

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4657-4672.doi: 10.11843/j.issn.0366-6964.2025.09.043
刘昕玥1(
), 李丹妮1, 宗颖2,3,4, 时坤2,3,4, 李健明2,3,4, 刁乃超2,3,4, 曾范利2,3,4,5,*(
), 杜锐2,3,4,5,*(
)
收稿日期:2024-12-02
出版日期:2025-09-23
发布日期:2025-09-30
通讯作者:
曾范利,杜锐
E-mail:2892144827@qq.com;zengfanli@jlau.edu.cn;durui@jlau.edu.cn
作者简介:刘昕玥(1999-),女,山东莱州人,硕士生,主要从事经济动物疫病防治研究,E-mail: 2892144827@qq.com
基金资助:
LIU Xinyue1(
), LI Danni1, ZONG Ying2,3,4, SHI Kun2,3,4, LI Jianming2,3,4, DIAO Naichao2,3,4, ZENG Fanli2,3,4,5,*(
), DU Rui2,3,4,5,*(
)
Received:2024-12-02
Online:2025-09-23
Published:2025-09-30
Contact:
ZENG Fanli, DU Rui
E-mail:2892144827@qq.com;zengfanli@jlau.edu.cn;durui@jlau.edu.cn
摘要:
前期研究发现,异源表达Rv3435c基因的耻垢分枝杆菌显著抑制巨噬细胞的炎症表达,为了探究Rv3435c基因在该过程行使的功能,使用二代测序分析Rv3435c重组耻垢分枝杆菌感染RAW264.7细胞的转录组差异。结果显示:通过RT-qPCR检测6、12、24、48 h的IL-1β、TNF-α、IL-6 mRNA表达水平,选择表达量最高的12 h作为转录组测序时间点。将测序获得的原始数据进行数据质量分析及比对结果统计,确定数据质量良好,进行样本关系分析,提示Rv3435c显著改变了小鼠巨噬细胞的基因表达情况。将数据过滤比对共得到14 077个差异基因,其中,有278个显著上调表达基因和118个显著下调表达基因。对差异基因进行GO注释,得到的差异基因主要富集在免疫系统过程、对外部刺激的反应、防御响应等方面。对差异基因进行KEGG通路富集分析,主要富集在环境信息处理、细胞过程、人类疾病、有机体系统几方面。通过蛋白互作分析筛选出核心基因10个,分别为IL-6、IL-1β、CCL2、Mmp9、Spp1、Itgam、Cdc20、Ccna2、Kif11、Ccnb2。对差异基因进行荧光定量PCR试验,验证转录组结果可信,根据筛选出的DEGs和RT-qPCR结果,推测SPP1是Rv3435c行使功能的潜在靶点,并通过RT-qPCR、ELISA、Western blot验证结果。本文为结核分枝杆菌Rv3435c基因的功能探索提供了重要的数据支持。
中图分类号:
刘昕玥, 李丹妮, 宗颖, 时坤, 李健明, 刁乃超, 曾范利, 杜锐. Rv3435c重组耻垢分枝杆菌感染小鼠RAW264.7巨噬细胞的转录组分析[J]. 畜牧兽医学报, 2025, 56(9): 4657-4672.
LIU Xinyue, LI Danni, ZONG Ying, SHI Kun, LI Jianming, DIAO Naichao, ZENG Fanli, DU Rui. Transcriptome Analysis of RAW264.7 Macrophages Infected with Rv3435c Recombinant Mycobacterium smegmatis[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4657-4672.
表 2
实时荧光定量PCR引物序列"
| 引物名称 Primer name | 序列(5′→3′) Sequence |
| CCL2 | F: ACCTGCTGCTACTCATTCACC R: TGAGCTTGGTGACAAAAACTAC |
| CCL3 | F: CCAGCCAGGTGTCATTTTCCTG R: ATTCAGTTCCAGGTCAGTGATG |
| CCL4 | F: CCAAGCCAGCTGTGGTATTCCT R: TTCAACTCCAAGTCACTCATGTACT |
| IL1RN | F: TCTCTCTCCTTCTCATCCTTCTGT R: GGCACCATGTCTATCTTTTCTTCT |
| PTGS2 | F: GCTCAGCCAGGCAGCAAATC R: CACCATAGAATCCAGTCCGGG |
| CCR2 | F: CCTCAGTTCATCCACGGCAT R: AGGGAGTAGAGTGGAGGCAG |
| SPP1 | F: TCCCTACAGTCGATGTCCCC R: TGTGGCATCAGGATACTGTTCAT |
| CXCL10 | F: GCCGTATTTTCTGCCTCAT R: TTTTTCATCGTGGCAATGATCTC |
| IGF1 | F: AGGCTATGGCTCCAGCATTC R: CGATAGGGACGGGGACTTCT |
| MTND2 | F: TTCGTCACACAAGCAACAGC R: GGGGCGAGGCCTAGTTTTAT |
| MTND1 | F: TCCGAGCATCTTATCCACGC R: GTATGGTGGTACTCCCGCTG |
表 3
重组耻垢分枝杆菌侵染下转录组测序数据统计"
| 样品名称 Sample | 原始度数 Raw reads No. | 原始碱基/bp Raw bases | 过滤读取编号 Clean reads number | 过滤数据/bp Clean data | 过滤度数/% Clean reads | Q30大小/bp Q30 | GC含量/% GC(%) | Q20含量/% Q20 | Q30含量/% Q30 |
| Ms-pMV361_1 | 45 267 206 | 6 835 348 106 | 44 170 312 | 6 653 345 332 | 97.58 | 6 424 197 947 | 48.31 | 97.79 | 93.98 |
| Ms-pMV361_2 | 46 295 026 | 6 990 548 926 | 45 367 066 | 6 831 943 984 | 98.00 | 6 590 259 271 | 48.31 | 97.92 | 94.27 |
| Ms-pMV361_3 | 40 132 684 | 6 060 035 284 | 39 248 806 | 5 905 695 590 | 97.80 | 5 677 418 957 | 48.28 | 97.69 | 93.69 |
| Ms-pMV361- Rv3 435c_1 | 47 080 516 | 7 109 157 916 | 46 071 820 | 6 941 076 740 | 97.86 | 6 663 366 706 | 48.44 | 97.72 | 93.73 |
| Ms-pMV361- Rv3 435c_2 | 46 194 334 | 6 975 344 434 | 45 053 236 | 6 783 376 551 | 97.53 | 6 500 746 215 | 48.41 | 97.50 | 93.20 |
| Ms-pMV361- Rv3 435c_3 | 41 735 852 | 6 302 113 652 | 40 638 676 | 6 121 139 659 | 97.37 | 5 875 725 971 | 48.64 | 97.50 | 93.23 |
表 5
差异基因中的前10位GO富集分析结果"
| 项目 Item | 中文名称 Chinese name | 调整后P值 Adjusted P-value |
| Immune system process | 免疫系统过程 | < 0.001 |
| Response to virus | 对病毒的响应 | < 0.001 |
| Defense response | 防御响应 | < 0.001 |
| Response to external stimulus | 对外部刺激的反应 | < 0.001 |
| Immune response | 免疫应答 | < 0.001 |
| Innate immune response | 先天免疫反应 | < 0.001 |
| Response to external biotic stimulus | 对外部生物刺激的反应 | < 0.001 |
| Response to biotic stimulus | 对生物刺激的反应 | < 0.001 |
| Response to other organism | 对其他微生物的反应 | < 0.001 |
| Defense response to other organism | 对其他生物体的防御反应 | < 0.001 |
表 6
差异表达基因的KEGG通路大类"
| 大类 Major categories | 中文名称 Chinese name | 调整后P值 Adjusted P-value |
| 环境信息处理 Environmental information processing | TNF信号通路 | < 0.001 |
| 细胞因子-细胞因子受体相互作用 | < 0.001 | |
| 病毒蛋白与细胞因子和细胞因子受体的相互作用 | < 0.001 | |
| 细胞过程 Cellular processes | 细胞周期 | < 0.001 |
| p53信号通路 | < 0.001 | |
| 人类疾病 Human diseases | 类风湿性关节 | < 0.001 |
| 炎脂质和动脉粥样硬化 | < 0.001 | |
| 有机体系统 Organic system | IL-17信号通路 | < 0.001 |
| Toll样受体信号通路 | < 0.001 |
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