【摘要】 通过重叠区扩增法(PCR-based accurate synthesis,PAS)人工合成猪附红细胞体ORF2基因,并使其在大肠杆菌(DE3)中高效表达。根据GenBank注册的AJ504999的ORF2基因序列,选择大肠杆菌偏爱的密码子,设计并合成11对寡核苷酸片段,用PAS方法,人工合成ORF2基因。基因克隆入pMD18-T载体,经测序证实正确后,连接到表达载体PET-28a,获得重组表达质粒PET-28a/ORF2,导入大肠杆菌BL21(DE3),在37℃,IPTG终浓度1mmol/L的条件下诱导表达,经SDS-PAGE分析,Western-blot鉴定,在35 000附近出现目的片段。同时通过生物信息学软件对其蛋白二级结构及抗原表位等方面进行预测。更多还原

【Abstract】 The ORF2 gene of Mycoplasma suis was obtained by PCR-based accurate synthesis(PAS) and put into E.coli(DE3) for expression.Base on the gene sequence of the ORF2 gene of the AJ504999 which registered in the GenBank,eleven pairs of oligonucleotides were synthesized with E.coli biased codons.The target fragment was amplified with PAS method and cloned into pMD18-T vector.After sequenced,the correctly gene was inserted into expression vector PET-28a.The recombinant plasmid PET-28a/ORF2 was transformed into E.coli BL21(DE3).Under the expression condition 1 mmol/L IPTG at 37℃,the protein appeared obviously at 35 000 by SDS-PAGE and Western-blot.At the same time,secondary structure,hydrophobicity and epitope of the target protein were predicted with bioinformatics software.更多还原