桥连双苯丙氨酸二肽增强灭活猪丁型冠状病毒在小鼠上的免疫效果分析

doi:10.11843/j.issn.0366-6964.2023.04.022

摘要/Abstract

摘要： 猪丁型冠状病毒（porcine deltacoronavirus，PDCoV）会引起新生仔猪腹泻、呕吐甚至死亡，对生猪养殖造成了严重危害。苯丙氨酸二肽及其衍生物可自组装成纳米超分子组装体，具有负载并递送药物的功能。本研究旨在探究桥连双苯丙氨酸二肽是否可以增强PDCoV的免疫原性。首先对桥连双苯丙氨酸二肽进行细胞毒性评价，之后作为佐剂（0.1 mg·mL^-1）等体积混合灭活PDCoV （PDCoV HNZK-02，TCID₅₀为10⁸·0.1 mL^-1），制备灭活疫苗。将灭活疫苗免疫小鼠（200 μL·只^-1），采用ELISA方法检测PDCoV N蛋白特异性IgG抗体、CCK-8试剂盒检测淋巴细胞增殖SI指数、乳酸脱氢酶（LDH）方法检测特异性细胞毒性T淋巴细胞（CTL）活性。小鼠免疫5周后，灌胃攻毒PDCoV （500 μL·只^-1），3 d后进行回肠等组织病毒载量检测。结果显示，桥连双苯丙氨酸二肽对猪睾丸细胞没有明显毒性作用，作为佐剂与灭活PDCoV混合免疫小鼠后，PDCoV N蛋白特异性IgG抗体水平、SI和CTL检测结果均高于灭活PDCoV组，肠道组织病毒载量显著低于灭活PDCoV组。桥连双苯丙氨酸二肽有良好的佐剂效果，具有成为新型安全有效佐剂的潜力。

关键词: 桥连双苯丙氨酸二肽, 佐剂, 猪丁型冠状病毒, 免疫评价

Abstract: Porcine deltacoronavirus (PDCoV) can cause diarrhea, vomiting and even lead to death of newborn piglets, which has serious harm to pig breeding. Phenylalanine dipeptide and its derivatives can be self-assembled into nanometer supramolecular assemblies, which have the function of loading and delivering drugs. This study was conducted to explore whether bridging diphenylalanine dipeptide can enhance the immunogenicity of PDCoV. Firstly, its cytotoxicity was evaluated. Then, it was used as adjuvant (0.1 mg·mL^-1) and mixed with equal volume of inactivate PDCoV (PDCoV HNZK-02, TCID₅₀=10⁸·0.1 mL^-1) to prepare inactivated vaccine. Mice (200 μL per mouse) were immunized with inactivated vaccine, and the specific IgG antibody was detected by ELISA method, the SI index of lymphocyte proliferation was detected by CCK-8 kit, and the activity of specific cytotoxic T lymphocytes (CTL) was detected by lactate dehydrogenase (LDH). After 5 weeks of immunization, mice were challenged with PDCoV (500 μL per mouse) by intragastric administration, and the viral load of ileum and other tissues was detected 3 days later. The results showed that bridged diphenylalanine dipeptide had no obvious toxic effect on porcine testicular cells. Mice immunized with inactivated PDCoV with bridging diphenylalanine dipeptide could produce high level of IgG, SI and CTL were higher than those of the inactivated PDCoV group, and the intestinal tissue viral load was significantly lower than that of the inactivated PDCoV group. Bridged diphenylalanine dipeptide showed a good adjuvant effect and has the potential to become a new safe and effective adjuvant.

Key words: bridging diphenylalanine dipeptide, adjuvant, porcine deltacoronavirus, evaluation of immune

中图分类号:

S852.4

王子, 王年祥, 田长明, 赵福杰, 刘林涛, 马梦瑶, 贾鑫浩, 刘国星, 郑兰兰. 桥连双苯丙氨酸二肽增强灭活猪丁型冠状病毒在小鼠上的免疫效果分析[J]. 畜牧兽医学报, 2023, 54(4): 1590-1597.

WANG Zi, WANG Nianxiang, TIAN Changming, ZHAO Fujie, LIU Lintao, MA Mengyao, JIA Xinhao, LIU Guoxing, ZHENG Lanlan. Using Mouse to Evaluate the Immune Effect of Bridged Diphenylalanine Dipeptide with Inactivated Porcine Deltacoronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1590-1597.

参考文献 32

[1]	WOO P C Y, LAU S K P, LAM C S F, et al. Discovery of seven novel Mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J]. J Virol, 2012, 86(7):3995-4008.
[2]	HU H, JUNG K, VLASOVA A N, et al. Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States[J]. J Clin Microbiol, 2015, 53(5):1537-1548.
[3]	MARTHALER D, RAYMOND L, JIANG Y, et al. Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus[J]. Emerg Infect Dis, 2014, 20(8):1347-1350.
[4]	LEE S, LEE C. COMPLETE genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J]. Genome Announc, 2014, 2(6):e01191.
[5]	SAENG-CHUTO K, LORSIRIGOOL A, TEMEEYASEN G, et al. Different lineage of porcine deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015[J]. Transbound Emerg Dis, 2017, 64(1):3-10.
[6]	JUNG K, HU H, EYERLY B, et al. Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J]. Emerg Infect Dis, 2015, 21(4):650-654.
[7]	MA Y M, ZHANG Y, LIANG X Y, et al. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States[J]. mBio, 2015, 6(2):e00064-15.
[8]	LI W T, HULSWIT R J G, KENNEY S P, et al. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility[J]. Proc Natl Acad Sci USA, 2018, 115(22):E5135-e5143.
[9]	JUNG K, VASQUEZ-LEE M, SAIF L J. Replicative capacity of porcine deltacoronavirus and porcine epidemic diarrhea virus in primary bovine mesenchymal cells[J]. Vet Microbiol, 2020, 244:108660.
[10]	LIANG Q Q, ZHANG H L, LI B X, et al. Susceptibility of chickens to porcine deltacoronavirus infection[J]. Viruses, 2019, 11(6):573.
[11]	BOLEY P A, ALHAMO M A, LOSSIE G, et al. Porcine deltacoronavirus infection and transmission in Poultry, United States[J]. Emerg Infect Dis, 2020, 26(2):255-265.
[12]	ZHANG H L, DING Q W, YUAN J, et al. Susceptibility to mice and potential evolutionary characteristics of porcine deltacoronavirus[J]. J Med Virol2022, 94(12):5723-5738.
[13]	ZHAO F J, LIU L T, WANG Z, et al. Development and immunogenicity evaluation of porcine deltacoronavirus inactivated vaccine with different adjuvants in mice[J]. Vaccine, 2022, 40(31):4211-4219.
[14]	LEDNICKY J A, TAGLIAMONTE M S, WHITE S K, et al. Independent infections of porcine deltacoronavirus among Haitian children[J]. Nature, 2021, 600(7887):133-137.
[15]	LI J L, XING R R, BAI S, et al. Recent advances of self-assembling peptide-based hydrogels for biomedical applications[J]. Soft Matter, 2019, 15(8):1704-1715.
[16]	LIU K, ZHANG H, XING R R, et al. Biomimetic oxygen-evolving photobacteria based on amino acid and porphyrin hierarchical self-organization[J]. ACS Nano, 2017, 11(12):12840-12848.
[17]	SHEN H L, XU W L, WU Z Y, et al. Vector-based RNAi approach to isoform-specific downregulation of vascular endothelial growth factor (VEGF)165 expression in human leukemia cells[J]. Leukem Res, 2007, 31(4):515-521.
[18]	CAO S Q, LIU Y P, SHANG H, et al. Supramolecular nanoparticles of calcitonin and dipeptide for long-term controlled release[J]. J Control Release, 2017, 256:182-192.
[19]	YAN X H, HE Q, WANG K W, et al. Transition of cationic dipeptide nanotubes into vesicles and oligonucleotide delivery[J]. Angew Chem Int Ed, 2007, 46(14):2431-2434.
[20]	刘青川, 王晨吟, 何书桃, 等. 苯丙氨酸二肽类化合物Y101对DHBV-DNA的影响[J]. 中国药理学通报, 2012, 28(10):1389-1393.LIU Q C, WANG C Y, HE S T, et al. Effects of phenylalanine dipeptide compound Y101 on DHBV-DNA[J]. Chinese Pharmacological Bulletin, 2012, 28(10):1389-1393. (in Chinese)
[21]	刘青川, 葛敏, 徐雪钰, 等. 苯丙氨酸二肽类化合物Y101对正常小鼠的免疫调节作用[J]. 解放军药学学报, 2011, 27(3):215-217.LIU Q C, GE M, XU X Y, et al. Immunifaction accommodation of phenylalanine dipeptide compound Y101 in normal mice[J]. Pharnmaceutical Journal of Chinese People's Liberation Army, 2011, 27(3):215-217. (in Chinese)
[22]	REN H J, YAN X G, LIU L T, et al. Identification of two novel B-cell epitopes on the nucleocapsid protein of porcine deltacoronavirus[J]. Virol Sin, 2022, 37(2):303-306.
[23]	JANETANAKIT T, LUMYAI M, BUNPAPONG N, et al. Porcine deltacoronavirus, Thailand, 2015[J]. Emerg Infect Dis, 2016, 22(4):757-759.
[24]	SUZUKI T, SHIBAHARA T, IMAI N, et al. Genetic characterization and pathogenicity of Japanese porcine deltacoronavirus[J]. Infect Genet Evolut, 2018, 61:176-182.
[25]	张帆帆, 宋德平, 周信荣, 等. 新现猪Delta冠状病毒RT-PCR检测方法的建立及其应用[J]. 中国农业科学, 2016, 49(7):1408-1416.ZHANG F F, SONG D P, ZHOU X R, et al. Establishment and application of a RT-PCR assay for detection of newly emerged porcine Deltacoronavirus[J]. Journal of Integrative Agriculture, 2016, 49(7):1408-1416. (in Chinese)
[26]	ZHAI S L, WEI W K, LI X P, et al. Occurrence and sequence analysis of porcine deltacoronaviruses in southern China[J]. Virol J, 2016, 13(1):136.
[27]	宋亚兵, 徐帅飞, 苏丹萍, 等. 2012年~2016年广东省猪丁型冠状病毒的流行病学调查及分析[J]. 中国预防兽医学报, 2018, 40(10):886-890.SONG Y B, XU S F, SU D P, et al. Epidemiological survey and analysis of porcine deltacoronavirus in Guangdong province from 2012 to 2016[J]. Chinese Journal of Preventive Veterinary Medicine, 2018, 40(10):886-890. (in Chinese)
[28]	DONG N, FANG L R, YANG H, et al. Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014[J]. Vet Microbiol, 2016, 196:98-106.
[29]	夏赟, 武梦玉, 张永辉. 免疫佐剂的发展现状与未来趋势[J]. 中国科学基金, 2020, 34(5):573-580.XIA Y, WU M Y, ZHANG Y H. Current status and future trends of immunologic adjuvants[J]. Bulletin of National Natural Science Foundation of China, 2020, 34(5):573-580. (in Chinese)
[30]	REED S G, ORR M T, FOX C B. Key roles of adjuvants in modern vaccines[J]. Nat Med, 2013, 19(12):1597-1608.
[31]	TOM J K, ALBIN T J, MANNA S, et al. Applications of immunomodulatory immune synergies to adjuvant discovery and vaccine development[J]. Trends Biotechnol, 2019, 37(4):373-388.
[32]	侯永利, 吴晓霞, 胡四海. 纳米佐剂在疫苗研究中的应用[J]. 微生物学免疫学进展, 2018, 46(5):80-85.HOU Y L, WU X X, HU S H. Applications of nanoparticle-based adjuvants on the vaccine[J]. Progress in Microbiology and Immunology, 2018, 46(5):80-85. (in Chinese)