东部马脑炎病毒E2基因与GM-CSF共表达质粒的构建及其免疫原性初步研究  

Construction of a plasmid co-expressing eastern equine encephalitis virus E2 gene and mouse GM-CSF gene and its immune effects

作　　者：尉继征[1] 林磊[1] 熊伟[1] 李佳明[1] 杨保安[1] 祝庆余[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2007年第1期28-31,53,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：北京市自然科学基金(No.7042051)

摘　　要：目的:利用粒细胞-巨噬细胞集落刺激因子(GM-CSF)作为基因佐剂,提高东部马脑炎病毒E2基因重组质粒的免疫效果。方法:分别扩增E2基因与GM-CSF基因,连接进入含有内部核糖体插入位点(IRES)的真核表达载体中,构建共表达质粒。Lipofectamine2000介导转染真核细胞BHK-21,反转录PCR检测E2和GM-CSF两个基因的转录,Western印迹法和间接免疫荧光法(IFA)检测细胞内E2基因表达产物的抗原性。通过肌肉注射免疫BALB/c小鼠,IFA法测定血清抗体效价,FACS测定免疫小鼠脾细胞中CD4+/CD8+淋巴细胞构成比,初步观察共表达质粒的免疫原性。结果:经酶切鉴定与序列测定证明重组共表达质粒中含有E2和GM-CSF两个基因且序列正确。转染细胞的反转录PCR可见E2和GM-CSF两个基因的转录。Western印迹结果可见转染细胞的裂解液在相对分子质量50000位置有特异性条带。将转染细胞制成荧光抗原片,经IFA检测可见胞浆中出现特异荧光。免疫小鼠的最高血清抗体效价为1∶160,但细胞免疫未观察到明显的提高。结论:构建的共表达质粒pE2-IRES-mGM-CSF具有良好的免疫原性,能有效刺激小鼠特异性体液免疫应答,提高了E2基因重组质粒诱导的血清抗体效价。Objective： To enhance the immune efficacy induced by E2 protein of eastern equine encephalitis virus （EEEV）by using granulocyte-macrephage colony stimulating factor （GM-CSF） as a genic adjuvant. Methods： E2 And mGM-CSF genes were amplified and cloned into eukaryotic expression vector containing internal ribosome entry site （IRES） to construct the plasmid co-expressius the two genes. After the co-expression plasmid pE2-IRES-mGM-CSF was transfected into BHK-21 ceils, E2 and mGM-CSF transcription was detected by RT-PCR. Western blotting and tests were carried out to observe the immunogenicity of expressed E2 protein. Finally, BALB/c mice were vaccinated with the recombinant. Anti-EEEV antibodies from immunized mice sera were detected by immunofluorescence assay and ratio oi CD4^ ＋ to CD8^ ＋ T lymphocytes was measured by FACS. Results： Construction of the recombinant plasmid was proved successful by restriction enzymic degradation and sequencing. Transcription of both E2 and mGM-CSF was detected by RT- PCR. The expressed E2 protein showed good antigenicity. The results showed that the recombinant had considerable immunogenicity, and the E2-specific humoral immune response was greatly enhanced by the co-expression of mGM-CSF, but cellular immune enhancement was not seen obviously. Conclusion：The co-expression plasmid shows good antigenicity of expressed E2 and EEEV-specific humoral immune response can be enhanced by GM-CSF expression.

关 键 词：东部马脑炎病毒 E2基因 粒细胞-巨噬细胞集落刺激因子 共表达 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学]