密码子优化和信号肽对猪圆环病毒ORF2基因在毕赤酵母表达中的影响  

Impact of codon optimization and signal peptide on the expression of PCV2 ORF2 gene in Pichia pastoris

作　　者：张春玲[1] 王瑞阳 钱忠辉[1] 赵本进[1] 张俊平[1] 葛宇燕 张子悦 丁卫星[1] ZHANG Chunling;WANG Ruiyang;QIAN Zhonghui;ZHAO Benjin;ZHANG Junping;GE Yuyan;ZHANG Ziyue;DING Weixing(Institute of Animal Husbandry&Veterinary Science,Shanghai Academy of Agricultural Sciences,Shanghai 201106,China)

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《上海农业学报》2024年第4期81-85,共5页Acta Agriculturae Shanghai

基　　金：上海市科技兴农项目(2022-02-08-00-12-F01454)。

摘　　要：为了在毕赤酵母中高效表达PCV2ORF2基因,对PCV2ORF2基因编码的187个氨基酸密码子进行毕赤酵母偏爱性优化,利用两步PCR全化学合成优化后的ORF2基因。将优化前后的PCV2ORF2基因片段插入毕赤酵母表达载体pPICZα-C,构建重组质粒pPICZα-C-ORF2和p PICZα-C-ORF2op。将线性化的重组质粒电转化毕赤酵母GS115,并筛选Zeocin抗性重组子。同时,也研究了信号肽对PCV2ORF2表达效率的影响。SDS-PAGE和Western-blot分析结果表明,只有经密码子优化且去除ORF2基因自身信号肽的重组子能够分泌表达PCV2 ORF2蛋白,目的蛋白分子质量约为30 ku,表达的重组ORF2蛋白可被PCV2阳性血清识别,具有生物学活性。In order to efficiently express porcine circovirus 2(PCV2)ORF2 gene in Pichia pastoris,the codons of 187 amino acid of PCV2 ORF2 novel phytase gene was chemically synthesized by the PCR-based two-step DNA synthesis according to yeast codon preference.Unoptimized or optimized PCV2 ORF2 genes,with or without encoding signal peptide were inserted into pPICZα-C expression vector.The recombinant plasmids were linearized and transformed into Pichia pastoris GS115.Recombinant strains were preliminarily selected by Zeocin resistance.Only the PCV2 ORF2 gene,which was optimized gene and removed encoding signal peptide,was successfully expressed.The expression of recombinant protein was approved by SDS-PAGE and Western-blot analysis.The molecular weight of the target protein is about 30 ku.That the recombinant expressed protein could be recognized by anti-PCV2 serum which means the recombinant ORF2 protein had bioactivity.

关 键 词：猪圆环病毒ORF2基因 密码子优化 稀有密码子 自身信号肽 毕赤酵母 

分 类 号：S852.65[农业科学—基础兽医学]