1 College of Biology & Pharmacy, Yulin Normal University, Yulin 537000, China; 2 Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology, Yulin 537000, China
Abstract：As a host cell chaperone protein, P58IPK (58 kD inhibitor of PKR) plays an important role in regulating the body's antiviral and immune response. In this study, CDS region of porcine (Sus scrofa) P58IPK gene was cloned and its prokaryotic expression vector was constructed. Purified P58IPK protein was used as an antigen to produce polyclonal antibody, which would provide basic materials for the further study on the interplay between Porcine circovirus type 2 (PCV2) and its host. A specific pair of primers with the usage of amplifying the partial P58IPK CDS sequence (containing no signal peptide sequence) by reverse transcription PCR (RT-PCR) was designed according to the complete genome sequence of porcine P58IPK (GenBank No. HQ287801.1). After double digestion by NdeⅠ/XbaⅠ restriction enzymes, the products were cloned into the prokaryotic expression vector pCzn1. After sequencing, the generated recombinant expression plasmid pCzn1-P58IPK was transformed into bacterium Arctic ExpressTMEscherichia coli. The expressed His-P58IPK recombinant proteins were denaturated, renaturated and purified by using His-Band Ni+ affinity chromatography, then were used as target antigens to immunize rabbits (Oryctolagus?cuniculus) to prepare polyclonal antibody (pAb). After the pAb was purified, its titer and specificity were detected by indirect ELISA and Western blot, respectively. The results showed that the length of the cloned P58IPK gene fragment was 1 425 bp, the expressed His-P58IPK recombinant protein with a molecular weight of about 54.44 kD mainly existed in a form of inclusion body. The titer of the pAb prepared from this recombinant protein was above 1∶512 000, and the specificity of this pAb was verified very well as it could not only recognize the His-P58IPK recombinant protein but also porcine P58IPK protein. These data indicated that the partial CDS region of porcine P58IPK gene has been successfully cloned, expressed, purified and the prepared polyclonal antibody could be used for the detection of porcine P58IPK protein. The obtained polyclonal antibody could be used as basic material for further studying of the function of porcine P58IPK protein and interaction mechanism between PCV2 virus and the host.
[1] 丛彦龙, 丁壮, 关振宏, 等. 2009. GST-P58IPK融合蛋白的表达、纯化及活性鉴定[J]. 中国兽医学报, 29(8): 1023-1027. (Cong Y L, Ding Z, Guan Z H, et al.2009. Expression, purification and identification of recombinant GST-P58IPK fusion protein[J]. Chinese Journal of Veterinary Medicine, 29(8): 1023-1027.) [2] 刘伟, 郭抗抗, 林鸷, 等. 2015. 猪Jiv基因的克隆表达与多克隆抗体制备[J]. 西北农林科技大学学报(自然科学版), 43(2): 7-13, 22.(Liu W, Guo K K, Lin Z, et al. 2010. Cloning and expression of swine Jiv gene and preparation of its polyclonal antibody[J]. Journal of Northwest Agricultural and Forestry University (Natural Science Edition), 43(2): 7-13, 22.) [3] 吕其壮. 2016. 猪圆环病毒2型ORF5蛋白功能分析和ORF4蛋白拮抗细胞凋亡机制研究[D]. 博士学位论文, 西北农林科技大学, 导师: 张彦明, pp. 116-117. (Lv Q Z.2016. Functional analysis of putative ORF5 protein of porcine circovirus type 2 and antiapoptotic mechanism of viral ORF4 protein[D]. Thesis for Ph.D., Northwest Agricultural & Forestry University, Supervisor: Zhang Y M, pp. 116-117.) [4] 王静. 2016. 猪圆环病毒2型ORF6基因功能的初步研究[D]. 硕士学位论文, 华中农业大学, 导师: 江云波, pp. 1-5. (Wang J.2016. Preliminary study on gene function ORF6 of Porcine circovirus type 2[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Jiang Y B, pp. 1-5.) [5] Finsterbusch T, Steinfeldt T, Doberstein K, et al.2009. Interaction of the replication proteins and the capsid protein of Porcine circovirus type 1 and 2 with host proteins[J]. Virology, 386(1): 122-131. [6] Gale M Jr, Blakely C M, Hopkins D H, et al.1998. Regulation of interferon-induced protein kinase PKR: Modulation of P58IPK inhibitory function by a novel protein, P52rIPK[J]. Molecular and Cellular Biology, 18(2): 859-871. [7] Goebl M, Yanagida M.1991. The TPR snap helix: A novel protein repeat motif from mitosis to transcription[J]. Trends in Biochemical Sciences, 16(5): 173-177. [8] Goodman A G, Fornek J L, Medigeshi G R, et al.2009. P58IPK: A novel 'CIHD' member of the host innate defense response against pathogenic virus infection[J]. PLOS Pathogens, 5(5): e1000438. [9] Goodman A G, Smith J A, Balachandran S, et al.2007. The cellular protein P58IPK regulates Influenza virus mRNA translation and replication through a PKR-mediated mechanism[J]. Journal of Virology, 81(5): 2221-2230. [10] Korth M J, Lyons C N, Wambach M, et al.1996. Cloning, expression, and cellular localization of the oncogenic 58 kD inhibitor of the RNA-activated human and mouse protein kinase[J]. Gene, 170(2): 181-188. [11] Lamb J R, Tugendreich S, Hieter P.1995. Tetratrico peptide repeat interactions: To TPR or not to TPR?[J]. Trends in Biochemical Sciences, 20(7): 257-259. [12] Lin Z, Liang W L, Kang K, et al.2014. Classical swine fever virus (CSFV) and p7 protein induce secretion of IL-1β in macrophages[J]. Journal of General Virology, 95(Pt 12): 2693-2699. [13] Luig C, Kother K, Dudek S E, et al.2010. MAP kinase-activated protein kinases 2 and 3 are required for Influenza A virus propagation and act via inhibition of PKR[J]. The FASEB Journal, 24(10): 4068-4077. [14] Lv Q Z, Guo K K, Xu H, et al.2015. Identification of putative ORF5 protein of Porcine circovirus type 2 and functional analysis of GFP-fused ORF5 protein[J]. PLOS ONE, 10(6): e0127859. [15] Lv Q Z, Guo K K, Zhang Y M.2004. Current understanding of genomic DNA of Porcine circovirus type 2[J]. Virus Genes, 49(1): 1-10. [16] Sharma K, Tripathi S, Ranjan P, et al.2001. Influenza A virus nucleoprotein exploits Hsp40 to inhibit PKR activation[J]. PLOS ONE, 6(6): e20215. [17] Zhang H M, Ye X, Su Y, et al.2010. Coxsackievirus B3 infection activates the unfolded protein response and induces apoptosis through down regulation of P58IPK and activation of CHOP and SREBP1[J]. Journal of Virology, 84(17): 8446-8459.
