CONSTRUCTION OF HUMAN COLORECTAL CARCINOMA cDNA PHAGE EXPRESSION LIBRARY AND ITS IDENTIFICATION BY PCR
- VernacularTitle:人源性大肠癌cDNA噬菌体库的构建及PCR鉴定
- Author:
Yuhu LIU
;
Zhenshu ZHANG
;
Jinbao WU
- Publication Type:Journal Article
- Keywords:
colorectal neoplasms;
phage;
cDNA library
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this experiment was to construct a human colorectal carcinoma cDNA phage expression library. Total RNA was extracted from the cancer tissue of human colorectal carcinoma, and the mRNA was purified. The single-strand and double-strand of cDNA were synthesized through reverse transcription-PCR and LD-PCR. cDNA fragments, after removal of those smaller than 500bp, were combined with ?TriplEx2 phage vector. The recombinant cDNA were packaged in vitro with MaxPlax TM Packaging Extract, then a small portion of packaged phage was used to infect E.coli XL1-blue for titration and determination of the percentage of recombinant clones. PCR method was used to identify the size of inserted cDNA. A human colorectal carcinoma cDNA phage library consisting of 2.07?10 6 pfu/ml recombinant bacteriophages was successfully constructed, the recombinant percentage was 94.5%, and the range of the fragment length of exogenous inserted cDNA was between 600bp～4kb, with an average of about 1.4kb. It met the universally accepted standards, and it could be useful in screening cDNA clones to find out the human colorectal carcinoma associated antigen genes.
