STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL CHARACTERIZATION OF HIV NEF AND VPU

相对较高的HIV-1和HIV-2阴性效应因子水平 (NEF)蛋白在昆虫细胞系SF-9中表达，并具有 杆状病毒载体。两种形式的杆状病毒产生HIV-1 Nef 通过亲和纯化，蛋白质纯度达到95%以上。 用Nef单抗(S-897-55S)柱层析。一个 细菌产生的HIV-2Nef蛋白的产量也相对较高 水平，并通过凝胶过滤和离子交换纯化到接近均匀的水平 层析法。研制了一种高效价兔抗血清。 全长HIV-2Nef蛋白，并用于鉴定23kD的Nef蛋白 用放射免疫沉淀法(RIP)检测HIV-2(NIHZ)感染的H9细胞。 HIV-1Nef单抗S-897-55S和HIV-2Nef兔 用抗血清研究Nef蛋白的体外齐聚反应。 此外，HIV-1杆状病毒和HIV-2细菌Nef蛋白 证明自身磷酸化活性，是有效的底物 体外检测蛋白激酶C活性。 我们还发现HTLV-IIIB感染的H9细胞株表达两个 不同种类的Nef蛋白(p25和p27)，由两个 不同的nef基因在不同的HIV-1基因变异体中表达。在……里面 此外，我们正在使用细菌表达的HIV-1 VPU蛋白来研究 其结合钙离子和/或结合钙通道激活剂的潜能 或抑制剂。

Relatively high levels of the HIV-1 and HIV-2-negative effector factor (Nef) proteins were expressed in the insect cell line, Sf-9, with a baculovirus vector. Two forms of the baculovirus produced HIV-1 Nef proteins were purified to greater than 95% homogeneity by affinity chromatography on a Nef monoclonal antibody (S-897-55S) column. A bacterially-produced HIV-2 Nef protein was also produced at relatively high levels and purified to near homogeneity by gel filtration and ion exchange chromatography. A high-titered rabbit antiserum was developed against the full-length HIV-2 Nef protein, and was used to identify a 23kD Nef protein in HIV-2 (NIHZ)-infected H9 cells by radioimmunoprecipitation (RIP) assay. Both the HIV-1 Nef monoclonal antibody S-897-55S and the HIV-2 Nef rabbit anti-serum were used to study the in vitro oligomerization of Nef proteins. In addition, both the HIV-1 baculovirus and HIV-2 bacterial Nef proteins demonstrate autophosphorylation activities and are efficient substrates in vitro for protein kinase C activity. We have also shown that the HTLV-IIIB-infected H9 cell line expresses two distinct species of Nef proteins (p25 and p27), which are coded by two different nef genes expressed in different genetic variants of HIV-1. In addition, we are using a bacterially-expressed HIV-1 vpu protein to study its potential for binding calcium and/or binding calcium channel activator or inhibitor agents.