血液肿瘤CD44基因检测及4亚型克隆原核表达

Detection of the expression of CD44 gene in leukemia and prokaryotic expression of CD44 isoform 4 subtype in E.coli

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 m RNA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p ET32a(+),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℃1m M的IPTG诱导2h后,CD44基因胞外区片段(h CD44om)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m RNA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。

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 fragment of h CD44 om was inserted into prokaryotic expressing vector p ET32a（＋） to construct p ET32-CD44 recombinant plasmid.Then, the expression of h CD44 om in BL21（DE3） cells was induced by IPTG, and the product was identified by Western blot. Results 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 h CD44 om expression vector, and found that the protein h CD44 om expressed in BL21（DE3） reaching the largest amount after 2 hours cultivation with 1 m M IPTG at37℃, 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 m RNA 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.