Construction of human promyelocytic cDNA library and its identification / 吉林大学学报(医学版)

ABSTRACT

Objective: To construct a cDNA library of human promyelocytic leukemia (HL60), and to elucidate the mechanism of related genes in the pathogenesis and development of acute promyelocytic leukemia (APL). Methods: The total RNA of HL60 cells was extracted by Trizol method. The mRNA of samples were isolated and purified by centrifugal column method. The first strand of the cDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR) while the second strand was synthesized by enzyme-catalyzed method. The cDNA fragments were recovered and then linked to the pPR3-N vector. The human promyelocytic cDNA library was constructed by homologous recombination in yeast strain Y187. The culture fluid was coated with LB plate to identify the library capacity, and PCR method was used to identify the size and distribution of the inserted fragment. Results: The RNA strand extracted from HL60 cells was clear, non-degradable and had low dispersion. The purified cell mRNA was used as template to synthesize the cDNA. After recovering the cDNA fragments, the cDNA fragments were successfully linked to the pPR3-N vector. The recombinant vector was transformed into yeast strain Y187, and the human promyelocytic cDNA library was successfully constructed by homologous recombination. The original electro transformation bacteria were diluted 100 times and 10 μL coating plate was taken, about 250 clones were generated. The total library capacity was 2. 5 × 106 CFU mL-1 and the total library capacity was 1. 25 × 107 CFU for 5 mL of transformed original bacterial solution. The average insertion fragment was more than 1 200 bp, and the positive rate was 100%. Conclusion: The human promyelocyic cDNA library is successfully constructed, and it lays a foundation for studying the pathogenesis and therapeutic mechanism of leukemia.