人PGK1基因真核表达载体的构建及其功能检测

Construction and functional detection of human PGK1 eukaryotic expression vector

目的构建带FLAG标签的人癌基因磷酸甘油酸激酶1(phosphoglycerate kinase1,PGK1)的真核表达载体,并验证其表达产物的生物学功能。方法采用PCR技术从人乳腺文库中扩增人PGK1基因,并将其克隆到真核载体pc DNA3. 0-FLAG上,酶切鉴定和测序鉴定后,通过Western印迹检测其表达;将FLAG-PGK1载体转染乳腺癌细胞ZR75-1,利用乳酸试剂盒测定乳腺癌细胞外乳酸含量,并通过CCK8法和划痕实验测定乳腺癌细胞的生长和迁移。结果从乳腺文库中,PCR扩增出约1260 bp DNA片段,克隆到pc DNA3. 0-FLAG载体上,测序结果与目的序列一致。Western印迹检测到目的基因表达,且位置正确。乳酸含量测定结果显示,PGK1可促进细胞乳酸含量增加;细胞生长曲线结果显示,转染FLAG-PGK1的乳腺癌细胞生长较空载体生长快;细胞划痕实验表明PGK1促进细胞迁移。结论成功构建了FLAG-PGK1的真核表达载体,其表达促进了乳腺癌细胞生长和迁移,同时也促进了乳酸的增加,为进一步研究PGK1在肿瘤发生发展中的作用奠定了基础。

Objective To construct the eukaryotic expression vector of human PGK1 with FLAG-tag,and determine the biological function of the PGK1 expression product. Methods Human PGK1 gene was amplified from human mammary c DNA library,and inserted into the eukaryotic expression vector of pc DNA3. 0-FLAG,followed by double enzyme digestion and sequencing confirmation. PGK1 expression was detected by Western blotting. The recombinant pc DNA3. 0-FLAGPGK1 plasmid was transfected into breast cancer ZR75-1 cells. The lactate content was detected by lactic acid kit,and breast cancer cell growth curve was investigated by CCK8 assay. Results The pc DNA3. 0-FLAG-PGK1 eukaryotic expression vector was successfully constructed by double digestion identification. Western blotting showed that the recombinant plasmid could express PGK1 protein in 293 T cells. The analysis of lactic acid content showed that cells transfected with FLAG-PGK1 had significantly higher lactic acid content than cells transfected with the empty vector. High expression of PGK1 could markedly enhance the proliferation and migration of breast cancer cells. Conclusion The pc DNA3. 0-FLAG-PGK1 eukaryotic expression is successfully constructed. PGK1 can promote the proliferation and migration of breast cancer cells. The PGK1 can promote lactate production.