猪繁殖与呼吸综合征病毒糖基化囊膜蛋白5原核表达载体的构建及免疫原性分析

Abstract

Abstract: In order to analyze the immunogenicity of glycosylated envelope protein 5 (GP5) from porcine reproductive and respiratory syndrome virus (PRRSV), ORF5 gene fragment was amplified by RT-PCR from the PRRSV (GenBank: HQ701732.1). Based on the ORF5 gene sequence, two pairs of primers were used to amplify two gene fragments, excluding the signal peptide sequence and transmembrane regions. The two gene fragments were cloned into the prokaryotic expression vector pET-28a（+）, and then the recombinant plasmid was transformed into Escherichia coli. The results of Western blotting and ELISA respectively showed that the expression of GP5 could be recognized by positive serum antibody of PRRSV, and corresponding antibodies of GP5 protein could produce in the immunized BALB/c mice. Therefore, the recombinant GP5 protein had good biological activity, and could provide fundamental data for the further study on the structure and function of GP5 protein of PRRSV.

Key words: porcine reproductive and respiratory syndrome virus; glycosylated envelope protein 5; recombinant protein; ELISA; Western blotting

LI Xin-xian, ZHANG Xiao-dan1, LIU Jing-jing, MIAO Zeng-min2, ZHANG Tao-tao, CHAI Tong-jie. Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus[J]. .

References

1 尹国友，孙婕，黄保续. 猪繁殖与呼吸综合征病毒ORF5基因的克隆与原核表达［J］. 动物医学进展，2009，30(12)：63～66.
2 孙建富，郝建伟，荣福龙，等. 猪繁殖与呼吸综合征病毒的分离与鉴定及ORF5基因的序列分析［J］. 中国畜牧兽医，2011，39(9)：138～142.
3 李国娟，柳纪省，李宝玉，等. 截短的猪繁殖与呼吸综合征ORF5基因及重组蛋白的原核表达［J］. 生物技术通报，2010，3：131～134.
4 郭宝清，陈章水，刘文兴，等. 从疑似PRRS流产胎儿分离PRRSV的研究［J］. 中国畜禽传染病，1996，18(2)：1～4.
5 鑫婷，侯绍华，贾红，等. 猪繁殖与呼吸综合征病毒的分子流行病学分析［J］. 中国畜牧兽医，2009，36(8)：115～121.
6 Blaha T. The “colorful” epidemiology of PRRS［J］. Vet Res, 2000, 31(1): 77～83.
7 Delputte P L, Vanderheijden N, Nauwynck H J, et al. Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages［J］. J Virol, 2002, 76(9): 4312～4320.
8 Duran J P, Climent I, Sarraseca J, et al. Baculovirus expression of proteins of porcine reproductive and respiratory syndrome virus strain Olot/91. Involvement of ORF3 and ORF5 proteins in protection［J］. Virus Genes, 1997, 14(1): 19～29.
9 Indik S, Valí〖AKcˇ〗ek L, Klein D, et al. Variations in the major envelope glycoprotein GP5 of Czech strains of porcine reproductive and respiratory syndrome virus［J］. Journal of General Virology, 2000, 81(10): 2497～2502.
10 Li G, Zeng Y, Yin J, et al. Cloning, prokaryotic expression, and biological analysis of recombinant chicken IFN-γ［J］. Hybridoma, 2010, 29(1): 1～6.
11 Liu B, Li G, Sui X, et al. Expression and functional analysis of porcine aminopeptidase N produced in prokaryotic expression system［J］. J Biotechnol, 2009, 141(1～2): 91～96.
12 Mengeling W L, Lager K M, Vorwald A C. Clinical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval［J］. Am J Vet Res, 1998, 59(1): 52～55.
13 Ren X, Wang M, Yin J, et al. Heterologous expression of fused genes encoding the glycoprotein 5 from PRRSV: A way for producing functional protein in prokaryotic microorganism［J］. Journal of Biotechnology, 2010, 147(2): 130～135.
14 Rodriguezm J, Sarraseca J, Fominaya J, et al. Identification of an immunodominant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus［J］. J Gen Virol, 2001, 82(5): 995～999.
15 Stevenson G W, VanAlstine W G, Kanitz C L, et al. Endemic porcine reproductive and respiratory syndrome virus infection of nursery pigs in two swine herds without current reproductive failure［J］. J Vet Diagn Invest, 1993, 5(3): 432～434.
16 Sur J H, Doster A R, Osorio F A. Apoptosis induced in vivo during acute infection by porcine reproductive and respiratory syndrome virus［J］. Vet Pathol, 1998, 35(6): 506～514.
17 Terpstra C, Wensvoort G, Pol J M A. Experimental reproduction of porcine epidemic abortion and respiratory syndrome (mystery swine disease) by infection with Lelystad virus: Koch’s postulates fulfilled［J］. Veterinary Quarterly, 1991, 13(3): 131～136.
18 Wensvoort G, Terpstra C, Pol J M A, et al. Mystery swine disease in the Netherlands: The isolation of Lelystad virus［J］. Veterinary Quarterly, 1991, 13(3): 121～130.
19 Yin J, Li G, Ren X, et al. Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes［J］. J Biotechnol, 2007, 127(3): 335～347.

[1]	KANG Pu, REN Zhaowen, CAI Rujian, LYU Zongji, HUANG Yuhao, WANG Xiaohu. Variation Regularity and Transmission of PRRSV-2 Lineage 8 Strains in Guangdong and Surrounding Provinces [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2001-2011.
[2]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[3]	CHEN Tianbao, WANG Yani, LIU Baoling, WANG Gang, WANG Xiaohu, LIU Xuhui, CAI Rujian, HE Dongsheng. Research Progress on the Anti-Porcine Reproductive and Respiratory Syndrome Virus Effect and Mechanism of Traditional Chinese Medicine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2114-2129.
[4]	LI Huawei, GUO Kewei, WANG Xuying, ZHANG Xiaoyu, LU Ziwei, LI Xinfeng, MA Hui, HOU Wenjing. Eukaryotic Expression and Subcellular Localization of Nonstructural Protein 7 of Porcine Reproductive and Respiratory Syndrome Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1160-1168.
[5]	MU Yijiao, YU Ruiming, ZHANG Liping, WANG Yonglu. Establishment and Application of ELISA for Detection of IgA Antibody Based on the Recombinant Full-length S2 Protein of Porcine Epidemic Diarrhea Virus Variant Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1185-1194.
[6]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[7]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[8]	HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3508-3519.
[9]	TANG Qingfeng, LI Ling, HUANG Jiaxiang, FANG Wenyuan, NONG Haoru, TANG Yan, LU Chengwei, WEI Panqiu, FENG Ling. Survey on Mycotoxin Contamination of Buffalo Milk in Different Seasons Under Different Feeding Modes in Guangxi [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3665-3675.
[10]	ZHANG Wenyan, WANG Yawen, YUAN Chen, FENG Yawen, TENG Zhaojian, SHANG Jialiang, LU Jicheng, SONG Qinye. Establishment of Indirect ELISA Method for Detection of African Swine Fever Virus Antibodies Based on Truncated p72 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2688-2697.
[11]	LIU Jingsong, FU Jingcheng, WEN Bingyan, YANG Yanbin, GUO Shuang, JIAO Xianqin, CHEN Jin, HUANG Ruochao, WANG Yueying, LI Heping. Establishment and Analysis of Expression System of Porcine Intestinal Mucosal Protection Factor HO-1 in Bacillus pumilus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(6): 2064-2071.
[12]	TIAN Guangyuan, WANG Yuchen, ZHOU Yaping, GUO Ting, ZHAO Hongmei, ZHAO Fengmiao, SUN Yajie, BIAN Yuchen, YU Jialiang, ZHOU Yuxia. Prokaryotic Expression and Indirect ELISA Method Establishment of P60_58aa-928aa Protein of Mycoplasma ovipneumoniae [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1960-1969.
[13]	LIU Qingqing, WU Peng, LI Peidong, ZHANG Jiangwei, CHEN Chuangfu, XIAO Chencheng. Construction of Recombinant Adenovirus Expressing Porcine Reproductive and Respiratory Syndrome Virus N Protein and Its Immunogenicity Analysis [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 1024-1031.
[14]	WANG Zhaoli, YANG Siyu, WU Yanmei, SONG Xiaoxiao, MU Xiang, ZHANG Tao. Effects of PRRSV-LPS Treatment on Neutrophil Adhesion to Porcine Pulmonary Microvascular Endothelial Cells [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 471-479.
[15]	WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.

Prokaryotic Expression Vector Construction and Immunogenicity Analysis of Glycosylated Envelope Protein 5 from Porcine Reproductive and Respiratory Syndrome Virus

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments