Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library
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1Institute of Infectious and Parasitic Diseases, Chongqing University of Medical Science, Chongqing 400016, China.
Objective:
To construct a lambda gt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China.
Methods:
Echinococcus multilocularis protoscolex mRNA was extracted using a Quickprep MicromRNA purification kit based on combining of the disruptive and protective properties of guanidinium thiocyanate (GTC) with the speed and selectivity of oligo (dT)-cellulose chromatography in a spum-column with some modification. Purified mRNA (1.8 micrograms) was submitted to reverse transcription using random hexamers [pd(N6)]. The double-strand blunt-ended cDNAs were ligated with an EcoRI/Notl adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector lambda gt11 EcoRI arms. After being packaged in vitro, lambda gt11 was put to an infectious bacteria Echinococcus coli (E. coli) strain Y1090; the recombinants were screened 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) clones could be derived from this lambda gt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb.
Conclusions:
A lambda gt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.
