禽内源性反转录病毒ALVE表观遗传学分析方法的建立及应用  

DEVELOPMENT AND APPLICATION OF ANALYTICAL METHODS FOR THE EPIGENETIC ALTERATIONS OF AVIAN ENDOGENOUS RETROVIRUS ALVE

作　　者：朱文奇 胡序明 陈世豪 刘洋洋 孙振 耿拓宇 宋成义 高波 王宵燕 秦爱建 崔恒宓 

机构地区：[1]扬州大学表观遗传学及表观基因组学研究所,扬州225009 [2]扬州大学教育部禽类预防医学重点实验室,扬州225009 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国动物传染病学报》2015年第5期15-20,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家973项目(2012CB517605);国家自然科学基金项目(81171965;81372237;91540117);江苏省畜牧学优势学科项目;中国博士后科学基金第57批面上资助项目(2015M571828)

摘　　要：根据NCBI公布的禽内源性反转录病毒ALVE1序列设计引物,以SPF鸡胚制备的成纤维细胞为生物材料,建立了ALVE基因序列分析、甲基化水平和转录表达检测方法。以鸡胚成纤维细胞(chicken embryo fibroblast cell,CEF)和巨噬细胞系(HD11)两种细胞的基因组为模板,通过常规PCR扩增获得了1912 bp的扩增产物,测序后与内源性ALVE1序列比对,核苷酸同源性高达98%以上,说明扩增产物为内源性病毒序列。同时,利用焦磷酸测序分析LTR片段甲基化水平,结果显示,LTR在非免疫细胞CEF中的甲基化水平显著高于巨噬细胞系HD11。利用常规RT-PCR和荧光定量PCR在两种细胞均检测到禽内源性反转录病毒LTR基因、gag基因、pol基因和env基因的转录表达。本研究为今后分析禽内源性反转录病毒ALVE功能提供了方法,同时也为深入研究禽内源性反转录病毒ALVE表观遗传学调控机制奠定了基础。In the present study, methods for analysis of genomic sequence, evaluation of DNA methylation and expression level of Avian endogenous retroviruses ALVE1 were established using chicken embryo fibroblast（CEF） cells and published NCBI information. A product of 1912 bp was amplified from CEF and avian macrophage（HD11） genomes using conventional PCR. The results showed that their sequences shared at least 98% homology to Avian endogenous retroviruses ALVE1, indicating that the amplification product was endogenous viral sequences. In addition, the expression of endogenous viral LTR, gag, pol and env genes were detected in CEF and HD11 cells using conventional RT-PCR and RT-q PCR. Methylation levels of LTR gene in CEF cells were significantly higher than those in HD11. The establishment of methods for analyzing the epigenetic pattern of Avian endogenous retrovirus ALVE would benefit to further research of its regulatory mechanisms.

关 键 词：禽内源性反转录病毒 DNA甲基化 表观遗传学 

分 类 号：S852.659.3[农业科学—基础兽医学]