猪附红细胞体ORF4基因片段的人工合成及基因表达

目的本研究通过人工合成猪附红细胞体ORF4基因，使其在大肠杆菌中高效表达。方法选择GenBank注册的AJ504999序列的ORF4基因，应用Graphical Codon Usage Analyser选择大肠杆菌偏爱的密码子，改变基因的transl_table，设计并合成3对寡核苷酸链，用PCR based accurate synthesis（PAS）法进行人工合成。经测序正确后，连接到表达载体PET28a，获得重组表达质粒PET28a/ORF4，导入大肠杆菌BL21(DE3)，在30℃，IPTG终浓度1mmol/L的条件下诱导表达，经SDS PAGE分析，在11 kDa附近出现目的片段。结果结果表明ORF4基因在大肠杆菌中能够得到高效的表达，通过生物信息学软件分析表明，该蛋白存在19个配基结合位点；在21~22位氨基酸可能存在信号肽裂解位点；为胞内蛋白，没有糖基化位点。结论ORF4基因能够在大肠杆菌中表达，推测其在M.suis蛋白质的形成，M.suis的生物功能方面有着重要的作用。

Cloning and expressing of synthesis fragment of Mycoplasma suis ORF4 gene

In this research,the ORF4 gene of the Mycoplasma suis was synthesized and put into E.coli for expression.Base on the gene sequence of the ORF4 gene of the AJ504999 which registered in the GenBank,according to the Graphical Codon Usage Analyser,three pairs of oligonucleotides were synthesized by using E.coli biased codons and changing transl_table.The fragment was amplified by PCR-based accurate synthesis（PAS）.Then it was inserted into expression vector PET28a after sequenced.The correctly recombinant plasmid PET28a/ORF4 was transformed into E.coli BL21.The recombinant was induced under 30℃ and 1 mmol/L concentration of IPTG.About 11kD a protein was appeared obviously by SDS-PAGE.The bioinformatics software analytic results showed that there were nineteen binding site residues,and a most likely cleavage site between position 21 and 22 AA in the ORF4 protein model.It was intracellular protein and had not glycosylation site.Consequently,ORF4 gene could be expressed in E.coli,suggesting that it has important roles in the formation of the protein and the biological function in M.suis.