融合蛋白TF-CTP-OD-HA和TF-CTP-OD1-HA的原核表达、纯化及其生物学活性

Prokaryotic expression,purification and biological activity of TF-CTP-OD-HA and TF-CTP-OD1-HA fusion proteins

目的原核表达、纯化融合蛋白TF-CTP-OD-HA和TF-CTP-OD1-HA,并检测其生物学活性。方法以pET32a(+)CTP-OD-HA为模板,PCR扩增OD序列中的第1-87 bp和第190-216 bp序列,并通过重叠PCR连接成为新序列,命名为OD1,克隆至pET32a(+)载体,构建原核表达质粒pET32a(+)CTP-OD1-HA;再分别以pET32a(+)CTP-OD-HA和pET32a(+)CTP-OD1-HA质粒为模板,PCR扩增CTP-OD-HA和CTP-OD1-HA片段,分别克隆至表达载体pCOLDTF-DNA,构建质粒pCOLD-TF-CTP-OD-HA和pCOLD-TF-CTP-OD1-HA,转化E.coli BL21,IPTG诱导表达TF-CTPOD-HA和TF-CTP-OD1-HA融合蛋白;表达的融合蛋白经镍离子亲和层析纯化和脱盐浓缩后,采用MTT法和DAPI染色检测两种融合蛋白对K562细胞的增殖抑制和诱导凋亡作用。结果重组原核表达质粒pCOLD-TF-CTP-OD-HA和pCOLD-TF-CTP-OD1-HA经菌落PCR及测序证实构建正确;表达的TF-CTP-OD-HA和TF-CTP-OD1-HA融合蛋白相对分子质量分别约为67 000和63 000,主要为可溶性表达,表达量分别占总菌体蛋白的35%和20%,脱盐浓缩后,纯度分别约为98%和95%,均可与鼠抗HA单克隆抗体特异性结合;TF-CTP-OD-HA和TF-CTP-OD1-HA对K562细胞的增殖抑制率分别为34%和31%,与PBS对照组比较,差异有统计学意义(P<0.001);TF-CTP-OD-HA和TFCTP-OD1-HA处理的K562细胞胞核均具有细胞凋亡的典型特征。结论原核表达并纯化了TF-CTP-OD1-HA融合蛋白,该蛋白具有与TF-CTP-OD-HA相似的生物学活性,为今后开发治疗慢性粒细胞白血病的小分子多肽药物提供了新的策略。

Objective To express fusion proteins TF-CTP-OD-HA and TF-CTP-OD1-HA in prokaryotic cells, purify the expressed products and determine their biological activities. Methods The cDNAs encoding 1-87 bp and 190 -216 bp of OD were by PCR using plasmid pET32a（＋）-CTP-OD-HA as a template and linked by overlap PCR The formed new cDNA fragment named as OD1 was inserted into plasmid pET32a （＋） to construct recombinant plasmid pET32a（＋）CTP-OD1-HA, The cDNAs encoding CTBOD-HA and CTP-OD1-HA were amplified by PCR using plasmids pET32a （＋）CTP-OD-HA and pET32a （＋）CTP- OD1-HA as templates, and inserted into expression vector pCOLD-TF-DNA respectively. The constructed recombinant plasmids pCOLD-TF-CTP-OD-HA and pCOLD-TF-CTP-OD 1-HA were transformed to E. coli BL21 for expression under induction of IPTG. The expressed fusion proteins TF-CTP-OD-HA and TF-CTP-OD1-HA were purified by nickel metal chelated affinity chromatography then desalted, concentrated and determined for biological activity in inhibiting the proliferation and inducing the apoptosis of K562 cells by MTIO and DAPI staining. Results Colony PCR and sequencing proved that recombinant plasmids pCOLD-TF-CTP-OD-HA ~I1 pCOLD-TF-CTP-OD1-HA were constructed correctly. The expressed fusion proteins TF-CTP-OD-HA and TF-CTP-OD1-HA, with relative molecular masses of about 67 000 and 63 000 respectively, main existed in soluble forms, contained 35% and 20% of total somatic protein and reached purities of about 98% andabout 95% after desahing respectively, which showed specific binding to specific mouse monoclonal antibody against HA. The inhibiting rates of TF-CTP-OD-HA and TF-CTP-OD1-HA to K562 cells were 34% and 31% respectively, which showed significant difference with those in PBS control group （P 〈 O. 001 ）. The K562 cells treated with TF-CTP-OD-HA and TF-CTP-OD1-HA showed typical characteristic of apnptosis. Conclusion TF-CTP-OD1-HA fusion protein was successfully expressed in prokaryotic cells and purified, which showed similar biological characters to those of TF-CTP- OD-HA. It provided a novel strategy for further development of sall molecular peptide drug for chronic myeloid leukemia.