鸡SAMD9L基因真核表达载体构建及其对ALV-J病毒复制的影响

Construction of eukaryotic expression vector of chicken SAMD9L gene and its effect on ALV-J replication

为分析鸡无菌α基序域蛋白9样(SAMD9L)蛋白的生物学特性,构建SAMD9L基因真核表达载体,并探究其对J亚型禽白血病病毒(ALV-J)复制的影响。利用ProtParam、Uniport、TMHMM和String等在线网站分析SAMD9L序列,获得亲疏水性、保守结构域、信号肽表达及互作蛋白等生物信息。构建携带His标签的SAMD9L重组表达载体并进行测序鉴定。采用qRT-PCR技术检测ALV-J感染对SAMD9L表达水平的影响;采用Western blot技术检测过表达SAMD9L对ALV-J复制的影响。结果表明:鸡SAMD9L无信号肽表达,无跨膜结构域,含有无菌α基序超家族保守结域(55—249位氨基酸),其互作蛋白预测为MX1、DDX60、ISG12-2和IFIT5等天然免疫抗病毒蛋白质。ALV-J感染36 h后显著诱导SAMD9L表达(P<0.001),而过表达SAMD9L则显著抑制ALV-J在DF-1细胞中的复制,推测鸡SAMD9L具有抗ALV-J病毒的能力。该研究成功构建了鸡SAMD9L基因真核表达载体,通过生物信息学分析了蛋白质特性,并初步探究SAMD9L与ALV-J感染之间的关系,为进一步阐明鸡SAMD9L的生物学功能及其抗J亚型禽白血病的分子机制奠定了基础。

The purpose of this study is to analyze the protein characteristics of SAMD9L and its interacting proteins through bioinformatics,construct a tag-fused eukaryotic expression vector,and analyze the effect of SAMD9L on ALV-J replication.By using the online websites including ProtParam,Uniport,TMHMM and String,we obtained the hydrophilicity,conserved domains,signal peptide expression,and interacting proteins of SAMD9L.Subsequently,the SAMD9L recombinant expression vector carrying the His tag was constructed and subjected to sequencing identification.qRT-PCR was used to analyse the expression level of SAMD9L in DF-1 cell infected with ALV-J at an interval time;Western blotting was used to analyze the protein expression level of ALV-J Env in the cells overexpressing of SAMD9L.The results showed that chicken SAMD9L was a stable hydrophilic protein without any signal peptide and transmembrane domain.It also contained a sterileαmotif(55-249 amino acids),and its interacting proteins mainly included some antiviral proteins such as MX1,DDX60,ISG12-2,and IFIT5.Our results show that ALV-J infection significantly induced the expression of SAMD9L at 36 h post-infection.Meanwhile,the overexpression of SAMD9L significantly inhibited the replication of ALV-J in DF-1 cells.In conclusion,the chicken SAMD9L recombinant vector constructed in this study can be expressed in chicken cells and its expression exhibits a role in inhibiting ALV-J replication.This study will provide the foundation for further studying the biological effects of SAMD9L and its resistance to ALV-J in chicken.