重组人类骨保护蛋白毕赤酵母分泌性表达株的构建及系列研究
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摘要
骨保护蛋白(Osteoprotegerin，OPG)及其配基(即核因子-κB受体激活因子配基，Receptor Activator of Nuclear Factor-κB Ligand，RANKL)对破骨细胞的分化和活性具有关键性调节作用，其中RANKL对破骨细胞的活性和形成起着决定性作用，OPG作为RANKL的可溶性假受体竞争性抑制其与破骨前体细胞或成熟破骨细胞膜受体(Receptor Activator of Nuclear Factor-κB，RANK)结合。成人体内两者均由成骨细胞、骨髓间充质细胞表达，作为破骨细胞局部调节终极环节，许多代谢性骨病均与成骨细胞系的这两种细胞因子表达异常有关，并且多数影响骨代谢的激素类因子是通过影响成骨细胞系的RANKL和OPG表达来调节破骨细胞的分化和活性的。OPG是目前所发现唯一的局部负向调节因子，因此对于骨吸收类疾病具有良好的药用潜质。
甲醇营养型酵母菌是一种理想的外源基因表达系统，兼有原核细胞良好的可操作性和真核系统的翻译后加工的双重特点。本实验根据OPG的结构特征，选择其功能片段(D1-D4)作为目的基因一，并选取IgG_1的Fc片段作为目的基因二，拟于P.pastoris酵母菌构建rhOPG-Fc融合蛋白的表达株。利用RT-PCR技术获得编码OPG(D1-D4)和IgG_1-Fc的基因，同时引入限制性内切酶XhoI和XbaI的酶切位点，通过XhoI和XbaI酶切作用以及噬菌体聚合酶T4作用，构建分泌型表达载体pPICZαB／OPG-Fc。经过测序证实：插入序列与预期结果相吻合，且接口处序列正确，未改变读码框架。利用该载体成功地构建和筛选得到了P.pastoris酵母菌高效分泌性表达株。X33／OPG-Fc菌株在甲醇诱导下(摇瓶发酵)与亲和层析纯化得到了目的蛋白-rhOPG-Fc，并经过SDS-PAGE电泳证实表达蛋白正确。并且以小鼠破骨细胞为作用体外实验对象，对摇瓶发酵获得的rhOPG-Fc进行生物活性验证和有效浓度测定，结果证实：于2×10~(-6)～2×10~(-8)g／ml，rhOPG-Fc对小鼠破骨前体细胞的增殖、破骨细胞分化和活性均有抑制作用。在此基础上，对X33／OPG-Fc菌株进行生物反应器发酵，并且对反应体系和纯化过程进行了优化，使rhOPG-Fc的产量达到0.2g／L。
以往研究表明：多数骨吸收类疾病的发生均与骨骼局部的OPG表达降
解放军总医院·军医进修学院
博士研究生学位论文
低有关，因此补充外源性OPG是治疗此类疾病的理想方法，本研究以骨巨
细胞瘤和原发性骨质疏松症作为骨吸收类疾病的代表，探讨thOPG一Fc作为
破骨细胞抑制剂的临床应用价值。首先建立了骨巨细胞瘤间质细胞系，在
此基础上研究肿瘤细胞的染色体核型异常与RANKL和OPG表达差异，进
而在体外条件下观察到毒性剂量的thoPG一Fc(2、1丫留ml)对骨巨细胞瘤
细胞的生长有抑制作用、在生理剂量下(2、10一7g/ml)对其有促成骨活性;
对雌性大鼠行卵粱切除术复制骨质疏松动物模型，通过随机对照研究观察:
体内条件下，thOPG一Fc可以抑制雌激素缺乏所引起的骨丢失。
综上所述，本研究构建的X33/o PG一F。菌株能够稳定、高效地表达目的
蛋白thOPG一Fc，该蛋白不仅生理活性等同于OPG全长肤链，而且作为嵌
合蛋白具有更好的生物相容性和体内稳定性。进而通过体内、体外实验证
实thOPG一Fc具有良好的抗骨吸收作用，而且可以抑制骨巨细胞瘤细胞生长
和促进其成骨活性，为thOPG一Fc的临床应用莫定了理论基础。
Bone is a dynamic tissue that is morphogenized and maintained by continuous formation and resorption. An imbalance between bone formation and resorption causes such metabolic bone diseases as osteopetrosis and osteoporosis. Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. Bone remodeling and bone loss are controlled by a balance between the tumor necrosis factor family molecule receptor activator of nuclear factor-KB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG). OPG is a key factor acting as a negative regulator against osteoclasts' diferantiation and activation, through competitively inhibiting RANKL binding to its membrane receptor -Receptor Activator of Nuclear Factor-KB (RANK). The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigatio
n of the regulation osteoclast differentiation and function. OPG is a member of TNFR superfamily, which active site is its domain 1-4, and a secretory glycoprotein. The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide OPG for the pre-clinical experiments, we are working on the high-throughput expression of human proteins. Therefore, cDNAs of OPG 22-201 aas and IgG1-Fc are cloned for secretory expression. The resulting fusion proteins carry affinity tags (IgG1-Fc) at the N- and C-terminus for the immunological detection and chromatographic purification of protein. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which prod
uce heterologous protein with a yield of 0.2g/L culture
volume or higher.
To assess the effect of different dosages of rhOPG-Fc on the differentiation and activation of osteoclasts, RAW264-7 cell line was employed as preosteoclast coculture with mouse osteoblasts for osteoclastogenesis. The results of in vitro experiments showed that rhOPG-Fc has different effects on preosteoclasts and osteoblasts at different dosages. At 2 × 10-6~2×10~8g/ml, rhOPG-Fc could disrupted differentiation and activation of osteoclasts and proliferation of preosteoclasts, and at 2 × 10-4g/ml or higher, has a toxic effect on bone cells and stomral cells from giant cell tumor of bone. The bone mass of ovariectomied rats that were abdominal injected rhOPG-Fc 5mg/kg/d × 14d did not decrease significantly 3 months after administration. These findings led us to conclude that the effect of rhOPG-Fc on anti-bone resorption is similar to OPG full-length protein.
甲醇营养型酵母菌是一种理想的外源基因表达系统，兼有原核细胞良好的可操作性和真核系统的翻译后加工的双重特点。本实验根据OPG的结构特征，选择其功能片段(D1-D4)作为目的基因一，并选取IgG_1的Fc片段作为目的基因二，拟于P.pastoris酵母菌构建rhOPG-Fc融合蛋白的表达株。利用RT-PCR技术获得编码OPG(D1-D4)和IgG_1-Fc的基因，同时引入限制性内切酶XhoI和XbaI的酶切位点，通过XhoI和XbaI酶切作用以及噬菌体聚合酶T4作用，构建分泌型表达载体pPICZαB／OPG-Fc。经过测序证实：插入序列与预期结果相吻合，且接口处序列正确，未改变读码框架。利用该载体成功地构建和筛选得到了P.pastoris酵母菌高效分泌性表达株。X33／OPG-Fc菌株在甲醇诱导下(摇瓶发酵)与亲和层析纯化得到了目的蛋白-rhOPG-Fc，并经过SDS-PAGE电泳证实表达蛋白正确。并且以小鼠破骨细胞为作用体外实验对象，对摇瓶发酵获得的rhOPG-Fc进行生物活性验证和有效浓度测定，结果证实：于2×10~(-6)～2×10~(-8)g／ml，rhOPG-Fc对小鼠破骨前体细胞的增殖、破骨细胞分化和活性均有抑制作用。在此基础上，对X33／OPG-Fc菌株进行生物反应器发酵，并且对反应体系和纯化过程进行了优化，使rhOPG-Fc的产量达到0.2g／L。
以往研究表明：多数骨吸收类疾病的发生均与骨骼局部的OPG表达降
解放军总医院·军医进修学院
博士研究生学位论文
低有关，因此补充外源性OPG是治疗此类疾病的理想方法，本研究以骨巨
细胞瘤和原发性骨质疏松症作为骨吸收类疾病的代表，探讨thOPG一Fc作为
破骨细胞抑制剂的临床应用价值。首先建立了骨巨细胞瘤间质细胞系，在
此基础上研究肿瘤细胞的染色体核型异常与RANKL和OPG表达差异，进
而在体外条件下观察到毒性剂量的thoPG一Fc(2、1丫留ml)对骨巨细胞瘤
细胞的生长有抑制作用、在生理剂量下(2、10一7g/ml)对其有促成骨活性;
对雌性大鼠行卵粱切除术复制骨质疏松动物模型，通过随机对照研究观察:
体内条件下，thOPG一Fc可以抑制雌激素缺乏所引起的骨丢失。
综上所述，本研究构建的X33/o PG一F。菌株能够稳定、高效地表达目的
蛋白thOPG一Fc，该蛋白不仅生理活性等同于OPG全长肤链，而且作为嵌
合蛋白具有更好的生物相容性和体内稳定性。进而通过体内、体外实验证
实thOPG一Fc具有良好的抗骨吸收作用，而且可以抑制骨巨细胞瘤细胞生长
和促进其成骨活性，为thOPG一Fc的临床应用莫定了理论基础。
Bone is a dynamic tissue that is morphogenized and maintained by continuous formation and resorption. An imbalance between bone formation and resorption causes such metabolic bone diseases as osteopetrosis and osteoporosis. Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. Bone remodeling and bone loss are controlled by a balance between the tumor necrosis factor family molecule receptor activator of nuclear factor-KB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG). OPG is a key factor acting as a negative regulator against osteoclasts' diferantiation and activation, through competitively inhibiting RANKL binding to its membrane receptor -Receptor Activator of Nuclear Factor-KB (RANK). The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigatio
n of the regulation osteoclast differentiation and function. OPG is a member of TNFR superfamily, which active site is its domain 1-4, and a secretory glycoprotein. The methylotrophic yeast Pichia pastoris has become a powerful host for the heterologous expression of proteins. In order to provide OPG for the pre-clinical experiments, we are working on the high-throughput expression of human proteins. Therefore, cDNAs of OPG 22-201 aas and IgG1-Fc are cloned for secretory expression. The resulting fusion proteins carry affinity tags (IgG1-Fc) at the N- and C-terminus for the immunological detection and chromatographic purification of protein. Expression is controlled by the tightly regulated and highly inducible alcoholoxidase 1 (AOX1) promoter. We have developed a cultivation and induction protocol amendable to automation to increase the number of clones screened for protein expression. Using the optimized feeding and induction protocol, we are now able to screen for and identify expression clones which prod
uce heterologous protein with a yield of 0.2g/L culture
volume or higher.
To assess the effect of different dosages of rhOPG-Fc on the differentiation and activation of osteoclasts, RAW264-7 cell line was employed as preosteoclast coculture with mouse osteoblasts for osteoclastogenesis. The results of in vitro experiments showed that rhOPG-Fc has different effects on preosteoclasts and osteoblasts at different dosages. At 2 × 10-6~2×10~8g/ml, rhOPG-Fc could disrupted differentiation and activation of osteoclasts and proliferation of preosteoclasts, and at 2 × 10-4g/ml or higher, has a toxic effect on bone cells and stomral cells from giant cell tumor of bone. The bone mass of ovariectomied rats that were abdominal injected rhOPG-Fc 5mg/kg/d × 14d did not decrease significantly 3 months after administration. These findings led us to conclude that the effect of rhOPG-Fc on anti-bone resorption is similar to OPG full-length protein.
引文
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