中国海南岛人恶性疟原虫基因文库的构建及环子孢子蛋白基因的筛选

本文介绍了以噬菌体λEMBL3为载体的中国海南岛人恶性疟原虫FCC1/HN分离株基因文库的构建。所得重组体的数目为3.3×10~4pfu,其相当于构建完整基因库理论计算值的6倍。作者以人工合成的巴西株恶性疟环子孢子蛋白基因重复序列区的24-mer寡核苷酸(AATGCAAACCCAAATGCAAACCCA)作探针,进行噬斑原位杂交,筛得三株阳性克隆。对其一重组体进行酶谱分析和Southern印迹实验,环子孢子蛋白基因被定位于HindⅢ和BamHI联合降解后所得的的4kb大小的片段上。

Construction of Plasmodium falciparum FCCl/HN Genomic Library and Isolation of the Gene Encoding the Circumsporozoite Protein

In order to isolate protective antigen for the development of malaria vaccines, the DNA of the P.falciparum isolated from Hainan Island in China, FCC1/ HN,was partially digested with Sau3Al.The 14 to 20kb fragments were collected, and inserted into the vector Lambda EMBL3 DNA between the BamH1 sites.Through ligation by T4 DNA ligase and in vitro packaging of bacteriophage, 3.3*******X104 recombinants were obtained, which was then amplified, in the construction of gene library for Three positive clones were selected by in situ hybridization with a synthetic 24-mer oligonucleotide (AATGCAAACCCAAATGCAAACCCA) probe, whose sequence was derived from the repeated unites in the CSP gene of P.falciparum isolated from Brazil.One of the clones was analysed and its cleavage sites for different restriction endonucleases were mapped.By Southern blotting, the CSP gene may localized in a 4kb fragment generated from BamHl and Hind**********III double digestion.