弓形虫昆山分离株速殖子期表达型cDNA文库的构建及鉴定

CONSTRUCTION AND ANALYSIS OF CDNA LIBRARY OF TOXOPLASMA GONDII

目的 构建弓形虫速殖子期表达的基因的完整长度cDNA文库。方法 用CF - 11纤维素柱快速提纯速殖子 ,以盐酸异硫氰酸胍 (AGPC)一步法抽提总RNA ,oligo -dT纤维素分离mRNA后 ,用ClonTech公司的SmartTMPCRcDNA文库构建试剂盒 ,构建了弓形虫昆山分离株的表达型文库。结果 获得 5× 10 6个独立克隆 ,重组率为 99% ,插入片段的平均长度为 1kb。

Aim\ To construct an expression full-length cDNA library of Toxoplasma gondii tachyzoite stage(Kunshan strain). Methods\ The purified parasites were obtained by passage through a column of CF-11 cellulose. After isolating total RNA from tachyzoites by AGPC method, poly-A+ RNA was isolated by standard oligo-dT affinity chromatography. The cDNA was synthesized with MMLV reverse transcriptase and digested by sfi1. After size-selected by chroma spin-400, the cDNA material was ligated into λTriplEx2 vctor arms, and packaged in vitro using extracts. Then the packaged composition was used to infect the host bacteria E. coli XL1 Blue. In order to determine the average length of the cDNA inserts, 16 plaques were randomly picked for PCR screening. Results\ The packaging efficiency and the recombinant rate were about 2 1×10\+6pfu/ml and 99% clones/μg cDNA,respectively. The average length of the cDNA inserts was about 1kb. Conclusion\ The results suggested that the filtration through cellulose columns method produced the better RNA quality and the cDNA library constructed has large content and good quality.