N端融合不同长度转位肽的重组粒酶B抑制细胞生长作用的比较

Growth Inhibitory Effects of Recombinant Granzyme B Containing Different N-terminal Translocating Peptides

采用重组PCR法将粒酶B基因的N端信号肽和酸性二肽编码序列去除 ,与两种不同长度的绿脓杆菌外毒素 (PE)转位肽序列分别连接 ,将它们插入pIND诱导表达载体 ,通过脂质体法与pVgRXR辅助质粒共转染HeLa细胞 ,建立了重组PEII GrBa基因的诱导表达细胞系。松甾酮A诱导后Western印迹检测到目的基因的表达 ,间接免疫荧光观察到表达细胞出现多核巨细胞的异常形态。两种表达的PEII GrBa融合蛋白均能够切割粒酶B的细胞内源性和外源性底物 ,并且使细胞生长速度减慢。其中 ,PEII (aa 2 80 35 8) GrBa的底物切割能力和生长抑制作用较强。流式细胞仪分析这种抑制作用可能与细胞周期的G2 期受到阻遏有关。上述结果证实了PEII GrBa融合蛋白仍然具有抑制细胞生长的作用 ,并且较短的转位肽对GrBa活性的影响较小 ,有助于进一步优化转位肽

Translocating protein and translocating peptides have therapeutic potential against tumors by exposing the cytotoxic domains of toxic proteins to the cell cytosol. The aim of this study is to investigate the effect of N terminally fused PE translocating peptides on granzyme B (GrBa) activity. PE II GrBa fusion protein genes were constructed by replacing N terminal signal and acidic dipeptide sequence of human granzyme B gene with two truncated translocating sequences of Pseudomonas exotoxin A (PE II aa 280 364/358) by recombinant PCR, and then cloned into pIND inducible expression vector. The resulting pIND PE II GrBa expression vectors were co transfected with assistant plasmid pVgRXR into HeLa cells through lipofectamine, followed by selection on G418 and zeocin. The resistant cells were collected and induced with ponasterone A. Western blot analysis demonstrated that ponasterone A induction caused the expression of PE II GrBa fusion proteins, and indirect immunofluorescence detected giant sized multinucleated cells, suggesting cytoskeletal and mitotic abnormalities as reported in our previous studies. Western blot, enzymatic activity assay and cell counting analysis indicated that two types of PE II GrBa fusion proteins were capable of cleaving both endogenous and exogenous substrates of granzyme B, and inhibiting the growth of cells. The PE II (aa 280 358) GrBa was shown to have higher serine protease activity and stronger growth inhibitory effect. Such inhibition was presumably associated with G 2 arrest as determined by cell cycle analysis. These data prove that PE II GrBa fusion proteins have cell inhibitory effect similar to GrBa, and that the shorter PE derived peptide exerts less influence on GrBa activity. This study helps to optimize the construction of recombinant protein comprising translocating peptides and cytotoxic molecules for tumor cell killing.