恶性疟原虫保护性抗原复合基因-痘苗病毒重组活疫苗株的构建

CONSTRUCTION OF A VACCINIA VIRUS VECTORED MULTI EPITOP VACCINE CANDIDATE FOR PLASMODIUM FALCIPARUM

将本实验室合成的一段编码恶性疟原虫不同发育阶段抗原表位基因，包括裂殖子表面抗原ＭＳＡ１、ＭＳＡ２、环子孢子蛋白ＣＳＰ及环状体感染红细胞表面蛋白ＲＥＳＡ以及来自白细胞介素－１（ＩＬ－１）和破伤风类毒素（ＴＴ）上Ｔ细胞激活位点等的复合抗原基因（ＨＧＦＳＰ），先定向克隆人ｐＳＫ载体的ＥｃｏＲＩ、ＢａｍＨＩ位点，后用ＥｃｏＲＩ单酶切、补平及用ＳａｃⅠ单酶切下的目的基因再定向克隆人痘苗病毒表达载体ＰＪ２－１６的ＳｍａⅠ、ＳａｃⅠ位点，转化ＴＧ－Ⅰ宿主菌，重组子经琼脂糖凝胶电泳、限制性内切酶分析及酶谱等鉴定，证实并筛选出了目的基因已插入到ＰＪ２－１６血凝素（ＨＡ）基因内的重组子，构建了恶性疟原虫抗原基因－痘苗病毒表达载体。将该重组表达载体与痘苗病毒天坛株经Ｌｉｐｏｆｅｃｔｉｍｉｎｅ处理，在Ｃｏｓ－７细胞内进行共转染和同源重组，转染物接种ＢＨＫ２１细胞，经鸡红细胞吸附试验挑选不吸附鸡红细胞的病毒斑（ＨＡ－），共筛选出两株ＨＡ－重组病毒，用抗该目的基因大肠杆菌表达蛋白抗体对重组病毒表达产物进行间接免疫荧光、Ｄｏｔ－ＥＬＩＳＡ及Ｗｅｓｔｅｒｎ－ｂｌｏｔ等试验证明，两株重组病毒中有一株可表达目的蛋白，蛋白分子量与按目的基因长度?

It has been proved in our previous studies that the immune sera obtained from rabbits immunized with multiepitope vaccine gene(HGFSP) expressed in E.Coli could inhibite the growth of cultured P.falciparum in vitro.In order to construct the live recombinant vaccinia virus,HGFSP gene was firstly inserted into the EcoRI and BamHI sites of pSK vector.After digestion with EcoRI,blunt ended by the treatment with Klenow enzyme,and digested with SacI,the HGFSP gene was cloned into the Smal and SacI sites of vaccinia virus insertion vector PJ2 16.Recombinant plasmid was identified by agarose gel electrophoresis,restriction enzyme analysis and enzyme map analysis.The results evidenced that HGFSP gene fragment was correctly inserted into the cloning site of hemagglutinin (HA) gene of vaccinia vector PJ2 16.This recombinant vaccinia plasmid was transfected into Cos 7 cells which were infected with wild type vaccinia virus by the help of Lipofectimine.Two recombinant vaccinia virus of P.f(HA-)were screened and cloned by chicken hemadsorption test in BHK21 cells.Indirect immunofluorecence assay(IFA),Western blot and Dot ELISA with the antibody against HGFSP protein expressed by E.coli showed that one of the two recombinant vaccinia viruses expressed the expected products in infected BHK21 cells.Western blot also showed that the molecular weight of two expressed protein bands was about 23 Kd,acoording with the theoretical molecular weight of HGFSP gene.Further identification of immunological characteristic of recombinantovirus is on the way.