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目的 :利用毕赤酵母表达系统表达牛O型口蹄疫外壳蛋白(FMDVVP1) ,并对表达的蛋白进行免疫原性鉴定。方法 :将FMDVvp1基因克隆到毕赤酵母Pichiapastoris分泌性表达载体pSuperY中 ,构建重组表达载体pSuperY/vp1,经测序证明vp1基因序列的正确性。将纯化的重组质粒经线性化酶切后 ,用电转化法将pSuperY/vp1导入毕赤酵母菌种SMD116 8H中。对表达产物用SDS PAGE和Westernblot进行分析 ,并用酵母表达的FMDVVP1蛋白免疫小鼠。结果 :以重组质粒pSuperY/vp1转化毕赤酵母菌后 ,能表达相对分子量 (Mr)为 6 6 0 0 0和4 30 0 0的FMDVVP1蛋白。动物免疫结果表明 ,FMDVVP1蛋白能诱导小鼠产生特异性的体液和细胞免疫应答。结论 :在毕赤酵母中成功地表达FMDVVP1蛋白 ,为研制新型FMDVVP1的基因工程疫苗奠定了基础
Objective: To express Bovine O-type Foot-and-Mouth Disease Virus (FMDVVP1) by using Pichia pastoris expression system and to identify the expressed protein. Methods: The FMDVvp1 gene was cloned into Pichia pastoris secretive expression vector pSuperY and the recombinant expression vector pSuperY / vp1 was constructed. The correctness of the sequence of vp1 gene was verified by sequencing. After the purified recombinant plasmid was linearized, pSuperY / vp1 was introduced into Pichia pastoris SMD116 8H by electroporation. The expressed product was analyzed by SDS PAGE and Western blot, and the mice were immunized with the yeast-expressed FMDVVP1 protein. Results: The recombinant plasmid pSuperY / vp1 was transformed into Pichia pastoris and expressed FMDVVP1 protein with relative molecular weights (Mr) of 60,000 and 40,000. Animal immunization results show that FMDVVP1 protein can induce specific humoral and cellular immune responses in mice. Conclusions: The successful expression of FMDVVP1 protein in Pichia pastoris laid the foundation for the development of a novel FMDVVP1 genetically engineered vaccine