人癌胚抗原-重组痘苗病毒的构建和制备

痘苗病毒的基因组庞大，结构复杂而特殊，不可能将外源基因直接插入它的基因组，必须利用一种特殊的痘苗病毒质粒，才能构建成功重组痘苗病毒．在分析了痘苗病毒质粒ｐＪ１２０〔含有我国天花疫苗－痘苗病毒天坛株７６１的启动子和胸苷激酶（ｔｈｙｍｉｄｉｎｅｋｉｎａｓｅ，简称ＴＫ基因），及含有人癌胚抗原（ｃａｒｃｉｎｏｅｍｂｒｙｎｉｃａｎｔｉｇｅｎ，简称ＣＥＡ）ｃＤＮＡ全序列的质粒ｐ９１０２３Ｂ－ｃｅａ－１７结构的基础上，设计出三步法构建了重组疫苗病毒质粒ｐＪ－ＣＥＡ．经酶切及ＰＣＲ鉴定ｐＪ－ＣＥＡ中ＣＥＡｃＤ－ＮＡ的存在，进一步用同源重组方法构建了表达人ＣＥＡ的重组痘苗病毒，并以人体成纤维细胞作为宿主细胞，对ＣＥＡ－重组痘苗病毒进行了大量培养．再次证实痘苗病毒是良好的真核表达载体，可以高效而准确地表达细胞膜糖蛋白ＣＥＡ．

Construction and Production of CEA recombinant Vaccinia Virus

A specific plasmid vector pJ120 was utilized to construct a transient recombinant pJ CEA.After transfection of vaccinia virus infected 143TK - cells with plasmid pJ CEA by liposome,homologous recombination occured between TK1 and TK2 sequences flanking CEA cDNA and Lac Z gene of pJ CEA,and the same TK sequence within the vaccinia virus genome.The recombinant vaccinia virus was constructed in the manner having the foreign gene CEA cDNA and Lac Z gene inserted into the vaccinia virus TK gene under the control of vaccinia virus promoter.Since recombinants had an interrupted TK gene,they could be selected on the basis of their TK - phenotype and then checked for the presence and expression of the foreign gene by PCR and radioimmunological method.By using vaccinia virus as a vector,a CEA recombinant vaccinia virus capable of highly expressing CEA was constructed.It further proved that vaccinia virus was an efficient vector for expressing eukaryotic protein and could maintain the characteristics of the protein.