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| The Soluble Expression of Porcine Circovirus Type 2 Cap Gene in Escherichia coli and Its Immunogenicity in Mice |
| LIU Pan-rao, ZHOU Xue-chen, ZHU Xue-jiao, BAI Juan, WANG Xian-wei, JIANG Ping |
| College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China |
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Abstract Porcine circovirus type 2(PCV2)Cap gene has been used as target gene for developing genetic engineering vaccine. However, it is usually expressed as the form of inclusion body in E.coli which having low protective efficacy against PCV2 challenge. The PCV2 ORF2 gene condons was optimized and fused with MPG gene. Then, they were cloned into expression vector pET28a and induced for expression in E.coli BL21(DE3) individually. SDS-PAGE and Western blot results confirmed that they could be expressed with the production of soluble proteins. After purification, the two kinds of proteins were mixed with GEL01, ISA206 and ISA15A adjuvant respectively. Mice experiment results showed that the GEL01 groups induced the highest levels of antibodies against PCV2 with ELISA and neutralization antibody assay. Following challenge with PCV2, the PCV2 virus loads in spleen in immunized groups except the ISA15A were significantly lower than challenge control group. It indicated that the vaccine prepared by the two recombinant proteins and GEL01 and ISA206 could induce immune protection in mice against PCV2 infection, which laid a foundation for the development of PCV2 subunit vaccine.
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Received: 12 October 2015
Published: 04 January 2016
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| [1] Ellis J, Hassard L, Clark E, et al. Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J, 1998, 39(1): 44-51.
[2] Opriessnig T, Meng X J, Halbur P G. Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest, 2007, 19(6): 591-615.
[3] Baekbo P, Kristensen C S, Larsen L E. Porcine circovirus diseases: a review of PMWS.Transbound Emerg Dis,2012, 59 (Suppl. 1):60-67.
[4] Hamel A L, Lin L L, Nayar G P. Nucleotide sequence of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. J Virol, 1998, 72(6): 5262-5267.
[5] Lv Q Z, Guo K K, Han X, et al. Identification of putative ORF5 protein of porcine circovirus type 2 and functional analysis of GFP-fused ORF5 protein. PLoS One,2015, 10(6): e0127859.
[6] Zhang H W, Qian P, Liu L F, et al. Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes. Viruses-Basel, 2014, 6(12): 4839-4855.
[7] 解庭波. 大肠杆菌表达系统的研究进展. 长江大学学报, 2008,5(3):77-82 Xie T B. The research progress of E.coli expression system . Journal of Yangze University(Natural Science Edition), 2008,5(3):77-82.
[8] Trundova M, Celer V. Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells. Virus Genes, 2007, 34(2):199-204.
[9] Wu P C, Lin W L, Wu C M, et al. Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development. Appl Microbiol Biotechnol, 2012, 95(6): 1501-1507.
[10] Liu Q, Willson P, Attoh-Poku S, et al. Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein. Protein Expression and Purification, 2001, 21(1): 115-120.
[11] Bucarey S A, Noriega J, Reyes P, et al. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts. Vaccine, 2009, 27(42): 5781-5790.
[12] Morris M C, Vidal P, Chaloin L, et al. A new peptide vector for efficient delivery of oligonucleotides into mammalian cells. Nucleic Acids Research, 1997,25(14): 2730-2736.
[13] Jrver P, Langel U. The use of cell-penetrating peptides as a tool for gene regulation. Drug Discov Today, 2004,9(9):395-402.
[14] Moschos S A, Jones S W, Perry M M, et al. Lung delivery studies using siRNA conjugated to TAT(48-60) and penetratin reveal peptide induced reduction in gene expression and induction of innate immunity. Bioconjugate Chemistry, 2007, 18(5): 1450-1459.
[15] Joliot A. Transduction peptides within naturally occurring proteins. Sci Stke, 2005, 2005(313):54.
[16] 朱琳,刘兰霞,宋丽萍,等. LHRH-MPG△NLS/siRNA纳米复合物的制备及稳定性观察. 生物医学工程与临床,2012,16(3): 283-285. Zhu L, Liu L X, Song L P, et.al. Study on the method for preparing stable LHRH-MPG△NLS/siRNA nanoparticles. Biomedical Engineering and Clinical Medicine,2012,16(3): 283-285.
[17] 吕慧,王先炜,李文良,等. 猪圆环病毒2型重组Cap蛋白间接ELISA抗体检测方法的建立. 畜牧与兽医,2009,41(11): 6-11. Lv H, Wang X W, Li W L, et.al. Development of an indirect ELISA for detecting antibody to porcine circovirus 2 using recombinant Cap protein. Animal Husbandry & Veterinary Medicine, 2009,41(11):6-11.
[18] 冯志新,姜平,王先炜,等.猪圆环病毒 2 型 TaqMan 实时 PCR 检测方法的建立. 中国病毒学,2006, 1(4): 371-374. Feng Z X, Jiang P, Wang X W, et al. Development of a real-time PCR method for detection of type 2 porcine circovirus. Virology Sinica, 2006,21(4):371-374.
[19] Beach N M, Meng X J. Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2). Virus Res, 2012, 164(1-2): 33-42. |
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