Regulation mechanism of Mycobacterium bovis PtpA protein on NF-κB signaling pathway
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摘要:目的
研究牛分枝杆菌Mycobacterium bovis PtpA蛋白对免疫应答相关的NF-κB信号通路的影响，以揭示PtpA蛋白在机体免疫应答中的作用。
方法构建PtpA基因真核表达载体FLAG-PtpA，并将其转染到HEK293T中，进行SDS-PAGE分析及Western blot检测。激活NF-κB信号通路后，通过双荧光素酶试验和qPCR方法探究PtpA蛋白对NF-κB信号通路的影响。
结果成功构建PtpA基因真核表达载体FLAG-PtpA，转染HEK293T后经SDS-PAGE分析，相对分子质量约为22 000处可见特异性蛋白条带。Western blot结果显示，表达产物可与一抗特异性结合，证明该蛋白是PtpA蛋白。双荧光素酶试验中，转染2~24 h，试验组与对照组萤火虫荧光素酶和海肾荧光素酶的相对荧光强度的比值差异显著(P<0.05)；转染2 h后，对照组萤火虫荧光素酶和海肾荧光素酶的相对荧光值是试验组的2.93倍，说明PtpA蛋白对NF-κB信号通路激活早期具有显著的抑制作用。qPCR结果显示，转染2 h后，对照组的IL-6、GM-CSF、BIRC-2和BIRC-3的表达量分别是试验组的3.93、3.42、2.17和2.30倍(P<0.01)；转染4 h后，对照组的IL-6、GM-CSF、BIRC-2和BIRC-3的表达量分别是试验组的4.26、3.93、2.36和2.50倍(P<0.01)，说明PtpA蛋白对NF-κB信号通路相关的细胞因子(IL-6、GM-CSF、BIRC-2、BIRC-3)在免疫早期具有显著抑制作用。
结论qPCR结果与双荧光素酶试验结果一致，表明牛分枝杆菌PtpA蛋白对NF-κB信号通路的影响主要发生在早期。本研究为后续研究有效的结核病防控药物提供了理论基础。
Abstract:ObjectiveTo investigate the effect of Mycobacterium bovis PtpA protein on NF-κB signaling pathway which is related to immune response, and reveal the role of PtpA protein in the body's response to immunity.
MethodThe PtpA gene eukaryotic expression vector FLAG-PtpA was transfected into HEK293T for SDS-PAGE analysis and Western blot detection. After activation of NF-κB signaling pathway, the effects of PtpA protein on NF-κB signaling pathway were investigated by dual luciferase assay and qPCR.
ResultThe eukaryotic expression vector FLAG-PtpA of PtpA gene was successfully constructed, transfected into HEK293T and analyzed by SDS-PAGE. The specific molecular band was visible with relative molecular mass of 22 000. Western blot results showed that the expression product specifically bound to the primary antibody, demonstrating that the protein was a PtpA protein. In the double luciferase assay, the ratios of the relative fluorescence intensities of firefly luciferase and Renilla luciferase in the test group and the control group were significantly different from 2 to 24 h after transfection (P<0.05). The relative fluorescence values of firefly luciferase and Renilla luciferase in the control group were 2.93 times higher than those in the experimental group 2 h after transfection, indicating that PtpA protein had a significant inhibitory effect on the early activation of NF-κB signaling pathway. qPCR results showed that the expression levels of IL-6, GM-CSF, BIRC-2 and BIRC-3 in the control group were 3.93, 3.42, 2.17 and 2.30 times respectively of those in the test group 2 h after transfection(P<0.01), and were 4.26, 3.93, 2.36 and 2.50 times respectively of those in the test group 4 h after transfection(P<0.01). These results indicated that PtpA protein had a significant inhibitory effect on NF-κB signaling pathway-associated cytokines (IL-6, GM-CSF, BIRC-2 and BIRC-3) in the early stage of immunization.
ConclusionqPCR results are consistent with the results of dual luciferase assay, indicating that the effect of M. bovis PtpA on NF-κB signaling pathway mainly occurs in the early stage. This study provides a theoretical basis for the follow-up study of effective tuberculosis prevention and control drugs.
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Keywords:
- bovine tuberculosis /
- Mycobacterium bovis /
- PtpA protein /
- NF-κB signaling pathway /
- cytokine /
- immune response
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图 1 PtpA基因的PCR扩增
1：PtpA基因的PCR扩增产物；M：DL2000 DNA marker；2：阴性对照
Figure 1. PCR amplification of the PtpA gene
1: PCR product of PtpA gene; M: DL2000 DNA marker;2: Negative control
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图 2 FLAG-PtpA重组质粒中PtpA基因的PCR扩增
1：阴性对照；M：DL2000 DNA marker；2：PtpA基因的PCR扩增产物
Figure 2. PCR amplification of PtpA gene in FLAG-PtpA recombinant plasmid
1: Negative control; M: DL2000 DNA marker; 2: PCR product of PtpA gene
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图 3 真核表达载体转染293T细胞
A1：293T未转染质粒(白镜)；A2：293T未转染质粒(荧光)；B1：293T转染FLAG-PtpA载体质粒+pEGFP-N1质粒(白镜)；B2：293T转染FLAG-PtpA载体质粒+pEGFP-N1质粒(荧光)
Figure 3. Eukaryotic expression vector transfected 293T cells
A1: 293T untransfected plasmid (white mirror); A2: 293T untransfected plasmid (fluorescent); B1: 293T transfected FLAG-PtpA plasmid and pEGFP-N1 plasmid (white mirror); B2: 293T transfected FLAG-PtpA plasmid and pEGFP-N1 plasmid (fluorescence)
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图 4 HEK293T细胞中FLAG-PtpA表达产物鉴定
M：低相对分子质量蛋白质标准；1:空白对照；2：HEK293T细胞中转染p3×FLAG-CMV-10质粒；3：HEK293T细胞中转染FLAG-PtpA质粒
Figure 4. Identification of FLAG-PtpA expression product in HEK293T cells
M: Low molecular weight protein standard; 1: Blank control; 2: p3×FLAG-CMV-10 plasmid transfected into HEK293T cells; 3: FLAG-PtpA plasmid transfected into HEK293T cells
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图 5 HEK293T表达FLAG-PtpA蛋白特异性分析
M：低相对分子质量蛋白质标准；1：HEK293T表达的FLAG-PtpA蛋白
Figure 5. HEK293T expression FLAG-PtpA protein specificity analysis
M: Low molecular weight protein standard; 1: FLAG-PtpA protein expressed by HEK293T
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图 6 PtpA蛋白对NF-κB信号通路激活的影响
“*”、“**”和“***”分别表示差异达到0.05、0.01和0.001的显著水平(t检验)
Figure 6. Effect of PtpA protein on activation of NF-κB signaling pathway
“*”, “**” and “***” indicate the difference reaches 0.05, 0.01 and 0.001 significance levels respectively (t test)
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图 7 细胞因子的mRNA表达量
“*”、“**”和“***”分别表示差异达到0.05、0.01和 0.001的显著水平(t检验)
Figure 7. mRNA expression of cell factors
“*”，“**” and “***” indicate the difference reaches 0.05，0.01 and 0.001 significance levels respectively (t test)
表 1 引物名称及序列
Table 1 Primer name and sequence
引物名称
Primer name引物序列(5′→3′)1)
Primer sequence产物大小/bp
Product sizeFLAG-PtpA-F AAGGAAAAAA GCGGCCGCGGTGTCTGATCCGCTGCACG 492 FLAG-PtpA-R CTAG TCTAGATCAACTCGGTCCGTT IL-6-F GGTGTTGCCTGCTGCCTTCC 100 IL-6-R GTTCTGAAGAGGTGAGTGGCTGTC GM-CSF-F TCCTGAACCTGAGTAGAGACACTGC 100 GM-CSF-R CAGGTCGGCTCCTGGAGGTC BIRC-2-F AGACACATGCAGCTCGAATGAGAAC 100 BIRC-2-R AACACCTCAAGCCACCATCACAAC BIRC-3-F CTGTGATGGTGGACTCAGGTGTTG 100 BIRC-3-R TGGCTTGAACTTGACGGATGAACTC 1) 有下划线的序列为Not I、Xba I酶切位点
1) Sequences with underlines are Not I and Xba I restriction sites
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