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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p ET28a-ans B-CGnRH3-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ans B-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p ET28a-ans B-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 a GnRH / M2 fusion protein of GnRH and M2 and study the inhibitory effect of DC vaccine primed by the fusion protein on the transplanted tumor of melanoma B16F10 mice . METHODS: The plasmid p ET28a-ans B-CGnRH3-hinge-MVP-M2 was constructed. The engineered plasmids transformed by this plasmid were expressed in inclusion bodies under the induction of lactose by fusion protein ans BC-GnRH3-hinge- After purification by sonication, washing and ethanol fractional precipitation, the protein polypeptide GnRH3-hinge-MVP-M2 was released by acid hydrolysis and was isolated by DEAE-52 anion exchange chromatography. DC was induced by sensitizing DC with this fusion polypeptide. According to the vaccination, CTX, GDC, GDC, DCC (BDC-sensitized DC) group were injected into B16F10 mice transplanted tumor model. ), GnRH / M2 fusion protein sensitized DC + cyclophosphamide group (GDCTX), tumor cell lysate sensitized DC + cyclophosphamide group (BDCTX) and saline group (NS) Transplanted tumor growth, CTL killing ability and T cell proliferation. Results: The p ET28a-ans B-C-GnRH3-hinge-MVP-M2 plasmid was successfully constructed and the fusion protein was highly expressed. The growth of tumor in GDC group was significantly slower than that in NS group (P <0.05), and similar to that in BDC group (P> 0.05). The tumor inhibitory effect in GDCTX group was further improved, but there was no significant difference compared with CTX group (P> 0.05). The killing effect of all experimental groups on B16F10 cells and the proliferation of T cells were better than the negative control group (P <0.05 or P <0.01), and there was no significant difference between GDC group and BDC group (P> 0.05). Conclusion: The DC vaccine primed by GnRH / M2 fusion polypeptide can effectively inhibit the growth of transplanted tumor of B16F10 melanoma in mice.