CN116103253A - 一种重组人超氧化物歧化酶及其制备方法和应用 - Google Patents
一种重组人超氧化物歧化酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种重组人超氧化物歧化酶及其制备方法和应用。一种重组人超氧化物歧化酶，所述重组人超氧化物歧化酶的氨基酸序列特征为SEQ ID NO:1；一种重组人超氧化物歧化酶的制备方法，包括制备重组载体、基因重组并筛选高抗性转化子、诱导表达rhSOD；一种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，所述抗氧化制剂为用于清除自由基、并抑制促炎细胞因子IL‑6、IL‑8和TNF‑α释放的制剂。本发明的重组人超氧化物歧化酶将人Cu/Zn‑SOD基因中非活性中心的Cys111密码子突变为Thr密码子，并且在N‑端添加6×His，在C‑端添加了自主设计的穿膜肽序列，可以引导SOD穿膜进入细胞内部，具有兼具抗氧化性能和细胞穿膜性能的优点，能广泛应用于化妆品、保健品、食品和药品等领域。
Description
技术领域
本发明涉及超氧化物歧化酶的技术领域，尤其是涉及一种重组人超氧化物歧化酶及其制备方法和应用。
背景技术
为了清楚机体内的氧自由基，减少其对基体的损害，恢复基体健康，常通过抗氧化剂来清除体内有害的氧自由基。现有的抗氧化剂包括超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、过氧化氢酶(CAT)和单胺氧化酶抑制剂等。其中，超氧化物歧化酶（SOD）是目前常用的酶性抗氧化剂，其具有抗氧化、防衰老、抗炎、防辐射三大功能，可广泛用于化妆品、保健品、食品、医疗等领域。
目前国内生产的SOD主要从天然生物体中提取，由于人血来源困难，而不宜推广使用。为此，授权公告号为CN103272223B的中国专利公开了一种抗氧化保健胶囊及其制备方法，以300g计，由以下组分制成的胶囊剂：20~30g的80wt%的人参皂苷粉、0.8~1.5g的牛血提取物SOD、15~25g的维生素C、25~35g的维生素E粉与余量的填充物。上述抗氧化剂中的SOD主要来源于牛血，能起到与人血制品SOD相似的抗氧化作用，但是非人源制品SOD用于人体有排异性，且和人血制品SOD都存在外源感染的风险，在各领域中应用受限。此外，天然SOD作为一种功能蛋白，具有药效高、刺激性小、毒性低、高活性、特异性强等优点，但蛋白质类药物的口服给药会在胃肠道发生降解，且胃肠黏膜的穿透性差、分子量大、制备困难、且存在抗原性，有待改进。
因此，如何研发出一种兼具抗氧化性能和细胞穿膜性能的重组人超氧化物歧化酶，并拓展其在抗氧化制剂中的应用领域，是目前亟需解决的问题。此外，还需设计一种制备重组人超氧化物歧化酶的方法，以提高其产率。
发明内容
针对现有技术存在的不足，本发明的第一个目的在于提供一种重组人超氧化物歧化酶，其具有兼具抗氧化性能和细胞穿膜性能的优点。
本发明的第二个目的在于提供一种重组人超氧化物歧化酶的制备方法，其具有毕赤酵母体外高效分泌表达、产率高的优点。
本发明的第三个目的在于提供一种重组人超氧化物歧化酶的应用，其达到了拓展其在抗氧化制剂中的应用领域的目的。
为实现上述第一个目的，本发明提供了如下技术方案：
一种重组人超氧化物歧化酶，所述重组人超氧化物歧化酶的氨基酸序列特征为SEQ ID NO:1。
通过采用上述技术方案，将人Cu/Zn-SOD基因中非活性中心的Cys111密码子突变为Thr密码子（二者均为亲水性中性氨基酸），保留第6位和57位的半胱氨酸，不影响其活性，但突变Cys111后可以防止二硫键的错配，以提高其空间结构的稳定性；根据毕赤酵母密码子偏爱性，对SOD基因密码子进行了优化，并且在N-端添加6×His， 以方便进行亲和纯化，在C-端添加了自主设计的穿膜肽序列（RRQRRQRRQRRQRRR，简称15RQ），可以引导SOD穿膜进入细胞内部。
为实现上述第二个目的，本发明提供了如下技术方案：
一种重组人超氧化物歧化酶的制备方法，包括以下步骤，
S1人工全基因合成所述重组人超氧化物歧化酶的核苷酸序列SEQ ID NO:2，并将其插入到pPICZαA载体的EcoRI和XbaI限制性酶切位点之间，得到重组载体pPICZαA-rhSOD；
S2将所述S1得到的重组载体线性化后导入毕赤酵母中，筛选高抗性转化子；
S3培养所述S2得到的转化子，并诱导表达rhSOD，表达完成后经后处理，得到所述重组人超氧化物歧化酶。
通过采用上述技术方案，本发明采用重组工程菌表达生产人源SOD，并将人工设计的穿膜肽15RQ（RRQRRQRRQRRQRRR）与SOD相连，所得产品分子量均一、结构稳定，采用毕赤酵母体外高效分泌表达，rhSOD产率高，且解决了人源SOD来源困难和血制品有外源感染的危险性的难题。
进一步地，在所述S3中，诱导表达的温度为20~37℃，优选为20、25、30、37℃。
进一步地，在所述S3中，后处理包括采用Ni-NTA亲和层析、超滤脱盐、调节pH、阳离子交换层析（SP-Sepharose FF）。
为实现上述第三个目的，本发明提供了如下技术方案：
一种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，所述抗氧化制剂为用于清除自由基、并抑制促炎细胞因子IL-6、IL-8 和TNF-α释放的制剂。
进一步地，所述自由基包括DPPH自由基（1，1-二苯基-2-三硝基苯肼）、ABTS自由基（2，2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸）、氧自由基和羟自由基。
进一步地，所述抗氧化制剂为用于预防或治疗与IL-6、IL-8 和TNF-α相关的疾病或病症的制剂，所述疾病或病症包括癌症、与高凝性相关的疾病或病状、与升高的血清CRP相关的疾病或病状、与低白蛋白血症相关的疾病或病状、炎症性病症、病毒性病症、消耗综合征、自身免疫病症或其任意组合。
更进一步地，所述抗氧化制剂为用于预防或治疗慢性阻塞性肺病的制剂。
慢性阻塞性肺病（COPD）是以不完全可逆的气流受限为特征的疾病，是闭塞性细支气管炎和肺实质破坏共同作用的结果，COPD到2030年将成为仅次于心脑血管疾病和肿瘤的全世界第三大死因，COPD患病率在40岁以上人群中高达8.2%，COPD不仅仅患病率高，致残、致死率也很高；rhSOD能专一地清除体内有害的自由基，以解除自由基氧化体内的某些组成成分而造成的机体损害，从而降低基体的氧化应激反应，并抑制促炎细胞因子IL-6、IL-8和TNF-α释放，从而降低COPD的发病率，并达到治疗和改善COPD的目的。
和/或，所述抗氧化制剂为用于预防或治疗人白血病细胞U937引起的白血病的制剂。
rhSOD能通过清除自由基和抑制炎症因子释放，来加强抗氧化系统作用，从而抑制内源性肿瘤启动子，来间接对人白血病细胞U937进行增值抑制和诱导凋亡，而不损害正常组织细胞，以达到预防和治疗白血病的目的。
和/或，所述抗氧化制剂为用于治疗烫烧伤和/或过敏引起的皮肤炎症的制剂。
rhSOD能通过专一地清除体内有害的自由基，避免肥大细胞和嗜碱粒细胞的氧化破坏，以解除自由基氧化体内的某些组成成分而造成的机体损害，如烫烧伤和/或过敏引起的皮肤炎症，不同于外擦炉甘石洗剂、外用皮质激素霜。但是皮质激素因为有多重副作用，rhSOD涂抹患处愈合是常规疗法的3倍，温和有效，而且疤痕明显减轻。
更进一步地，所述抗氧化制剂为化妆品、保健品、食品和药品中的一种。
更进一步地，所述抗氧化制剂的剂型为散剂、 片剂、颗粒剂、胶囊剂、微囊剂、丸剂、滴丸、软胶囊、口服液、糖浆剂、分散片、喷雾剂 、气雾剂、栓剂、静脉注射剂、肌肉注射剂、软膏剂、吸入剂和凝胶剂中的一种。
最进一步地，所述抗氧化制剂的剂型为吸入剂，所述抗氧化制剂由包含以下重量份的原料经灌装压入抛射剂二氧化碳制成，重组人超氧化物歧化酶1.5份，蔗糖5份，聚乙二醇1份，对羟基苯甲酸乙酯0.02份，丙二醇5份，去离子水加至100份。
本发明的重组人超氧化物歧化酶（rhSOD）吸入剂具有以下优点：（1）蛋白质结构更稳定，并且很好的穿膜作用，容易进入细胞里面，发挥清除氧自由基的作用；（2）药物密闭于容器中，能保持药物清洁无菌状态，并能增加药物的稳定性；（3）使用方便，药物可避免胃肠道的破坏和肝脏的首过效应，提高药物的生物利用度；（4）可以用定量阀门准确控制剂量；（5）外用气雾剂使用时对创面的机械刺激性小；（6）对于口腔、气道、以及肺局部治疗的疾病药物可直接到达病灶部位，可以降低给药剂量和毒副反应等。
或者，所述抗氧化制剂的剂型为喷雾剂，所述抗氧化制剂由包含以下重量份的原料制成，重组人超氧化物歧化酶1.0份，甘油5份，山梨醇1份，维生素C 0.10份，维生素E0.10份，尼泊金酯0.2份，薄荷醇0.1份，香精0.1份，10mM氯化铜的PBS缓冲液至100份。
本发明通过将rhSOD制备成喷雾剂具有以下优势：（1）药物直接到达作用部位，并且有穿膜效果，分布均匀，起效快；（2）使用方便，奏效迅速；（3）能保持药物清洁和无菌状态，并能提高药物的稳定性。由于药物装在密闭于不透明的容器中，能避免与空气、水分、和光线的接触，因此减少了污染与变质的可能，不易被污染；（4）不经过胃肠道系统，可以完全避免胃肠道的破坏作用和肝脏的首过效应，提高药物的生物利用度；（5）减少局部涂药的疼痛（如烧伤和敏感皮肤病患者）与感染；（6）由于气雾剂中的药物是以雾状喷出的，所以可减少对创面的刺激性；（7）喷出的雾粒微小，能直达作用部位或吸收部位，且分布均匀，给药剂量较小，故副作用也小；（8）定量阀门控制剂量比较准确，并可以单剂量或多剂量给药。
综上所述，本发明的有益技术效果为：
1.本发明的重组人超氧化物歧化酶将人Cu/Zn-SOD基因中非活性中心的Cys111密码子突变为Thr密码子，并且在N-端添加6×His，在C-端添加了自主设计的穿膜肽序列（RRQRRQRRQRRQRRR，简称15RQ），可以引导SOD穿膜进入细胞内部，具有兼具抗氧化性能和细胞穿膜性能的优点；
2.本发明的方法采用重组工程菌表达生产人源SOD，并将人工设计的穿膜肽15RQ（RRQRRQRRQRRQRRR）与SOD相连，所得产品分子量均一、结构稳定，采用毕赤酵母体外高效分泌表达，rhSOD产率高，且解决了人源SOD来源困难和血制品有外源感染的危险性的难题；
3.本发明拓展重组人超氧化物歧化酶在清除自由基、抑制促炎细胞因子IL-6、IL-8 和TNF-α释放的化妆品、保健品、食品和药品等领域的应用。
附图说明
图1是本发明实施例1的重组人超氧化物歧化酶的结构示意图；
图2是本发明实施例2的制备方法的流程图；
图3是本发明实施例3的不同浓度咪唑洗脱重组人超氧化物歧化酶的SDS-PAGE电泳图；其中，M为蛋白质marker，1为50mM咪唑洗脱，2为100mM咪唑洗脱，3为200mM咪唑洗脱，4为300mM咪唑洗脱；
图4是本发明实施例3的（A）重组人超氧化物歧化酶表达及纯化电泳和（B）Western-blotting鉴定图；其中，M为蛋白质marker，1和4为菌体破碎后的上清液；2和5为200mM咪唑洗脱液，3和6为300mM咪唑洗脱液；
图5是本发明实施例3的重组人超氧化物歧化酶穿膜效果图；其中，0.5mg/ml蛋白浓度，作用于PC12细胞，A和B为15RQ-SOD-EGFP作用于PC12细胞（4h，8h)；C和D为不含穿膜肽的SOD-EGFP作用于PC12细胞（4h，8h)；
图6是本发明实施例4的各组大鼠肺组织中IL-6、IL-8、TNF-α含量变化图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与作用更加清楚及易于了解，下面结合附图和具体实施方式对本发明作进一步阐述。
实施例1：参照图1，为本发明公开的一种重组人超氧化物歧化酶，其氨基酸序列特征为SEQ ID NO:1。
实施例2：参照图2，为本发明公开的一种重组人超氧化物歧化酶的制备方法，与实施例1的不同之处在于，包括以下步骤，
S1人工全基因合成所述重组人超氧化物歧化酶的核苷酸序列SEQ ID NO:2，并将其插入到pPICZαA载体的EcoRI和XbaI限制性酶切位点之间，得到重组载体pPICZαA-rhSOD；
具体的，将人Cu/Zn-SOD基因中非活性中心的Cys112密码子突变为Ser密码子（二者均为亲水性中性氨基酸），保留第7位和58位的半胱氨酸，不影响其活性，但突变Cys112后可以防止二硫键的错配，以提高其空间结构的稳定性；根据毕赤酵母密码子偏爱性，对rhSOD基因密码子进行了优化，采用全基因合成的方法获得SODC112S，获得pUC57-rhSODC112S质粒，并且通过重叠延伸PCR的方法在N-端添加6×His， 以方便进行亲和纯化，在C-端添加了自主设计的穿膜肽序列（RRQRRQRRQRRQRRR，简称15RQ），可以引导SOD穿膜进入细胞内部；为此设计了3条引物：
Primer 1（TGAATTCAAAAGACACCATCATCATCATCACATGGCGACCAAAGCAGTTT）
Primer 2（CTGTCTGCGCTGTCTTCTTTGCCGTCGTTGGGCGATTCCTATAAC）
Primer 3 （GTTCTAGATCAACGCCTCCGCTGGCGACGCTGTCTGCGCTGTC）
对rhSOD基因密码子进行了优化，由生工生物工程(上海)公司采用全基因合成的方法获得含有SODC112S基因的重组质粒体pUC57-rhSODC112S；以pUC57-rhSODC112S质粒为模板，采用Primer 1 与Primer 2对其进行PCR扩增，获得PCR产物6×His-rhSODC112S；在以PCR产物6×His-rhSODC112S为模板，采用Primer 1 与Primer 3对其进行PCR扩增，即可获得6×His-rhSODC112S-15RQ基因片段；
采用EcoRI与XbaI对6×His-rhSODC112S-15RQ基因片段与空载体pPICZαA分别进行双酶切，酶切产物进行胶回收，用T4连接酶16oC 连接过夜，获得重组质粒pPICZαA-rhSODC112S；
采用ECORI与XbaI对融合片段与空载体pPICZαA分别进行双酶切，酶切产物进行胶回收，用T4连接酶16oC 连接过夜，获得重组质粒pPICZαA-rhSOD；
S2将所述S1得到的重组载体线性化后导入毕赤酵母中，筛选高抗性转化子；
重组质粒经过Sac I单酶切后，然后通过电转化方法（1500 V，4 mS）转入毕赤酵母X-33宿主菌中，再利用含0.1mg/ml Zeocin的YPDS平板进行置于30℃培养中， 待长出单克隆之后，再利用梯度浓度的Zeocin（0.2、0.4、0.6、0.8、1.0 、1.2 mg/ml）平板进行筛选，保留高抗性克隆，并通过菌落PCR进行鉴定；
S3培养所述S2得到的转化子，并诱导表达rhSOD，表达完成后经后处理，得到所述重组人超氧化物歧化酶；
种子液培养、300L中试罐扩大培养的具体步骤：挑取单克隆，接种到4个装有250mlBMGY的1L三角烧瓶中，作为一级种子液，培养17-20 h；按1:10的比例二级接种于30 L发酵罐中，实际装量为10 L，培养4~8 h左右，将所有10 L二级种子液按1:10接种上罐（发酵液起始体积为100L~120 L），发酵过程中培养阶段的pH设为5.0、温度30℃、DO>30%，待溶氧上升后以10~15转度流加50%甘油，待溶氧再次上升后用100%甲醇以转度1开始诱导，诱导开始pH设为6.0，其他参数不变，在诱导过程中逐渐提速，并分别在诱导0小时和24小时加入占总体积1%（M:V）的Polypeptone作为保护剂，诱导后每隔2 h留样，诱导72 h结束，样品检测SDS-PAGE（并扫描）、蛋白含量；
通过离心（4℃，8000rpm，30min），收集发酵液上清，然后通过Ni-NTA亲和层析（0.5M NaCl，20mM Tris-HCl，pH8.0/0.4M 咪唑，0.5M NaCl，20mM Tris-HCl，pH8.0），5K超滤（20mM Tris-HCl，pH8.0），阳离子交换层析（SP-Sepharose FF，20mM Tris-HCl，pH8.0/0.15M NaCl，20mM Tris-HCl，pH8.0），3K超滤脱盐（10mM PBS，pH7.4），浓缩，最终样品的缓冲液为磷酸氢二钠/磷酸二氢钠缓冲液（10mM PB，pH7.4）。
在S3中，将所构建的转化子在不同温度（20、25、30、37℃）下进行诱导表达，并将转化子破碎后的沉淀和上清液分别进行蛋白质电泳，表达产物基本在上清液中，分子量为19kD，与理论分子量一致，并且随着诱导温度的增加，所表达的蛋白量也相应增加。
由于在重组人超氧化物歧化酶N端设计了6个His标签，因此可以利用Ni-NTA进行亲和层析，然后再进行超滤脱盐，调溶液pH值为8.0，由于重组蛋白的PI为9.2，因此重组人超氧化物歧化酶带正电，可以利用SP-SepharoseFF填料进行阳离子交换层析，再经过超滤浓缩换盐，就可以得到较纯的重组人超氧化物歧化酶，结果如图3所示。
参照图4，重组人超氧化物歧化酶经过表达、纯化，然后进行转膜、一抗、二抗杂交、显色，结果发现在19kD处有一条带十分明显的杂交条带，与SDS-PAGE条带位置一致。
为了验证重组人超氧化物歧化酶的穿膜效果，以PC12细胞为模型，加入DMEM稀释的重组15RQ-SOD-EGFP融合蛋白（不含穿膜肽的SOD-EGF作为对照），至终浓度分别为0.5mg/ml，37℃、5%CO2培养，分别于4h、8h在荧光显微镜下各观察一次。然后用PBS洗涤3次，4%多聚甲醛固定15min，用含0.5%TritonX的PBS(v/v)通透5min，0.1μg/mlDAPI染色1min，PBS洗涤，于荧光显微镜下观察重组蛋白进入细胞内部情况。观察发现，4h后就有一部分细胞发出绿色荧光，8h后细胞内部绿色荧光显著增加。而不含穿膜肽的SOD-EGFP却无绿色荧光，如图5所示，说明15RQ-SOD穿膜效果良好。
实施例3：为本发明公开的种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，与实施例2的不同之处在于，所述抗氧化制剂为用于清除自由基、并抑制促炎细胞因子IL-6、IL-8 和TNF-α释放的制剂。
其中，自由基包括DPPH自由基（1，1-二苯基-2-三硝基苯肼）、ABTS自由基（2，2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸）、氧自由基和羟自由基。
抗氧化制剂为用于预防或治疗与IL-6、IL-8 和TNF-α相关的疾病或病症的制剂。疾病或病症包括癌症、与高凝性相关的疾病或病状、与升高的血清CRP相关的疾病或病状、与低白蛋白血症相关的疾病或病状、炎症性病症、病毒性病症、消耗综合征、自身免疫病症或其任意组合。
抗氧化制剂为化妆品、保健品、食品和药品中的一种。
抗氧化制剂的剂型为散剂、 片剂、颗粒剂、胶囊剂、微囊剂、丸剂、滴丸、软胶囊、口服液、糖浆剂、分散片、喷雾剂 、气雾剂、栓剂、静脉注射剂、肌肉注射剂、软膏剂、吸入剂和凝胶剂中的一种。
实施例4：为本发明公开的种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，与实施例3的不同之处在于，抗氧化制剂为用于预防或治疗慢性阻塞性肺病的制剂。
其中，抗氧化制剂的剂型为吸入剂，其由包含以下重量份的原料经灌装压入抛射剂二氧化碳制成，重组人超氧化物歧化酶1.5份，蔗糖5份，聚乙二醇1份，对羟基苯甲酸乙酯0.02份，丙二醇5份，去离子水加至100份。
上述重组人超氧化物歧化酶吸入剂的制备步骤包括，
1）设备与原料前处理；
2）原辅料质检与品控，抛射剂、无菌注射用水的制备；容器、阀门系统的处理与装配；
3）主成分的配制；
4）称量与配制；
5）罐装；
6）抛射剂的罐装；
7）器盖的接轧；
8）漏气检查；重量和压力的检查；产品包装。
利用人超氧化物歧化酶吸入剂抗氧化特性，通过解除或减少氧化应激作用治疗或改善慢性阻塞性肺病，进行抗炎性能检测 ，检测数据如图6所示。
正常组：选择8周大的小鼠，正常饲养。
COPD小鼠模型：选择8周大的小鼠，每天在烟雾中暴露3个小时，每周7天，共进行2周，建立COPD小鼠模型。
实验组：除对照组给予雾化生理盐水之外，其余8组给予本发明rhSOD吸入剂。吸入治疗3天后检测每组小鼠的肺部炎症因子含量。
结果：与正常组比，模型组肺组织匀浆中促炎细胞因子IL-6、IL-8、TNF-α 极显著升高（P﹤0.01）；与模型组比，rhSOD治疗组肺组织匀浆中 IL-8、TNF-α 极显著降低（P﹤0.01）。
实施例5：为本发明公开的种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，与实施例3的不同之处在于，抗氧化制剂为用于预防或治疗人白血病细胞U937引起的白血病的制剂。
其中，抗氧化制剂的剂型为喷雾剂，其由包含以下重量份的原料制成，重组人超氧化物歧化酶1.0份，甘油5份，山梨醇1份，维生素C 0.10份，维生素E 0.10份，尼泊金酯0.2份，薄荷醇0.1份，香精0.1份，10mM氯化铜的PBS缓冲液至100份。
上述重组人超氧化物歧化酶喷雾剂的制备步骤包括，
1）原辅料前处理：无菌注射用水的制备，原辅料质检与品控；
2）容器的前处理：将乳化锅清洗干净，并消毒灭菌后烘干备用；喷雾瓶的灭菌消毒处理；
3）称量与配制（浓配和稀配）；
4）过滤：采用800目滤布过滤出料。进行pH以及含量检测；
5）灌封：澄明度，装样量检测；检漏；灯检；外包装。
通过重组人超氧化物歧化酶喷雾剂抑制白血病细胞增殖U937，检测数据如表1所示。
人白血病U937细胞于含5%CO2的37℃培养箱中培养传代，用含10%小牛血清及100U/ml青霉素、100U/ml链霉素的RMPI-1640培养液培养；用0.25%胰酶-0.02%EDTA消化传代。将对数生长期的人白血病细胞U937用胰酶消化后直接稀释为相应浓度，将细胞接种于96孔板，每孔加100μ1。次日加入3000U/ml浓度rhSOD及相应的新鲜培养基，每孔加100μ1，并设一个溶剂对照组，每组至少设三个平行孔。于37℃继续培养48h后，每孔加20μ15mg/ml的MTT溶液。离心除掉孔内的培养液，然后加入 100μL DMSO，室温下在震荡器上震荡 15min，使胞内的甲瓒结晶被 DMSO 溶解，并转移到胞外，将无色的 DMSO 着色为蓝紫色。在分光光度仪上测定各孔在 490nm波长处的光密度值，各孔细胞的活性可以用光密度值（OD）代表。细胞的增殖率 % = 实验组吸光值/对照组吸光值的比值*100%；抑制率（%）=（对照组平均OD值-给药组平均OD值）/对照组平均OD值*100%。用酶标仪在参考波长630nm，检测波长570nm条件下测定光密度值（OD），以溶剂对照处理的肿瘤细胞为对照组。
表1
| 48 h、U937增殖率（%） | 96 h、U937增殖率（%） | |
| 对照组 | 100±1.9 | 100±2.1 |
| rhSOD实验组 | 42.1±1.7 | 25±1.5 |
| 抑制率 % | 57.9% | 75 % |
结果：对照组U937白血病细胞的增殖率高于rhSOD处理组；随着作用时间的延长，rhSOD 对U937 细胞增殖抑制率呈上升趋势。rhSOD可以显著的抑制白血病细胞U937的增殖。
实施例6：为本发明公开的种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，与实施例3的不同之处在于，抗氧化制剂为用于治疗烫烧伤和/或过敏引起的皮肤炎症的制剂。rhSOD涂抹患处愈合是常规疗法的3倍，温和有效，而且疤痕明显减轻。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 杭州浦泰生物科技有限公司
<120> 一种重组人超氧化物歧化酶及其制备方法和应用
<130> 2022
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 175
<212> PRT
<213> Artificial Sequence
<400> 1
His His His His His His Met Ala Thr Lys Ala Val Cys Val Leu Lys
1 5 10 15
Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys Glu Ser
20 25 30
Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr Glu Gly
35 40 45
Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala Gly Cys
50 55 60
Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His Gly Gly
65 70 75 80
Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val Thr Ala
85 90 95
Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val Ile Ser
100 105 110
Leu Ser Gly Asp His Ser Ile Ile Gly Arg Thr Leu Val Val His Glu
115 120 125
Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr Lys Thr
130 135 140
Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile Ala Gln
145 150 155 160
Arg Arg Gln Arg Arg Gln Arg Arg Gln Arg Arg Gln Arg Arg Arg
165 170 175
<210> 2
<211> 528
<212> DNA
<213> Artificial Sequence
<400> 2
caccatcatc atcatcacat ggcgaccaaa gcagtttgtg tacttaaggg cgacggccca 60
gtacaaggaa ttataaattt tgagcagaaa gaatccaacg gcccggtcaa ggtgtggggg 120
tcaatcaaag ggctgactga gggactacac ggatttcacg tacatgagtt cggtgataac 180
actgcgggct gcacctcggc aggtccacac ttcaacccct tgagcaggaa gcatggtggt 240
cctaaggacg aagagcgaca cgtcggggat ttaggtaatg tcacagccga caaggacggt 300
gtcgctgacg tgtcaattga agactccgtt atttcgttgt ctggcgatca tagcatcata 360
ggccgtacat tagtggttca cgagaaagca gatgatctgg ggaaaggggg aaatgaagaa 420
tctacgaaaa cgggaaatgc tggcagtcgg ctcgcgtgtg gggttatagg aatcgcccaa 480
cgacggcaaa gaagacagcg cagacagcgt cgccagcgga ggcgttga 528
Claims (10)
1.一种重组人超氧化物歧化酶，其特征在于：所述重组人超氧化物歧化酶的氨基酸序列特征为SEQ ID NO:1。
2.根据权利要求1所述的一种重组人超氧化物歧化酶的制备方法，其特征在于：包括以下步骤，
S1人工全基因合成所述重组人超氧化物歧化酶的核苷酸序列SEQ ID NO:2，并将其插入到pPICZαA载体的EcoRI和XbaI限制性酶切位点之间，得到重组载体pPICZαA-rhSOD；
S2将所述S1得到的重组载体线性化后导入毕赤酵母中，筛选高抗性转化子；
S3培养所述S2得到的转化子，并诱导表达rhSOD，表达完成后经后处理，得到所述重组人超氧化物歧化酶。
3.根据权利要求1所述的一种重组人超氧化物歧化酶在制备抗氧化制剂中的应用，其特征在于：所述抗氧化制剂为用于清除自由基、并抑制促炎细胞因子IL-6、IL-8 和TNF-α释放的制剂。
4.根据权利要求3所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述自由基包括DPPH自由基、ABTS自由基、氧自由基和羟自由基。
5.根据权利要求4所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂为用于预防或治疗与IL-6、IL-8 和TNF-α相关的疾病或病症的制剂，所述疾病或病症包括癌症、与高凝性相关的疾病或病状、与升高的血清CRP相关的疾病或病状、与低白蛋白血症相关的疾病或病状、炎症性病症、病毒性病症、消耗综合征、自身免疫病症或其任意组合。
6.根据权利要求5所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂为用于预防或治疗慢性阻塞性肺病、人白血病细胞U937引起的白血病、烫烧伤和/或过敏引起的皮肤炎症、或其任意组合的制剂。
7.根据权利要求3所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂为化妆品、保健品、食品和药品中的一种。
8.根据权利要求3所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂的剂型为散剂、 片剂、颗粒剂、胶囊剂、微囊剂、丸剂、滴丸、软胶囊、口服液、糖浆剂、分散片、喷雾剂 、气雾剂、栓剂、静脉注射剂、肌肉注射剂、软膏剂、吸入剂和凝胶剂中的一种。
9.根据权利要求7所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂的剂型为吸入剂，所述抗氧化制剂由包含以下重量份的原料经灌装压入抛射剂二氧化碳制成，重组人超氧化物歧化酶1.5份，蔗糖5份，聚乙二醇1份，对羟基苯甲酸乙酯0.02份，丙二醇5份，去离子水加至100份。
10.根据权利要求7所述的一种重组人超氧化物歧化酶的应用，其特征在于：所述抗氧化制剂的剂型为喷雾剂，所述抗氧化制剂由包含以下重量份的原料制成，重组人超氧化物歧化酶1.0份，甘油5份，山梨醇1份，维生素C 0.10份，维生素E 0.10份，尼泊金酯0.2份，薄荷醇0.1份，香精0.1份，10mM氯化铜的PBS缓冲液至100份。
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