恶性疟原虫(海南株)cDNA表达文库的构建及初步鉴定

CONSTRUCTION AND PRIMARY CHARACTERIZATION OF ERYTHROCYTIC PLASMODIUM FALCIPARUM( FCC/HN) c DNA EXPRESSION LIBRARY

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。

AIM:To construct a c DNA expression library from erythrocytic Plasmodium falci- parum(FCC/HN) .METHODS:1 2 2 4μg total RNA and35 .84μg poly(A) +m RNA have been successively obtained from the blood- stage Plasmodium falciparum mainly consisting of trophozoites using“single- step method”and by chromatography on oligo- (d T) cellulose. With the reverse transcriptase(M- MLV) ,1 0 μg m RNA was synthesized into1 .75 μg blunt c DNA.After the c DNA was ligated to Eco R I(Not I,Sal I) adapter and purified by fraction- ation,2 0 0 ng c DNA of about5 0 0 bp- 7kb was collected.Of which5 0 ng c DNA was ligated intoλgt1 1 phage particles and was packaged with the packagen extract system in vitro. This library was characterized primarily by PCR.RESUL TS:A c DNA library containing1 0 6re- combinants has been constructed.CONCLUSION:The capacity of this library and the size of c DNA is suitable for further study.