CAJ | 학술논문

目的对融合的粒细胞-巨噬细胞集落刺激因子（ gramulocyte-macrophase colony-stimu-latingfactor， GM-CSF）基因与EB病毒（Epstein-Barr virus）基因LMP2A进行鉴定并对rBCG进行免疫学特性研究。方法用免疫印迹法（ Western blot ）检测rBCG中融合基因表达；将表达融合基因的rBCG注射小鼠，用酶联免疫吸附试验（ ELISA）检测血清特异性抗体表达水平，乳酸脱氢酶法检测细胞免疫功能，观察rBCG的作用；建立小鼠EBV阳性的肿瘤模型并观察rBCG的治疗效果。结果 rBCG能分泌性表达目的蛋白，该目的蛋白能被GM-CSF抗体及LMP2 A抗体识别；ELISA结果显示rBCG能刺激小鼠产生相应抗体，注射小鼠的细菌数量为5×108个时，抗体最大滴度为1∶27900[（326．5±7．8） pg/ml]；rBCG组对EBV阳性胃癌细胞（GT39细胞）的杀伤率为（89．6±6．8）％，显著大于对照组（P＜0．05）。 PBS及BCG对照组的肿瘤体积明显大于rBCG治疗组，3组肿瘤体积分别为（1964．0±548．7） mm3、（1268．7±72．4） mm3、（168．6±78．8） mm3。结论 GM-CSF与LMP2A融合基因重组卡介苗成功表达目的蛋白，免疫小鼠后产生体液免疫和细胞免疫；rBCG能有效抑制小鼠体内的肿瘤生长。
목적대융합적립세포-거서세포집락자격인자（ gramulocyte-macrophase colony-stimu-latingfactor， GM-CSF）기인여EB병독（Epstein-Barr virus）기인LMP2A진행감정병대rBCG진행면역학특성연구。방법용면역인적법（ Western blot ）검측rBCG중융합기인표체；장표체융합기인적rBCG주사소서，용매련면역흡부시험（ ELISA）검측혈청특이성항체표체수평，유산탈경매법검측세포면역공능，관찰rBCG적작용；건립소서EBV양성적종류모형병관찰rBCG적치료효과。결과 rBCG능분비성표체목적단백，해목적단백능피GM-CSF항체급LMP2 A항체식별；ELISA결과현시rBCG능자격소서산생상응항체，주사소서적세균수량위5×108개시，항체최대적도위1∶27900[（326．5±7．8） pg/ml]；rBCG조대EBV양성위암세포（GT39세포）적살상솔위（89．6±6．8）％，현저대우대조조（P＜0．05）。 PBS급BCG대조조적종류체적명현대우rBCG치료조，3조종류체적분별위（1964．0±548．7） mm3、（1268．7±72．4） mm3、（168．6±78．8） mm3。결론 GM-CSF여LMP2A융합기인중조잡개묘성공표체목적단백，면역소서후산생체액면역화세포면역；rBCG능유효억제소서체내적종류생장。
Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .