E3泛素连接酶FBXL8对A亚群禽白血病病毒复制的影响

Effect of E3 Ubiquitin Ligase FBXL8 on the Replication of Avian Leukosis Virus Subgroup A

【目的】制备鸡E3泛素连接酶FBXL8多克隆抗体,探究鸡源FBXL8对A亚群禽白血病病毒(Avian leukosis virus subgroup A,ALV-A)在鸡胚成纤维细胞(DF-1)内复制的影响。【方法】以DF-1细胞cDNA为模板,通过PCR扩增FBXL 8基因,构建鸡源FBXL8原核表达重组质粒pET-28a-FBXL8,将其转化大肠杆菌表达菌株RIL进行诱导表达及纯化,将表达产物His-FBXL8免疫10月龄新西兰大白兔制备多克隆抗体,利用Western blotting和间接ELISA分别检测抗体特异性及效价;针对已公布的FBXL 8基因序列设计过表达引物及靶向FBXL 8基因的干扰序列,构建过表达质粒psi-flag-FBXL8及干扰质粒pLKO.1-FBXL8-shRNA,使用Western blotting检测其表达效果。将构建成功的过表达质粒和干扰质粒转染DF-1细胞,24 h后感染ALV-A,通过半定量PCR、Western blotting分别检测ALV-A的结构蛋白P27、GP85表达水平。【结果】PCR成功扩增出大小为1182 bp的FBXL 8基因片段,并成功构建FBXL8原核表达重组质粒pET-28a-FBXL8,诱导表达的融合蛋白His-FBXL8大小为43 ku。Western blotting和间接ELISA结果显示,制备的FBXL8兔多克隆抗体可特异性识别外源蛋白,且抗体效价为1∶64000。成功构建了FBXL8过表达质粒和干扰质粒,其在DF-1细胞中能够实现对FBXL8的过表达和敲低。半定量PCR和Western blotting检测结果显示,过表达FBXL8可抑制ALV-A复制,而敲低FBXL8则促进其复制。【结论】本研究制备的FBXL8多克隆抗体具有良好特异性及反应性;在DF-1细胞中,FBXL8负向调控ALV-A复制,研究结果为进一步深入探究FBXL8生物学特性提供了重要参考,并为揭示FBXL8抗ALV-A感染分子机制奠定了基础。

【Objective】The objective of this study was to prepare chicken E3 ubiquitin ligase FBXL8 polyclonal antibody and to investigate the impact of chicken-derived FBXL8 on the replication of Avian leukosis virus subgroup A(ALV-A)in chicken embryonic fibroblasts(DF-1).【Method】The FBXL 8 gene was amplified by PCR using DF-1 cell cDNA as a template,followed by construction of the recombinant plasmid pET-28a-FBXL8.The recombinant plasmid pET-28a-FBXL8 was transformed into E.coli expression strain RIL for induction and purification.The recombinant protein His-FBXL8 was utilized to immunize 10-month-old New Zealand White rabbits in order to prepare polyclonal antibodies.Western blotting and indirect ELISA were used to identify the immunogenicity and titer of the polyclonal antibody.Overexpression primers and interference sequences targeting FBXL 8 gene were designed according to the published FBXL 8 gene sequence.Furthermore,the overexpression plasmid psi-flag-FBXL8 and interference plasmid pLKO.1-FBXL8-shRNA were constructed.Western blotting was used to detect the expression effect.Successful overexpression plasmid and interference plasmid were constructed to transfect DF-1 cells,followed by infection with ALV-A after 24 hours.The expression levels of structural proteins P27 and GP85 of ALV-A were detected by semi-quantitative PCR and Western blotting.【Result】The FBXL 8 gene fragment with the size of 1182 bp was successfully amplified by PCR,and the prokaryotic expression recombinant plasmid pET-28a-FBXL8 was successfully constructed.The induced expression fusion protein His-FBXL8 was 43 ku.Western blotting and indirect ELISA results showed that the prepared rabbit polyclonal antibody of FBXL8 could specifically recognize foreign protein,and the titer of the antibody was 1∶64000.FBXL8 overexpression plasmid and interference plasmid were successfully constructed,which could effectively overexpress and knock down FBXL8 in DF-1 cells.Semi-quantitative PCR and Western blotting results showed that overexpression of FBXL8 inhibited the replication of ALV-A,while knockdown of FBXL8 promoted its replication.【Conclusion】The FBXL8 polyclonal antibody had good specificity and reactivity.In DF-1 cells,FBXL8 negatively regulated the replication of ALV-A.The results provided an important reference for further exploring the biological characteristics of FBXL8,and laid a foundation for revealing the molecular mechanism of FBXL8 against ALV-A infection.