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Prokaryotic expression and potential application of the truncated PCV-2 capsid protein

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Virologica Sinica

Abstract

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

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Authors and Affiliations

  1. Animal Infectious Diseases Research Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Zhong-zi Lou, Zhi-yong Li, Jian-qiang Li, Xi Lan, Xue-rui Li, Xiang-ping Yin & Ji-xing Liu

  2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Zhong-zi Lou, Zhi-yong Li, Jian-qiang Li, Xi Lan, Xue-rui Li, Xiang-ping Yin & Ji-xing Liu

  3. Key Laboratory of Grazing Animal Diseases of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Zhong-zi Lou, Zhi-yong Li, Jian-qiang Li, Xi Lan, Xue-rui Li, Xiang-ping Yin & Ji-xing Liu

  4. Key Laboratory of Veterinary Public Health of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Zhong-zi Lou, Zhi-yong Li, Jian-qiang Li, Xi Lan, Xue-rui Li, Xiang-ping Yin & Ji-xing Liu

  5. College of Veterinary Medicine, Shandong Agricultural University, Taian, 271018, China

    Gang Wang & Si-dang Liu

Authors
  1. Zhong-zi Lou
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  2. Zhi-yong Li
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  3. Gang Wang
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  4. Jian-qiang Li
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  5. Xi Lan
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  6. Xue-rui Li
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  7. Xiang-ping Yin
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  8. Ji-xing Liu
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  9. Si-dang Liu
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Corresponding author

Correspondence to Ji-xing Liu.

Additional information

Foundation item: This work was supported by the special studies for social welfare researches in institutes (2005DIB4J041)

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Lou, Zz., Li, Zy., Wang, G. et al. Prokaryotic expression and potential application of the truncated PCV-2 capsid protein. Virol. Sin. 25, 86–97 (2010). https://doi.org/10.1007/s12250-010-3111-7

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  • DOI: https://doi.org/10.1007/s12250-010-3111-7

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