旋毛虫肌幼虫ES抗原基因TSPGⅡ在真核细胞中的表达

EXPRESSION IN COS7 CELL LINE OF STRUCTURAL GENE TSPG ENCODING ES ANTIGEN FROM TRICHINELLA SPIRALIS MUSCLE LARVAE

用限制性内切酶ＥｃｏＲⅠ和ＨｉｎｄⅢ从克隆载体ｐＵＴＳⅡ中切出一个０．９８８ｋｂＴＳＰＧⅡ基因，经ＥｃｏＲⅠ和ＢｓｔＸⅠ酶切鉴定；表达载体用相同的酶切，经琼脂糖凝胶电泳，分别回收ＴＳＰＧⅡ基因和ｐＳＶ·ＳＰＯＲＴⅠ表达载体，用Ｔ４ＤＮＡ连接酶定向连接，转化大肠杆菌感受态细胞，挑取重组子，经ＥｃｏＲⅠ，ＢｓｔＸⅠ和ＨｉｎｄⅢ酶切鉴定，构建了重组表达质粒ｐＳＶ·ＴＳⅡ。利用脂质体作载体，将重组表达质粒转染ｃｏｓ７细胞，４８～６０ｈ后用酶联免疫吸附试验检测，显示表达产物能与猪旋毛虫病的阳性血清反应。

TSPG gene encoding ES antigen from T. spiralis muscle larvae was expressed by SV40 promoter of expression vector pSVSPORT in cos7 cells. Cloning vector pUTS was digested by EcoR and eletrophoresed in agarose gel to recover a 0.988 kb TSPG gene fraginent. TSPG gene was inserted into vector pSVSPORT which was predigested by EcoR and Hind , and ligased to the downstream of SV40 promoter. Thus recombinant expression plamids of pSVTS were constructed by T4 DNA ligase before they were transformed into E. coli DH 5 a. Identified by EcoR , BstX and Hind , pSVTs were prepared by alkaline lysis. Cos7 cells were transfected with liposome for 60 hr. The result of ELTSA showed that expression products can react with positive sera from swine infected with T. Spiralias.