hASB-8基因是对肿瘤细胞生长具有明显抑制作用的人类新基因。其编码蛋白属于人ASB蛋白家族中的一个成员，与小鼠中的ASB-8蛋白同源性达96%。保守结构域分析显示hASB-8在N端包含4个Ankyfin re-peats，在C端包含了一个SOCS box。利用酵母双杂交技术，筛选了人的胎盘（Placenta）cDNA 文库，获得了与KASB-8相互作用的2个蛋白，Elongin C和CDK4 binding protein；并在二倍体酵母体内进行了验证。这些试验提示hASB-8蛋白可能介导肿瘤细胞中靶蛋白和泛素复合体之间的相互作用，并与肿瘤细胞靶蛋白转录调节有关。

利用同源重组的方法，构建了具有高拷贝数和高稳定性的新型酿酒酵母表达质粒。对酵母附加体型载体pHC11进行一系列改造，得到质粒pHC11R，并用适当的酶切线性化；利用PCR反应得到人IFNa2b表达单元片段，它的两端和线性化的pHC11R的两端具有50bp左右的同源区段。上述两个片段共转化酿酒酵母，在酵母体内经同源重组后得到表达质粒pHC11R-IFNa2b，该表达质粒与pHC11-IFNoa2b相比去除了质粒上用于在大肠杆菌中复制和筛选所需的序列，在宿主菌中的稳定性和拷贝数均有明显提高，同时外源基因的表达量也获得了一定程度的提高。在此基础上，进一步构建了带有不同表达元件的pHR系列载体，使得外源基因能够通过同源重组在酿酒酵母中快速、方便地克隆和表达。

CDC16Hs是细胞周期末期促进复合物（APC）的亚基。利用基于LexA的酵母双杂交系统，把它作为诱饵蛋白筛选人胎脑文库，发现它与DNA双链断端修复蛋白Ku80的羧基端相互作用。CDC16Hs和全长Ku80的结合通过pull down实验在体外得到验证。

将编码人的血管生成素样蛋白4（ANGPTL4）的cDNA克隆至分泌表达载体pPIC9k上，在Pichia Pas-toris酵母宿主菌SMD1168中获得分泌表达；用MTT方法检测的细胞生长抑制实验结果表明，发酵液中分泌表达的重组蛋白ANGPTL4对人脐静脉内皮细胞的生长具有一定的抑制作用。

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n=1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n=1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex 75 gel filtration and analyzed by SDSPAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1 (n=1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex™ 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n=1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.