目的：制备促性腺激素释放激素（gonadotropin releasing hormone，GnRH）与M2的融合蛋白（GnRH/M2），研究由该融合蛋白致敏而成的DC疫苗对黑色素瘤B16F10细胞小鼠移植瘤的抑制作用。方法：构建表达载体pET28a-ansB-C-GnRH3-hinge-MVP-M2质粒，该质粒转化的工程菌在乳糖的诱导下，融合蛋白ansB-C-GnRH3-hinge-MVP-M2以包涵体形式表达，经超声破碎、洗涤和乙醇分级沉淀纯化后，通过酸水解将蛋白多肽GnRH3-hinge-MVP-M2释放出来，并通过DEAE-52阴离子交换层析进行分离。将此融合多肽致敏DC获得DC疫苗。构建黑色素瘤B16F10细胞小鼠移植瘤模型，按接种疫苗不同，分为：环磷酰胺组（CTX）、GnRH/M2融合蛋白致敏DC组（GDC）、肿瘤细胞裂解物致敏DC组（BDC）、GnRH/M2融合蛋白致敏DC+环磷酰胺组（GDCTX）、肿瘤细胞裂解物致敏DC+环磷酰胺组（BDCTX）和生理盐水组（NS），观察GnRH/M2疫苗对模型小鼠的移植瘤生长、CTL杀伤能力和T细胞增殖的作用。结果：成功构建pET28a-ansB-C-GnRH3-hinge-MVP-M2质粒并高效表达融合蛋白。GDC组移植瘤生长明显慢于NS组（P<0.05），且与BDC组相似（P>0.05）；GDCTX组抑瘤效果虽进一步提高，但与CTX组相比差异无统计学意义（P>0.05）。各实验组对B16F10细胞的杀伤作用和对T细胞增殖作用均优于阴性对照组（P<0.05或P<0.01），且GDC组与BDC组间差异不显著（P>0.05）。结论：初步证明融合多肽GnRH/M2致敏的DC疫苗能有效抑制黑色素瘤B16F10细胞小鼠移植瘤的生长。

Objective:To prepare fusion protein of gonadotropin releasing hormone (GnRH) and M2 (GnRH/M2), and investigate inhibitory effect of DC vaccine sensitized with the fusion protein on melanoma B16F10 cell xenografts. Methods: An expression vector of pET28a-ansB-C-GnRH3-hinge-MVP-M2 plasmid was constructed and transferred into engineering bacteria. Under the induction of lactose, fusion protein of ansB-C-GnRH3-hinge-MVP-M2 was expressed as inclusion body in the transferred engineering bacteria. Then the fusion protein was purified by means of ultrasonic broken, washings and ethanol fractionation precipitation. After purification, protein polypeptide GnRH3-hinge-MVP-M2 was released by acid hydrolysis and isolated with DEAE-52 anion exchange chromatography. DCs were sensitized with the fusion polypeptide to obtain DC vaccine. Mouse models with melanoma B16F10 xenografts were established and divided into cyclophosphamide group (CTX), DC sensitized with GnRH/M2 fusion protein group (GDC), DC sensitized with tumor cell lysate group (BDC), DC sensitized with GnRH/M2 fusion protein plus cyclophosphamide group (GDCTX), DC sensitized with tumor cell lysate plus cyclophosphamide group (BDCTX) and normal saline group (NS), according to different vaccination. Effects of GnRH/M2 vaccine on growth of the xenografts, killing ability of CTL and proliferation of T cells in the mouse models were observed. Results: The pET28a-ansB-C-GnRH3-hinge-MVP-M2 plasmid was successfully constructed and the fusion protein efficiently expressed. Growth of the xenografts in GDC group was more slower than that in NS group obviously (P<0.05), and similar to that in BDC group (P>0.05). Although inhibitory effect on tumor in GDCTX group was further increased, but there not was any significant differences comparing with CTX group (P>0.05). Killing effect on B16F10 cells and proliferation effect on T cells in various treatment groups were significantly better than those in negative control groups (P<0.05 or P<0.01), but differences of them between GDC and BDC groups were not obvious (P>0.05). Conclusion: It was preliminarily proved that the DC vaccine sensitized with the fusion polypeptide GnRH/M2 could effectively inhibit growth of the melanoma B16F10 xenografts in the mouse model.

国家自然科学基金资助项目（No.81172973，81373232）；大学生创新药物研制能力提高项目（No.J1030830），江苏高校优势学科建设工程资助项目（PAPD）；江苏省杰出人才引导项目（No. BK20140029）