弓形虫P30抗原基因的体外扩增、克隆及原核表达

IN VITRO AMPLIFICATION, CLONING AND PROTOCARYONCELL EXPRESSION OF THE GENE ENCODING THE MAJOR TOXOPLASMA GONDII ANTIGENP30

根据已知弓形虫主要表面抗原Ｐ３０的基因序列，用已合成的一对引物，通过聚合酶链反应，从弓形虫ＲＨ、ＺＳ１和ＺＳ２株中扩增了Ｐ３０的编码基因，经纯化及相应酶切后插入质粒ｐｃＤＮＡ３中并转化大肠杆菌ＴＧ１。经含氨苄青霉素ＬＢ培养基初筛后，挑菌扩增双酶切鉴定，阳性克隆子在ＴＧ１中表达，产物经ＳＤＳ－ＰＡＧＥ分析显示，Ｐ３０基因在大肠杆菌中高效表达。

In present study, two pairs of primers synthesized according to the sequence of Toxoplasma gondiiP30 gene were used to amplify the P30 gene fragment by PCR from Toxoplasma RH,ZS1 and ZS2 strains. By purification and digestion with restriction endonuclease (EcoRⅠ and HindⅢ), the amplified gene fragments and plasmid pcDNA3 were ligated. After transformation with the recombinant vector into the  E. coli TG1, the genetically engineered bacteria clones were selected and identified by restriction endonuclease. The expressing products of the positive bacteria clones were analyzed by SDSPAGE. The result showed that a novel wide band could be seen at about 35 kDa in PAGE.