高致病性猪繁殖与呼吸综合征病毒TA-12株ORF6基因的克隆、鉴定及真核表达

生物技术通报 ›› 2013, Vol. 0 ›› Issue (11): 159-163.

高致病性猪繁殖与呼吸综合征病毒TA-12株ORF6基因的克隆、鉴定及真核表达

王彤彤¹, 汪晓飞¹, 李心安¹, 田国宁², 刘立涛¹, 王广文¹, 李宁¹, 王方昆¹, 李红梅¹, 赵孝民¹, 肖一红¹

1.山东农业大学动物科技学院，泰安 271018；2.潍坊出入境检验检疫局，潍坊 261041

收稿日期:2013-05-02 出版日期:2013-11-14 发布日期:2013-11-14
作者简介:王彤彤，女，硕士研究生，研究方向：免疫病理学；E-mail：wangtong.1021@163.com
基金资助:
国家自然科学基金项目(30901063)

Cloning，Identification and Eukaryotic Expression of ORF6 Gene from Highly Pathogenic PRRSV Strain TA-12

Wang Tongtong¹, Wang Xiaofei¹, Li Xin'an¹, Tian Guoning², Liu Litao¹, Wang Guangwen¹, Li Ning¹, Wang Fangkun¹, Li Hongmei¹, Zhao Xiaomin¹, Xiao Yihong¹

(1. College of Animal Science and Technology，Shandong Agricultural University，Taian 271018；2. Weifang Entry-Exit Inspection And Quarantine Bureau, Weifang 261041)

Received:2013-05-02 Published:2013-11-14 Online:2013-11-14

摘要/Abstract

摘要： 旨在获得 HP-PRRSV TA-12(GenBank登录号：HQ416720)分离株ORF6蛋白的高效表达，将HP-PRRSV TA-12分离株的ORF6基因克隆到载体PfastBac HTB，并经转座进入E. coli含有杆状病毒质粒Bacmid的DH10Bac^TM细胞株，通过蓝白斑筛选和PCR鉴定出阳性重组杆粒，再将提取的重组杆粒Bacmid-ORF6转染昆虫细胞sf9，使其表达ORF6蛋白。经Western blot鉴定，在感染重组杆状病毒后第4天ORF6蛋白表达量最大，达到0.05 μg/μL。表达的ORF6蛋白主要存在于细胞质中。

关键词: 高致病性猪繁殖与呼吸综合征病毒, ORF6蛋白, 真核表达

Abstract: To obtain the efficient expression of ORF6 proteins of HP-PRRSV strain TA-12, the eukaryotic expression plasmid PfastBac HTB-ORF6 was constructed and transformed into DH10Bac^TM to get Bacmid-ORF6. The recombined plasmid was screened and identified by blue-white color selection and PCR using specific primers. Then the Bacmid-ORF6 was transfected into sf9 cells for expression. The results showed that the protein was expressed and the highest expression amount of 0.05 μg/μL was found at four days post infection using Western blot, and the expressed protein was mainly located in cytoplasm. The successful expression of the ORF6 protein has laid good foundation for the study of its potential function in the process of PRRSV infection.

Key words: HP-PRRSV, ORF6 protein, Eukaryotic expression

王彤彤, 汪晓飞, 李心安, 田国宁, 刘立涛, 王广文, 李宁, 王方昆, 李红梅, 赵孝民, 肖一红. 高致病性猪繁殖与呼吸综合征病毒TA-12株ORF6基因的克隆、鉴定及真核表达[J]. 生物技术通报, 2013, 0(11): 159-163.

Wang Tongtong, Wang Xiaofei, Li Xin'an, Tian Guoning, Liu Litao, Wang Guangwen, Li Ning, Wang Fangkun, Li Hongmei, Zhao Xiaomin, Xiao Yihong. Cloning，Identification and Eukaryotic Expression of ORF6 Gene from Highly Pathogenic PRRSV Strain TA-12[J]. Biotechnology Bulletin, 2013, 0(11): 159-163.

参考文献

[1] Tong GZ, Zhou YJ, Hao XF, et al. Emergence of the high pathogenic porcine reproductive and respiratory syndrome in China[J]. Emerging Infectious Diseases, 2007, 13(9)：1434-1436.
[2] Zhou YJ, Hao XF, Tan ZJ, et al. Highly virulent porcine reproductive and respiratory syndrome virus emerged in China[J]. Transbound Emerge Dis, 2008, 55(3-4)：152-164.
[3] Tian K, Yu X, Zhao T, et al. Emergence of fatal PRRSV variants：unparalleled outbreaks of a typical PRRS in China and molecular dissection of the unique hallmark[J]. PLoS One, 2007, 6：1-10.
[4] Zhou Z, Ni J, Cao Z, et al. The epidemic status and genetic diversity of 14 highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV)isolates from China in 2009[J]. Veterinary Microbiology, 2011, 150：257-269.
[5] Allende R, Laegreid WWL, Kutish GF, et al. Porcine reproductive and respiratory syndrome virus：description of persistence in individual pigs upon experimental infection[J]. J Virol, 2000(74)：10834-10837.
[6] Zhou EM, Clavijo A, Jiang Z, et al. Induction of auto-anti-idiotypic antibodies specific for antibodies to matrix and envelope glycoprotein from pigs experimentally infected with porcine reproductive and respiratory syndrome virus[J]. Veterinary Immunology and Immunopathology, 2004, 101：49-59.
[7] Oleksiewicz MB, Butner A, Toft P, et al. Emergence of porcine reproductive and respiratory syndrome virus deletion mutants：relation with the porcine antibody response to a hyper variable site in the ORF3 structural glycoprotein[J]. J Virology, 2000, 267：135-142.
[8] Drew TW, Paul Lowings J, Yapp F. Variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the UK[J]. Veterinary Microbiology, 1997(55)：209-221.
[9] Delputte PL, Vanderheijden N, Nauwynck HJ, et al. Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparin-like receptor on porcine alveolar macrophages[J]. J Virol, 2002, 76：4312-4320.
[10] Bastos RG, Dellagostin OA, Barletta RG, et al. Immune responses of pigs inoculated with Mycobacterium bovis BCG expressing a truncated form of GP5 and M protein of porcine reproductive and respiratory syndrome virus[J]. Vaccine, 2004, 22：467-474.
[11] Nakhai B, Pal R, Sridhar P, et al. The alpha subunit of human chorionic gonadotropin hormone synthesized in insect cells using a baculovirus vector is biologically active[J]. FEBS Lea, 1991, 283：104-108.
[12] Jones I, Morikawa Y. Baculovirus vectors for expression in insect cells[J]. Curropin Biotechnical, 1996, 7：512-516.
[13] Tjia ST, zu Alteuschildesche GM, Doerfler W. Autographa califomica nuclear polyhedrosis virus(AcNPV)DNA does not persist in mass culture of mammalian cells[J]. Virology, 1983, 125：107-117.
[14] Voikman LE, Goldsmith PA. In vitro survey of Autographa californica nuclear polyhedrosis virus interaction with nontarget vertebrate host cells[J]. Appl Environ Microbiol, 1983, 45：1085-1093.
[15] Carbonell LF, Klowden MJ, Miller LK. Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells[J]. J Viral, 1985, 56：153-160.
[16] Murtaugh MP, Xiao PZ, Zuckermann F. Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection[J]. Viral Immunol, 2002, 15：533-547.
[17] Lopez OJ, Osorio FA. Role of neutralizing antibodies in PRRSV protective immunity[J]. Vet Immunol Immunopathol, 2004, 102：155-163.
[18] Dea S, Gagnon CA, Mardassi H, et al. Antigenic variability among North American and European strains of porcine reproductive and respiratory syndrome virus as defined by no clonal antibodies to the matrix protein[J]. J Clinical Microbiol, 1996, 34：1488-1493.