Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (10): 4646-4659.doi: 10.11843/j.issn.0366-6964.2024.10.036
• Basic Veterinary Medicine • Previous Articles Next Articles
Mengli WU1,2(
), Hualin SUN1,2, Jifei YANG1,2, Yaru ZHAO1,2, Guiquan GUAN1,2, Hong YIN1,2,3, Qingli NIU1,2,*()
Received:2023-11-29
Online:2024-10-23
Published:2024-11-04
Contact:
Qingli NIU
E-mail:15238649312@163.com;niuqingli@caas.cn
CLC Number:
Mengli WU, Hualin SUN, Jifei YANG, Yaru ZHAO, Guiquan GUAN, Hong YIN, Qingli NIU. Construction of a Passaged Porcine Alveolar Macrophage Cell Line Stably Expressing the Porcine BRD4-BD1/2 Protein and Its Effects on ASFV Proliferation[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4646-4659.
Table 1
Primer Sequence Information"
| 引物名称 Primer name | 引物序列(5’→3’) Sequence(5’→3’) |
| CP204L: F | GCGGTAGAATTGTTACGACCGCT |
| CP204L: R | CCTCCGATGAGGGCTCTTGC |
| B646L: F | AGTTATGGGAAACCCGACCC |
| B646L: R | CCCTGAATCGGAGCATCCT |
| sus GAPDH: F | GCGAGATCCCGCCAACATCA |
| sus GAPDH: R | GTCCCTCCACGATGCCGAAG |
Fig. 1
Screening for BRD4 functional domains affecting ASFV replication The impact of ASFV replication was assessed following transfection with different doses (1, 2, 4 μg) of BRD4 and its various truncations. The results demonstrated that BRD4-BD1/2 notably enhances the replication of ASFV. The specific sizes of BRD4 and truncations are as follows: the full-length BRD4 is approximately 250 ku; BRD4-BD1/2 is around 100 ku; and BRD4-BD1 is about 30 ku. A. Schematic diagram of each structural domain of BRD4; B. ASFV p30 and p72 protein expression levels after dose transfection of BRD4 and its truncates; C. mRNA levels of ASFV B646L gene post-transfection with varying doses of BRD4 and its truncated forms; D. mRNA levels of ASFV CP204L gene after transfection with different doses of BRD4 and its truncated versions. ns. P>0.05, *. P≤0.05 indicates a significant difference between data, **. P≤0.01, ***. P≤0.001, ****. P≤0.000 1"
Fig. 2
Identification of recombinant plasmids by double digestion M. DL2000 DNA marker; 1 Enzymatic products of pLVX-IRES-Puro-3x flag-BRD4-BD1/2 by EcoRⅠ, BamHⅠ. Through the double digestion experiment with EcoRⅠ and BamHⅠ restriction enzymes, we observed bands of approximately 5 000 bp corresponding to the vector and around 2 000 bp for the target gene. These results indicate the successful construction of the lentiviral expression vector pLVX-IRES-Puro-3x Flag-BRD4-BD1/2"
Fig. 3
Stable expression of BRD4-BD1/2 gene in 3D4/21-BRD4-BD1/2 cell line After infection and puromycin selection by the lentiviral packaging system, total protein was extracted from the positive clone cells after continuous passage. Western Blot analysis showed stable expression of BRD4-BD1/2 protein in the passaged 3D4/21-BRD4-BD1/2 cell line"
Fig. 4
Cell viability of the 3D4/21-BRD4-BD1/2 cell line was assessed at various time points using the CCK-8 assay Cellular viability of 3D4/21-BRD4-BD1/2 cells in comparison to 3D4/21-WT cells was evaluated at various time points (12, 24, 36, and 48 hours) and generations (F10, F20, F30)using the CCK-8 assay. The results indicated no significant differences between the cell lines at the time above intervals (ns, P>0.05), suggesting that overexpression of BRD4-BD1/2 does not significantly affect the viability of 3D4/21 cells under the conditions tested. A. CCK-8 detection of 3D4/21-BRD4-BD1/2 cell line activity at different time points; B. CCK-8 detection of 3D4/21-BRD4-BD1/2 cell line activity at different generations"
Fig. 5
Detection of the effects of ASFV-EGFP infection on BRD4-BD1/2-3D4/21 and 3D4/21-WT cell lines by fluorescence microscopy 3D4/21-BRD4-BD1/2 and 3D4/21-WT cells were infected with ASFV-EGFP at varying multiplicities of infection (MOIs) of 0, 0.1, 1, and 2. Fluorescence intensity was monitored at 36 hours post-infection. The results indicated that the 3D4/21-BRD4-BD1/2 cells exhibited increased fluorescence intensity compared to the WT cells, suggesting that the 3D4/21-BRD4-BD1/2 cell line can enhance ASFV replication"
Fig. 6
The impact of two cell lines on ASFV gene transcription and protein expression under various MOI conditions of ASFV infection Both cell types were infected with varying MOIs of ASFV CN/GS/2018 (0, 0.1, 1, 2), and the transcription levels of CP204L, B646L/GAPDH were measured using RT-qPCR, along with the analysis of viral protein expressions p30 and p72 through Western blot. The results revealed that the 3D4/21-BRD4-BD1/2 cell line exhibited significantly higher transcription of CP204L and B646L genes, as well as the expression of p30 and p72 proteins, compared to the 3D4/21-WT cells. A. Expression levels of ASFV proteins p30 and p72 at different MOIs; B. Grayscale analysis of ASFV p30 and p72 protein expression at different MOIs; C. mRNA levels of ASFV B646L gene at different MOIs; D. mRNA levels of ASFV CP204L gene at different MOIs. ns. P>0.05; *. P≤0.05;***. P≤0.001;****. P≤0.000 1"
Fig. 7
The influence of two cell lines on ASFV gene transcription and protein expression at different time points during ASFV infection The gene transcription and protein expression profiles at various time points (12, 24, 36, and 48 hpi) post-ASFV CN/GS/2018 infection were analyzed in 3D4/21-BRD4-BD1/2 and 3D4/21-WT cell lines. RT-qPCR technique was utilized to measure the expression levels of CP204L, B646L, and GAPDH mRNA in both cell types. The results indicated a significant increase in the transcription of CP204L and B646L genes at 36 hpi in the 3D4/21-BRD4-BD1/2 cell line (P < 0.01). Additionally, Western blot analysis revealed that the expression of ASFV proteins p30 and p72 was also significantly higher in the 3D4/21-BRD4-BD1/2 cells compared to the 3D4/21-WT cells. A. ASFV p30 and p72 protein expression levels at different time points of infection with ASFV CN/GS/2018; B. Gray value analysis of ASFV p30 and p72 protein expression levels at different time points of infection with ASFV CN/GS/2018; C. mRNA levels of ASFV B646L at different time points; D. mRNA of ASFV CP204L at different time points levels. ns. P > 0.05; *. P≤0.05;***. P≤0.001;****. P≤0.000 1"
Fig. 8
Titer determination of ASFV CN/GS/2018 strain infected 3D4/21 and 3D4/21-BRD4-BD1/2 cells The HAD50 determination method measured the viral titers of ASFV CN/GS/2018 in 3D4/21-BRD4-BD1/2 and 3D4/21-WT cell lines post-infection. The results showed that the HAD50 values for ASFV in the 3D4/21-BRD4-BD1/2 cell line were significantly higher than those in the 3D4/21-WT cell line (P < 0.05). ns. P>0.05; *. P≤0.05;***. P≤0.001;****. P≤0.000 1"
Fig. 9
Analysis of ASFV CN/GS/2018 replication in different generations of 3D4/21-BRD4-BD1/2 cell lines Comparative analysis of ASFV CN/GS/2018 replication capabilities in the 10th, 20th, and 30th generations of 3D4/21-BRD4-BD1/2 cell lines and the control 3D4/21-WT cells. Cells were seeded in 12-well plates and post-infection cell samples were collected at 12, 24, 36, and 48 hours to detect ASFV proteins p30 and p72 using Western blot, indicative of viral replication. The expression of BRD4-BD1/2 in stable cell lines of different generations was examined simultaneously using immunofluorescence. The results from sections A-D indicate that the 3D4/21-BRD4-BD1/2 cell line from the described passages can enhance ASFV replication at the same infection time points compared to WT cells. The results shown in Figure E demonstrate that BRD4-BD1/2 is stably expressed across different passages of the stable expression cell line"
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