内皮抑素30肽基因真核表达载体的构建及实验研究

目的构建改构内皮抑素抗肿瘤相关肽（30肽）的真核表达载体pVAX1,检测该重组载体的生物学活性。方法在30肽基因的5′端加入胶原蛋白ⅩⅧ信号肽编码序列,通过PCR扩增获得目的基因30肽,并连接到质粒pVAX1中,构建表达分泌型内皮抑素的重组质粒pVAX1-30E,然后将重组质粒pVAX1-30E直接注入小鼠肿瘤组织。通过ELISA小鼠体内抑瘤实验检测目的基因的表达及其活性。结果ELISA实验表明构建的分泌型内皮抑素重组质粒pVAX1-30E能在肿瘤细胞中表达30肽,免疫组化结果表明瘤组织中表达的30肽能抑制肿瘤微血管的新生,而体内抑瘤实验表明在肿瘤部位直接注射重组质粒能抑制肿瘤生长,抑瘤率为28.19%。结论通过向瘤组织中直接注射分泌型内皮抑素重组质粒pVAX1-30E可以抑制小鼠体内肿瘤微血管新生和肿瘤生长而实现其抗肿瘤活性。

Construction of Eukaryotic Expression Vector of 30-Peptide of Human Endostatin and Its Anti-Tumor Activity

Objective The anti-tumor peptide of human endostatin was cloned into eukaryotic expression vector pVAX1 and its bioactivity was assayed.Methods／The nucleotide sequence encoding 1-30 amino acids（30-peptide,sequence RGIRGAD was changed to RGDRDG） of human endostatin was synthesized and inserted into plasmid pVAX1.The signal peptide coding sequences of collagen XVIII was also linked to the 5＇-end of target gene.Then the recombinant plasmid was directly injected into the tumor tissue in mice.ELLISA,immunohistochemistry and tumor inhibition test in mice were conducted to observe the expression and anti-tumor activity of the 30-peptide.Results ELLISA showed the target peptide was expressed via the secretory recombinant plasmid in tumor cells.Immunohistochemistry revealed that 30-peptide could inhibit angiogenesis in tumor tissue.Tumor inhibition test in mice indicated that 30-peptide provided anti-tumor activity via injection of its recombinant plasmid into tumor tissue.Conclusions／The 30-peptide show its antitumor activity in mice via injection of its secretory recombinant plasmid in tumor.