[Construction of Echinococcus granulosus genomic library].
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 x 10(6) 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 Sau3Al, 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. It was estimated that the E. granulosus genome was about 1.5 x 10(8) bp. A perfect E.g. genomic library is expected to contain 4.6 x 10(4) independent recombinants at minimum. The constructed genomic library has enough independent recombinants (1.2 x 10(6)). 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.
Chen
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W
Institute of Parasitic Diseases, Chinese Academy of Preventive Medicine.
Xue
H C
HC
Qiu
L S
LS
D000818:Animals; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant; D004446:Echinococcus / Q000235:genetics*; D015698:Genomic Library
D004274:DNA, Recombinant; D004247:DNA