分泌型重组多肽sPD-1-CellⅠ(P/C-1)调节免疫细胞功能活性及抗肿瘤效应的研究

目的构建双结构域重组多肽sPD-1-Cell Ⅰ（P/C-1）分泌型真核表达载体，并研究体内外表达重组多肽在调节免疫细胞功能活性及抗肿瘤方面的作用和机制。方法用PCR方法分别扩增出编码PD-1胞外结构域的cDNA与编码CH50 CellⅠ结构域的cDNA，3片段连接法插入分泌型真核表达载体pSecTagA中，获得真核表达载体pP／C-1；脂质体介导体外转染BHK细胞，G418筛选出阳性克隆，RT-PCR及免疫印迹检测重组多肽P／C-1的表达，MTT法检测表达产物对脾淋巴细胞和腹腔巨噬细胞杀伤活性的影响，建立小鼠H22肝细胞癌移植瘤模型，裸DNA肌肉注射法分别于体内转染sPD-1、CH50和P／C-1基因，观察并比较各处理因素对小鼠的肿瘤生长的抑制作用。结果酶切及测序鉴定结果表明重组多肽P／C-1真核表达载体构建成功，在BHK细胞培养上清中检测到重组多肽P/C-1的表达，后者能显著增强脾淋巴细胞和腹腔巨噬细胞对H22细胞的杀伤作用，体内实验表明，P/C-1转染对肿瘤生长的抑制作用强烈而持久，显著优于sPD-1和CH50治疗组。结论重组多肽P／C-1真核表达载体构建成功，表达产物P／C-1兼有sPD-1和CH50的生物学功能，能够激活巨噬细胞和脾淋巴细胞，发挥二者协同抗肿瘤作用，从而将非特异性和特异性抗肿瘤免疫有机地结合起来，为肿瘤基因治疗提供新的思路。

Regulatory effect of secretory recombinant peptide PD-1-Cell Ⅰ (P/C-1) on the function of immune cells and its anti-tumor effect

Objective To construct a secretory eukaryotic expression vector of recombinant peptide P/C-1 containing two function domains and to investigate the anti-tumor function of P/C-1 expressed in vivo or in vitro by regulating the activity of immune cells. Methods The cDNA encoding PD-1 extracellular domain and CH50 Cell I domain were amplified by PCR, then the PCR products were cloned into secretory eukaryotic expression vector pSecTagA by ligation, so the recombinant plasmid pP/C-1 was constructed. The plasmid pP/C-1 was trans-fected into BHK cells by lipofectin-mediated method, the expression of P/C-1 was identified by RT-PCR and Western blot, and the effect of P/C-1 expressed in vitro on the cytotoxicity of spleen cells and macrophages was measured. After the inoculation of H22 hepatic carcinoma cells, the expression vectors of P/C-1, CH50 and sPD-1 were injected into muscle of mouse, and the tumor growth rate was recorded. Results Restriction enzyme digestion and DNA sequence analysis revealed that the insert fragments were sPD-1 and Cell I coding genes, the expression of P/C-1 by BHK cells was found in culture medium. The recombinant peptide of P/C-1 expressed by BHK cells enhanced the cytotoxicity of spleen cells and macrophages against H22 cells. The result of tumor treatment experiment showed that P/C-1 expressed in vivo could suppress the growth of H22 hepatic carcinoma more effectively than sPD-1 and CH50. Conclusion P/C-1 expressed in vivo could increase the anti-tumor activity of body and the constructed plasmid vector of recombinant peptide P/C-1 may provide a novel method of tumor gene therapy.