EXPRESSION OF HIV-2 ENV GENE PRODUCTS IN E.COLI

HIV-2 ENV 基因产物在大肠杆菌中的表达

• 批准号: 3896378
• 负责人: T S PAPAS
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3896378
• 关键词: AIDS /HIV diagnosis; AIDS /HIV test; HIV infections; antiviral antibody; chimeric proteins; gene deletion mutation; gene expression; genetic manipulation; genetic promoter element; genetic transcription; genetic translation; genome; glycoprotein biosynthesis; human immunodeficiency virus 1; human immunodeficiency virus 2; membrane proteins; serotyping; transfection; transposon /insertion element; viral vaccines; virus antigen; virus classification; virus envelope; virus genetics; virus protein; western blottings

Human immunodeficiency virus, type-2 (HIV-2), the West African counterpart of the acquired immunodeficiency syndrome (AIDS) virus, is related to, but is quite distinct from HIV-1. The HIV-2 NIH-Z genome is approximately 9.4 kb long with an env gene of about 2.7 kb which codes for an envelope protein of 856 amino acids. The gpl6O envelope protein is matured into a 120 Kd exterior glycoprotein (gpl2O) and a transmembrane protein of 35 Kd (gp35). The gpl2O and gp35 envelope proteins of HIV-2 NIH-Z show about 32% and 39% homology, respectively, with the human T-lymphotropic virus, type-IIIB (HTLV-IIIB) strain of HIV-1. The chief objectives of this project are (1) to develop a diagnostic antigen for accurately detecting antibodies to HIV-2 in all infected human sera and (2) to use the bacterially-expressed HIV-2 env gene products in some vaccine applications. We have expressed the env gene of the NIH-Z isolate of HIV-2 as five overlapping fragments in E. coli. The env open reading frames (ORFS) were initially expressed as tripartite fusions in between the 12 amino-terminal codons of the lambda cII gene and the lacZ (Beta-galactosidase) gene of E. coli that are under the transcriptional control of the lambda PL promoter of the expression plasmid pWS50. The 0-galactosidase portion of the fusion protein was then deleted at the DNA level by generating in-frame translational stops or translational frame-shift mutations. One of the ORFs spanning the amino acid residues 536 through 705 of the HIV-2 env gene directed the synthesis of a 20 Kd protein that was serologically specific for identifying antibodies to HIV-2. This antigenic protein is produced at levels approximately 5% of the total cellular proteins. -No significant immunological cross-reactivity was observed with HIV-1-positive sera or with normal control sera in immmunoblot assays with a crude preparation of this protein. Work is now in progress on the immunological characterization of the gene products of the remaining four HIV-2 env ORFS.

2型人体免疫机能丧失病毒（HIV-2），西非对应物 获得性免疫缺陷综合征（艾滋病）病毒，是有关，但 与HIV-1截然不同。HIV-2 NIH-Z基因组约为9.4 7 kb长，编码包膜的env基因约为2.7kb 蛋白质由856个氨基酸组成。gp 160包膜蛋白被成熟为 120 Kd的外部糖蛋白（gp 120）和35 Kd的跨膜蛋白 （gp35）。HIV-2NIH-Z的gp 120和gp 35包膜蛋白显示约32%的 与人嗜T淋巴细胞病毒的同源性分别为39%， HIV-1的IIIB型（HTLV-IIIB）毒株。该项目的主要目标 是（1）开发用于准确检测抗体的诊断抗原 在所有感染的人血清中检测HIV-2，以及（2）使用 在某些疫苗应用中，细菌表达的HIV-2 env基因产物。 我们表达了HIV-2的NIH-Z分离株的env基因， 重叠片段E.杆菌env开放阅读框（ORFs）为 最初表达为12个氨基末端之间的三重融合体， 大肠杆菌λ cII基因和lacZ（β-半乳糖苷酶）基因的密码子。 在λ PL启动子的转录控制下， 表达质粒pWS 50的构建。融合的0-半乳糖苷酶部分 然后在DNA水平上通过产生符合读框的 翻译停止或翻译移码突变。其中一个ORF 跨越HIV-2 env基因的氨基酸残基536至705 指导合成了一个20 Kd的蛋白质， 用于鉴定HIV-2抗体。这种抗原性蛋白质是在 约占总细胞蛋白质的5%。- 无重大影响 观察到与HIV-1阳性血清或 在免疫印迹试验中，用正常对照血清， 这种蛋白质。目前正在进行免疫学表征的工作， 其余四个HIV-2 env ORFs的基因产物。