重组人干扰素-γ在毕赤酵母中的表达及对乳腺癌抑制作用的研究
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摘要
人干扰素-γ（human interferon-γ，hIFN-γ）是一种常见的天然糖蛋白，属于干扰素（Interferon，IFN）家族的一员，具有多种生物活性。根据蛋白来源和结构不同可将IFN家族分为Ⅰ、Ⅱ、Ⅲ型，而hIFN-γ属于Ⅱ型IFN，其主要通过作用于巨噬细胞及T细胞来调节机体免疫来达到抗病毒的效果，而体外研究发现IFN-γ可抑制体外培养癌细胞的增殖并诱导其凋亡，同时对多种移植瘤模型亦有效，表明其有较好的抗肿瘤潜能。以上提示IFN有巨大的临床应用前景。
乳腺癌发病率和死亡率均较高，是最常见的女性恶性肿瘤，预后极差。其异常生物学行为是导致难以治愈的关键因素，故探讨有效的抑制乳腺癌增殖、侵袭和转移的策略，对降低乳腺癌患者术后复发及延长生存期有重要意义。研究表明IFN-γ具有抗肿瘤活性，本研究以此为研究基础，探讨IFN-γ在抑制乳腺癌增殖、侵袭及转移方面的作用。
获得大量的高纯度的hIFN-γ对临床研究及其机制研究有重要意义。但由于天然hIFN-γ来源有限，故需借助生物工程技术进行大规模制备。先前报道指出可采用腺病毒、大肠杆菌、酿酒酵母及中国仓鼠卵巢细胞来制备重组hIFN-γ（rhIFN-γ），但操作工艺复杂、生产成本高及产量和生物活性降低限制了以上表达系统的应用。本研究应用巴斯德毕赤酵母（Pichia pastoris）表达系统制备出与hIFN-γ组分相似的rhIFN-γ制剂，同时通过体内和体外实验检测了该生物制剂的生物学活性，取得以下结果：
一、rhIFN-γ毕赤酵母表达载体的构建、表达、纯化及活性检测
1.成功构建了重组人干扰素-γ的分泌型表达载体pPICZ-rhIFN-γ，利用巴斯德毕赤酵母表达系统稳定分泌表达了rhIFN-γ。
2.免疫印迹及N末端测序显示rhIFN-γ成功表达，从毕赤酵母培养液分离和浓缩后，采用阳离子交换色谱系统进行纯化。从5L的培养基中共获得总量为0.601g的rhIFN-γ。TRICINE-SDS-PAGE电泳及高效液相色谱显示获得rhIFN-γ的纯度为95.6%。
二、rhIFN-γ的抗肿瘤活性
采用乳腺癌常用细胞株MDA-MB-231及乳腺癌皮下移植瘤模型分别在体外、体内水平研究rhIFN-γ的抗肿瘤作用。
1.体外水平：rhIFN-γ能呈时间依赖的方式抑制癌细胞生长、提高凋亡率及凋亡指数、阻滞细胞周期并抑制PI3K/Akt信号通路的激活。
2.体内水平：rhIFN-γ能降低乳腺癌皮下移植瘤模型的成瘤率、降低血清及瘤内血管内皮生长因子水平，显著降低肿瘤组织中的MMP-2和MMP-9的表达。
本研究的创新之处在于：(1）建立了利用巴斯德毕赤酵母高效分泌表达rhIFN-γ的真核表达系统；(2）通过Tricine-SDS-PAGE、免疫印迹及氨基酸直接测序技术鉴定rhIFN-γ，阳离子交换色谱系统纯化得到高纯度rhIFN-γ；(3）在体外水平、体内水平证实rhIFN-γ具有广泛的抗肿瘤作用。
Human interferon-γ (hIFN-γ) is a common natural glycoprotein, a member ofinterferon (Interferon, IFN) family, possessing a variety of biological activities.hIFN-γ can achieve the anti-viral effect mainly by acting on macrophages and T cellsto regulate immune. In vitro studies have found that IFN-γ can inhibit the proliferationof cancer cells in vitro and induce apoptosis. IFN-γ also can be effective for a varietyof xenograft model, indicating a better anti-tumor potential. The incidence andmortality rates of breast cancer are high and it is the most common female cancer withpoor prognosis. The abnormal biological behaviors are key factors leading todifficulty to cure for breast cancer. The above indicates hIFN-γ may become a strategyfor breast cancer treatment, may reduce the recurrence and prolong survival ofpatients with breast cancer. Large quantity high-purity hIFN-γ is important for clinicalresearch and their mechanisms. Due to the limitation of acquiring natural hIFN-γ, itrequires the use of bio-engineering technology for large-scale preparation. Theprevious expression systems are improper for their complex process operation, highproduction costs and lower yields. If the efficient bio-engineering vector can beemployed for obtaining rhIFN-γ with the similar functions as natural hIFN-γ, thesource problem of hIFN-γ can be well solved.
In this study, the Pichia pastoris expression system was employed to producerhIFN-γ with similar components as hIFN-γ. Meanwhile, the biological activity ofrhIFN-γ was evaluated by in vivo and in vitro studies,including the following aspects:1Preparation and optimized fermentation conditions of rhIFN-γ
The sequence of hIFN-γ was designed according to GeneBank (accessionnumber: BC070256).After the gene synthesis, hIFN-γ was connected to pPICZvector and then transferred to construct engineered bacteria. The successful-expreesedengineered bacteria were hereby selected. The optimal condition for Pichia pastoris toexpress rhIFN-γ was pH of6.0at the temperature of28℃.And the optimal inductiontime point was the6thday.A Total amount of0.601g purified rhIFN-γ was obtained from5L medium. The Tricine-SDS-PAGE electrophoresis and high performanceliquid chromatography showed a purity of95.6%for rhIFN-γ.2Studies of in vitro inhibitory effect of rhIFN-γ
The human breast cancer MDA-MB-231cells were treated with rhIFN-γ at thedose of0,100,500and1000U/mL, respectively. MTT shows the rhIFN-γ can inhibitcell proliferation of MDA-MB-231cells at24th,48thand96thh after treatment. TheHoechst staining and flow cytometry indicate that rhIFN-γ can increase the apoptoticindex and apoptosis rate in a dose-dependent manner. The DHE staining showsrhIFN-γ can reduce the ROS levels in a dose-dependent manner. The western blotshow rhIFN-γ can decrease levels of Bcl-2and p-Akt/Akt, and increase levels ofCleaved caspase-3and Bax in a dose-dependent manner.3Studies of in vivo inhibitory effect of rhIFN-γ
The breast cancer xenografts in nude mice model were treated with rhIFN-γ atthe dose of0,100,500and1000U/g, respectively. The rhIFN-γ can decerase the tumorsize in a dose-dependent manner. The immunohistochemical staining shows thatrhIFN-γ can reduce the expression of MMP-2and MMP-9in the tumor tissue in adose-dependent manner. Elisa shows rhIFN-γ can reduce the VEGF levels of tumortissue and serum in a dose-dependent manner. The western blot shows rhIFN-γ candecrease the level of p-Akt/Akt in a dose-dependent manner.
In conclusion, Pichia pastoris stably secret,and express rhIFN-γ and the newmethod for large-scale purification of the recombinant protein was established. The invivo and in vitro studies proved rhIFN-γ can inhibit the development of breast cancer.
乳腺癌发病率和死亡率均较高，是最常见的女性恶性肿瘤，预后极差。其异常生物学行为是导致难以治愈的关键因素，故探讨有效的抑制乳腺癌增殖、侵袭和转移的策略，对降低乳腺癌患者术后复发及延长生存期有重要意义。研究表明IFN-γ具有抗肿瘤活性，本研究以此为研究基础，探讨IFN-γ在抑制乳腺癌增殖、侵袭及转移方面的作用。
获得大量的高纯度的hIFN-γ对临床研究及其机制研究有重要意义。但由于天然hIFN-γ来源有限，故需借助生物工程技术进行大规模制备。先前报道指出可采用腺病毒、大肠杆菌、酿酒酵母及中国仓鼠卵巢细胞来制备重组hIFN-γ（rhIFN-γ），但操作工艺复杂、生产成本高及产量和生物活性降低限制了以上表达系统的应用。本研究应用巴斯德毕赤酵母（Pichia pastoris）表达系统制备出与hIFN-γ组分相似的rhIFN-γ制剂，同时通过体内和体外实验检测了该生物制剂的生物学活性，取得以下结果：
一、rhIFN-γ毕赤酵母表达载体的构建、表达、纯化及活性检测
1.成功构建了重组人干扰素-γ的分泌型表达载体pPICZ-rhIFN-γ，利用巴斯德毕赤酵母表达系统稳定分泌表达了rhIFN-γ。
2.免疫印迹及N末端测序显示rhIFN-γ成功表达，从毕赤酵母培养液分离和浓缩后，采用阳离子交换色谱系统进行纯化。从5L的培养基中共获得总量为0.601g的rhIFN-γ。TRICINE-SDS-PAGE电泳及高效液相色谱显示获得rhIFN-γ的纯度为95.6%。
二、rhIFN-γ的抗肿瘤活性
采用乳腺癌常用细胞株MDA-MB-231及乳腺癌皮下移植瘤模型分别在体外、体内水平研究rhIFN-γ的抗肿瘤作用。
1.体外水平：rhIFN-γ能呈时间依赖的方式抑制癌细胞生长、提高凋亡率及凋亡指数、阻滞细胞周期并抑制PI3K/Akt信号通路的激活。
2.体内水平：rhIFN-γ能降低乳腺癌皮下移植瘤模型的成瘤率、降低血清及瘤内血管内皮生长因子水平，显著降低肿瘤组织中的MMP-2和MMP-9的表达。
本研究的创新之处在于：(1）建立了利用巴斯德毕赤酵母高效分泌表达rhIFN-γ的真核表达系统；(2）通过Tricine-SDS-PAGE、免疫印迹及氨基酸直接测序技术鉴定rhIFN-γ，阳离子交换色谱系统纯化得到高纯度rhIFN-γ；(3）在体外水平、体内水平证实rhIFN-γ具有广泛的抗肿瘤作用。
Human interferon-γ (hIFN-γ) is a common natural glycoprotein, a member ofinterferon (Interferon, IFN) family, possessing a variety of biological activities.hIFN-γ can achieve the anti-viral effect mainly by acting on macrophages and T cellsto regulate immune. In vitro studies have found that IFN-γ can inhibit the proliferationof cancer cells in vitro and induce apoptosis. IFN-γ also can be effective for a varietyof xenograft model, indicating a better anti-tumor potential. The incidence andmortality rates of breast cancer are high and it is the most common female cancer withpoor prognosis. The abnormal biological behaviors are key factors leading todifficulty to cure for breast cancer. The above indicates hIFN-γ may become a strategyfor breast cancer treatment, may reduce the recurrence and prolong survival ofpatients with breast cancer. Large quantity high-purity hIFN-γ is important for clinicalresearch and their mechanisms. Due to the limitation of acquiring natural hIFN-γ, itrequires the use of bio-engineering technology for large-scale preparation. Theprevious expression systems are improper for their complex process operation, highproduction costs and lower yields. If the efficient bio-engineering vector can beemployed for obtaining rhIFN-γ with the similar functions as natural hIFN-γ, thesource problem of hIFN-γ can be well solved.
In this study, the Pichia pastoris expression system was employed to producerhIFN-γ with similar components as hIFN-γ. Meanwhile, the biological activity ofrhIFN-γ was evaluated by in vivo and in vitro studies,including the following aspects:1Preparation and optimized fermentation conditions of rhIFN-γ
The sequence of hIFN-γ was designed according to GeneBank (accessionnumber: BC070256).After the gene synthesis, hIFN-γ was connected to pPICZvector and then transferred to construct engineered bacteria. The successful-expreesedengineered bacteria were hereby selected. The optimal condition for Pichia pastoris toexpress rhIFN-γ was pH of6.0at the temperature of28℃.And the optimal inductiontime point was the6thday.A Total amount of0.601g purified rhIFN-γ was obtained from5L medium. The Tricine-SDS-PAGE electrophoresis and high performanceliquid chromatography showed a purity of95.6%for rhIFN-γ.2Studies of in vitro inhibitory effect of rhIFN-γ
The human breast cancer MDA-MB-231cells were treated with rhIFN-γ at thedose of0,100,500and1000U/mL, respectively. MTT shows the rhIFN-γ can inhibitcell proliferation of MDA-MB-231cells at24th,48thand96thh after treatment. TheHoechst staining and flow cytometry indicate that rhIFN-γ can increase the apoptoticindex and apoptosis rate in a dose-dependent manner. The DHE staining showsrhIFN-γ can reduce the ROS levels in a dose-dependent manner. The western blotshow rhIFN-γ can decrease levels of Bcl-2and p-Akt/Akt, and increase levels ofCleaved caspase-3and Bax in a dose-dependent manner.3Studies of in vivo inhibitory effect of rhIFN-γ
The breast cancer xenografts in nude mice model were treated with rhIFN-γ atthe dose of0,100,500and1000U/g, respectively. The rhIFN-γ can decerase the tumorsize in a dose-dependent manner. The immunohistochemical staining shows thatrhIFN-γ can reduce the expression of MMP-2and MMP-9in the tumor tissue in adose-dependent manner. Elisa shows rhIFN-γ can reduce the VEGF levels of tumortissue and serum in a dose-dependent manner. The western blot shows rhIFN-γ candecrease the level of p-Akt/Akt in a dose-dependent manner.
In conclusion, Pichia pastoris stably secret,and express rhIFN-γ and the newmethod for large-scale purification of the recombinant protein was established. The invivo and in vitro studies proved rhIFN-γ can inhibit the development of breast cancer.
引文
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