猪圆环病毒3型Cap蛋白在毕赤酵母中的优化表达  

Optimized expression of porcine circovirus type 3 Cap protein in Pichia pastoris

作　　者：李丁 李炳坤 武奇 张雪花[1,2,3] 梅梅 LI Ding;LI Bingkun;WU Qi;ZHANG Xuehua;MEI Mei(Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose,Yangzhou University,Yangzhou 225009,China;Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,Nanjing 210014,China;College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所,江苏南京210014 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [3]江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地,江苏南京210014 [4]江西农业大学生物科学与工程学院,江西南昌330045

出　　处：《畜牧与兽医》2022年第12期80-86,共7页Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年基金项目(32002316);江苏省自然科学基金青年基金项目(BK20220745)。

摘　　要：以猪圆环病毒3型(porcine circovirus type 3,PCV3)Cap蛋白为模式抗原,探索病毒衣壳蛋白在毕赤酵母中高效表达的方法。体外构建多拷贝毕赤酵母表达载体,转入GS115感受态细胞后获得含有1、2、4、8拷贝目的基因的毕赤酵母菌株。对上述酵母菌株进行摇瓶发酵,利用SDS-PAGE、Western blot和灰度分析等方法检测目的蛋白表达情况。消除GS-8CAP菌株博来霉素(Zeocin)抗性后获得抗生素敏感型菌株(GS-8CAP-ZeoS),向该菌株中继续转入8拷贝酵母表达质粒从而获得GS-16CAP菌株,评估GS-16CAP的目的蛋白产量。结果显示:GS-8CAP菌株目的蛋白产量与GS-1CAP相比提高487.6%;GS-16CAP菌株目的蛋白产量是GS-8CAP的1.64倍,是GS-1CAP的9.47倍;通过与梯度稀释的牛血清白蛋白(BSA)进行蛋白条带比较,评估GS-16CAP菌株目的蛋白产量约为113 mg/L。研究表明,多拷贝技术能够有效提高Cap蛋白在毕赤酵母中的产量。本研究将为毕赤酵母高效表达其他病毒衣壳蛋白提供重要参考,并为PCV3亚单位疫苗的开发奠定基础。This study was to establish an efficient method of expressing virus capsid protein in yeast. Porcine circovirus type 3(PCV3) Cap protein was heterologously expressed in Pichia pastoris. Multi-copy expression vectors were constructed in vitro and transferred into GS115 competent cells to obtain yeast strains containing 1, 2, 4 and 8 copies of the target genes. The recombinant yeast strains were fermented in a shaking flasks, and the target protein was identified by SDS-PAGE, Western blot and gray-scale analyses. Methanol-induced resistance elimination experiment was performed, and linearized pMCO-AOX-8 CAP was transferred to the Zeocin-sensitive strain GS-8 CAP-ZeoSto obtain a GS-16 CAP strain. The target protein yield of GS-16 CAP was finally evaluated. The results showed that the target protein yield of the GS-8 CAP strain was increased by 487.6%, compared with that of GS-1 CAP. The target protein yield of the GS-16 CAP strain was 1.64 times that of GS-8 CAP and 9.47 times that of GS-1 CAP. By comparing the protein bands with serially diluted BSA, the yield of the target protein in the GS-16 CAP strain was estimated to be 113 mg/L. In conclusion, the multi-copy technology effectively improved the production of PCV3 Cap in Pichia pastoris. This study provided new essential information for other virus capsid proteins to be expressed in yeast.

关 键 词：PCV3 Cap 毕赤酵母 重组蛋白产量 多拷贝 

分 类 号：S858.2[农业科学—临床兽医学]