密码子优化人PD-L1基因在巴斯德毕赤酵母中的表达与鉴定  

EXPRESSION AND IDENTIFICATION OF THE HUMAN PD-L 1 GENE WITH THE OPTIMIZED CODON IN PICHIA PASTORIS

作　　者：战映璇 王环[2] 汤潜[2] 玄超[2] 于建立 张霞 王清[2] ZHAN Yingxuan;WANG Huan;TANG Qian;XUAN Chao;YU Jianli;ZHANG Xia;WANG Qing(Qingdao University Faculty of Medicine,Qingdao 266071,China)

机构地区：[1]青岛大学医学部,山东青岛266071 [2]青岛大学附属医院检验科 [3]中国科学院青岛生物能源与过程研究所 [4]深圳市国创纳米抗体技术有限公司

出　　处：《精准医学杂志》2022年第4期342-346,共5页Journal of Precision Medicine

基　　金：山东省重点研发计划(2019GSF108108)。

摘　　要：目的利用巴斯德毕赤酵母获得具有可溶性、免疫原性的重组PD-L1蛋白。方法根据巴斯德毕赤酵母表达系统的密码子使用偏好,优化人PD-L1基因序列,将优化后的序列构建至质粒pPIC9K,合成重组质粒pPIC9K-PDL1。使用SacⅠ-HF将pPIC9K-PDL1线性化,然后通过电击法将pPIC9K-PDL1上的PD-L1基因序列整合至巴斯德毕赤酵母基因组中,依次使用MD平板(无组氨酸)和遗传霉素G418从电击处理过的菌液中筛选重组巴斯德毕赤酵母菌株,然后使用PCR和测序对筛选到的菌株进行鉴定。利用甲醇体积分数为0.005的BMMY培养基诱导重组巴斯德毕赤酵母菌株表达,使用免疫印迹法检测菌株培养基上清液中的表达产物。结果筛选得到了稳定表达PD-L1蛋白的重组巴斯德毕赤酵母菌株,表达的PD-L1相对分子质量约为44700,可以与抗PD-L1抗体特异性结合。结论成功构建了稳定表达PD-L1蛋白的重组巴斯德毕赤酵母菌株,菌株表达的PD-L1蛋白可用于开发血清PD-L1检测试剂盒。Objective To obtain the soluble and immunogenic recombinant PD-L1 protein with Pichia pastoris.Me-thods The human PD-L 1 gene sequence was optimized based on codon usage preference in the expression system of Pichia pastoris,and the optimized sequence was synthesized and ligated into pPIC9K plasmid to obtain the recombinant plasmid pPIC9K-PDL1.SacⅠ-HF was used to linearize pPIC9K-PDL1,and then the PD-L 1 gene sequence on pPIC9K-PDL1 was integrated into the genome of Pichia pastoris by electroporation.MD plates(without histidine)and geneticin G418 were used to screen out recombinant Pichia pastoris strains from the bacterial solution treated by electroporation,and then PCR and sequencing were used for the identification of the strains screened out.The expression of recombinant Pichia pastoris strains was induced by BMMY medium containing methanol at a volume fraction of 0.005,and Western blotting was used to measure the expression products in medium supernatant.Results A recombinant Pichia pastoris strain stably expressing PD-L1 protein was screened out,and the PD-L1 protein expressed had a relative molecular weight of about 44700 and could specifically bind to anti-PD-L1 antibody.Conclusion This study successfully constructs a recombinant Pichia pastoris strain stably expressing PD-L1 protein,and PD-L1 protein expressed by the strain can be used to develop serum PD-L1 diagnostic kits.

关 键 词：毕赤酵母 质粒构建 重组蛋白质类 程序性死亡配体-1 免疫印迹法 试剂盒 诊断 

分 类 号：R394.3[医药卫生—医学遗传学] R392.116[医药卫生—基础医学]