發(fā)布時(shí)間：2019-05-22 16:58

【摘要】：【目的】干擾素(Interferon,IFN)是一類具有抗病毒、抗腫瘤和調(diào)節(jié)機(jī)體免疫功能等重要生物學(xué)功能的細(xì)胞因子,在臨床上具有廣泛應(yīng)用前景。為了使重組豬干擾素-α(rPoIFN-α)能夠更快的從實(shí)驗(yàn)室走向產(chǎn)業(yè)化和臨床應(yīng)用,對rPoIFN-α的工業(yè)化表達(dá)及其分離純化條件的優(yōu)化和生物安全性評價(jià)具有重要的現(xiàn)實(shí)意義�！痉椒ā坷米詣�(dòng)發(fā)酵罐培養(yǎng)重組P.pastoris菌株,獲得高效表達(dá)的rPoIFN-α,硫酸銨鹽析沉淀后,通過離子交換和分子篩層析法分離純化,Marc-145/VSV系統(tǒng)測定活性。通過兔抗rPo IFN-α多克隆抗體和鼠抗rPoIFN-α單克隆抗體建立檢測rPoIFN-α濃度的雙抗體夾心ELISA方法。選擇30日齡和2日齡仔豬,肌肉注射不同劑量的rPoIFN-α,利用建立雙抗體夾心ELISA檢測不同時(shí)間點(diǎn)仔豬血清中rPoIFN-α濃度的變化,研究IFN在豬體內(nèi)的代謝規(guī)律。通過動(dòng)物毒性試驗(yàn)、基因殘留檢測以及致仔豬產(chǎn)生抗體試驗(yàn)等,對基因工程rPoIFN-α進(jìn)行安全性評價(jià)�！窘Y(jié)果】以P.pastoris作為菌種自動(dòng)發(fā)酵罐高效表達(dá)rPoIFN-α,50%飽和度硫酸銨鹽析、Q Sepharose FF陰離子交換和Superdex 200分子篩層析技術(shù)純化,瓊脂糖G-25層析脫鹽,可獲得純度為97.75%rPoIFN-α,活性可達(dá)到1.00×108 U/μL。利用兔多克隆抗r PoIFN-α抗體和鼠單克隆抗r PoIFN-α抗體,建立了雙抗夾心ELISA方法,檢測PoIFN-α濃度范圍為2.5~0.0781μg/mL。以1、10、50mg/頭三種不同劑量的r PoIFN-α肌肉注射30日齡和2日齡仔豬,雙抗夾心ELISA檢測不同時(shí)間點(diǎn)血清中r PoIFN-α濃度,繪制消長曲線,顯示在注射后60min血清中IFN濃度達(dá)到高峰,IFN在血清中的濃度與其注射劑量成正相關(guān),在30日齡仔豬血清中的存在時(shí)間將近24h,而在2日齡仔豬血清中的存在時(shí)間只有3h,存在時(shí)間與注射劑量無明顯相關(guān)性。小鼠急性毒性試驗(yàn)表明,10mg/只以下劑量不會(huì)對小鼠產(chǎn)生任何毒性作用;基因殘留試驗(yàn)和誘發(fā)仔豬抗體證明,基因工程rPoIFN-α不攜帶重組基因和抗生素抗性基因,也不誘發(fā)仔豬產(chǎn)生rPoIFN-α抗體。但在仔豬毒性試驗(yàn)中,組織切片觀察表明,高劑量(50mg/頭)rPo IFN-α可導(dǎo)致仔豬肺泡壁增厚、脾臟紅髓比例增加,肝臟脂肪樣病變等輕微病理變化。
[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-偽 (rPoIFN- 偽) to move from laboratory to industrialization and clinical application more quickly, It is of great practical significance to optimize the industrial expression of rPoIFN- 偽, 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- 偽. 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- 偽 concentration was established by rabbit anti-rPoIFN- 偽 polyclonal antibody and mouse anti-rPoIFN- 偽 monoclonal antibody. Different doses of rPoIFN- 偽 were injected intramuscular in 30-day-old and 2-day-old piglets. The changes of rPoIFN- 偽 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- 偽 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- 偽. 50% saturated ammonium sulfate salting out, Q Sepharose FF anion exchange and Superdex 200 molecular sieves chromatography purification, agarose G 鈮，

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