纳尔逊湾正呼肠孤病毒非结构蛋白μNS和σNS表达特性的分析

doi:10.13481/j.1671-587X.20210416

摘要/Abstract

摘要： 目的

检测非结构蛋白μNS和σNS之间的相互作用及其结合区域，阐明纳尔逊湾正呼肠孤病毒（NBV）的致病机制。

方法

采用Lipo3000转染试剂将pCAG M3和pEFHAσNS质粒分别和共同转染至BHK细胞中，免疫荧光法检测μNS和σNS蛋白在细胞中的定位和分布；用NBV感染BHK细胞，采用Western blotting法检测μNS和σNS蛋白在感染细胞中的表达。构建σNS蛋白N末端10~60个氨基酸（aa）残基缺失的质粒，免疫荧光法检测μNS和σNS蛋白在细胞内共定位的结合区域，酵母双杂交实验验证μNS和σNS蛋白相互作用的结合区域。

结果

亚细胞定位显示，NBV的μNS蛋白在无其他病毒蛋白的情况下会形成包涵体结构，而σNS蛋白弥散地分布在整个细胞质中。Western blotting检测证实μNS和σNS蛋白在感染细胞中表达。采用偶联抗体进行免疫染色，共聚焦显微镜下观察到μNS和σNS蛋白共定位于细胞质中。利用酵母双杂交实验检测到与μNS蛋白相互作用的σNS蛋白的基本区域位于σNS蛋白 N末端区域的60个aa残基区域内。

结论

NBV自噬相关蛋白σNS蛋白可以通过与μNS蛋白相互作用而共同表达于病毒包涵体中，σNS蛋白与μNS蛋白的结合区域位于σNS蛋白N末端的60个aa残基区域内。

关键词: 纳尔逊湾正呼肠孤病毒, 蛋白互作, 非结构蛋白, 免疫荧光法, 酵母双杂交实验

Abstract: Objective

To detect the interaction between non-structural proteins μNS and σNS and their binding regions，and to clarify the pathogenic mechanism of Nelson Bay orthoreovirus （NBV）.

Methods

The pCAG M3 and pEFHAσNS plasmids were transfected into the BHK cells separately and co-transfected with Lipo3000 transfection reagent. Immunofluorescence method was used to detect the expressions and location of μNS and σNS proteins in the cells，NBV was used to infect the BHK cells， and Western blotting method was used to detect the expressions of μNS and σNS proteins in the transfected cells.A plasmid with 10－60 amino acid（aa） residues deleted from the N-terminal of σNS protein was constructed.Immunofluorescence method was used to detect the co-localized binding region of μNS and σNS proteins in the cells. Yeast two-hybrid experiment was used to verify the binding region of μNS and σNS protein interaction.

Results

The subcellular location results showed that the μNS protein of NBV formed an inclusion body structure in the absence of other viral proteins， while the σNS protein was diffusely distributed throughout the cytoplasm.The Western blotting results confirmed that μNS and σNS proteins were expressed in the infected cells. Immunostaining was carried out with the conjugated antibody. It was observed under confocal microscope that the μNS and σNS proteins co-localized in the cytoplasm. The results of yeast two-hybrid experiment showed that the basic region of the σNS protein interacting with the μNS protein was located at 60 aa in the N-terminal region of the σNS protein within the residue region.

Conclusion

NBV autophagy-related protein σNS can be co-expressed in the viral inclusion bodies by interacting with μNS protein. The binding region of σNS and μNS protein is located within the 60 aa residue region of the N-terminal of σNS protein.

Key words: Nelson Bay orthoreoviruses, protein interaction, non-structural protein, immunofluorescence, yeast two-hybrid experiment

中图分类号:

R373

李藤菲,王诗雨,蒋欣如,孙淼,李婷婷,林家锋,王颖,李永刚,陶晓莉. 纳尔逊湾正呼肠孤病毒非结构蛋白μNS和σNS表达特性的分析[J]. 吉林大学学报(医学版), 2021, 47(4): 934-942.

Tengfei LI,Shiyu WANG,Xinru JIANG,Miao SUN,Tingting LI,Jiafeng LIN,Ying WANG,Yonggang LI,Xiaoli TAO. Analysis on expression characteristics of non-structural protein μNS and σNS of Nelson Bay orthoreovirus[J]. Journal of Jilin University(Medicine Edition), 2021, 47(4): 934-942.

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Primer	Sequence
pEFHAσNS	F 5'-CGCCGAATTCGGATCaAATGCGTGTCGGTGTCTCGAGAAA-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N10	F 5'-CGCCGAATTCGGATCaAGGTACTGGGTCTCAGACCATACTAC-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N20	F 5'-CGCCGAATTCGGATCaATTTGCTCTTTTACGTTGTGTACTATCAGC-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N30	F 5'-CGCCGAATTCGGATCaAGATTCACGTCAAGCTACTCGCGCT-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N40	F 5'-CGCCGAATTCGGATCaATCTCATTTCCCAGGTTTGGGACGC-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N50	F 5'-CGCCGAATTCGGATCaAAAATGTCTGTCACCGCTGACTGCC-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
pEFHAσNS△N60	F 5'-CGCCGAATTCGGATCaAGCAGACCGTACTGGTCCGCCACGTT-3' R 5'-CGACATCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'

Primer	Sequence
μNS	F 5'-GGATTCCCGGGGATCaAATGCGTGTCGGTGTCTCGAGAAA-3' R 5'-GCAGGTCTACGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
σNS	F 5'-ATCGATAGGGATCCaAATGCGTGTCGGTGTCTCGAGAAA-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-10aa	F 5'-ATCGATAGGGATCCaAGGTACTGGGTCTCAGACCATACTAC-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-20aa	F 5'-ATCGATAGGGATCCaATTTGCTCTTTTACGTTGTGTACTATCAGC-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-30aa	F 5'-ATCGATAGGGATCCaAGATTCACGTCAAGCTACTCGCGCT-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-40aa	F 5'-ATCGATAGGGATCCaATCTCATTTCCCAGGTTTGGGACGC-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-50aa	F 5'-ATCGATAGGGATCCaAAAATGTCTGTCACCGCTGACTGCC-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'
ΔS3-60aa	F 5'-ATCGATAGGGATCCaAGCAGACCGTACTGGTCCGCCACGTT-3' R 5'-CGAGCTCGATGGATCTTATGCGATGCGGCCTGGTCTGACA-3'

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