mB7AP-exCD40L融合蛋白的分子设计及其在Pichia pastoris中的表达和生物活性  

Molecular Designing, Expression in Pichia pastoris and Bioactivities of mB7AP-exCD40L Fusion Protein

作　　者：陈晋[1] 储以微[1] 邵先安[1] 任学芳[1] 张洪勇[1] 高海峰[1] 何球藻[1] 熊思东[1] 

机构地区：[1]复旦大学上海医学院免疫学系-教育部分子医学重点实验室-上海基因免疫与疫苗研究中心,上海200032

出　　处：《复旦学报（医学版）》2005年第4期401-406,F003,共7页Fudan University Journal of Medical Sciences

基　　金：上海科技发展基金(03JC14085);国家重点基础研究发展计划(2001CB510005);上海市科委优秀学科带头人计划(04XD14003)资助

摘　　要：目的小鼠B7反义肽与小鼠CD40L胞外区融合蛋白(mB7AP-exCD40L)的分子模拟设计及其酵母表达体系的建立,研究其酵母表达产物的生物活性。方法分子模拟设计mB7AP-exCD40L;构建pPIC9K-mB7AP-exCD40L质粒并用电击法转化PichiapastorisGS115。Westernblot鉴定融合蛋白的表达,混合淋巴细胞反应(MLR)研究mB7AP-exCD40L对淋巴细胞增殖的影响。结果构建的重组Pichiapastoris实现了mB7AP-exCD40L的分泌表达,表达蛋白质的相对分子质量约2.6×103,对MLR具有抑制作用。结论分子模拟设计的mB7AP-exCD40L在Pichiapastoris表达体系成功表达,为进一步研究mB7AP-exCD40L在抗移植排斥反应中的作用奠定了基础。Purpose To design the mB7AP-exCD40L fusion protein and express it by the yeast expression system,and then study the bioactivities of the fusion protein. Methods Insight Ⅱ software package was used to design the mB7AP-exCD40L fusion molecule.The cDNA encoding the murine extra-cellular portion of CD40L was amplified by RT-PCR from murine splenocytes and cloned into T-vector (Pmd-18-T Vector),and then subcloned into the Pichia pastoris expression vector pPIC9K.The Pichia pastoris expression vector encoding exCD40L and pPIC9K-exCD40L was successfully constructed.Meanwhile,a B7AP-linker sequence of 82 bp was synthesized.The pPIC9K-exCD40L was digested by EcoR Ⅰ and ligated to the mB7AP-linker gene to construct pPIC9K-mB7AP-exCD40L expression vector.After the pPIC9K or pPIC9K-mB7AP-exCD40L was transfected into Pichia pastoris GS115,the mB7AP-exCD40L fusion protein was analyzed by Western blot.The mixed lymphocyte reaction (MLR) was performed to evaluate the bioactivities of the mB7AP-exCD40L fusion protein. Results Recombinant Pichia pastoris expression vectors encoding mB7AP-exCD40L fusion protein were constructed correctly.The mB7AP-exCD40L fusion protein secreted from the transfected yeast cells was also found in the supernatants and able to inhibit the lymphocytes-proliferation of MLR. Conclusions The mB7AP-exCD40L fusion protein was successfully expressed by Pichia pastoris expression system and can inhibit the MLR,it may be applied in the field of transplantation.

关 键 词：PASTORIS 融合蛋白 生物活性 分子设计 混合淋巴细胞反应 重组Pichia Western 抗移植排斥反应 酵母表达体系 分子模拟 淋巴细胞增殖 相对分子质量 CD40L B7反义肽 GS115 表达产物 blot 分泌表达 抑制作用 胞外区 电击法 

分 类 号：R392.2[医药卫生—免疫学] R392.11[医药卫生—基础医学]