CAJ | 학술논문

目的：构建GM-CSF和EB病毒LMP2A融合基因的重组腺病毒，对重组腺病毒进行鉴定及免疫学研究。方法：采用RT-PCR及Western blot方法检测重组腺病毒GC2A基因表达，用ELISA方法对vAd-GC2A刺激小鼠产生的抗体水平进检测及分析，乳酸脱氢酶法检测其对小鼠的细胞免疫功能的影响，采用t检验方法对重组腺病毒的免疫效果进行初步分析和评价。结果：PCR扩增重组腺病毒GC2A片段大小为1961 bp，与预期结果一致；蛋白质印迹法检测结果显示，重组腺病毒vAd-GC2A表达的蛋白能被血清GM-CSF抗体（或LMP2A抗体）识别；ELISA检测结果表明，vAd-GC2A能刺激机体产生抗体，随着免疫时间的延长，重组腺病毒刺激机体产生的抗体增长较快；重组腺病毒诱导的CTL对EBV阳性肿瘤细胞的杀伤率为（66.7±6.9）％，而腺病毒5型野毒株和PBS的杀伤率分别为（24.3±2.5）％和（32.4±3.1）％（P≤0.05）。结论：构建的重组腺病毒可表达GC2A基因，vAd-GC2A在小鼠体内可激活体液免疫和细胞免疫功能，有望成为预防和治疗EB病毒阳性肿瘤的新手段。
목적：구건GM-CSF화EB병독LMP2A융합기인적중조선병독，대중조선병독진행감정급면역학연구。방법：채용RT-PCR급Western blot방법검측중조선병독GC2A기인표체，용ELISA방법대vAd-GC2A자격소서산생적항체수평진검측급분석，유산탈경매법검측기대소서적세포면역공능적영향，채용t검험방법대중조선병독적면역효과진행초보분석화평개。결과：PCR확증중조선병독GC2A편단대소위1961 bp，여예기결과일치；단백질인적법검측결과현시，중조선병독vAd-GC2A표체적단백능피혈청GM-CSF항체（혹LMP2A항체）식별；ELISA검측결과표명，vAd-GC2A능자격궤체산생항체，수착면역시간적연장，중조선병독자격궤체산생적항체증장교쾌；중조선병독유도적CTL대EBV양성종류세포적살상솔위（66.7±6.9）％，이선병독5형야독주화PBS적살상솔분별위（24.3±2.5）％화（32.4±3.1）％（P≤0.05）。결론：구건적중조선병독가표체GC2A기인，vAd-GC2A재소서체내가격활체액면역화세포면역공능，유망성위예방화치료EB병독양성종류적신수단。
Objective:To construct GM-CSF and EB virus LMP2A fusion gene recombinant adenovirus and identificate it , using recombinant adenovirus to do immunologic study.Methods:With RT-PCR and Western blot to detect GC 2A gene expression of the recombinant adenovirus , with ELISA to analysis antibodies of vAd-GC2A stimulation in murine body , lactate dehydrogenase assay to detect the mouse cellular immunity effect , thereby the recombinant adenovirus immune effect was initially analysis.T-test method was used to do preliminary analysis and evaluation of the immune effect of recombinant adenovirus .Results:GC2A fragment amplified by PCR, the size was 1 961 bp and it was the expected.The results showed, the recombinant adenovirus vAd-GC2A proteins can be recognized by sera antibody of GM-CSF ( LMP2A); the results of ELISA showed that vAd-GC2A can stimulate the generation of antibodies.EBV-positive tumor cell killing rate (%) by CTL induced recombinant adenovirus was (66.7 ±6.9)%, at the same time the adenovirus type 5 wild strains and PBS were (24.3 ±2.5)%and(32.4 ±3.1)%only(P≤0.05).Conclusion:Fusion gene has been successfully constructed , vAd-GC2A in mice have humoral and cellular immune function.The data suggest that the vAd-GC2A should play a potent role in preventing the positive EB virus tumor.