【作者】 楼朝平；

【导师】 孔维；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 Cul5蛋白作为E3复合物的一个支架蛋白,全长为784个氨基酸。在体外表达Cul5全长蛋白比较困难,我们通过PCR的方法获得Cul5蛋白N末端138个氨基酸的表达基因,克隆至pRSETB表达载体中,进行融合表达,获得His-融合的Cul5蛋白,经Ni-NTA亲和纯化树脂纯化获得高纯度的Cul5可溶性蛋白,SDS-PAGE鉴定蛋白的分子量为18KD左右。以纯化的Cul5蛋白作为抗原免疫雌性Balb/c小鼠,小鼠经鉴定产生抗体后,取脾细胞与处在对数生长期的SP2/0骨髓瘤细胞在浓度为50%的聚乙烯二醇(PEG)的作用下融合,经HAT培养基选择性培养,间接ELISA法筛选阳性杂交瘤,获得了4株特异性分泌抗Cul5蛋白单克隆抗体的杂交瘤细胞。通过初步纯化杂交瘤细胞上清,得到高效价的抗Cul5蛋白单克隆抗体。更多还原

【Abstract】 HIV-1 Vif is an important accessory protein that is crucial for viral replication and viral infectivity. Vif is necessary for efficient package of infectious viral particles and can increase the infectivity of the virions by 10-1000 times. Researches found that HIV Vif-deficient can survive in nonpermissive cells such as human lympholeukocyte, macrophage, HUT78 cell line and T leucoma cell line (CEM), but can not produce infectious viral particles. While in permissive cells, such like SupT1, CEM SS 293T, HeLa CD4、COS7, they can both replicate and produce the infectious particles. Through followed researches, people found there is a host antiviral factor APOBEC 3G, a human cytidine deaminase in those nonpermissive cells, which can distinctly degrade the viral infectivity. However, this function can be suppressed by HIV-1 Vif.As an antivirus factor in the human body, APOBEC3G’s ability can be antagonized by the protein HIV-1 Vif.Vif binds EloC through BCBox motif, and then the Zinc finger at the N terminal of Vif binds the repeat sequence at the N terminal of Cullin5 through hydrophobic interaction, and finally inhibit APOBEC3G’s antivirus activity by degradation through the ubiquitin pathway.HIV-1 Vif is an essential co-protein for HIV-1 to replicate in vivo and to keep its pathogenesis. Initially, its gene was found as an ORF formed after multiple cuts of HIV-1 mRNA. Once the protein was called orfA or sor, then finally named Vif (virus infectivity factor) according to its property. It is a 192-aa coded alkaline protein (MW 23kDa). HIV-1 Vif has the key function during the process of producing infective viruses, by increasing the infectivity 10~1,000 times with its present. Vif can bind with APOBEC3G in the cell, preventing the recruit of APOBEC3G, therefore the cytosine in filial minus DNA will not be deaminated and the cDNA produced afterwards will not be degraded. In one word, the infectivity of viruses will not be affected by APOBEC3G. Now research has proved that Vif antagonizes APOBEC3G through irreversible binding of these two proteins and rapid degradation afterwards. Further research shows, Vif binds EloC through BCBox motif, and then the Zinc finger at the N terminal of Vif binds the repeat sequence at the N terminal of Cul5 through hydrophobic interaction, and finally inhibit APOBEC3G’s antivirus activity by degradation through the ubiquitin pathway.Our main work is: obtaining the expression gene with 138 amino acids at N terminal of Cul5 protein by PCR method, constructing the gene to pRSETB prokaryotic expression vector, inducing the expression by IPTG, getting the homogenous Cul5 fusion protein by His purification and identifying the 18KD protein by SDS-PAGE.We immunized female Balb/c mice with the purified Cul5 protein. The dosage is 100ug/indivadule/time, the intermission of immunization is two weeks. After several immunizations, the mice were identified to have produced antibody. We increase the dose of protein to 500ug to reinforce the effect of immunization 3 days before cell fusion. Obtain the spleen cells and fusion them with SP2/0 myeloma cells at log phage with PEG (concentration of 50%).Monoantibody is a shaping cellular experimental technology, however, still with many difficulties. We did serial of conditional optimization before cell fusion: 1. Screen the myeloma cells by 8-azaguanine and obtain round and full cells. 2. Optimize the quantity of cells with some regulation and the final cell density is 104. 3. Optimize the fusion condition. Generally, while cell fusion, 0.7ml~PEG should be dropwised in 1minites. Dropwise PEG of 0.7ml~1ml within 30seconds, effect with 30~90min, and then the fusion effect can be increased by about 20%. After condition optimization, the masculine rate of cell fusion is 14%. After the selective culture with HAT medium, we screen the masculine myeloma by indirect ELISA method and obtain 4 stocks of fusion cells which can express the monoantibody of Cullin5 protein with specifity. The antibody titer of cell supernatant is 1:100. After identification of monoantibody typing reagent, the 4 stocks are all IgG1 subgroup.更多还原