人MUC1重复序列与GM—CSF融合表达的重组卡介苗疫苗对乳腺癌生长的抑制作用

Inhibitory effects of recombinant bacillus Calmette-Guerin vaccines coexpressing tandem repeats of MUC1 and GM-CSF on the growth of breast cancer

目的构建一种新的基于人MUCl重复序列与GM—CSF融合表达的重组卡介苗疫苗，并观察其对乳腺癌生长的预防性抑制作用。方法分步克隆人MUC1重复序列1、4、8串联体基因（MVNTR1／4／8），构建人MUC1重复序列串联体与GM—CSF基因融合的大肠一分枝杆菌表达载体pDE22-MVNTR1／4／8-CSF，酶切鉴定及序列分析后，将构建的穿梭质粒电穿孔转染卡介苗，构建重组卡介苗疫苗rBCG．MVNTR1／4／8-CSF，SDS—PAGE和Western Blot检测MVNTR与GM-CSF融合蛋白的表达。在hu。PBL．SCID鼠模型评价其对乳腺癌生长的抑制作用，并通过免疫组化染色检测其免疫诱导的T细胞反应。结果SDS—PAGE和Western Blot结果说明：人MUC1重复序列与GM．CSF基因融合的重组卡介苗疫苗分别有MVNTR1／4／8与GM—CSF融合蛋白的表达。rBCG—MVNTR4／8-CSF免疫组在MCF-7乳腺癌细胞接种42d后，肿瘤成瘤率分别为37．5％和25％，而PBS和BCG—pDE22对照组均为100％（P〈0．05）。与对照组相比，rBCG—MVNTR4／8-CSF预防接种显著抑制MCF-7乳腺癌细胞生长（P〈0．01），且生长抑制作用随着VNTR的增加而增强。肿瘤细胞接种70d后，rBCG—MVNTR4／8-CSF组小鼠生存率分别为75％和87．5％，而BCG—pDE22对照组的生存率为12．5％（P〈0．05）。rBCG—MVNTR4／8．CSF免疫组瘤体组织中可见CD4’和CD8’T淋巴细胞浸润。结论重组卡介苗疫苗rBCG—MVNTR4／8-CSF预防接种可显著抑制乳腺癌生长。

Objective To construct a recombinant bacillus Calmette-Gu6rin（BCG） vaccines based on different tandem repeats of MUC1 and GM-CSF, rBCG-MVNTR1/4/8-CSF, and to observe the ability of three recombinant BCG vaccines in the inhibition of breast cancer. Methods After MUC1 variable-number tandem repeats （MVNTR1/4/8） were cloned in a stepwise manner, the E. coli-Mycobacteria shuttle expression vector pDE22-MVNTR1/4/8-CSF were constructed by fusing MVNTR1/4/8 and GM-CSF, and then used to transform competent BCG by electroporation after identification by restriction endonuclease digestion analysis and DNA sequencing. A novel breast cancer vaccines, rBCG-MVNTR1/4/8-CSF was constructed. The expression of fused MVNTR1/4/8-CSF protiens was analyzed by SDS-PAGE and Western blot. The ability of rBCG vaccines inhibiting the growth of breast cancer was observed in hu-PBL-SCID mice. The specific T cell responses in mice were assessed by immunohistochemistry. Results The expression of recombinant MVNTR1/4/8-CSF fusion proteins were detected by SDS-PAGE and Western Blot in rBCG-MVNTR1/4/8-CSF vaccines, respectively. Tumor incidence in mice prophylactic immunized with rBCG-MVNTR4-CSF （37. 5% ） or rBCG-MVNTR8-CSF （25%） significantly decreased compared with PBS and BCG-pDE22 control （ 100% ） at 42 days after tumor implantation （P 〈 0. 05 ）. MCF-7 tumor growth inhibition in rBCG- MVNTR4/8-CSF-immunized mice was more significant than that in controls （P 〈 0.01 ）.The inhibition effect of three rBCG vaccines on breast tumor growth appeared to rise with increase of numbers of the tandem repeats of MUC1. Survival rate was 75% of mice in the rBCG-MVNTR4-CSF-treated group and 87. 5% of mice in the rBCG-MVNTR8-CSF-treated groups at 70 days after tumor implantation; however, survival rate was only 12. 5% in control group（ P 〈 0. 05）. The CIM-positive and CDS-positive lymphocytes were detected only in rBCG-MVNTR4/8-CSF-immunized mice. Conclusions rBCG-MVNTR4/8-CSF immunization inhibits breast cancer growth in mice.