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目的构建携带融合蛋白基因PSA-IZ-mCD40L(PmL)和GM-CSF基因的重组质粒载体pUDK-PmL-IR-GM,并评价其小鼠体内免疫激活作用。方法采用类似基因合成的方法将前列腺特异性抗原基因、异亮氨酸拉链基因和鼠源性CD40配体基因连接并扩增获得融合蛋白基因PmL。将GM-CSF、PmL和核糖体内部进入位点基因依次克隆到pUDK载体,获得重组质粒pUDK-PmL-IR-GM。分别于第1、11、21和35天免疫小鼠,末次免疫后10d分离小鼠脾脏T淋巴细胞。采用流式细胞术检测T淋巴细胞亚群;MTT法检测前列腺特异性抗原刺激下的T淋巴细胞增殖效应;非放射性细胞杀伤检测试剂盒检测对LNCaP细胞的特异性杀伤作用。结果 PCR成功扩增GM-CSF基因和融合蛋白基因PmL,并将其克隆至pUDK载体上,以核糖体内部进入位点连接,得到重组质粒pUDK-PmL-IR-GM。pUDK-PmL-IR-GM免疫小鼠后,T淋巴细胞性能检测显示:CD4+T淋巴细胞与CD8+T淋巴细胞的比值较对照组明显升高(P<0.05);前列腺特异性抗原刺激后,其增殖能力明显增强(与阴性对照相比,P<0.01),且明显高于其他免疫组(P<0.01);其对LNCaP细胞杀伤效率大于25%,且明显强于pUDK免疫组(P<0.05)。结论成功构建重组质粒载体pUDK-PmL-IR-GM,并证实其能在一定程度激活小鼠T淋巴细胞的特异性免疫反应。
Objective To construct a recombinant plasmid vector pUDK-PmL-IR-GM carrying the fusion protein gene PSA-IZ-mCD40L (PmL) and GM-CSF gene and evaluate its immune activation in vivo. Methods A similar gene synthesis method was used to ligate the prostate specific antigen gene, isoleucine zipper gene and murine CD40 ligand gene to obtain the fusion protein gene PmL. The genes of GM-CSF, PmL and ribosome internal access site were cloned into pUDK vector in turn, and the recombinant plasmid pUDK-PmL-IR-GM was obtained. Mice were immunized on days 1, 11, 21 and 35, respectively, and spleen T lymphocytes were isolated 10 days after the last immunization. The T lymphocyte subsets were detected by flow cytometry. The proliferation of T lymphocytes stimulated by prostate specific antigen was detected by MTT assay. The cytotoxicity of T lymphocytes was detected by non-radioactive cell killing assay kit. Results The recombinant plasmid pUDK-PmL-IR-GM was successfully amplified by PCR. The recombinant plasmid pUDK-PmL-IR-GM was cloned into pUDK vector and ligated into the internal site of ribosome. After the mice were immunized with pUDK-PmL-IR-GM, the T lymphocyte function test showed that the ratio of CD4 + T lymphocytes to CD8 + T lymphocytes was significantly higher than that of the control group (P <0.05). After stimulation with prostate specific antigen (P <0.01), which was significantly higher than that of other immunized groups (P <0.01). The cytotoxicity of LNCaP cells was more than 25%, which was significantly higher than that of pUDK immunized group <0.05). Conclusion The recombinant plasmid vector pUDK-PmL-IR-GM was successfully constructed and proved to activate the specific immune response of mouse T lymphocytes to some extent.