Transient expression of recombinant S1 protein of Porcine Epidemic Diarrhea Virus in Nicotiana benthamiana

Ho Thi Thuong; Le Thu Ngoc; Nguyen Thu Giang; Trinh Thai Vy; Phan Trong Hoang; Pham Bich Ngoc; Vu Huyen Trang; Chu Hoang Ha

doi:10.15625/1811-4989/14614

Author affiliations

Authors

Ho Thi Thuong Institute of Biotechnology
Le Thu Ngoc Institute of Biotechnology
Nguyen Thu Giang Institute of Biotechnology
Trinh Thai Vy Institute of Biotechnology
Phan Trong Hoang LEIBNIZ-Institute for Plant Genetic and Crop Plant Research
Pham Bich Ngoc Institute of Biotechnology
Vu Huyen Trang Institute of Biotechnology
Chu Hoang Ha Institute of Biotechnology

DOI:

https://doi.org/10.15625/1811-4989/14614

Keywords:

Protein S1 tái tổ hợp, PEDV, biểu hiện tạm thời, vắc-xin thực vật, Agrobacterium tumefacines

Abstract

Porcine Epidemic Diarrhea (PED) is an infectious disease with high mortality especially in suckling piglets. Among the structural proteins of Porcine Epidemic Diarrhea Virus (PEDV), the S protein (including sub-domain S1 and S2), is a homotrimer protein that plays an important role in attaching the viruses to the cell receptors. In particular, the S1 protein is considered as an important sub-component in the development of effective vaccines against PEDV. In this study, for the purpose of expressing S1 in the original form of trimmer and oligomer of trimer based on S-tag and S-protein interactions, the DNA encoding for S1 protein was fused with GCN4pII or GCN4pII-Stag, was then inserted to the pRTRA cloning vector under the control of the 35S CaMV promoter. After that, the whole cassete was inserted into the pCB301 vector and transformed into Agrobacterium tumefaciens for transient expression on Nicotiana benthamiana. The expression of recombinant S1 proteins in tobacco was determined by Western blot. The results showed that the expression levels of S1 trimer and S1 trimer S-tag proteins were equal in plants, which also indicated that S-tag fusion did not affect the expression level of the S1 protein. However, the expression level of S1 proteins was relatively low, reaching 0.005% of total soluble protein. In addition, the expression of S1 trimer S-tag protein and Sprotein-tp protein by co-transformation of two A. tumefaciens strains containing corresponding vectors in plants were also determined by Western blot. This is a premise study for the development of subunit vaccines in plants that prevent the spread of PEDV.

Downloads

Download data is not yet available.

References

Asai, T, Wims, LA, Morrison, SL (2005). An interaction between S tag and S protein derived from human ribonuclease 1 allows site-specific conjugation of an enzyme to an antibody for targeted drug delivery. J Immunol Methods 299: 63–76.

Bae, JL, Lee, JG, Kang, TJ, Jang, HS, Jang, YS and Yang, MS (2003)

Induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen. Vaccine 21: 4052-4058.

Chang, SH, Bae, JL, Kang, TJ, Kim, J, Chung, GH, Lim, CW, et al. (2002). Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus. Mol Cells 14: 295-9.

Chen, Q, Lai, H, Hurtado, J, Stahnke, J, Leuzinger, K, and Dent, M (2013). Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins. Adv Tech Biol Med 1: 103. doi: 10.4172/atbm.1000103

Chiou, HY, Huang, YL, Deng, MC, Chang, CY, Jeng, CR, Tsai, PS et al. (2015). Phylogenetic Analysis of the Spike (S) Gene of the New Variants of Porcine Epidemic Diarrhoea Virus in Taiwan. Transbound Emerg Dis 64: 157–166. doi: 10.1111/tbed.12357

Conrad, U., Plagmann, I., Malchow, S., Sack, M., Floss, D.M., Kruglov, A.A., Nedospasov, S.A., Rose-John, S., Scheller, J. (2011). ELPylated anti-human TNF therapeutic single-domain antibodies for prevention of lethal septic shock. Plant Biotechnol J. 9, 22–31 doi: 10.4172/2379-1764.1000103

Cruz, DJ, Kim, CJ, Shin, HJ (2008). The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus. Virus Res 132:192¬6.

Deng, F, Ye, G, Liu, Q, Navid, MT, Zhong, X, Li, Y, et al. (2016). Identification and comparison of receptor binding haracteristics of the spike protein of two porcine pidemic diarrheavirus strains. Viruses. doi: 10.3390/v8030055.

Jung, K & Saif, L J (2015). Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet J 204: 134–143.

Huy, NX, Kim, SH, Yang, MS and Kim, TG (2012) Immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: M cell targeting ligand fusion protein expressed in transgenic rice calli. Plant Cell Rep 31: 1933-1942.

Khamis, Z (2016). Producing a Subunit Vaccine for Porcine Epidemic Diarrhea Virus. Electronic Thesis and Dissertation. Repository: 4296.https://ir.lib.uwo.ca/etd/4296

Kim YK, Lim SI, Lim JA, Cho IS, Park EH, Le VP, Hien NB, Thach PN, Quynh do H, Vui TQ, Tien NT, An DJ (2015). A novel strain of porcine epidemic diarrhea virus in Vietnamese pigs. Arch Virol 160(6):1573-7.

Kim, JS, Raines, RT (1993). Ribonuclease S-peptide as a carrier in fusion proteins. Protein Sci 2: 348–356.

Kun, M, Bing, Y, Jing, X, XiangJie, Q, HongZhuan, Z, FuZhou, X, ChengYang, X, FengPing, Y, LiQuan, Z and XiangLong, L (2014) Expression of core neutralizing epitope gene of porcine epidemic diarrhea virus in maize. JASFT (Beijing) 16, 28-35.

Langel, SN, Paim, FC, Lager, KM, Vlasova, AN, Saif, LJ (2016). Lactogenic immunity and vaccines for porcine epidemic diarrhea virus (PEDV): historical and current concepts. Virus Res 226: 93–107.

Lee, C (2015). Porcine epidemic diarrhea virus: An emerging and re-emerging epizootic swine virus. Virol J 12:193.

Makadiya, N, Brownlie, R, van den Hurk, J, Berube, N, Allan, B, Gerdts, V, Zakhartchouk, A (2016). S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen. Virol J 13:57.

Oldham, J (1972) Letter to the editor. Pig Farming 10:72-3.

Park, SJ, Kim, HK, Song, DS, An, DJ, Park, BK (2012). Complete genome sequences of a Korean virulent porcine epidemic diarrhea virus and its attenuated counterpart. J Virol 86: 5964.

Phan, HT, Ho, TT, Chu, HH, Vu, TH, Gresch, U, Conrad, U (2017). Neutralizing immune responses induced by oligomeric H5N1-hemagglutinins from plants. Vet Res 48: 53.

Phan, HT, Pohl, J, Floss, DM, Rabenstein, F, Veits, J, Le, BT, Chu, HH, Hause, G, Mettenleiter, T, Conrad, U (2013). ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice. Plant Biotechnol J 11: 582-93.

Sato, T, Takeyama, N, Katsumata, A, Tuchiya, K, Kodama, T, Kusanagi, K (2011). Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo. Virus Genes 43: 72–78.

Song, D, Park, B (2012). Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines. Virus Genes 44: 167-75.

Suzuki, T, Murakami, S, Takahashi, O, Kodera, A, Masuda, T, Itoh, S, et al. (2015). Molecular characterization of pig epidemic diarrhoea viruses isolated in Japan from 2013 to 2014. Infect Genet Evol 36:363– 8.

Tian, Y, Yu, Z, Cheng, K, Liu, Y, Huang, J, Xin, Y, Li, Y, et al. (2013). Molecular characterization and phylogenetic analysis of new variants of the porcine epidemic diarrhea virus in Gansu, China in 2012. Viruses 5: 1991–2004.

Topp, E, Irwin, R, McAllister, T, Lessard, M, Joensuu, JJ, Kolotilin, I, et al. (2016). The case for plantmade veterinary immunotherapeutics. Biotechnol Adv 34:597–604. doi: 10.1016/j.biotechadv.2016.02.007

Wang X, Chen J, Shi D, Shi H, Zhang X, Yuan J, Jiang S, Feng L (2016) Immunogenicity and antigenic relationships among spike proteins of porcine epidemic diarrhea virus subtypes G1 and G2. Arch Virol 161:537–47.

Xiang C, Han P, Lutziger I, Wang K, and Oliver D (1999). A mini binary vector series for plant transformation. Plant Mol Biol 40:711.