CAJ | 학술논문

目的：检测血液肿瘤细胞株K562、HL-60及临床急慢性粒细胞白血病患者外周血细胞CD44分子表达、分型情况，通过基因重组技术原核表达CD44分子。方法根据CD44 mRNA剪切模式设计引物，PCR扩增CD44基因，测序比对分析分子亚型，然后酶切插入原核表达载体pET32a(+)，转化大肠杆菌BL21(DE3)，IPTG诱导表达后进行Western blot分析。结果9例临床标本均为CD44isoform4型，K562细胞株中未能检测到CD44，HL-60细胞株CD44为isoform4变异型；在37℃1 mM的IPTG诱导2h后，CD44基因胞外区片段(hCD44om)的蛋白表达量最大，约占菌体总蛋白的48.6%，Western blot检测显示为特异性的CD44蛋白。结论 AML和CML外周血标本分子为CD44isoform4型，HL-60细胞株为CD44isoform4变异型，可能为mRNA新的剪切体及基因组不稳定突变所致；在大肠埃希菌中成功表达CD44isoform4型蛋白胞外区多肽，为后续单克隆抗体研制奠定了基础。
목적：검측혈액종류세포주K562、HL-60급림상급만성립세포백혈병환자외주혈세포CD44분자표체、분형정황，통과기인중조기술원핵표체CD44분자。방법근거CD44 mRNA전절모식설계인물，PCR확증CD44기인，측서비대분석분자아형，연후매절삽입원핵표체재체pET32a(+)，전화대장간균BL21(DE3)，IPTG유도표체후진행Western blot분석。결과9례림상표본균위CD44isoform4형，K562세포주중미능검측도CD44，HL-60세포주CD44위isoform4변이형；재37℃1 mM적IPTG유도2h후，CD44기인포외구편단(hCD44om)적단백표체량최대，약점균체총단백적48.6%，Western blot검측현시위특이성적CD44단백。결론 AML화CML외주혈표본분자위CD44isoform4형，HL-60세포주위CD44isoform4변이형，가능위mRNA신적전절체급기인조불은정돌변소치；재대장애희균중성공표체CD44isoform4형단백포외구다태，위후속단극륭항체연제전정료기출。
Objective To detect the CD44 expression and molecular classification of K562, HL-60 and samples from clinical patients with acute or chronic myeloid leukemia, and prokaryotic expression of CD44 molecule by gene recombination technology. Methods RT-PCR was applied to amplify CD44 followed by DNA sequencing and alignment with BLASTX algorithm. The frag-ment of hCD44om was inserted into prokaryotic expressing vector pET32a (+) to construct pET32-CD44 recombinant plasmid. Then, the expression of hCD44om in BL21(DE3) cells was induced by IPTG, and the product was identified by Western blot. Re-sults Nine cases of clinical samples were CD44 isoform4 type, no CD44 was detected in K562 cells, and the sequence of CD44 of HL-60 was most close to CD44 isoform 4 subtype. On the other hand, we successfully cloned hCD44om expression vector, and found that the protein hCD44om expressed in BL21 (DE3) reaching the largest amount after 2 hours cultivation with 1 mM IPTG at 37℃, accounting for about 48.6% of total protein. The result of Western blot showed the CD44 has good specificity. Conclusion The CD44 of HL-60 cell line is a variant of CD44 isoform 4 which may be caused by a newly mRNA splicing, and CD44 gene of human has been successfully cloned and expressed in E. coli BL21(DE3), which laid a foundation for further developing a targeted treatment study on acute myeloid leukemia.