates of infected mononuclear cells obtained from AIDS patients in geographically distinct regions will be used as the source of viral isolates. The HIV genome segment comprising nucleotide residues 6597-6955 which encode the entire V3 loop will be amplified by PCR using primer #1 (5'-CGCTGCAGAAGAAGAGGTAGTAATTAGAT) and primer #2 (5'-CCCTGCAGTAGAAAAATTCCCCTCTACAA). The amplified DNA will be electroeluted from 5%-10% polyacrylamide gradient gels and then subcloned into the pst I site in pGEM-blue plasmids. The inserts will be expanded in Escherichia coli and then sequenced by means of dideoxynucleotide chain termination. The sequence data obtained from the Ugandan isolates will be compared with each other and with those of standard American strains currently in use for vaccine design and development.