Construction and primary characterization of Echinococcus multilocularis protoscolex cDNA expression library
10.3760/j.issn:0366-6999.2001.02.003
- VernacularTitle:多房棘球绦虫原头节cDNA表达文库的构建及初步鉴定
- Author:
Shuping LI
1
;
Yatang CHEN
Author Information
1. 重庆医科大学
- Keywords:
echinococcus multicularis * protoscolex * λgt11 * cDNA library * clone
- From:
Chinese Medical Journal
2001;114(2):124-127
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a λgt11 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 μg) was submitted to reverse transcription using random hexamers ［pd(N6)］. The double-strand blunt-ended cDNAs were ligated with an EcoRI/NotI adaptor to form a cohesive EcoRI end. Subsequently the synthesized cDNA was inserted into vector λgt11 EcoRI arms. After being packaged in vitro, λgt11 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 1×106 clones could be derived from this λgt11 cDNA library. PCR results indicated that the insertion DNAs were about 1.48 kb. Conclusions A λgt11 cDNA expression library consisting of a million recombinant clones has been constructed from Echinococcus multicularis protoscolex mRNA. Further studies on this library are deserved.
