Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (12): 5751-5761.doi: 10.11843/j.issn.0366-6964.2024.12.037
• Basic Veterinary Medicine • Previous Articles Next Articles
LIU Weijiao1(
), HE Qing1, JIANG Yifan1, CAO Siyu1, ZHANG Shirui2, LÜ Jialu2, LONG Ping2, YANG Lingchen1, ZHOU Chuan2, WANG Naidong1,*()
Received:2024-01-16
Online:2024-12-23
Published:2024-12-27
Contact:
WANG Naidong
E-mail:1149283137@stu.hunau.edu.cn;naidongwang@hunau.edu.cn
CLC Number:
LIU Weijiao, HE Qing, JIANG Yifan, CAO Siyu, ZHANG Shirui, LÜ Jialu, LONG Ping, YANG Lingchen, ZHOU Chuan, WANG Naidong. The Role of Porcine Lck Protein in PCV2 Replication in vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5751-5761.
Table 1
Primer sequence and length"
| 引物 Primer | 引物序列(5′→3′) Primer sequence (5′→3′) | 引物长度/nt Primer length | 退火温度/℃ Tm |
| Lck-SH3-His-F-01 | TTCCATATGCATCATCATCATCATCATGGTTCCCCACTGC AAGACAACCT | 50 | 54.05 |
| Lck-SH3-R-02 | CGCGGATCCTTATGTTTGCTTTGGCCACGAA | 31 | 58.81 |
| Fyn-SH3-His-F-01 | TTCCATATGCATCATCATCATCATCATGGTTCCAACAGG AGTGACGCTGTTTGTG | 55 | 59.86 |
| Fyn-SH3-R-01 | CGCGGATCCTTACTCTTCTGCCTGGATGGAGTC | 33 | 61.57 |
Fig. 1
Bioinformatics analysis of porcine LCK protein A. Similarity alignment of acid amino sequence of Lck gene among different species(1. Pig; 2. Human; 3. Mouse; 4. Rat; 5. Dog; 6. Rabbit; 7. Horse; 8. Sheep; 9. Panda; 10. Cow); B. Prediction results of porcine Lck protein tertiary structure; C. Prediction results of porcine Lck-SH3 doamin protein tertiary structure(Rose red: β fold sheet; Blue: alpha spiral); D. Amino acid sequence comparison of porcine SFKs SH3 domain"
Fig. 2
Expression and purification of pET-28a(+)-Lck-SH3、pET-28a(+)-Fyn-SH3 domain A. Expression and purification of Lck-SH3 protein; B. Expression and purification of Fyn-SH3 protein; M. Protein marker; Total. Total protein; Sup. Supernatant, soluble protein in total protein; FT. Flow-through, protein not bound to nickel column packing; W. Wash, protein eluted by low concentration imidazole; E. Elution, protein eluted with high imidazole concentration"
Fig. 3
The specificity of mouse Lck polyclonal antibody was detected by Western blot A. Western blot based on His monoclonal antibody; B. Western blot based on mouse negative serum (1∶3 000); C. Western blot based on mouse Lck-SH3 antiserum (1∶3 000). M. Protein molecular weight standard"
Fig. 4
Effect of PCV2 early infection on the expression and phosphorylation of Lck protein in cells A. Identification of exogenous Lck protein using IFA; B. Identification of exogenous and exogenous Lck protein in HEK 293 T cells and PK-15 cells using Western blot; C. Identification of Lck protein expression in different tissues and cells of pig using Western blot; D.The effect of early infection of PCV2 on the expression and phosphorylation of Lck protein was detected using Western blot, and the levels of proteins were quantified with Image J analysis software(Fig.E, Fig.F). *. P < 0.05; **. P < 0.01;ns. P>0.05"
Fig. 5
Effect of Lck overexpression on replication of PCV2 in cells A. The effect of overexpression of Lck on the expression of Cap protein was detected by Western bolt; B. The effect of overexpression of Lck on the expression of Cap protein was detected by RT-qPCR; C. The effect of overexpression of Lck or Lck-SH3 on the expression of Cap protein was detected by IFA, and the fluorescence value was measured by Image J software (Fig. D). *. P < 0.05; **. P < 0.01"
Fig. 6
Effect of Lck specific inhibitor A770041 on PCV2 replication A. CCK8 assay was used to detect cytotoxicity of A770041 with different concentrations in PK-15 cells; B. The effect of Lck specific inhibitor A770041 on virus copy number of viral genomes was calculated using RT-qPCR; C. Virus titer of PCV2 was determined using TCID50 assay; D. The effect of overexpressed Lck on the expression of Cap protein was detected using IFA, and the fluorescence value was measured using Image J software (Fig. E). *. P < 0.05; **. P < 0.01."
| 1 |
GAMAGE L N A , ELLIS J , HAYES S . Immunogenicity of bacteriophage lambda particles displaying porcine Circovirus 2 (PCV2) capsid protein epitopes[J]. Vaccine, 2009, 27 (47): 6595- 6604.
doi: 10.1016/j.vaccine.2009.08.019 |
| 2 |
SEGALÉS J , KEKARAINEN T , CORTEY M . The natural history of porcine circovirus type 2:from an inoffensive virus to a devastating swine disease?[J]. Vet Microbiol, 2013, 165 (1-2): 13- 20.
doi: 10.1016/j.vetmic.2012.12.033 |
| 3 |
SEGALÉS J . Best practice and future challenges for vaccination against porcine circovirus type 2[J]. Expert Rev Vac, 2015, 14 (3): 473- 487.
doi: 10.1586/14760584.2015.983084 |
| 4 |
ALARCON P , RUSHTON J , WIELAND B . Cost of post-weaning multi-systemic wasting syndrome and porcine circovirus type-2 subclinical infection in England - an economic disease model[J]. Prev Vet Med, 2013, 110 (2): 88- 102.
doi: 10.1016/j.prevetmed.2013.02.010 |
| 5 | 苏芮, 王东亮, 陈指龙, 等. PCV2 ORF1~ORF4基因所编码蛋白功能的研究进展[J]. 经济动物学报, 2020, 24 (1): 46- 51. |
| su b , wang d l , chen z l , et al. research progress on major function proteins encoded by PCV2 ORF1-ORF4 Gene[J]. Journal of Economic Animal, 2020, 24 (1): 46- 51. | |
| 6 |
WANG N D , ZHAN Y , WANG A B , et al. In silico analysis of surface structure variation of PCV2 capsid resulting from loop mutations of its capsid protein (Cap)[J]. J Gen Virol, 2016, 97 (12): 3331- 3344.
doi: 10.1099/jgv.0.000634 |
| 7 |
MACDONALD A , CROWDER K , STREET A , et al. The hepatitis C virus NS5A protein binds to members of the Src family of tyrosine kinases and regulates kinase activity[J]. J Gen Virol, 2004, 85 (3): 721- 729.
doi: 10.1099/vir.0.19691-0 |
| 8 |
TRAN T , HOFFMANN S , WIESEHAN K , et al. Insights into human Lck SH3 domain binding specificity: different binding modes of artificial and native ligands[J]. Biochemistry, 2005, 44 (45): 15042- 15052.
doi: 10.1021/bi051403k |
| 9 |
SAKSELA K , CHENG G , BALTIMORE D . Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4[J]. EMBO J, 1995, 14 (3): 484- 491.
doi: 10.1002/j.1460-2075.1995.tb07024.x |
| 10 |
TRIBLE R P , EMERT-SEDLAK L , SMITHGALL T E . HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction[J]. J Biol Chem, 2006, 281 (37): 27029- 27038.
doi: 10.1074/jbc.M601128200 |
| 11 |
KENNEY S P , MENG X J . An SH3 binding motif within the nucleocapsid protein of porcine reproductive and respiratory syndrome virus interacts with the host cellular signaling proteins STAMI, TXK, Fyn, Hck, and cortactin[J]. Virus Res, 2015, 204, 31- 39.
doi: 10.1016/j.virusres.2015.04.004 |
| 12 |
BOMMHARDT U , SCHRAVEN B , SIMEONI L . Beyond TCR signaling: emerging functions of Lck in cancer and immunotherapy[J]. Int J Mol Sci, 2019, 20 (14): 3500.
doi: 10.3390/ijms20143500 |
| 13 |
WAGNER M J , SMILEY J R . Herpes simplex virus requires VP11/12 to induce phosphorylation of the activation loop tyrosine (Y394) of the Src family kinase Lck in T lymphocytes[J]. J Virol, 2009, 83 (23): 12452- 12461.
doi: 10.1128/JVI.01364-09 |
| 14 |
CHASE A J , WOMBACHER R , FACKLER O T . Intrinsic properties and plasma membrane trafficking route of Src family kinase SH4 domains sensitive to retargeting by HIV-1 Nef[J]. J Biol Chem, 2018, 293 (20): 7824- 7840.
doi: 10.1074/jbc.RA118.002794 |
| 15 |
MELLORS J W , GUO S , NAQVI A , et al. Insertional activation of STAT3 and LCK by HIV-1 proviruses in T cell lymphomas[J]. Sci Adv, 2021, 7 (42): eabi8795.
doi: 10.1126/sciadv.abi8795 |
| 16 |
STRASNER A B , NATARAJAN M , DOMAN T , et al. The Src kinase Lck facilitates assembly of HIV-1 at the plasma membrane[J]. J Immunol, 2008, 181 (5): 3706- 3713.
doi: 10.4049/jimmunol.181.5.3706 |
| 17 |
ZHANG J X , LEI X N , WANG D L , et al. Inhibition of Abl or Src tyrosine kinase decreased porcine circovirus type 2 production in PK15 cells[J]. Res Vet Sci, 2019, 124, 1- 9.
doi: 10.1016/j.rvsc.2019.01.024 |
| 18 | ZHAN Y , YU W T , CAI X , et al. The carboxyl terminus of the porcine circovirus type 2 capsid protein is critical to virus-like particle assembly, cell entry, and propagation[J]. J Virol, 2020, 94 (9): e00042- 20. |
| 19 |
LIU Y G , XIA X S . Molecular characterization, tissue expression, polymorphism and association of porcine LCK gene[J]. Mol Biol Rep, 2012, 39 (4): 4023- 4028.
doi: 10.1007/s11033-011-1183-6 |
| 20 | 张永德, 林勇, 冯鹏霏, 等. 尼罗罗非鱼Lck多克隆抗体的制备及鉴定[J]. 南方农业学报, 2018, 49 (11): 2304- 2310. |
| ZHANG D Y , LIN Y , FENG P F , et al. Preparation and identification of Lck polyclonal antibody in Nile tilapia[J]. Journal of Southern Agriculture, 2018, 49 (11): 2304- 2310. | |
| 21 | 张佳鑫. Src和Abl酪氨酸激酶家族介导的猪圆环病毒2型感染机制的初步研究[D]. 长沙: 湖南农业大学, 2018. |
| ZHNAG J X. Study on porcine circovirus 2 infection mechanism mediated by Src and Abl tyrosine kinase family[D]. Changsha: Hunan Agricultural University, 2018. (in Chinese) | |
| 22 |
SALMOND R J , FILBY A , QURESHI I , et al. T-cell receptor proximal signaling via the Src-family kinases, Lck and Fyn, influences T-cell activation, differentiation, and tolerance[J]. Immunol Rev, 2009, 228 (1): 9- 22.
doi: 10.1111/j.1600-065X.2008.00745.x |
| 23 |
MA T , CHEN X R , OUYANG H S , et al. HMGCR inhibits the early stage of PCV2 infection, while PKC enhances the infection at the late stage[J]. Virus Res, 2017, 229, 41- 47.
doi: 10.1016/j.virusres.2016.12.010 |
| 24 |
OUYANG T , NIU G Y , ZHANG Y F , et al. Porcine HMGCR inhibits porcine circovirus type 2 infection by directly interacting with the viral proteins[J]. Viruses, 2019, 11 (6): 544.
doi: 10.3390/v11060544 |
| 25 |
LIU X W , WANG Y , HAN C , et al. TGF-β from the porcine intestinal cell line IPEC-J2 induced by porcine circovirus 2 increases the frequency of treg cells via the activation of ERK (in CD4+ T cells) and NF-κB (in IPEC-J2)[J]. Viruses, 2022, 14 (11): 2466.
doi: 10.3390/v14112466 |
| 26 | WANG T T , DU Q , NIU Y Y , et al. Cellular p32 is a critical regulator of porcine circovirus type 2 nuclear egress[J]. J Virol, 2019, 93 (23): e00979- 19. |
| 27 |
ISLAM M A , GROßE-BRINKHAUS C , PRÖLL M J , et al. PBMC transcriptome profiles identifies potential candidate genes and functional networks controlling the innate and the adaptive immune response to PRRSV vaccine in Pietrain pig[J]. PLoS One, 2017, 12 (3): e0171828.
doi: 10.1371/journal.pone.0171828 |
| 28 |
FERNANDES L T , TOMÁS A , BENSAID A , et al. Exploratory study on the transcriptional profile of pigs subclinically infected with porcine circovirus type 2[J]. Anim Biotechnol, 2009, 20 (3): 96- 109.
doi: 10.1080/10495390902885785 |
| 29 |
PAGANO M A , TIBALDI E , PALÙ G , et al. Viral proteins and Src family kinases: Mechanisms of pathogenicity from a "liaison dangereuse"[J]. World J Virol, 2013, 2 (2): 71- 78.
doi: 10.5501/wjv.v2.i2.71 |
| 30 |
WEIS D D , KJELLEN P , SEFTON B M , et al. Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri tip[J]. Protein Sci, 2006, 15 (10): 2402- 2410.
doi: 10.1110/ps.052016406 |
| 31 |
ISAKOV N , BIESINGER B . Lck protein tyrosine kinase is a key regulator of T-cell activation and a target for signal intervention by Herpesvirus saimiri and other viral gene products[J]. Eur J Biochem, 2000, 267 (12): 3413- 3421.
doi: 10.1046/j.1432-1327.2000.01412.x |
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