|
|
|
| Preparation and identification of human cytomegalovirus US31 protein specific antibody |
| CHEN Bi1, 2, XIE Shangdan1, XIANG Chaoli1, SHI Xinyu3, GUO Gangqiang3, OUYANG Jingzhong1, SHEN Xian1 |
| 1.The Second Clinical Medical College, Wenzhou Medical University, Wenzhou 325035, China; 2.Department of Surgical Oncology, Wenzhou People’s Hospital, Wenzhou 325000, China; 3.School of Basic Medical Sciences, Wenzhou Medical University, Wenzhou 325035, China. |
|
| Cite this article: |
|
CHEN Bi,XIE Shangdan,XIANG Chaoli, et al. Preparation and identification of human cytomegalovirus US31 protein specific antibody[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2019, 49(8): 547-551.
|
|
|
|
Abstract Objective: To explore the preparation and identification of human cytomegalovirus (HCMV) US31 protein and its polyclonal antibody. Methods: After HCMV US31 gene was optimized by prokaryotic codon for whole gene synthesis and cloned to pET21a (+) prokaryotic expression vector, pET21a (+)/HCMV US31 recombinant plasmid was constructed, with identified sequence. Recombinant plasmid was transformed into Escherichia coli BL21 (DE3), target protein expression induced by IPTG, and US31 protein purified by Ni-NTA affinity chromatography. Immunized New Zealand rabbits were used to prepare polyclonal antibodies, and antibody titer and specificity was detected by ELISA, Western blot and immunofluorescence method. Results: High expression of HCMV US31 fusion protein was induced in Escherichia coli, and high purity target protein was obtained by nickel column affinity purification, and the specific IgG antibody was induced by immune New Zealand rabbits, with the valence being 1:49 600. ELISA, Western blot and immunofluorescence have confirmed that the prepared specific antibodies were able to identify US31 proteins expressed in cells. Conclusion: HCMV US31 protein and specific antibodies were successfully prepared, which laid foundations for further study of the biological function of US31 protein.
|
|
|
| |
|
|
|
[1] BURGDORF S W, CLARK C L, BURGDORF J R, et al. Mutation of glutamine to arginine at position 548 of IE2 86 in human cytomegalovirus leads to decreased expression of IE2 40, IE2 60, UL83, and UL84 and increased transcription of US8-9 and US29-32[J]. J Virol, 2011, 85(21): 11098-11110.
[2] GUO G, YE S, XIE S, et al. The cytomegalovirus protein US31 induces inflammation through mono-macrophages in systemic lupus erythematosus by promoting NF-κB2 activa-tion[J]. Cell Death Dis, 2018, 9(2): 104.
[3] 李建蓉, 黄汉菊. HCMV基因及其产物在感染中的作用[J].国外医学(病毒学分册), 2005, 12(6): 175-178.
[4] BOECKH M, LJUNGMAN P. How we treat cytomegalovirus in hematopoietic cell transplant recipients[J]. Blood, 2009, 113(23): 5711-5719.
[5] PIGNATELLI S, DAL MONTE P, ROSSINI G, et al. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains[J]. Rev Med Virol, 2004, 14(6): 383-410.
[6] 魏中南, 胡洪波, 彭巧英. 人巨细胞病毒AD169株UL115基因编码gL蛋白序列分析及B细胞表位预测[J]. 临床血液学杂志(输血与检验版), 2012, 25(8): 505-507.
[7] 何雨. 高危人群HCMV和BKV感染情况的调查及其与肾病关系的研究[D]. 南宁: 广西医科大学, 2010.
[8] BATE S L, DOLLARD S C, CONNON M J. Cytomegalovirus seroprevalence in the United States: the national health and nutrition examination surveys[J]. Clin Infect Dis, 2010, 50(11): 1439-1447.
[9] ENGSTRAND M, TOURNAY C, PEYRAT M A, et al. Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants[J]. Transplantation, 2000, 69(11): 2243-2250.
[10] 王羽, 廖翔, 岳俊杰, 等. 福氏志贺菌5a型M90T株GAPDH蛋白多克隆抗体的制备和鉴定[J]. 细胞与分子免疫学杂志, 2014, 30(4): 407-410.
[11] 余晓霞, 刘华峰, 陈孝文, 等. EBV LMP1和CMVpp65在肾脏疾病肾组织中的表达[J]. 临床与实验病理学杂志, 2011, 27(3): 257-260. |
|
|
|
