密码子优化的肺炎支原体P1蛋白在大肠杆菌中的克隆表达

目的:获得密码子优化的肺炎支原体P1黏附蛋白优势表位抗原基因,并在大肠杆菌中表达,为临床诊断试剂和疫苗研制打下基础。方法:采用生物信息学分析肺炎支原体P1蛋白的抗原表位,筛选特异性P1蛋白优势表位区;采用大肠杆菌优势密码子,设计上述P1蛋白优势表位基因序列;采用退火PCR技术合成上述基因,并利用载体pGEX-4T-2实现P1优势表位抗原在大肠杆菌中的表达;采用ELISA法对纯化的P1抗原活性进行测定。结果:肺炎支原体P1蛋白特异性抗原表位主要位于1154~1521 aa,获得的P1优化密码子基因平行突变37个稀有密码子和2个终止密码子;在大肠杆菌中表达的GST-P1融合蛋白的相对分子质量为65.9×103,纯化后重组抗原能与肺炎支原体感染者血清发生特异性的免疫反应。结论:采用密码子优化基因合成技术实现了肺炎支原体P1优势表位抗原在大肠杆菌中的高效表达,为肺炎支原体感染的诊断试剂研究提供了重要参考。

Cloning and Expression of Codon-Optimized P1 Protein Gene of Mycoplasma pneumoniae in Escherichia coli

Objective: To obtain codon-optimized P1 adhesion protein of predominant epitope gene of Mycoplas ma pneumoniae(Mp),to achieve the expression of the above gene in Escherichia coli for the study of clinical diag nostic reagents and vaccines.Methods: The predominant epitopes of P1 protein were selected by bioinformatic analysis and the DNA sequences was designed according to of high-usage codons of E.coli.Above gene was ex pressed in E.coli by vector pGEX-4T-2,and its immunoreactivity was measured by ELISA.Results: Specific epit opes of P1 protein of Mp were mainly located at 1154~1521 aa.37 rare codons and 2 stop codons were mutated in the acquirement of the P1 optimized DNA sequence.The molecular weight of GST-P1 fusion protein expressed in E.coli was 65.9 kD.Purified recombinant antigen displayed specific immune response with sera of patients in fected with Mp.Conclusion: In this study,the P1 predominant epitopes of Mp was expressed in E.coli by codon optimization,which provides an important reference for the study of diagnostic reagents of Mp infection.