|
|
Effect of Vesicle Transport Protein SEC22B Knockdown on the Replication of Bovine viral diarrhea virus |
ZHANG Cheng-Yuan, YUAN Yuan-Yuan, GUO Yan-Ting, YANG Li, RAN Duo-Liang, FU Qiang, SHI Hui-Jun* |
College of Animal Medicine, Xinjiang Agricultural University, Urumqi 830052, China |
|
|
Abstract The infection of bovine viral diarrhea is becoming more and more serious, which has caused serious losses to the breeding industry. At present, the lack of effective treatment leads to the complicated infection pattern in China, and the research on the pathogenesis and virus-host interaction is still unclear. To investigate the effect of vesicle transport protein (SEC22B) deletion on Bovine viral diarrhea virus (BVDV) replication, according to the SEC22B gene sequence in GenBank database, small guide RNA (sgRNA) was designed using Benchling platform and cloned into LentiCRISPR V2 vector for lentivirus packaging. The SEC22B gene in madin-darby bovine kidney cells (MDBK) was knocked down (KD) by CRISPR/Cas9 gene editing technique, and identified by Western blot. After BVDV infection with SEC22B KD cells at different times, the changes of 5' UTR mRNA level and accumulation of double-stranded RNA (dsRNA) were detected by qPCR and immunofluorescence staining, respectively. Reed-Muench method was used to measure the titer change of progeny virus after BVDV infection at different time. The results showed that compared with scramble cells, the protein expression level of SEC22B in SEC22B KD cells was significantly decreased by Western blot. Compared with scramble cells infected with BVDV, the level of 5' UTR mRNA and dsRNA of SEC22B KD cells were extremely significantly decreased (P<0.01) after BVDV infection, and the titer of progeny virus was extremely significantly decreased (P<0.01). The results showed that knockdown SEC22B gene significantly inhibited the replication of BVDV. This study provides a target for the establishment of new methods for the prevention and control of BVDV.
|
Received: 14 April 2022
|
|
Corresponding Authors:
* shihuijunmm@163.com
|
|
|
|
[1] 封华, 陈晨, 王义琴, 等. 2009. 植物可溶性 N-乙基马来酰亚胺敏感因子连接物复合体(SNAREs)及其生物学功能研究进展[J]. 遗传, 31(5): 471-478. (Feng H, Chen C, Wang Y Q, et al. 2009. Research progress on soluble N-ethyl maleimide sensitive factor linker complex (SNAREs) and its biological function[J]. Genetics, 31(5): 471-478. ) [2] 郭妍婷. 2021. 牛病毒性腹泻病毒离子通道蛋白 p7 的互作蛋白筛选及功能研究[D]. 硕士学位论文, 新疆农业大学, 导师 : 付强, 史慧君, pp. 16-19. (Guo Y T. 2021. Screening and functional study of the interaction protein of Bovine viral diarrhea virus ion channel protein P7 [D]. Thesis for M. S., Xinjiang Agricultural University, Su-pervisor: Fu Q, Shi H J, pp. 16-19) [3] 胡新艳, 郭妍婷, 赵新艳, 等. 2020. 紧密连接蛋白 Occludin 影响牛病毒性腹泻病毒感染[J]. 中国兽医学报, 40(11): 2119-2126. (Hu X Y, Guo Y T, Zhao X Y, et al. 2020. Effects of tight junction protein Occludin on Bo-vine viral diarrhea virus infection[J]. Chinese Journal of Veterinary Medicine, 40(11): 2119-2126. ) [4] 李新培, 周伟光, 关平原, 等. 2018. 牛病毒性腹泻病毒致病机制研究进展[J]. 中国畜牧兽医, 045(008): 2303-2311. (Li X P, Zhou W G, Guan P Y, et al. 2018. Re-search progress on pathogenic mechanism of Bovine vi-ral diarrhea virus[J]. China Animal Husbandry and Vet-erinary, 045(008): 2303-2311. ) [5] 李艳萍, 王立群, 李慧霞, 等. 2020. 一株源于商品胎牛血清的 BVDV-2 型的分离鉴定及基因组序列分析[J]. 畜牧兽医学报, 051(002): 320-328. (Li Y P, Wang L Q, Li H X, et al. 2020. Isolation, identification and genome se-quence analysis of a BVDV-2 strain derived from com-mercial fetal bovine serum[J]. Journal of Animal Sci-ence and Veterinary Medicine, 051(002): 320-328. ) [6] 田瑞鑫, 李胜男, 胡新艳, 等. 2020. 牛 miR-29b 影响牛病毒性腹泻病毒感染 BALB/c 小鼠的作用[J]. 中国兽医学报, 40(02): 264-271. (Tian R X, Li S N, Hu X Y, et al. 2020. Effect of bovine Mir-29b on Bovine viral diarrhea virus infection in BALB/C mice[J]. Chinese Journal of Veterinary Medicine, 40(02): 264-271. ) [7] 王晓华. 2011. 植物细胞物质转运中分泌囊泡动态和胞间连丝组成蛋白的细胞分子生物学研究[D]. 博士学位论文, 中国科学院研究生院, 导师: 林金星, 王钦丽, pp. 6-8. (Wang X H. 2011. Dynamics of secretory vesicles and plasmodesmata component proteins in plant cellular ma-terial transport [D]. Thesis for Ph. D., Graduate School of Chinese Academy of Sciences, Supervisor: Lin J X, Wang Q L, pp. 6-8. ) [8] Cai H Q, Ferro N S, Reinisch K, et al. 2007. Coats, tethers, Rabs, and SNAREs work together to mediate the intra-cellular destination of a transport vesicle[J]. Develop-mental Cell, 12: 671-682. [9] Cebrian I, Visentin G, Blanchard N, et al. 2011. Sec22b regu-lates phagosomal maturation and antigen cross presenta-tion by dendritic cells[J]. Cell, 147(6): 1355-1368. [10] Largo E,Gladue D P, Torralba J, et al. 2018. Mutation-in-duced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses[J]. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1860(5): 1015-1021. [11] Lei C, Yang J, Hu J, et al. 2021. On the calculation of TCID50 for quantitation of virus infectivity[J]. Virologica Sinica, 36(1): 141-144. [12] Mosena A C S, Falkenberg S M, Ma H, et al. 2020. Multivari-ate analysis as a method to evaluate antigenic relation-ships between BVDV vaccine and field strains[J]. Vac-cine, 38(36): 5764-5772. [13] Sun W, Tian B X, Wang S H, et al. 2020. The function of SEC22B and its role in human diseases[J]. Cytoskele-ton, 77(8): 303-312. [14] Wang T, Li L, Hong W. 2017. SNARE proteins in membrane trafficking[J]. Traffic, 18(12): 767-775. [15] Wu S R J, Khoriaty R, Kim S H. et al. 2019. SNARE protein SEC22B regulates early embryonic development[J]. Sci-entific Reports, 9(1): 11434. [16] Zhao C, Shen X, et al. Wu R. 2017. Classical swine fever virus nonstructural protein p7 modulates infectious virus pro-duction[J]. Scientific Reports, 7(1): 12995. |
|
|
|