Expression of Echinococcus granulosus adult-worm EgM123 fused with cholera toxin B and its antigenicity
QI Wen-jing1, 2, HE Li2, WANG Tian2, ZHENG Xue-ting2, GUO Bao-ping2, ZHANG Wen-bao2, KUANG Ling1, LI Jun2
1.College of Veterinary Medicine Xinjiang Agricultural University, Urumqi 830052, China; 2.State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Clinical Medicine Institute, The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, China
Abstract:We aimed to express Echinococcus granulosus EgM123 fused with cholera toxin B (CTB-EgM123) in Escherichia coli and to determine its antigenicity in both mice and dogs. The EgM123 was amplified and subcloned into pET28a containing CTB sequence, which was then transformed into E. coli BL21. The expression of the fused protein CTB-EgM123 was induced by IPTG. The expressed protein was analyzed and identified by SDS-PAGE and Western-Blotting. BalB/c mice and Beagles were immunized, and the serum antibody titers of mice and dogs and antibody isotypes in serum and intestinal mucus were analyzed by ELISA. PCR and sequencing analysis confirmed that the size of CTB-EgM123 was 804 bp and the open reading frame of pET28a/CTB-EgM123 was correct. The high yield of fused protein in form of inclusion body was induced by 0.4 mM of IPTG for 5 hours at 37 °C. SDS-PAGE showed that CTB-EgM123 was size of 37 kDa. Mice were induced with serum titer >320,000 with IgG2a being predominant in the serum. The fused protein was also induced dogs producing higher titer and longer production of antibodies compared with these dogs vaccinated with EgM123 (P<0.05). We found CTB-EgM123 was successfully expressed in E. coli, and the purified protein could induce mice and dogs producing high titer of antibodies in serum and intestinal tissues, indicating the fused recombinant protein has good antigenicity in terms of stimulating high levels of humoral and mucosal immune responses, which is essential for dog vaccine development against E. granulosus infection.
齐文静,何黎,王甜,郑雪婷,郭宝平,张文宝,况玲,李军. 细粒棘球绦虫EgM123蛋白融合霍乱毒素B亚单位疫苗的构建表达及免疫原性研究[J]. 中国人兽共患病学报, 2019, 35(7): 647-654.
QI Wen-jing, HE Li, WANG Tian, ZHENG Xue-ting, GUO Bao-ping, ZHANG Wen-bao, KUANG Ling, LI Jun. Expression of Echinococcus granulosus adult-worm EgM123 fused with cholera toxin B and its antigenicity. Chinese Journal of Zoonoses, 2019, 35(7): 647-654.
[1] Mcmanus DP, Zhang W, Li J, et al.Echinococcosis[J]. Lancet, 2003, 362(9392):1295-1304. DOI: 016/S0140-6736(03)14573-4 [2] 齐颜凤, 伍卫平. 棘球蚴病流行病学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(2):143-148. [3] 严俊, 胡桃, 雷正龙. 全国重点寄生虫病的防控形势与挑战[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(6):412-417. [4] Budke CM, Peter D, Torgerson PR.Global socioeconomic impact of Cystic echinococcosis[J]. Emerg Infect Dis, 2006, 12(2):296-303. DOI: 10.3201/eid1202.050499 [5] 赵莉, 张旭, 张壮志,等. 包虫病诊断技术与预防疫苗的研究进展[J]. 疾病预防控制通报, 2013, (2):84-87. [6] Zhang W, Li J, Lin R, et al.Recent advances in the immunology and serological diagnosis of echinococcosis[M]// Serological Diagnosis of Certain Human, Animal and Plant Diseases. InTech, 2012:24-41. [7] Zhang W, Li J, You H, et al.A gene family from Echinococcus granulosus differentially expressed in mature adult worms[J]. Mol Biochem Parasit, 2003, 126(1):25-33. DOI: 10.1016/S0166-6851(02)00241-4 [8] Zhang W, Mcmanus DP.Vaccination of dogs against Echinococcus granulosus: a means to control hydatid disease?[J]. Trends Parasitol, 2008, 24(9):419-424. DOI: 10.1016/j.pt.2008.05.008 [9] 王慧, 齐文静, 何黎,等. 细粒棘球绦虫成虫相关基因EgM123的克隆、表达及鉴定[J]. 中国病原生物学杂志, 2017, (1):38-41. [10] 任学毅, 朱成钢, 张耀洲. 霍乱毒素B亚单位的研究进展[J]. 中国药学杂志, 2003, 38(12):897-900. [11] Sun J B, Czerkinsky C, Holmgren J.Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit[J]. Scand J Immunol, 2010, 71(1):1-11. DOI: 10.1111/j.1365 -3083.2009.02321 [12] Zhang W, Zhang Z, Yimit T, et al.A pilot study for control of hyperendemic Cystic hydatid disease in China[J]. PLoS Negl Trop Dis, 2009, 3(10):e534. DOI: 10.1371/journal.pntd.0000534 [13] Zhang ZZ, Guo G, Li J, et al.Dog vaccination with EgM proteins against Echinococcus granulosus[J]. Infect Dis Poverty, 2018, 7(1):61. DOI: 10.1186/s40249-018-0425-4 [14] Dertzbaugh MT, Peterson DL, Macrina FL.Cholera toxin B-subunit gene fusion: structural and functional analysis of the chimeric protein[J]. Infect Immun, 1990, 58(1):70-79. [15] Tochikubo K, Isaka M, Yasuda Y, et al.Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally co-administered bovine serum albumin[J]. Vaccine, 1998, 16(2/3):150-155. DOI: 10.1016/S0264-410X(97)00194-1 [16] Oh IG, Jawale CV, Lee JH.The B subunits of cholera and Escherichia coli heat-labile toxins enhance the immune responses in mice orally immunised with a recombinant live P-fimbrial vaccine for avian pathogenic E. coli[J]. Acta Vet Hung, 2014, 62(3): 293-303. DOI: 10.1556/AVet.2014.006 [17] Hou J, Liu Y, Hsi J, et al.Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice[J]. Hum Vaccin Immunother, 2014, 10(5): 1274-1283. DOI: 10.4161 /hv.28371 [18] 周虎. 新型多房棘球蚴亚单位疫苗CTB-Emy162的研究[D]. 西宁:青海大学, 2016. [19] Lee J, Yoo JK, Sohn HJ, et al.Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants[J]. Parasitol Res, 2015, 114(4): 1377-1385. DOI: 10.1007/s00436-015-4316-3 [20] Brunner R, Jensen-Jarolim E, Pali-schöll I. The ABC of clinical and experimental adjuvants-a brief overview[J]. Immunol Lett, 2010, 128(1): 29-35. DOI: 10.1016/j.imlet.2009.10.005 [21] Coccia EM, Remoli ME, Di GC, et al.Cholera toxin subunit B inhibits IL-12 and IFN-γ production and signaling in experimental colitis and Crohn’s disease[J]. Gut, 2005, 54(11):1558-1564. DOI: 10.1136/ gut.2004.062174 [22] Holmgren J, Adamsson J, Anjuère F, et al.Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA[J]. Expert Rev Vaccines, 2005, 97(2):181-188. DOI: 10.1016/j.imlet.2004.11.009 [23] Khabiri AR, Bagheri F, Assmar M, et al.Analysis of specific IgE and IgG subclass antibodies for diagnosis of Echinococcus granulosus[J]. Parasite Immunol, 2010, 28(8):357-362. DOI: 10.1111/j.1365-3024.2006. 00837.x