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
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.