王玉麒Yu-Chie Wang陳俊維June-Wei Chen2019-09-052007-8-12019-09-052006http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22GN0692430231%22.&%22.id.&http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/104103本研究主要探討重組人類γ干擾素於大腸桿菌與植物細胞內的生合成狀況，以及生產高應用性與高附加價值的γ干擾素單株與多株抗體。在大腸桿菌的實驗中，我們採用四種不同的重組γ干擾素表現質體來測試細胞內重組γ干擾素表現的情形，結果顯示利用pET28a質體建構的表現質體具備最高的重組γ干擾素生合成能力，且利用BL21(DE3) 菌株做為寄主時，30至300 μM的IPTG濃度能使細胞內的重組γ干擾素表現量達到最高，此時每公升菌液可表現出約200 mg的重組γ干擾素，佔大腸桿菌細胞內總蛋白質的46.5%，若利用固相金屬親和性層析法進行純化，則純化後可得到純度約95%重組γ干擾素，回收率約為48.4%。若利用低溫誘導或復性策略來取得水溶性重組γ干擾素，則發現水溶性重組γ干擾素會在緩衝液中迅速變性沉澱，雖然甘油與精胺酸（arginine-HCl）等添加劑能夠延緩水溶性重組γ干擾素的沉澱，但實驗顯示該水溶性重組γ干擾素仍沒有顯著的抗病毒活性。植物細胞的部份則採用水稻及蕃茄原生質體來進行重組γ干擾素生合成的初步探討，在挑選出具備最高活性的玉米ZmUbq-1啟動子後，建構出以ZmUbq-1啟動子調控表現的基因表現卡匣供轉形實驗使用，而暫時性表現實驗的結果顯示基因轉錄作用雖正常進行，但重組γ干擾素無法於水稻原生質體中有效生合成與累積。而在γ干擾素單株抗體生產部份，本實驗利用細菌生合成之重組γ干擾素生產出九株適用於ELISA實驗的單株抗體，而其中三個單株抗體更可兼用於免疫染色實驗（Western blot），且抗體效價很好，在0.25 μg/ml的抗體濃度下，即可清楚辨識到0.3至1 ng以下的γ干擾素。在多株抗體的生產部分，則收集到總體積超過200 ml的多株抗體血清，實驗結果顯示多株抗體血清的效價亦相當高且具有一致性，即使將血清稀釋三十萬倍後，仍可明顯辨識到5 ng以下的γ干擾素。The present studies describe our attempts to express the recombinant human interferon-gamma (rh-IFNγ) and to generate antibodies directed to rh-IFNγ. In the experiments to express rh-IFNγ in E. coli, we have scrutinized four different expression vectors and found that pET28a was most suitable for rh-IFNγ expression in E. coli strain BL21(DE3). By adding inducer IPTG to final concentration of 30~300 μM, the rh-IFNγ was expressed in E. coli at a high level of near 200 mg/L, which accounted for 46.5% of total proteins in the cell. By adsorption to and elution from an immobilized nickel column, the collected rh-IFNγ was near 95% pure and the recovery yield was about 48.4%. To prepare soluble rh-IFNγ for antiviral assay, the recombinant protein was expressed at 16°C and either glycerol or arginine was included as protein stabilizer. Nevertheless, no significant antiviral effect has been demonstrated to the rh-IFNγ thus far. In the experiments to express rh-IFNγ in plant cells, we have constructed and tested a number of gene expression cassettes. The results of transient expression assays revealed that maize ZmUbq-1 promoter and CaMV 35S promoter had the highest activity to drive gene expression in rice and tomato protoplasts, respectively. However, rice protoplasts transformed with IFNγ gene driven by ZmUbq-1 promoter were found to accumulate no detectable amount of rh-IFNγ protein. In the experiments to generate monoclonal antibodies directed to rh-IFNγ, a total of 9 cell lines were successfully screened. Among them, six lines are effective for ELISA, while the other three lines worked well for both ELISA and immunoblot assay. In addition, we have also collected more than 200 ml high-titer rabbit anti-sera against rh-IFNγ, which could detect 5 ng antigen in an immunoblot assay even being diluted by 300000-fold.重組人類gamma干擾素重組蛋白質生產單株抗體生產多株抗體原生質體轉形recombinant interferon-gammarecombinant proteinmonoclonal antibody productionpolyclonal antibody productionprotoplast transformation