多房棘球绦虫原头节cDNA表达文库的构建及初步鉴定

摘要

Objective　To construct a λgt11 cDNA expression library of Echinococcus multilocularis protoscolex isolated in China. rnMethods　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.rnResults　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. rnConclusions　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.%目的　构建中国分离株多房棘球绦虫原头节cDNA表达文库。rn方法　mRNA的提取采用Quicprep MicromRNA purification kit并做适当改进，主要利用异硫氰酸胍的破坏和保护双重作用及oligo（dT）-纤维素柱层析来提取、纯化mRNA。以约1.8 μg的mRNA为模板、六聚体随机引物逆转录，再用衔接头EcoRI/NotI修饰逆转录所得的cDNA钝端以使其两端形成粘性EcoRI位点，然后克隆入噬菌体λgtll的EcoRI臂，体外包装后转染大肠杆菌Y1090，蓝白斑筛选重组体，PCR鉴定插入片段大小。rn结果　重组率几乎100%；库容量约1×106，插入片段长为1.47 Kb。rn结论　已成功构建中国分离株多房棘球绦虫原头节cDNA表达文库，可进行进一步的深入研究。