Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector

HUANG Cuiyuan; WANG Jiaojiao; LI Wenqiang; LIU Li; WANG Wei; ZENG Ping; ZHANG Jing

doi:10.7619/jcmp.20240262

Journal of Clinical Medicine in Practice > 2024 > 28(8): 22-28. > DOI: 10.7619/jcmp.20240262

HUANG Cuiyuan, WANG Jiaojiao, LI Wenqiang, LIU Li, WANG Wei, ZENG Ping, ZHANG Jing. Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector[J]. Journal of Clinical Medicine in Practice, 2024, 28(8): 22-28. DOI: 10.7619/jcmp.20240262

Citation:

PDF (4172 KB)

Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector

HUANG Cuiyuan^{1, 2, 3},
WANG Jiaojiao^{1, 2, 3, 4},
LI Wenqiang^{1, 2, 3},
LIU Li^{1, 2, 3},
WANG Wei^{1, 2, 3},
ZENG Ping^{1, 2, 3},
ZHANG Jing^{1, 2, 3, ,}

1.
Central Laboratory, the First Clinical Medical School of China Three Gorges University, Yichang Central People's Hospital, Yichang, Hubei, 443003
2.
Hubei Key Laboratory of Ischemic Cardiovascular Diseases, Yichang, Hubei, 443003
3.
Hubei Provincial Clinical Research Center for Ischemic Cardiovascular Disease, Yichang, Hubei, 443003
4.
Department of Cardiovascular Medicine, the First Clinical Medical School of China Three Gorges University, Yichang Central People's Hospital, Yichang, Hubei, 443003

More Information

Received Date: January 15, 2024
Revised Date: March 01, 2024
Available Online: May 05, 2024

Graphical Abstract

Abstract

Abstract

Objective
To construct and identify an overexpressing recombinant adenovirus vector carrying the mouse nucleotide-binding oligomerization domain-like receptor protein 6 (NLRP6) gene (NM_133 946.2).
Methods
The coding sequence of the mouse NLRP6 gene was synthesized, digested with enzymes, and inserted into the adenovirus vector (GL2003). The transformed cells were then cultured in strain DH-5α, transformants were selected, and after identification by polymerase chain reaction (PCR), samples were sent for sequencing and comparison. The expression of NLRP6 protein in the recombinant plasmid was validated using the Western blot. The recombinant adenovirus vector Ad-NLRP6 was obtained using the Admax packaging system, then transduced into HEK293 cells, amplified, purified, and the viral titer was determined afterwards.
Results
The successful integration of NLRP6 into the expression plasmid was confirmed through double enzyme digestion and DNA sequencing. Concurrent detection of the FLAG-tagged protein indicated a significant increase in the expression level of NLRP6. Recombinant adenovirus carrying NLRP6 were packaged and produced, followed by infecting HEK293 cells with the virus. The cellular expression of green fluorescent protein in Ad-NLRP6 indicated successful infection. The virus was collected, amplified, purified, and the viral titer was determined to be 4×10¹⁰ PFU/mL.
Conclusion
The overexpression recombinant adenovirus vector carrying the mouse NLRP6 gene has been successfully constructed, providing an effective tool for further exploring the molecular function of NLRP6.
- NLRP6 gene,
- recombinant adenoviral vector,
- inflammation,
- metabolic disease

FullText(HTML)

References (29)

References

[1]	CHENUET P, MARQUANT Q, FAUCONNIER L, et al. NLRP6 negatively regulates type 2 immune responses in mice[J]. Allergy, 2022, 77(11): 3320-3336. doi: 10.1111/all.15388
[2]	BARNETT K C, LI S R, LIANG K X, et al. A 360° view of the inflammasome: mechanisms of activation, cell death, and diseases[J]. Cell, 2023, 186(11): 2288-2312. doi: 10.1016/j.cell.2023.04.025
[3]	LI R Z, ZHU S. NLRP6 inflammasome[J]. Mol Aspects Med, 2020, 76: 100859. doi: 10.1016/j.mam.2020.100859
[4]	ZHI F, LI B W, ZHANG C X, et al. NLRP6 potentiates PI3K/AKT signalling by promoting autophagic degradation of p85α to drive tumorigenesis[J]. Nat Commun, 2023, 14(1): 6069. doi: 10.1038/s41467-023-41739-z
[5]	ANGOSTO-BAZARRA D, MOLINA-LÓPEZ C, PELEGRÍN P. Physiological and pathophysiological functions of NLRP6: pro-and anti-inflammatory roles[J]. Commun Biol, 2022, 5(1): 524. doi: 10.1038/s42003-022-03491-w
[6]	SCHNEIDER K M, MOHS A, GUI W F, et al. Imbalanced gut microbiota fuels hepatocellular carcinoma development by shaping the hepatic inflammatory microenvironment[J]. Nat Commun, 2022, 13(1): 3964. doi: 10.1038/s41467-022-31312-5
[7]	TWEEDELL R E, KANNEGANTI T D. It's just a phase: NLRP6 phase separations drive signaling[J]. Cell Res, 2022, 32(2): 113-114. doi: 10.1038/s41422-021-00594-7
[8]	HAO Y C, DINA C, SMALL A M, et al. Dyslipidemia, inflammation, calcification, and adiposity in aortic stenosis: a genome-wide study[J]. Eur Heart J, 2023, 44(21): 1927-1939. doi: 10.1093/eurheartj/ehad142
[9]	HUANG C Y, LIU Q H, TANG Q, et al. Hepatocyte-specific deletion of Nlrp6 in mice exacerbates the development of non-alcoholic steatohepatitis[J]. Free Radic Biol Med, 2021, 169: 110-121. doi: 10.1016/j.freeradbiomed.2021.04.008
[10]	何苗, 曹羽, 王佳, 等. 妊娠期糖尿病患者血清NLRP3炎性小体信号通路的临床研究[J]. 实用临床医药杂志, 2023, 27(16): 89-95. doi: 10.7619/jcmp.20231655
[11]	徐东蕊, 王小龙, 马文超, 等. 核苷酸结合寡聚化结构域样受体蛋白3炎症小体对冠状动脉支架植入术后支架内再狭窄的影响[J]. 实用临床医药杂志, 2021, 25(19): 16-19. doi: 10.7619/jcmp.20211930
[12]	李林昊, 孙月华, 屈沛杰, 等. 小鼠Toll样受体2重组慢病毒的构建及表达测定[J]. 巴楚医学, 2021, 4(4): 91-95. https://www.cnki.com.cn/Article/CJFDTOTAL-BMJJ202104021.htm
[13]	SEREGIN S S, GOLOVCHENKO N, SCHAF B, et al. NLRP6 function in inflammatory monocytes reduces susceptibility to chemically induced intestinal injury[J]. Mucosal Immunol, 2017, 10(2): 434-445. doi: 10.1038/mi.2016.55
[14]	GREMEL G, WANDERS A, CEDERNAES J, et al. The human gastrointestinal tract-specific transcriptome and proteome as defined by RNA sequencing and antibody-based profiling[J]. Gastroenterol, 2015, 50(1): 46-57.
[15]	MUKHERJEE S, KUMAR R, TSAKEM LENOU E, et al. Deubiquitination of NLRP6 inflammasome by Cyld critically regulates intestinal inflammation[J]. Nat Immunol, 2020, 21(6): 626-635. doi: 10.1038/s41590-020-0681-x
[16]	FAN W T, DING C C, LIU S H, et al. Estrogen receptor β activation inhibits colitis by promoting NLRP6-mediated autophagy[J]. Cell Rep, 2022, 41(2): 111454. doi: 10.1016/j.celrep.2022.111454
[17]	CHENG C, HU Y, LI Y N, et al. Qing-Chang-Hua-Shi granule ameliorates DSS-induced colitis by activating NLRP6 signaling and regulating Th17/Treg balance[J]. Phytomedicine, 2022, 107: 154452. doi: 10.1016/j.phymed.2022.154452
[18]	GAO H L, ZHOU H, ZHANG Z Q, et al. Vitamin Dalleviates inflammation in ulcerative colitis by activating the VDR-NLRP6 signaling pathway[J]. Front Immunol, 2023, 14: 1135930. doi: 10.3389/fimmu.2023.1135930
[19]	WU Y, WANG K, WANG X M, et al. The role of the gut microbiome and its metabolites in metabolic diseases[J]. Protein Cell, 2021, 12(5): 360-373. doi: 10.1007/s13238-020-00814-7
[20]	MAMANTOPOULOS M, RONCHI F, VAN HAUWERMEIREN F, et al. Nlrp6-and ASC-dependent inflammasomes do not shape the commensal gut microbiota composition[J]. Immunity, 2017, 47(2): 339-348, e4. doi: 10.1016/j.immuni.2017.07.011
[21]	YDENS E, DEMON D, LORNET G, et al. Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes[J]. Neuroinflammation, 2015, 12: 143. doi: 10.1186/s12974-015-0367-8
[22]	MOUND A, GOORMACHTIGH G, BRAY F, et al. The NLRP6 protein is very faintly expressed in several normal and cancerous epithelial cells and may be confused with an unrelated protein[J]. PLoS One, 2023, 18(1): e0279028. doi: 10.1371/journal.pone.0279028
[23]	MENG C C, ZHANG Y, ZHANG L Y, et al. Effects of NLRP6 in cerebral ischemia/reperfusion (I/R) injury in rats[J]. Mol Neurosci, 2019, 69(3): 411-418. doi: 10.1007/s12031-019-01370-4
[24]	ZHANG Y, JIANG N, ZHANG L Y, et al. NLRP6 expressed in astrocytes aggravates neurons injury after OGD/R through activating the inflammasome and inducing pyroptosis[J]. Int Immunopharmacol, 2020, 80: 106183. doi: 10.1016/j.intimp.2019.106183
[25]	SHEN C, LI R Z, NEGRO R, et al. Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome[J]. Cell, 2021, 184(23): 5759-5774, e20. doi: 10.1016/j.cell.2021.09.032
[26]	ZHANG L, ZHANG X Z, ZHAO W, et al. NLRP6-dependent pyroptosis-related lncRNAs predict the prognosis of hepatocellular carcinoma[J]. Front Med, 2022, 9: 760722. doi: 10.3389/fmed.2022.760722
[27]	SANCHES R C O, SOUZA C, MARINHO F V, et al. NLRP6 plays an important role in early hepatic immunopathology caused by Schistosoma mansoni infection[J]. Front Immunol, 2020, 11: 795. doi: 10.3389/fimmu.2020.00795
[28]	JI X Y, LI L L, LU P, et al. NLRP6 exerts a protective role via NF-kB with involvement of CCL20 in a mouse model of alcoholic hepatitis[J]. Biochem Biophys Res Commun, 2020, 528(3): 485-492. doi: 10.1016/j.bbrc.2020.05.171
[29]	MAINZ R E, ALBERS S, HAQUE M, et al. NLRP6 inflammasome modulates disease progression in a chronic-plus-binge mouse model of alcoholic liver disease[J]. Cells, 2022, 11(2): 182.

Cited By

Cited by

Periodical cited type(6)

1.	杨虹，季红珍，黄萍，陈善美. 腹部联合阴道超声用于生育“二孩”或“三孩”瘢痕子宫孕妇孕早期胎盘植入诊断的价值. 生殖医学杂志. 2024(06): 813-816 .
2.	谢贤海，郑宗杰. 经阴道彩色多普勒超声心动图诊断宫颈部胎盘植入的效果. 医疗装备. 2023(05): 66-69 .
3.	时明芳，常书娟，张聪祎. 超声评分法联合血浆人胎盘催乳素水平检测对前置胎盘合并胎盘植入的诊断价值. 中国医药导报. 2023(09): 97-100 .
4.	高莉，张玉琴，薛祖兵，王小漫，徐庆. 产前不同超声特征对瘢痕子宫胎盘植入的评价分析. 生殖医学杂志. 2023(05): 759-762 .
5.	孙雪，高霞，刘博，周春花，李云鹏. 孕妇胎盘内部血流参数与妊高症关系研究. 西部医学. 2023(05): 765-769 .
6.	张敬丽，谷小乐，元丽芝. 二维超声及彩色多普勒超声诊断孕晚期胎盘植入的价值. 临床医学. 2022(10): 81-83 .

Other cited types(0)

Get Citation

PDF

XML

Article views (145) PDF downloads (6) Cited by(6)

Turn off MathJax

Article Contents

Abstract

References

Construction and identification of nucleotide-binding oligomerization domain-like receptor protein 6 recombinant adenoviral overexpression vector

Abstract

References

Cited by

Periodical cited type(6)

Other cited types(0)

Catalog

Export File

Citation

Format

Content