NUCLEOTIDE SEQUENCE OF UGANDAN HIV ISOLATES

描述：(改编自申请人的摘要)调查员提出 采用聚合酶链式反应(PCR)、DNA克隆和测序 尝试确定HIV基因组编码的多样性的技术 V3在中国流行地区约300株有代表性的分离株中循环 乌干达。艾滋病患者感染单个核细胞的裂解物 在地理上不同的地区将被用作病毒的来源 分离株。由6597-6955核苷酸残基组成的HIV基因组片段 哪个编码整个V3环的片段将通过使用1号引物的聚合酶链式反应进行扩增 (5‘-CGCTGCAGAAGAAGAGGTAGTAATTAGAT)和引物#2 (5‘-CCCTGCAGTAGAAAAATTCCCCTCTACAA)。扩增出的DNA将被 从5%-10%的聚丙烯酰胺梯度凝胶中电洗脱，然后亚克隆 进入pGEM-蓝色质粒中的Pst I位点。插入物将在 用双脱氧核苷酸链法进行序列测定 终止。从乌干达分离株获得的序列数据将是 相互比较以及与标准美国品系的比较 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.