关键词: 弓形虫, mRNA, cDNA文库, 克隆

Abstract: ATM: To construct a cDNA library of Toxoplasma gondii. METHOD: We extracted the RNA of Toxoplasma gondii (RH strain)by using a single step acid guanidinium thiocyanate method and isolated the poly(A) +RNA by using messenger RNA affinity membrane containing poly U to synthesize ds cDNAs.After ligating the cDNA into the adaptor of EcoRI/NotI and then into λgt11 DNA arms of the expressive vector,the ligated products were packaged in vitro and used to infect the E.coli Y 1090 .The 32 P dCTP genomic DNA of T.gondii RH strain hybridized the λgt11 recombinant plaques in situ .RESULTS:The ds cDNAs were found to be 0.5-2 kb in size.The cDNA library of T.gondii constructed showed 6.97×10 5 recombinant plaqes.The recombination rate and the cloning efficiency were 98.73% and 6.97×10 6 clones/μg cDNA,respectively.For in situ hybridization of the λgt11 recombinant plaques,the rate of hybridization was 95.2%.CONCLUSION:The method of extracting the RNA of T.gondii is simple and efficient.The cDNA library constructed has large content and good quality.