摘要
细粒棘球绦虫DNA经Sau3AI部分水解,并用电洗脱方法从琼脂糖凝胶中回收15-23kb大小的片段。得到的片段与λ噬菌体置换载体EMBL3 DNA重组,采用体外包装系统构建了细粒棘球绦虫的基因库,获得了1.2×10~6个重组子。通过与探针的分子杂交,从库中分离到6个细粒棘球绦虫γDNA基因的阳性克隆。
Using EMBL3 phage DNA as a vector, a perfect genomic library of Echinococcus granulosus from sheep from Xinjiang has been constructed, which contains 1.2×06 independent recombinants.The main procedure of construction comprised: 1. extraction of Echinococcus granulosus genomic DNA, 2. partial digestion of the extracted E. granulosus genomic DNA with restriction enzyme Sau3A1, 3. harvest of 15-23 kb DNA fragments by electric elution equipment, 4. preparation of EMBL3 phage vector DNA, 5. cleavage of EMBL3 vector DNA with two restriction enzymes Bam HI and Eco RI to remove the central fragment, 6. ligation of the harvested 15-23 kb E. granulosus genomic DNA and the cleavaged vector DNA with T4 DNA ligase, 7. the package reaction of ligated recombinant DNA in vitro with the package protein and with Q359 strain (Spi-) to screen recombinants, 8. identification of the genomic library, 9. hybridization in situ by pSM889 probe, obtaining 6 positive recombinant clones. (Figs. 1-6)It was estimated that the E. granulosus genome was about 1.5×108 bp. A perfect E.g. genomic library is expected to contain 4.6×104 independent recombinants at minimum The construced genomic library has enough independent recombinants (1.2×106).This is the first time to establish an E. granulosus genomic library in China. Previous work showed that there existed differences in many aspects, including pathogenicity, among various isolates of E. granulosus from different hosts and areas. We plan to employ this library to screen the E. granulosus intraspecies DNA probe by hybridization in situ. This kind of probe is envisaged to be of advantage for epidemiological investigation of the hydatid disease in China. It also provides a base for researching E. granulosus at the molecular level.
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
1991年第2期98-101,共4页
Chinese Journal of Parasitology and Parasitic Diseases
基金
国家自然科学基金~~
关键词
细粒棘球绦虫
基因库
Echinococcus granulosus, genomic library.