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目的：建立弓形虫ｃＤＮＡ文库。方法：用异硫氰酸胍酸性一步法提取弓形虫（ＲＨ株）ＲＮＡ，含ＰｏｌｙＵ的ｍＲＮＡ亲和膜分离Ｐｏｌｙ（Ａ）＋ＲＮＡ，进而合成双链ｃＤＮＡ。将ｃＤＮＡ与ＥｃｏＲＩ／ＮｏｔＩ接头连接，再与表达载体λｇｔ１１ＤＮＡ臂连接，经体外包装，感染大肠杆菌Ｙ１０９０。以３２ｐ标记的弓形虫ＲＨ株基因组ＤＮＡ为探针，对重组噬菌斑进行原位杂交。结果：ｃＤＮＡ产物分布在０．５－２ｋｂ，得到６．９７×１０５重组子，重组率为９８．７３％，克隆效率为６．９７×１０６克隆／μｇｃＤＮＡ。噬菌斑原位杂交的阳性斑点占重组克隆９５．２％。结论：用异硫氰酸胍酸性一步法提取弓形虫ＲＮＡ简便有效，已建立了一个容量大、质量好的弓形虫ｃＤＮＡ文库。
Objective: To establish a Toxoplasma cDNA library. Methods: Poly (A) + RNA was extracted from Polygonatum gondii RH strain RNA by Polyglutamic acid (MoU) affinity chromatography using one-step guanidinium isothiocyanate (AA) method and double stranded cDNA was synthesized. The cDNA was ligated to the EcoRI / NotI linker, ligated to the lambda gt11 DNA arm of the expression vector, and then inoculated in vitro to infect E. coli Y1090. Genomic DNA of 32p-labeled Toxoplasma gondii RH strain was used as a probe to in-situ hybridize the recombinant plaques. Results: The cDNA products were distributed in the range of 0.5-2kb, yielding 6.97 × 105 recombinants with a recombination rate of 98.73% and a cloning efficiency of 6.97 × 106 clones / μg cDNA. Positive plaques in situ hybridization accounted for 95.2% of recombinant clones. Conclusion: It is simple and effective to extract Toxoplasma gondii RNA with guanidine isothiocyanate one step, and a large and good Toxoplasma gondii cDNA library has been established.