重组猪α干扰素的分离纯化与安全性研究
发布时间：2019-05-22 16:58
【摘要】：【目的】干扰素(Interferon,IFN)是一类具有抗病毒、抗肿瘤和调节机体免疫功能等重要生物学功能的细胞因子,在临床上具有广泛应用前景。为了使重组猪干扰素-α(rPoIFN-α)能够更快的从实验室走向产业化和临床应用,对rPoIFN-α的工业化表达及其分离纯化条件的优化和生物安全性评价具有重要的现实意义。【方法】利用自动发酵罐培养重组P.pastoris菌株,获得高效表达的rPoIFN-α,硫酸铵盐析沉淀后,通过离子交换和分子筛层析法分离纯化,Marc-145/VSV系统测定活性。通过兔抗rPo IFN-α多克隆抗体和鼠抗rPoIFN-α单克隆抗体建立检测rPoIFN-α浓度的双抗体夹心ELISA方法。选择30日龄和2日龄仔猪,肌肉注射不同剂量的rPoIFN-α,利用建立双抗体夹心ELISA检测不同时间点仔猪血清中rPoIFN-α浓度的变化,研究IFN在猪体内的代谢规律。通过动物毒性试验、基因残留检测以及致仔猪产生抗体试验等,对基因工程rPoIFN-α进行安全性评价。【结果】以P.pastoris作为菌种自动发酵罐高效表达rPoIFN-α,50%饱和度硫酸铵盐析、Q Sepharose FF阴离子交换和Superdex 200分子筛层析技术纯化,琼脂糖G-25层析脱盐,可获得纯度为97.75%rPoIFN-α,活性可达到1.00×108 U/μL。利用兔多克隆抗r PoIFN-α抗体和鼠单克隆抗r PoIFN-α抗体,建立了双抗夹心ELISA方法,检测PoIFN-α浓度范围为2.5~0.0781μg/mL。以1、10、50mg/头三种不同剂量的r PoIFN-α肌肉注射30日龄和2日龄仔猪,双抗夹心ELISA检测不同时间点血清中r PoIFN-α浓度,绘制消长曲线,显示在注射后60min血清中IFN浓度达到高峰,IFN在血清中的浓度与其注射剂量成正相关,在30日龄仔猪血清中的存在时间将近24h,而在2日龄仔猪血清中的存在时间只有3h,存在时间与注射剂量无明显相关性。小鼠急性毒性试验表明,10mg/只以下剂量不会对小鼠产生任何毒性作用;基因残留试验和诱发仔猪抗体证明,基因工程rPoIFN-α不携带重组基因和抗生素抗性基因,也不诱发仔猪产生rPoIFN-α抗体。但在仔猪毒性试验中,组织切片观察表明,高剂量(50mg/头)rPo IFN-α可导致仔猪肺泡壁增厚、脾脏红髓比例增加,肝脏脂肪样病变等轻微病理变化。
[Abstract]:[objective] Interferon (Interferon,IFN) is a kind of cytokines with important biological functions, such as antiviral, antitumor and regulating immune function, and has a wide range of clinical application prospects. In order to enable recombinant pig interferon-伪 (rPoIFN- 伪) to move from laboratory to industrialization and clinical application more quickly, It is of great practical significance to optimize the industrial expression of rPoIFN- 伪, the optimization of isolation and purification conditions and the evaluation of biosafety. [methods] the recombinant P.pastoris strain was cultured in an automatic fermenter to obtain the highly expressed rPoIFN- 伪. After ammonium sulfate salting out precipitation, it was separated and purified by ion exchange and molecular sieve chromatography, and the activity was determined by Marc-145/VSV system. A double antibody sandwich ELISA method for the detection of rPoIFN- 伪 concentration was established by rabbit anti-rPoIFN- 伪 polyclonal antibody and mouse anti-rPoIFN- 伪 monoclonal antibody. Different doses of rPoIFN- 伪 were injected intramuscular in 30-day-old and 2-day-old piglets. The changes of rPoIFN- 伪 concentration in serum of piglets at different time points were detected by establishing double antibody sandwich ELISA to study the metabolism of IFN in pigs. The safety of genetic engineering rPoIFN- 伪 was evaluated by animal toxicity test, gene residue detection and antibody induced antibody test. [results] P.pastoris was used as automatic fermenter to express rPoIFN- 伪. 50% saturated ammonium sulfate salting out, Q Sepharose FF anion exchange and Superdex 200 molecular sieves chromatography purification, agarose G 鈮，
本文编号：2483090
[Abstract]:[objective] Interferon (Interferon,IFN) is a kind of cytokines with important biological functions, such as antiviral, antitumor and regulating immune function, and has a wide range of clinical application prospects. In order to enable recombinant pig interferon-伪 (rPoIFN- 伪) to move from laboratory to industrialization and clinical application more quickly, It is of great practical significance to optimize the industrial expression of rPoIFN- 伪, the optimization of isolation and purification conditions and the evaluation of biosafety. [methods] the recombinant P.pastoris strain was cultured in an automatic fermenter to obtain the highly expressed rPoIFN- 伪. After ammonium sulfate salting out precipitation, it was separated and purified by ion exchange and molecular sieve chromatography, and the activity was determined by Marc-145/VSV system. A double antibody sandwich ELISA method for the detection of rPoIFN- 伪 concentration was established by rabbit anti-rPoIFN- 伪 polyclonal antibody and mouse anti-rPoIFN- 伪 monoclonal antibody. Different doses of rPoIFN- 伪 were injected intramuscular in 30-day-old and 2-day-old piglets. The changes of rPoIFN- 伪 concentration in serum of piglets at different time points were detected by establishing double antibody sandwich ELISA to study the metabolism of IFN in pigs. The safety of genetic engineering rPoIFN- 伪 was evaluated by animal toxicity test, gene residue detection and antibody induced antibody test. [results] P.pastoris was used as automatic fermenter to express rPoIFN- 伪. 50% saturated ammonium sulfate salting out, Q Sepharose FF anion exchange and Superdex 200 molecular sieves chromatography purification, agarose G 鈮，
本文编号：2483090
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