重组猪α干扰素抗猪繁殖与呼吸综合征病毒的应用研究
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摘要
利用本实验室成功构建的猪α干扰素原核达载体PETIFN-α,在大肠杆菌BL21中表达了猪α干扰素,表达产物为一融合蛋白。SDS-PAGE电泳后在约40.2 kDa处出现一条带,同预期结果一致。经过优化IPTG的浓度和诱导时间,猪α干扰素的表达量明显提高,表达量约占细菌总蛋白量的20%。应用镍琼脂糖凝胶树脂层析柱吸附纯化猪α干扰素融合蛋白,不同浓度咪唑缓冲液洗脱吸附蛋白,SDS-PAGE电泳鉴定表明,400mM咪唑的缓冲液洗脱的猪α干扰素蛋白,纯度最高,达到蛋白纯化的预期目的,可以用于抗病毒活性试验。
用细胞病变抑制法在Marc-145细胞上测定重组猪α干扰素活性,测定重组猪α干扰素抗PRRSV的病毒活性为5.0×103U/mg。将干扰素做不同浓度梯度稀释后,在单层Marc-145细胞上做猪α干扰素抑制猪繁殖与呼吸综合征病毒试验,测得干扰素对Marc-145细胞的无毒浓度为0.20mg/ml,最小抑制浓度为0.14mg/ml。结果表明,重组猪α干扰素在体外对PRRSV有较低的抑制作用。
重组猪α干扰素体内抑制PRRSV试验研究表明,使用重组干扰素配合抗生素可以治疗猪病毒病,缩短治疗疗程2-3 d,治愈率高于不含干扰素的药物治疗组但差异不显著,使用干扰素治疗,停药后不易复发。干扰素的预防试验结果表明,与对照Ⅱ组相比,在断奶后第14天,健康仔猪断奶应激造成的腹泻率降低。
From the recombinant expression vectors of pGEXIFN-α,which Constructed and appraised correctly, The fusion protein were expressed after the recombinant expression vectors transformed in BL21 of E.coli. There was a strip occurring at the location labled 40.2 kD when expression products were identifited by SDS-PAGE. The strip was specific, because its molecular weight was just the same as the tag in the pET32a of procaryon expression vector and the protein deduced according to porcine IFN-αgene. Optimizeing to the concentration of IPTG and the time of IPTG induceing, the produces of IFN-αincreased. The proteins were purityed by NTA and had only one strip by SDS-PAGE. The expression of the porcine IFN-αgene quantity was twenty percent of the bacterium total protein. Purifing the porcine protein of IFN-α, Using the different density imidazole buffer solution of NTA, the protein of the porcines of IFN-αwere adsorption and purification. By SDS-PAGE, the results indicated that the porcine proteins of IFN-α, the purity was highest,by 400 mM density imidazole buffer solution of NTA adsorbed. The anticipeated aim of the protein purification was achieved. So the purified porteins may use in the anti-virus activity test.
By inhibiting the cytopathic effect, the porcine protein of IFN-αwas detected, on the Marc-145 cell, the result indicated that activity of recombinant of porcine protein of IFN-αagainst PRRSV was 5.0×103 U/mg. The experiment of the porcine protein of IFN-αsuppressed the PRRSV in vitro. The experiment obtained that the smallest virulent dentisty to the Marc-145 cell was 0.20mg/ml. The result of the experiment indicated that the porcine protein of IFN-αhad the function of the inhibitoried to anti-PRRSV in vitro. But the functions were not too obvious, by diluting the porcine protein of IFN-αat different concentration gradient, using the inhibiting the cytopathic effect, the function of the porcine protein of IFN-αwere to be dected. The density of the smallest of the blocked denstity was 0.14mg/ml.
The results of the recombinant of porcine protein of IFN-αagainst PRRSV in vivo indicated: using the recombinant of porcine protein of IFN-αand the medicines could treat the desease pigs effectively, reducing the treatment course 2?3d, the cure rate was higher than the group of only using the medicine, and the group used the recombinant of porcine protein of IFN-αwas not easy to recur. Through the prevention of the experiment of the recombinant of porcine protein of IFN-α, which studied the diarrhea experimental, the result indicated : after being weaned 14d, comparied to the experimental group and comparisonⅡ, the experimental group was more lower than the comparison group, which was possibley related to the result of the organism immunologic function enhancement; to the antibody titer’s and the masculine gender rate of swine plague’s immune body was enhanced .
用细胞病变抑制法在Marc-145细胞上测定重组猪α干扰素活性,测定重组猪α干扰素抗PRRSV的病毒活性为5.0×103U/mg。将干扰素做不同浓度梯度稀释后,在单层Marc-145细胞上做猪α干扰素抑制猪繁殖与呼吸综合征病毒试验,测得干扰素对Marc-145细胞的无毒浓度为0.20mg/ml,最小抑制浓度为0.14mg/ml。结果表明,重组猪α干扰素在体外对PRRSV有较低的抑制作用。
重组猪α干扰素体内抑制PRRSV试验研究表明,使用重组干扰素配合抗生素可以治疗猪病毒病,缩短治疗疗程2-3 d,治愈率高于不含干扰素的药物治疗组但差异不显著,使用干扰素治疗,停药后不易复发。干扰素的预防试验结果表明,与对照Ⅱ组相比,在断奶后第14天,健康仔猪断奶应激造成的腹泻率降低。
From the recombinant expression vectors of pGEXIFN-α,which Constructed and appraised correctly, The fusion protein were expressed after the recombinant expression vectors transformed in BL21 of E.coli. There was a strip occurring at the location labled 40.2 kD when expression products were identifited by SDS-PAGE. The strip was specific, because its molecular weight was just the same as the tag in the pET32a of procaryon expression vector and the protein deduced according to porcine IFN-αgene. Optimizeing to the concentration of IPTG and the time of IPTG induceing, the produces of IFN-αincreased. The proteins were purityed by NTA and had only one strip by SDS-PAGE. The expression of the porcine IFN-αgene quantity was twenty percent of the bacterium total protein. Purifing the porcine protein of IFN-α, Using the different density imidazole buffer solution of NTA, the protein of the porcines of IFN-αwere adsorption and purification. By SDS-PAGE, the results indicated that the porcine proteins of IFN-α, the purity was highest,by 400 mM density imidazole buffer solution of NTA adsorbed. The anticipeated aim of the protein purification was achieved. So the purified porteins may use in the anti-virus activity test.
By inhibiting the cytopathic effect, the porcine protein of IFN-αwas detected, on the Marc-145 cell, the result indicated that activity of recombinant of porcine protein of IFN-αagainst PRRSV was 5.0×103 U/mg. The experiment of the porcine protein of IFN-αsuppressed the PRRSV in vitro. The experiment obtained that the smallest virulent dentisty to the Marc-145 cell was 0.20mg/ml. The result of the experiment indicated that the porcine protein of IFN-αhad the function of the inhibitoried to anti-PRRSV in vitro. But the functions were not too obvious, by diluting the porcine protein of IFN-αat different concentration gradient, using the inhibiting the cytopathic effect, the function of the porcine protein of IFN-αwere to be dected. The density of the smallest of the blocked denstity was 0.14mg/ml.
The results of the recombinant of porcine protein of IFN-αagainst PRRSV in vivo indicated: using the recombinant of porcine protein of IFN-αand the medicines could treat the desease pigs effectively, reducing the treatment course 2?3d, the cure rate was higher than the group of only using the medicine, and the group used the recombinant of porcine protein of IFN-αwas not easy to recur. Through the prevention of the experiment of the recombinant of porcine protein of IFN-α, which studied the diarrhea experimental, the result indicated : after being weaned 14d, comparied to the experimental group and comparisonⅡ, the experimental group was more lower than the comparison group, which was possibley related to the result of the organism immunologic function enhancement; to the antibody titer’s and the masculine gender rate of swine plague’s immune body was enhanced .
引文
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[3]连京华,李玉峰,高广尧,等.兽用干扰素的研究进展[J].山东农业科学,2005,(1): 78～80.
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[5] Lefevre F, Guillomot M, D'Andrea S,et al. Interferon-delta: the first member of a novel type I interferon family[J]. Biochimie. 1998 Aug-Sep;80(8-9): 779～788.
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[8]姜怀志,李雪,马宁.干扰素对绵羊妊娠识别影响的研究进展[J].家畜生态学报,2005 ,26(3):71～73.
[9] Chinsangaram J, Piccone ME, Grubman MJ.Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon[J].J Virol. 1999 Dec;73(12):9891～9898.
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[12]李军,曾芸.病毒诱导I型干扰素产生的机制[J].《生命的化学》,2006 ,26(5 ):395～398.
[13] Seth RB et al. suppression of alpha/beta interferon[J]. Cell Res, 2006, 16(2): 141～147.
[14] Oganesyan G et al.produce of interferon[J]. Nature, 2006, 439(7073): 208～211.
[15] Hacker H et al. interferon[J].Nature, 2006, 439(7073): 204～207.
[16] Sarkar SN et al. interferon[J].Nat Struct Mol Biol, 2004,11(11): 1060～1067.
[17] Meylan E et al. produce of interferon[J]. Nat Immunol, 2004, 5(5): 503～507.
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