建立胰腺癌黏液蛋白核芯肽-连续重复序列核酸疫苗的实验研究

The construction of MUCI-VNTR DNA vaccine for pancreatic cancer

目的构建胰腺癌黏液蛋白核芯肽连续重复序列(MUC1-VNTR)核酸疫苗,研究疫苗所诱生的免疫应答。方法将重组人VNTR基因定向克隆入真核表达载体pcDNA3．1／Myc-his(+) A质粒的多克隆位点中,构建胰腺癌MUC1-VNTR核酸疫苗。C57BL／6(H-2b)小鼠2次免疫2周后取血检测抗VNTR抗体,取脾细胞体外以VNTR合成肽特异刺激培养6 d后进行细胞毒性T淋巴细胞 (CTL)杀伤试验。结果疫苗组小鼠脾细胞CTL的特异性杀伤率(34．8±3．1)％显著高于载体对照组[(9．2±0．8)％,P<0．01]和空白对照组[(6．1±0．6)％,P<0．01]。CTL对未经VNTR合成肽孵育的靶细胞EL4-VNTR杀伤较低(P<0．01)。VU 3C6可抑制CTL对经VNTR合成肽孵育的靶细胞E14-VNTR+的杀伤活性(P<0．01)。疫苗组小鼠血清抗VNTR抗体等效浓度(2324±238)μg/ml 高于载体对照组(1896±533)μg／ml和空白对照组(1736±142)μg／ml,P<0．01。结论本实验构建的胰腺癌MUC1—VNTR核酸疫苗免疫C57BL／6小鼠后可诱生VNTR特异的CTL应答和抗体应答。

Objective To construct MUC1-VNTR DNA vaccine pancreatic cancer. Methods The recombinant gene of VNTR was synthesized and cloned into MCS in the pcDNA3. 1/Myc-his （ ＋ ） A vector, pcDNA3.1-VNTR/Myc-his（ ＋ ） A was injected twice into C57BL/6（ H-2^b） female mice （V group, n=15）. Mice inoculated with either the empty plasmid vector （D group, n = 15） or 0.9% NaCl （NS group, n = 15 ） were used as control. Two weeks later, both humoral and cellular immunity of the mice were studied. Results The recombinant plasmid pcDNA3.1-VNTR/Myc-his （ ＋ ） A encoded the whole exact translation frame region of the pcDNA3. 1/Myc-his （ ＋ ） A vector and the recombinant gene of human VNTR. The transfected COS7 cells expressed transgene products at 48 hours after transfection. Intramuscular delivery of the recombinant plasmid into C57BL/6 mice resulted in more efficient induction of CTL lysis specific against VNTR polypeptide than the D group and the NS group（ P 〈 0.01 ）. Anti-VNTR specific antibodies were found with a higher level in mice inoculated with the vaccine than the other two groups（ P 〈 0. 01 ）. Conclusions The recombinant plasmid pcDNA3.1-VNTR/Myc-his （ ＋ ） A constructed exactly expresses VNTR polypeptide in the transfected mammalian cells and induces VNTR specific CTL and antibodies response.