Primary culture of cat intestinal epithelial cell and construction of its cDNA library / 中国血吸虫病防治杂志

ABSTRACT

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.