| Objective: Identify the human hepatocarcinoma cell strain with the highest reactivity to hALR and construct the phage-displayed library with its cDNA as the basis of the selection of special protein binding hALR from this library.Methods: The reactivity to hALR of HepG2, 7701 and QGY was compared by 3H-TdR methods in vitro. The mRNA of QGY with the highest reactivity to hALR was isolated with PloyATtract System 1000 kit. The phage-displayed library with cDNA fragments of QGY was constructed with T7SelectlO-3 OrientExpress cDNA Cloning System and Random Primer kit. The quality of this library was evaluated by phage titer assay and PCR.Results: hALR's different effect on the proliferation of QGY, HepG2, and 7701 was well demonstrated. And the dose-dependent relationshiop between hALR and the proliferation of three cell strains was manifested. Particularly, it was found that QGY exerted the highest reactivity to hALR and followed the surest dose-dependent way in vitro. The phage-displayed library with cDNA of QGY was successfully constructed. According to the result of phage titer assay, approximate 2xl07 primary recombinants were measured in the cDNA library. It was indicated by PCR that the size of the cDNA inserts was from 0.2 to 2kb, the average 0.6kb. Thorough information about mRNA of QGY cells could be soundly represented by this library.Conclusion: It was showed that QGY had the highest reactivity to the hALR among three strains detected. The highly quality phage-displayed library with cDNA of QGY was successfully constructed. |
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