鸡c-myc原癌基因反义RNA的鉴定及其抗ALV-J能力的分析

Identification of chicken c-myc proto-oncogene antisense RNA and analysis of its anti-ALV-J ability

原癌基因c-myc被鉴定为J亚群禽白血病病毒(ALV-J)诱导髓样细胞瘤的常见整合位点,其异常激活可能是ALV-J最重要的分子致病机制。为了鉴定c-myc反义RNA对c-myc基因表达的影响及其在ALV-J增殖中的作用,本研究通过cDNA末端快速扩增(RACE)技术、PCR扩增5’-race和3’-race后对其编码属性的分析鉴定结果显示,鸡c-myc原癌基因反义RNA全长621 bp,且不编码蛋白;该反义RNA序列定位于鸡2号染色体,且源自c-myc外显子3反向序列,因此将其命名为ch-MYC-AS1。将ch-MYC-AS1连接载体pcDNA3.1,构建真核表达质粒pcDNA3.1-ch-MYC-AS1,将其转染至鸡巨噬细胞细胞系HD11,培养48 h后利用荧光定量PCR和western blot检测ch-MYC-AS1过表达对c-myc表达的影响,结果显示,过表达ch-MYC-AS1可以显著抑制c-myc原癌基因在HD11中的表达。以MOI 5的ALV-J(JS09GY3株)感染HD11细胞4 h后,将pcDNA3.1-ch-MYC-AS1质粒转染该细胞,48 h后采用IDEXX禽白血病抗原检测试剂盒检测细胞上清中ALV-J p27蛋白的表达水平,结果显示过表达ch-MYC-S1显著减少细胞上清中ALV-J p27蛋白的表达;进一步通过荧光定量PCR、western blot检测过表达ch-MYC-AS1对ALV-J复制的影响,结果显示,ALV-J env基因mRNA转录水平和蛋白表达水平在ch-MYC-AS1转染后48 h的HD11细胞中均显著下调(P<0.01);利用间接免疫荧光技术检测DF-1细胞上清中ALV-J的病毒效价,结果显示:与对照组相比,HD11细胞上清中ALV-J的病毒效价在ch-MYC-AS1转染后48 h明显降低。上述结果表明过表达ch-MYC-AS1可以显著抑制ALV-J在HD11细胞中的增殖。本研究首次揭示了原癌基因c-myc反义RNA的抗病毒功能,为进一步探究c-myc基因在ALV-J致病机理中的作用提供了参考依据。

The c-myc proto-oncogene is identified as a common integration site for avian leukemia virus subgroup J-induced myeloid cell tumors,and its abnormal activation may be the most important molecular pathogenic mechanism of ALV-J.In order to identify the effect of c-myc antisense RNA on the expression of c-myc gene and its role in the proliferation of ALV-J,this study used the rapid amplification of cDNA ends(RACE) technology,PCR amplification and coding attribute analysis,and the analysis and identification results showed that the full-length sequence of the chicken c-myc proto-oncogene antisense RNA was 621 bp and does not encode protein;this antisense RNA sequence is located on chicken chromosome 2 and derived from the antisense sequence of proto-oncogene exon 3,and thus named as ch-MYC-AS1.The eukaryotic expression plasmid pcDNA3.1-ch-MYCAS1 was constructed by cloning ch-MYC-AS1 into vector pcDNA3.1 and transfected into chicken macrophage cell line HD11.After 48 hours of cell culture,fluorescence quantitative PCR and western blot technologies were used to detect the influence of ch-MYC-AS1 overexpression on the expression of c-myc proto-oncogene.The results showed that overexpression of ch-MYC-AS1 significantly inhibited the expression of c-myc proto-oncogene in HD11.HD11 cells were infected with ALV-J(strain JS09 GY3) with MOI 5 for 4 hours,and then transfected with pcDNA3.1-ch-MYC-AS1 plasmid.The influence of ch-MYC-AS1 overexpression on ALV-J replication was detected by fluorescence quantitative PCR,western blot,ELISA and indirect immunofluorescence experiments after 48 hours of transfection.The results showed that overexpression of ch-MYC-AS1 significantly inhibited the ALV-J proliferation in HD11 cells.This study for the first time reveals the antiviral function of the proto-oncogene c-myc antisense RNA,and provides a reference for further exploring the role of c-myc gene in the pathogenic mechanism of ALV-J.