恶性疟原虫基因文库构建及特异DNA探针的应用研究

Construction of Plasmodium Genomic Library and Applications of the Specific DNA Probe

将恶性疟原虫（Ｐｌａｓｍｏｄｉｕｍｆａｌｃｉｐａｒｕｍ，Ｐ．ｆ）Ｆｃｃ－１／ＨＮ株基因组ＤＮＡ用ＨｉｎｄⅢ消化与ｐＢＲ３２２重组后导入大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）ＨＢ１０１，转化效率为１．５×１０６转化子／μｇＤＮＡ，建成基因文库。用Ｐ．ｆ基因组ＤＮＡ原位杂交，证明了该文库的有效性，又筛出特异克隆。克隆之一ｐＢＦ２ＤＮＡ用３２Ｐ和光敏生物素标记作探针，可分别从间日疟原虫（Ｐ．ｖｉｖａｘ，Ｐ．ｖ）、恶性疟原虫（Ｐ．ｆ）、伯氏疟原虫（Ｐ．ｂｅｒｇｈｅｉ，Ｐ．ｂ）、食蟹猴疟原虫（Ｐ．ｃｙｎｏｍｏｌｇｉ，Ｐ．ｃ）和人白细胞ＤＮＡ样本中特异地检出Ｐ．ｆＤＮＡ，显示该探针具有Ｐ．ｆ的种特异性；该探针可检出Ｐ．ｆ基因组ＤＮＡ的灵敏度为１０ｐｇ和０．００１％原虫血症，显示其高度敏感性；两种标记探针检测采自疟区病人的血样与镜检结果基本一致。

DNA of Fcc1/HN gene was digested with Hind and recombined with pBR322 and then transferred into E.coli HB 101 for the construction of genomic library. The transfer rate was shown to be 1.5 106 clones per g DNA.This library was provedto be effective when P.f genomic DNA was used for hybridization with the specific clone screened. One of the clone pBF2 DNA was labelled with32P and lightsensitivebiotin and used as probe, which could specifically distinguishthe P.f DNA form DNA of human leukocyte and some other kindsof plasmodium DNA, such as BXP.v, P.b and P.c in which the probewas shown to have high specificity for P.f. The sensitivity ofthis probe in the detection of P.f genomic DNA was 5 g and aparasitemia as 0.001% . The results of the detection with theprobe labelled by the two methods was in agreement with the results with microscope,demonstrating a very promisingDapplicability of the probe.