E3泛素连接酶RNF185对A亚群禽白血病病毒体外复制的影响

doi:10.16431/j.cnki.1671-7236.2022.11.027

摘要/Abstract

摘要： 【目的】环指蛋白185（ring finger protein 185，RNF185）是Ring家族E3泛素连接酶的一员。试验旨在探明鸡源RNF185对A亚群禽白血病病毒（Avian leukemia virus subgroup A，ALV-A）体外复制的影响，为进一步研究鸡源RNF185对ALV-A致病性的影响和介导的致瘤机制提供参考。【方法】参考GenBank中已公布的鸡RNF185基因（登录号：NP_001007841）序列分别设计RNF185过表达引物和RNF185单链shRNA，通过PCR扩增RNF185基因及合成双链shRNA，将其分别克隆至真核表达载体psi-flag和干扰载体pLKO.1-GFP，并使用PCR、双酶切和测序等方法验证重组质粒。使用Western blotting验证重组质粒的表达和干扰效果，并使用CCK-8试剂盒检测重组质粒对DF-1细胞活性的影响。在RNF185过表达和干扰后的DF-1细胞中分别接种10⁴半数组织感染剂量（TCID₅₀） ALV-A （rHB2015012）病毒液，于接种完成后3、4和5 d收取细胞，并使用Western blotting检测ALV-A的病毒载量。【结果】 PCR成功扩增出RNF185基因，单链shRNA经退火后形成双链shRNA，并成功构建重组质粒psi-flag-RNF185、pLKO.1-RNF185-1和pLKO.1-RNF185-2。Western blotting结果表明，构建的过表达和干扰质粒能够在DF-1细胞中稳定表达和干扰RNF185。CCK-8结果表明，过表达和干扰RNF185不影响DF-1细胞的活性。RNF185过表达可以促进ALV-A在DF-1细胞中的复制，干扰RNF185表达可以抑制ALV-A在DF-1细胞中的复制。【结论】本研究构建了可以在DF-1细胞中高效表达和干扰RNF185的过表达质粒和干扰质粒，RNF185对DF-1细胞增殖无明显影响，能够促进ALV-A （rHB2015012）在体外的复制。

关键词: 环指蛋白185(RNF185); A亚群禽白血病病毒(ALV-A); 病毒复制

Abstract: 【Objective】 Ring finger protein 185 (RNF185) is a member of Ring family E3 ubiquitin ligases.This study was aimed to explore the effect of chicken-derived RNF185 on the replication of Avian leukemia virus subgroup A (ALV-A) in vitro, and study the effect of chicken-derived RNF185 on the pathogenicity and mediated tumorigenic mechanism of ALV-A.【Method】 Referring to the sequence of RNF185 gene in chicken (accession No.:NP_001007841) published in GenBank, RNF185 overexpression primers and RNF185 single-stranded shRNA were designed, respectively.The RNF185 gene and double-stranded shRNA were synthesized by PCR and cloned into eukaryotic expression vectors psi-flag and interference vector pLKO.1-GFP, respectively.The constructed recombinant plasmids were verified by PCR, double restriction digestion and sequencing, and the CCK-8 kit was used to detect the effect of the recombinant plasmid on the viability of DF-1 cells.10⁴ tissue culture infective dose 50% (TCID₅₀) ALV-A (rHB2015012) virus solution was inoculated into DF-1 cells after RNF185 overexpression and interference, respectively, the cells were harvested at 3, 4 and 5 d after inoculation, and the viral load of ALV-A was detected by Western blotting.【Result】 The RNF185 gene was successfully amplified by PCR and the single-stranded shRNA was annealed to form double-stranded shRNA.The recombinant plasmids psi-flag-RNF185, pLKO.1-RNF185-1 and pLKO.1-RNF185-2 were successfully constructed.Western blotting results showed that the constructed overexpression and interference plasmid could stably express and interfere with RNF185 in DF-1 cells.The CCK-8 results indicated that overexpression and interference of RNF185 did not affect the activity of DF-1 cells.Overexpression of RNF185 could promote the replication of ALV-A in DF-1 cells, and interfere with the expression of RNF185 could inhibit the replication of ALV-A in DF-1 cells.【Conclusion】 This study constructed an overexpression plasmid and an interference plasmid that could highly express and interfere with RNF185 in DF-1 cells.RNF185 had no significant effect on the proliferation of DF-1 cells, confirming that RNF185 could promote the replication of ALV-A (rHB2015012) in vitro.

Key words: ring finger protein 185 (RNF185); Avian leukemia virus subgroup A (ALV-A); virus replication

中图分类号:

S852.65⁺7

刘晶, 陈雪阳, 曾扬, 杨玉莹. E3泛素连接酶RNF185对A亚群禽白血病病毒体外复制的影响[J]. 中国畜牧兽医, 2022, 49(11): 4364-4371.

LIU Jing, CHEN Xueyang, ZENG Yang, YANG Yuying. Effect of E3 Ubiquitin Ligase RNF185 on Replication of Avian Leukemia Virus Subgroup A in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4364-4371.

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