TRIM39.2过表达对鸡巨噬细胞转录表达的影响

doi:10.11843/j.issn.0366-6964.2025.01.017

摘要/Abstract

摘要：

旨在探究鸡主要组织相容性复合体(major histocompatibility complex，MHC)定位的TRIM39.2过表达对鸡巨噬细胞转录表达谱的影响。本研究通过RT-qPCR构建鸡TRIM39.2基因组织表达谱，克隆鸡TRIM39.2基因并构建携带标签的重组表达载体。分别转染空载体和TRIM39.2真核表达载体至HD11细胞内，收获RNA制备文库并进行转录组测序及生物信息学分析。筛选差异表达基因，随后进行GO和KEGG富集分析，并用RT-qPCR方法进一步验证转录组测序结果。结果表明，与其他组织相比，脾脏中TRIM39.2的表达最高。成功克隆了鸡TRIM39.2基因，并构建了携带Myc标签的重组表达载体pCAGGS-Myc-TRIM39.2，并验证了重组载体成功在鸡HD11细胞中表达TRIM39.2重组蛋白。转录组测序分析获得了33个显著差异表达基因。GO分析主要富集于细胞因子的活性特性及其参与介导的信号传导途径。KEGG分析则主要富集于细胞因子与受体间的相互作用及RIG-I样受体信号等通路。鸡TRIM39.2基因在巨噬细胞介导的免疫应答和信号传导中具有重要的调控作用，尤其对RIG-I样受体介导的I型干扰素信号通路具有正调控作用，本研究为进一步丰富鸡RIG-I样受体信号通路调控网络奠定基础。

关键词: TRIM39.2, 鸡巨噬细胞, 转录组, GO, KEGG

Abstract:

The study aimed to explore the effect of overexpression of TRIM39.2, localized in the chicken major histocompatibility complex (MHC), on the transcription expression of chicken macrophages. The tissue expression profile of chicken TRIM39.2 gene was constructed using RT-qPCR.The chicken TRIM39.2 gene was cloned and a recombinant expression vector carrying the tag was constructed. HD11 cells were transfected with either an empty vector or a TRIM39.2 eukaryotic expression vector.RNA was harvested to prepare a library, and transcriptome sequencing and bioinformatics analysis were performed. Differentially expressed genes were screened, followed by GO and KEGG enrichment analysis, and RT-qPCR method was used to further verify the transcriptome sequencing results. The results showed that the expression of TRIM39.2 was the highest in the spleen compared to other tissues. The chicken TRIM39.2 gene was successfully cloned, and a recombinant expression vector carrying a Myc tag, pCAGGS-Myc-TRIM39.2, was constructed, and confirmed to express TRIM39.2 recombinant protein in chicken HD11 cells.Transcriptome sequencing analysis identified 33 significantly differentially expressed genes. GO analysis was primarily enriched in the active characteristics of cytokines and their involvement in signal transduction pathways.KEGG analysis mainly focused on the interactions between cytokines and receptors, as well as pathways such as RIG-I-like receptor signaling pathway. The chicken TRIM39.2 gene plays a significant regulatory role in macrophage-mediated immune responses and signal transduction, particularly in positively regulating the type Ⅰ interferon signaling pathway mediated by RIG-I-like receptors. This study lays the foundation for further exploration of the regulatory mechanism of RIG-I-like receptor signaling pathway in chickens.

Key words: TRIM39.2, chicken macrophages, transcriptome, GO, KEGG

中图分类号:

S831.2

吴双, 尹娜, 余莫涵, 平玉宇, 白皓, 陈世豪, 常国斌. TRIM39.2过表达对鸡巨噬细胞转录表达的影响[J]. 畜牧兽医学报, 2025, 56(1): 178-188.

WU Shuang, YIN Na, YU Mohan, PING Yuyu, BAI Hao, CHEN Shihao, CHANG Guobin. The Effect of TRIM39.2 Overexpression on the Transcriptional Expression of Chicken Macrophages[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(1): 178-188.

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基因 Gene	引物序列(5′→3′) Primers sequence	GenBank登录号 GenBank accession No.
TRIM39.2 (载体构建)	F: $\underline{{\rm{ggatcttccagagat}}}$ATGGATGAAGATAACCCAGCTGA R: $\underline{{\rm{ctgccgttcgacgat}}}$TCACGGGGACAGCGTGAA	NM_001006196.3
TRIM39.2	F: GTATCAGGCTCTCACTTCAC R: ACCTCATTGTTGGCATCG	NM_001006196.3
β-ACTIN	F: GAGAAATTGTGCGTGACATCA R: CCTGAACCTCTCATTGCA	NM_205518.2
SAMD9L	F: TCAGAATATCTCAGCAGGAAG R: AGGAACAAGCATAAGGACAT	XM_004939297
LYZ	F: TGTGTGCCGCAAAATTCGAG R: CCAGCGGCTGTTGATCTGTA	NM_205281.2
IFNW1	F: CTTGCCCACAACAAGACGTG R: TGTTTTGGAGTGTGTGGGCT	NM_001024836
MDA5	F: GTGTCCGCTTGTCAGATT R: AGGTGAGGCTGTAAGTCC	NM_001193638.2
CCL19	F: TCAAGGCAATTAGGCTCTG R: CACCACAGCAGGACATAG	NM_001302168.1
TRIM25	F: GAGGAAGAGCAGGAGGCG R: GCTGATGGTGGCGGAAG	NM_001318458.2
TBK1	F: AGACTGGCACAACACTTCCC R: CCATACTGGCATCCCCATCC	NM_001199558.2
IRF7	F: GAGCCTCCTCCCTCAACAGT R: AGGGACACAGGAAGGGAGTG	NM_205372.2
IFNβ	F: AGCTCTCACCACCACCTTCTC R: TGGCTGCTTGCTTCTTGTCCTT	NM_002176.4
OASL	F: ACATCCTCGCCATCATCGA R: GCGGACTGGTGATGCTGACT	NM_001397447.1
MX1	F: CCGCAACACAGAAATACAG R: TTATCTTGTGGCTGGTTCC	NM_204609.2

样品 Sample	原始数据/条 Raw reads	有效数据/条 Clean reads	Q20/%	Q30/%
MOCK1	44 575 852	44 401 146	99.7	99.61
MOCK2	46 024 180	45 838 076	99.72	99.60
MOCK3	39 214 078	39 000 552	99.68	99.46
TRIM39.2-1	37 936 706	37 817 626	99.72	99.69
TRIM39.2-2	46 822 660	46 438 132	99.68	99.18
TRIM39.2-3	40 622 054	40 429 634	99.71	99.53

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