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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?
The gene fragment encoding the antigenic epitopes of Plasmodium falciparum at different developmental stages including merozoite surface antigen MSA1, MSA2, circumsporozoite protein CSP, and the erythrocyte surface protein RESA of the ring body, (HGFSP) such as interleukin-1 (IL-1) and T cell activation site on tetanus toxoid (TT). The EcoRI and BamHI sites of the human pSK vector were firstly targeted and then digested with EcoRI And the target gene excised by Sac I enzyme was used to redirect the SmaI and SacI sites of the human vaccinia virus expression vector PJ2-16 to transform the host strain TG-Ⅰ. The recombinant was analyzed by agarose gel electrophoresis and restriction enzyme analysis And zymogram identification, confirmed that the target gene has been inserted into PJ2-16 hemagglutinin (HA) gene recombinants, constructed Plasmodium falciparum antigen gene - vaccinia virus expression vector. The recombinant expression vector and vaccinia Tiantan strain were treated with Lipofectimine and co-transfected and homologous recombined in Cos-7 cells. The transfectant was inoculated with BHK21 cells, and the chicken erythrocyte adsorption test was used to select the non-adsorbed chicken erythrocytes HA-), two strains of HA-recombinant virus were screened out. Indirect immunofluorescence analysis of the recombinant virus expression product against E. coli expressing protein of the target gene, Dot-ELISA and Western-blot showed that among the two recombinant viruses One can express the target protein, protein molecular weight and the length of the gene of interest?