GM-CSF和EB病毒LMP2 A融合基因的表达及抗肿瘤特性研究

Expression and anti-tumor characterization of fusion gene GM-CSF and LMP2A of EB virus gene

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1 961 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。

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.