CAO Rui-bing, BAO Jing-jing, ZHOU Hai-xia, ZHENG Qi-sheng, ZHOU Bin and CHEN Pu-yan*. Prokaryotic Expression of Porcine Interferon -βand Its Inhibition Effect on Porcine Epidemic Diarrhea Virus[J]. Virologica Sinica, 2004, 19(4): 364-368.
Citation: CAO Rui-bing, BAO Jing-jing, ZHOU Hai-xia, ZHENG Qi-sheng, ZHOU Bin, CHEN Pu-yan*. Prokaryotic Expression of Porcine Interferon -βand Its Inhibition Effect on Porcine Epidemic Diarrhea Virus .VIROLOGICA SINICA, 2004, 19(4) : 364-368.

猪β-干扰素的原核表达及其对猪流行性腹泻病毒的抑制作用研究

  • 本研究通过PCR技术克隆了猪β干扰素全基因,设计引物亚克隆猪β干扰素成熟蛋白编码基因并对, 5’端1个稀有密码子进行了大肠杆菌偏嗜性改造。构建了猪IFN-β原核单纯表达载体pRLC-poIFNβ,实现了poIFN-β在大肠杆菌中的表达, 表达产物约占菌体总蛋白的17.3%。表达产物以包涵体形式存在,用含6mol/L 盐酸胍的变性液溶解及含GSH-GSSG复性液复性处理,复性后的表达产物经凝胶层析纯化后,MDBK细胞-VSV病变抑制法测定结果表明,重组猪β干扰素具有良好的抗病毒活性,约为5.6x105U/mg。用重组猪β干扰素处理猪肾传代细胞PK-15后,细胞病变抑制法(CPE50)测定结果表明:重组猪β干扰素可显著抑制猪流行性腹泻病毒(PEDV)的感染。

Prokaryotic Expression of Porcine Interferon -βand Its Inhibition Effect on Porcine Epidemic Diarrhea Virus

  • The porcine IFN-βgene was amplified by polymerase chain reaction. The amplified fragment was cloned into pGEM-T-easy vector and then sequenced. The result indicated that the cloned gene was a complete porcine IFN-βgene with the codes for signal peptide ,which had the identities of 100% with the poIFN-βgene published in the GenBank. Another pair of primers was designed to sub-clone the gene coding porcine IFN-βmature protein and one rare codon near 5` terminus was modified to the biased codons of E.coli. After sequencing, the sub-cloned IFN-βgene was successfully inserted to expression vector pRLC and expressed in E.coli, the expressed protein was about 17.3% of the total cell protein. Recombinant porcine IFN-βexpressed as inclusion body, which was dissolved in 6 mol/L guanidine chloride and subsequently re-natured by diluting with refolding buffer containing GSH and GSSG. In order to obtain pure protein, the re-natured poIFN-βwas purified by Sepha- crylS-200 chromatography. As a result, the purified product was verified to be of high cytokine activity by inhibiting the cyto-pathogenic effect, which is about 5.6x105U/mg. In addition, the inhibition effect of recombinant poIFN-βon PEDV was determined using CPE50 method. The results indicated that high concentration of recombinant poIFN-βcould completely inhibit PEDV on PK-15 cell line.

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    Prokaryotic Expression of Porcine Interferon -βand Its Inhibition Effect on Porcine Epidemic Diarrhea Virus

    • 1. Key Laboratory of Animal Disease Diagnosis and Immunology, Ministry of Agriculture, Nanjing Agricultural University , Nanjing 210095 ,China

    Abstract: The porcine IFN-βgene was amplified by polymerase chain reaction. The amplified fragment was cloned into pGEM-T-easy vector and then sequenced. The result indicated that the cloned gene was a complete porcine IFN-βgene with the codes for signal peptide ,which had the identities of 100% with the poIFN-βgene published in the GenBank. Another pair of primers was designed to sub-clone the gene coding porcine IFN-βmature protein and one rare codon near 5` terminus was modified to the biased codons of E.coli. After sequencing, the sub-cloned IFN-βgene was successfully inserted to expression vector pRLC and expressed in E.coli, the expressed protein was about 17.3% of the total cell protein. Recombinant porcine IFN-βexpressed as inclusion body, which was dissolved in 6 mol/L guanidine chloride and subsequently re-natured by diluting with refolding buffer containing GSH and GSSG. In order to obtain pure protein, the re-natured poIFN-βwas purified by Sepha- crylS-200 chromatography. As a result, the purified product was verified to be of high cytokine activity by inhibiting the cyto-pathogenic effect, which is about 5.6x105U/mg. In addition, the inhibition effect of recombinant poIFN-βon PEDV was determined using CPE50 method. The results indicated that high concentration of recombinant poIFN-βcould completely inhibit PEDV on PK-15 cell line.

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