A组链球菌M蛋白二价DNA疫苗表达载体的构建与鉴定

Construction of a divalent DNA vaccine expression vector for M protein of group A of streptococcus

目的构建含A组链球菌M蛋白基因(emm基因)1型和3型特异性抗原决定簇基因的重组质粒。方法用PCR技术从40381137(T1)型和31281187(T3)型A组链球菌中分别扩增emm基因,插入T 载体后测序、blast后分别确定为emm1和emm3基因型,再从emm1和emm3基因分别扩增105 bp目的基因,用聚合酶链反应(polymerase chain reaction,PCR)技术将两目的片段连接起来。插入真核表达载体 pSecTaqB中,阳性质粒克隆经酶切和测序鉴定。结果从40381137(T1)和31281187(T3)A组链球菌株的基因组成功克隆emm基因,经测序及blast后分别确认为emm1型和emm3型;用pst I和BamH I酶切重组质粒pSemml-3、测序均显示插入正确。结论成功构建A组莲球菌的emm1型和emm3型二价真核表达载体,为下一步研究表达功能及表达蛋白的生物活性打下基础。

Objectives To construct a recombinant vector containing fragments of emm 1 genes and emm 3 gene that are the type specific epitopes of streptococcus pyogenes. Methods The emm 1 gene and emm 3 gene were amplified respectively by PCR from genomic DNA of 40381137 （T1） and 31281187 （T3） streptococcus isolates and identified by blast to CDC data bank. Then, 105 bp fragments were amplified by PCR from emm 1 and emm 3 genes, and were ligated by PCR. The ligated fragment was inserted into eukaryotic expression vector pSecTaqB. The positive clones were screened and identified by agarose gel electrophoresis endonuelease digestion and sequencing. Results 1. The emm 1 gene and emm 3 gene were amplified and identified by blast to CDC data bank. 2. The positive clones of recombinant plasmld pSemml-3 was identified that the code frame was right, Conclusions We succeeded to construct a DNA expression vector containing divalent emm genes of streptococcus pyogenes M protein. It＇s the base to further study the expression ability and the biology active of expression protein.