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胸膜肺炎放线杆菌apxⅠA基因在不同宿主系统
中的表达及密码子偏好性分析
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黄琦1,2，谢芝勋1*，庞耀珊1
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（1．广西兽医研究所 生物技术实验室，广西 南宁530001; 2．广西大学 动物科技学院，广西 南宁530001）
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摘要：
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用PCR方法从胸膜肺炎放线杆菌（APP）血清1型参考株APP259的基因组DNA中，扩增了约954bp的apxⅠA基因片段，分别构建了大肠杆菌表达载体pGEX-4T-1/apxⅠA和毕赤酵母表达载体pPICZαA/apxⅠA。对目的基因密码子偏好性进行了分析，发现其中有4个大肠杆菌稀有密码子，占1.2%；有49个毕赤酵母稀有密码子，占15.4%。利用所构建的大肠杆菌表达载体pGEX-4T-1/apxⅠA和毕赤酵母表达载体pPICZαA/apxⅠA分别进行表达。结果显示，目的基因在大肠杆菌中能以包涵体形式进行高效表达，其融合蛋白占菌体总蛋白的32%；在毕赤酵母中则以较低水平分泌表达至培养基的上清液中，40倍浓缩后方能检测到少量目的蛋白。证实，目的基因序列中的密码子偏好性影响其在大肠杆菌和毕赤酵母中的表达。 |
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关键词：
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胸膜肺炎放线杆菌；apxⅠA基因；宿主系统；表达；密码子偏好性 |
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中图分类号：S 852.619：Q 786 文献标识码：
A 文章编号：1673-4696(2008)02-0101-05 |
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Expression of apxⅠA gene of Actinobacillus pleuropneumoniae
in different host systems and analysis of its codon bias |
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HUANG Qi1，2,XIE Zhi-xun1,PANG
Yao-shan1 |
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(1.Laboratory of Biotechnology,Guangxi Veterinary Research Institute,Nanning 530001,China；
2.College of Animal Science and Technology,Guangxi University,Nanning 530001,China) |
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Abstract: The gene fragment apxⅠA of approximately 954bp in length was amplified from genomic DNA of Actinobacillus pleuropneumoniae(APP) serovar 1 reference strain APP259 by PCR to construct recombinant prokaryotic expression vector pGEX-4T-1/apxⅠA and recombinant eukaryotic expression vector pPICZαA/apxⅠA.Analysis of codon bias of the apxⅠA gene showed that there were only 4 rare codons of Escherichia coli(1.2%) and 49 rare codons of Pichia pastoris(15.4%).The apxⅠA gene in the two recombinant vectors was expressed.SDS-PAGE analysis revealed that the apxⅠA gene in the pGEX-47-1/apxⅠA recombinant prokaryotic expression vector was highly expressed as inclusion body in E.coli and accounted for up to 32% of the total amount of bacterial proteins.The apxⅠA gene in the pPICZαA/apxⅠA produced low level interest protein in P.pastoris by secreting to the supernatant and the product concentrated for 40 folds was detected by SDS-PAGE.The results confirmed that the codon bias of apxⅠ A gene might be the critical factor influencing its higher expression in E.coli but lower expression in P.pastoris. |
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Key words: Actinobacillus pleuropneumoniae;apxⅠA gene;host system;expression;codon bias |
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