Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library

Authors
Citation
Sp. Li et Yt. Chen, Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library, CHIN MED J, 114(2), 2001, pp. 124-127
Citations number
10
Categorie Soggetti
General & Internal Medicine
Journal title
CHINESE MEDICAL JOURNAL
ISSN journal
03666999 → ACNP
Volume
114
Issue
2
Year of publication
2001
Pages
124 - 127
Database
ISI
SICI code
0366-6999(200102)114:2<124:CAPCOE>2.0.ZU;2-V
Abstract
Objective To construct a lambda gt11 cDNA expression library of Echinococcu s multilocularis protoscolex isolated in China. Methods Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive a nd protective properties of guanidinium thiocyanate (GTC) with the speed an d selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 mug) was submitted to reverse transcr iption using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subse quently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arm s. After being packaged in vitro, lambda gt11 was put to an infectious bact eria Echinococcus coli (E. coli) strain Y1090; the recombinants were screen ed by color selection. PCR amplification was performed to evaluate the size of insertion DNA fragments. Results The recombinant ratio was nearly 100% and approximately 1 x 10(6) c lones could be derived from this lambda gt11 cDNA library. PCR results indi cated that the insertion DNAs were about 1.48 kb. Conclusions A lambda gt11 cDNA expression library consisting of a million r ecombinant clones has been constructed from Echinococcus multicularis proto scolex mRNA. Further studies on this library are deserved.