P[1]型A群牛轮状病毒VP8重组蛋白的表达与免疫原性研究

doi:10.16431/j.cnki.1671-7236.2022.05.038

Abstract

Abstract: 【Objective】 The purpose of this study was to express P[1] Bovine rotavirus A (BRVA) VP8 recombinant protein by prokaryotic expression system,and evaluate its reactivity and immunogenicity.【Method】 The codon preference was optimized according to the VP8 gene sequence of domestic epidemic strain RVA/Cow-tc/CHN/SDA2/2018/G6P[1] (GenBank accession No.:MN937517),the prokaryotic expression system of P[1] BRVA VP8 E.coli was constructed,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,the expression was induced by IPTG,and the recombinant protein was purified by nickel agarose affinity chromatography.Western blotting was used to identify the specific recognition ability of recombinant protein and rabbit anti-G6P[1] BRVA serum,and P[1] BRVA VP8 recombinant protein was used to immunize rabbits.Two weeks after the first immunization,the immunization was strengthened once and twice in total.The immunogenicity was evaluated by indirect ELISA and neutralization test.【Result】 The prokaryotic expression system of P[1] type BRVA VP8 recombinant protein was successfully constructed,SDS-PAGE showed that the recombinant protein was highly expressed with a size of 20 ku,and the recombinant protein existed in soluble form.Western blotting results showed that the recombinant protein could specifically bind to rabbit anti-G6P[1] BRVA serum.Indirect ELISA results showed that P[1] type BRVA VP8 recombinant protein could induce antibody production in rabbits.The antibody began to appear and increased rapidly in the 1st week after the first immunization,peaked in the 3rd week and remained at a high level until to the 5th week.The neutralization test results showed that the neutralization titers of immunized rabbit serum to G6P[1] and G8P[1] BRVA were 1∶17 783 and 1∶64,respectively.【Conclusion】 This study successfully constructed a prokaryotic expression system to express P[1] BRVA VP8 recombinant protein,and confirmed that it had good reactivity and immunogenicity,which laid a foundation for the development of BRVA subunit vaccine.

Key words: Bovine rotavirus A (BRVA); P[1]; VP8 protein; prokaryotic expression; immunogenicity

CLC Number:

S82.65^＋3

JIANG Xiaoming, TANG Cheng, YUE Hua. Expression and Immunogenicity of P[1] Bovine Rotavirus A VP8 Recombinant Protein[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1970-1976.

References

[1] GENTSCH J R,LAIRDA R,BIEFELT B,et al.Serotype diversity and reassortment between human and animal Rotavirus strains:Implications for Rotavirus vaccine programs[J].Journal of Infectious Diseases,2005,192(Suppl 1):S146-S159.
[2] VLASOVA A N,AMIMO J O,SAIF L J.Porcine rotaviruses:Epidemiology,immune responses and control strategies[J].Viruses,2017,9(3):48.
[3] JIN S L,BING C,HAN C G,et al.Faecal shedding of Rotavirus vaccine in Chinese children after vaccination with Lanzhou lamb rotavirus vaccine[J].Scientific Reports,2018,8(1):1001.
[4] 常继涛,于力.牛轮状病毒引起的犊牛腹泻研究进展[J].中国奶牛,2016,2:22-25. CHANG J T,YU L.Research progress of calf diarrhea caused by Bovine rotavirus[J].China Dairy,2016,2:22-25.(in Chinese)
[5] 常继涛,于力.牛轮状病毒基因重配二价减毒疫苗接种怀孕母牛的免疫原性评价[J].中国预防兽医学报,2013,35(6):481-484. CHANG J T,YU L.Evaluationg the immunogenicity of Bovine rotavirus reassortant bivalent attenuated vaccine in pregnant cows[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(6):481-484.(in Chinese)
[6] SURADHAT S,YOO D,BABIUK L A,et al.DNA immunization with a Bovine rotavirus VP4 gene induces a Th1-like immune response in mice[J].Viral Immunology,1997,10:117-127.
[7] KIM Y,NIELSEN P R,HODGINS D,et al.Lactogenic antibody responses in cows vaccinated with recombinant Bovine rotavirus-like particles (VLPs) of two serotypes or inactivated Bovine rotavirus vaccines[J].Vaccine,2002,20:1248-1258.
[8] WIGDOROVITZ A,MOZGOVZJ M,SANTOS M J D,et al.Protective lactogenic immunity conferred by an edible peptide vaccine to Bovine rotavirus produced in transgenic plants[J].Journal of General Virology,2004,85:1825-1832.
[9] ZHAO Q H,WEN Y L,YANG Y U,et al.Expression and immunoreactivity of a Human group A rotavirus VP4[J].Virologica Sinica,2007,22(4):287-293.
[10] YI J L,MIAO G X,LIN Q Y,et al.Expression and characterization of a novel truncated Rotavirus VP4 for the development of a recombinant Rotavirus vaccine[J].Vaccine,2018,36(16):2068-2092.
[11] CIARLET M,LUDERT J E,ITURRIZAOMARA M,et al.Initial interaction of Rotavirus strains with N-acetylneuraminic (sialic) acid residues on the cell surface correlates with VP4 genotype,not species of origin[J].Journal of Virology,2002,76(8):4087-4095.
[12] DORMITZER P R,SUN Z Y J,WAGNER G,et al.The Rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site[J].Embo Journal,2014,21(5):885-897.
[13] MONNIER N,HIGO-MORIGUCHI K,SUN Z Y,et al.High-resolution molecular and antigen structure of the VP8^* core of a sialic acid-independent human Rotavirus strain[J].Journal of Virology,2006,80:1513-1523.
[14] 贾连智,李廷栋,葛胜祥.轮状病毒VP4亚单位疫苗研究进展[J].生物工程学报,2017,7:1075-1084. JIA L Z,LI T D,GE S X.Research progress of Rotavirus VP4 subunit vaccine[J].Journal of Bioengineering,2017,7:1075-1084.(in Chinese)
[15] DUNN S J,FIORE L,WERNER R L,et al.Immunogenicity,antigenicity,and protection efficacy of Baculovirus expressed VP4 trypsin cleavage products,VP5(1)^* and VP8^* from Rhesus rotavirus[J].Archives of Virology,1995,140:1969-1978.
[16] PADILLA-NORIEGA L,FIORE L,RENNELS M B,et al.Humoral immune responses to VP4 and its cleavage products VP5^* and VP8^* in infants vaccinated with Rhesus rotavirus[J].Journal of Clinical Microbiology,1992,30:1392-1397.
[17] 王珍贤,汤承,岳华.牛Nebovirus VP1蛋白的表达及免疫原性研究[J].西南民族大学学报(自然科学版),2021,47(1):11-18. WANG Z X,TANG C,YUE H.Expression and immunogenicity of Bovine nebovirus VP1 protein[J].Journal of Southwest Minzu University (Natural Science Edition),2021,47(1):11-18.(in Chinese)
[18] AOKI S T,SETTEMBRE E C,TRASK S D,et al.Structure of Rotavirus outer-layer protein VP7 bound with a neutralizing Fab[J].Science,2009,324(5933):1444-1447.
[19] 魏晓曼,闻晓波,冉旭华,等.猪轮状病毒Gottfried株和SB-1A株截短VP8基因的原核表达、蛋白纯化及免疫原性分析[J].中国生物制品学杂志,2013,26(8):1049-1053. WEI X M,WEN X B,RAN X H,et al.Prokaryotic expression,protein purification and immunogenicity of truncated VP8 genes of Porcine rotavirus gottfried and SB-1A strains[J].Chinese Journal of Biologicals,2013,26(8):1049-1053.(in Chinese)
[20] YAN N,LI R,WANG Y W,et al.High prevalence and genomic characteristics of G6P[1] Bovine rotavirus A in yak in China[J].Journal of General Virology,2020,101:701-711.
[21] LIU X Y,YAN N,YUE H,et al.Detection and molecular characteristics of Bovine rotavirus A in dairy calves in China[J].Journal of Veterinary Science,2021,22(5):e69.
[22] TRASK S D,MCDONALD S M,PATTON J T.Structural insights into the coupling of virion assembly and Rotavirus replication[J].Nature Reviews Crobiology,2012,10(3):165-177.
[23] NASIRI K,NASSIRI M,TAHMOORESPOUR M,et al.Design and construction of chimeric VP8-S2 antigen for Bovine rotavirus and Bovine coronavirus[J].Advanced Pharmaceutical Bulletin,2016,6(1):91-98.
[24] KIM H S,LEE B,HAN S Y,et al.Expression of Bovine rotavirus VP8 and preparation of IgY antibodies against recombinant VP8[J].Acta Virologica,2017,61(2):143-149.
[25] ESPOSITO D,CHATTERJEE D K.Enhancement of soluble protein expression through the use of fusion tags[J].Current Opinion Biotechnology,2006,17(4):353-358.
[26] RICHARDSON V,HERNANDEZ-PICHARDO J,QUINTANARr-SOLARES M,et al.Effect of Rotavirus vaccination on death from childhood diarrhea in Mexico[J].The Massachusetts Medical Society,2010,362(4):299-305.
[27] 张俭伟,赵宏坤,杨少华,等.牛轮状病毒灭活疫苗的免疫原性研究[J].西南农业学报,2008,1:200-203.ZHANG J W,ZHAO H K,YANG S H,et al.Immunogenicity of inactivated Bovine rotavirus vaccine[J].Southwest China Journal of Agricultural Sciences,2008,1:200-203.(in Chinese)
[28] WEI S C,CHE T J,SONG C J,et al.Subunit vaccine preparation of Bovine rotavirus and its efficacy in mice[J].Iranian Journal of Immunology,2015,12(3):188-197.
[29] GUNAYDIN G,ZHANG R,HAMMARSTROM L,et al.Engineeres Lactobacillus rhamonosus GG expressing IgG-binding domains of protein G:Capture of hyperimmune bovine colostrum antibodies and protection against diarrhea in a mouse pup Rotavirus infection model[J].Vaccine,2014,32(4):470-477.
[30] KUROKI M,IKEMORI Y,YOKOYAMA H,et al.Passive protection against Bovine rotavirus-induced diarrhea in murine model by specific immunoglobulins from chicken egg yolk[J].Veterinary Microbiology,1993,37(1-2):135-146.
[31] LENTZ E M,MOZGOVOJ M V,BELLIDO D,et al.VP8 antigen produced in tobacco transplastomic plants confers protection against Bovine rotavirus infection in a suckling mouse model[J].Journal of Biotechnology,2011,156(2):100-107.

Expression and Immunogenicity of P[1] Bovine Rotavirus A VP8 Recombinant Protein

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