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目的 :构建恶性疟原虫 c DNA表达文库。方法 :采用“一步法”提取红内期恶性疟原虫12 2 4 μg RNA,经 Oligo( d T)纤维素柱纯化得到 35.84 μg poly ( A) +m RNA。取 10 μg m RNA反转录得到 1.75μg平端双链 c DNA,与含 Eco RI,Not I,Sal I酶切位点的衔接头连接后 ,经分级分离柱纯化回收到 2 0 0 ng大小主要在 50 0 bp- 7kb左右的 c DNA片段。取 50 ng c DNA与λgt11噬菌体臂连接后体外包装 ,完成建库。并采用 PCR方法初步鉴定该文库。结果 :已构建一个含 10 6个重组子的 c DNA表达文库。结论 :文库容量及插入 c DNA片段的大小适合于进一步研究。
Objective: To construct Plasmodium falciparum c DNA expression library. Methods: One-step method was used to extract 122.4 μg of RNA from Plasmodium falciparum in the red stage. 35.84 μg poly (A) + m RNA was purified by Oligo (dT) cellulose column. Reverse transcription of 10 μg m RNA resulted in 1.75 μg of blunt-ended c DNA, which was ligated with the adapter containing Eco RI, Not I and Sal I restriction sites and purified by fractionated column to recover 200 ng In the 50 0 bp - 7kb about c DNA fragments. Take 50 ng c DNA and λgt11 phage arm connected in vitro packaging, to complete the construction of the library. The library was preliminarily identified by PCR. Results: A cDNA expression library containing 10 6 recombinants was constructed. Conclusion: The capacity of the library and the size of inserted c DNA fragments are suitable for further study.