CN100390280C - 表达重组全长人肝再生增强因子的基因及重组全长人肝再生增强因子的制备方法和用途 - Google Patents
表达重组全长人肝再生增强因子的基因及重组全长人肝再生增强因子的制备方法和用途 Download PDFInfo
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- CN100390280C CN100390280C CNB200510087027XA CN200510087027A CN100390280C CN 100390280 C CN100390280 C CN 100390280C CN B200510087027X A CNB200510087027X A CN B200510087027XA CN 200510087027 A CN200510087027 A CN 200510087027A CN 100390280 C CN100390280 C CN 100390280C
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Abstract
本发明属于生物工程产品的生产方法和应用领域，根据全长人肝再生增强因子(ALR，Augmenter of Liver Regeneration)的氨基酸序列，人工合成表达重组全长人肝再生增强因子的基因序列，将其插入到质粒pPIC9K中，转化入毕赤酵母GS115中，建立了重组全长人肝再生增强因子酵母表达及蛋白纯化的生产工艺，最终获得重组全长人肝再生增强因子纯品。该纯品能够在体外促进肝癌细胞系HepG2的DNA合成，在体内促进肝部分切除术后大鼠肝脏的再生，促进CCl4中毒后大鼠肝细胞DNA合成与修复，提高CCl4中毒后大鼠存活率，可能用于慢性肝炎、重型肝炎及其肝硬化的治疗。
Description
技术领域
本发明属于生物工程产品的生产方法和应用领域，具体涉及一种通过毕赤酵母表达重组全长人肝再生增强因子的DNA核苷酸序列及其表达载体，以及利用上述DNA表达载体制备所述重组全长人肝再生增强因子的方法和重组全长人肝再生增强因子的药学用途。
背景技术
肝脏是人体内再生能力最为强大的器官之一，肝脏的再生在多种肝脏疾病如急性/慢性病毒性肝炎、重型肝炎的发病与治疗中具有重要的意义，因此多年来人们对于肝脏再生的机理、调节机制进行了持续的研究，目前已经发现的具有调节作用的因子多达数十种，如肝细胞生长因子(HGF)、表皮生长因子(EGF)、转化生长因子-α(TGF-α)等。由于多数因子的作用缺乏肝特异性，因此不可能是肝脏再生的关键调节因子。
1975年，Labrecque等人首次报道在刚刚断乳的大鼠肝和肝部分切除大鼠的再生肝中存在一种热稳定的特异性刺激肝细胞增殖的物质，称为肝刺激物质(HSS)。之后的研究发现，在狗、小鼠等其它动物中也存在相同性质的物质。
1994年，Hagiya等人从成年大鼠部分肝切除后的再生肝中提取肝刺激物质，经过复杂的步骤后获得了一个全长1.2kb的cDNA，包含375bp的开发读框，编码125个氨基酸的小分子蛋白，将其命名为肝再生增强因子(ALR，Augment er of LiverRegeneration)。
在大鼠ALR分子克隆的基础上，1999年我们利用同源引物PCR技术获得了人ALR的基因组序列(Genbank号为AF 146394)，根据预测的375bp的开放读框，可以编码125个氨基酸的小分子蛋白。之后Lisowsky发现此基因有3种不同的剪切形式，125个氨基酸为不成熟形式，而204个氨基酸的形式为成熟形式，在组织中所占的比率最高，并且具有最强的生物学活性。全长hALR的分子量为23kDa，主要分布于肝脏、肾脏。hALR的主要生物学活性为促进肝再生。在肝部分切除大鼠模型中进行的实验表明，肝再生增强因子能促进肝细胞DNA合成，使3H-TdR掺入量提高2.5倍。对肝部分切除后肝再生增强因子mRNA的动态研究发现，术后12h mRNA即显著增加，并于术后24h达到峰值。在门腔静脉吻合模型中，重组肝再生增强因子能够使肝体积保持正常，防止肝细胞萎缩，而对照组肝组织出现肝萎缩，肝细胞粗面内质网和核糖体减少，线粒体变大且嵴破裂。肝再生增强因子对于肝细胞损伤具有保护作用。在CCl4模拟体内重肝损伤模型中，肝再生增强因子能够明显抑制CCl4诱导损伤肝细胞的LDH释放，在体内实验中重组肝再生增强因子能够使治疗组的存活率提高20％，血清LDH水平降低34％，ALT水平降低38.3％。此外，肝再生增强因子还能够提高糖尿病大鼠进行胎鼠胰腺移植的成功率，并且在精子生成中发挥作用。
发明内容
本发明的第一个目的在于公开一种通过毕赤酵母表达重组全长人肝再生增强因子的DNA序列。
本发明的第二个目的在于公开包含上述DNA序列的表达载体。
本发明的第三个目的在于公开制备重组全长人肝再生增强因子的方法。
本发明的第四个目的在于公开重组全长人肝再生增强因子的医学用途。
本发明的目的是通过以下方法实现的：
1、从Genebank里选取Genebank号为CAB 87993的全长人肝再生增强因子氨基酸序列如SEQ ID NO.1，根据密码子图表分析其对应的DNA核苷酸序列，并根据毕赤酵母偏爱密码子原则重新设计适合毕赤酵母表达的全长人肝再生增强因子的DNA序列，将所得序列表交由“上海博亚生物技术公司”进行DNA序列合成，将合成后的DNA序列交由“上海申友生物技术有限责任公司”进行DNA序列测序、鉴定，得到如SEQ ID NO.2所述的DNA核苷酸序列；
2、将SEQ ID NO.2所述的核苷酸序列经EcoR I酶酶切，连接入质粒pPIC9K，得到表达载体pPIC9K-whALR；
3、将表达载体pPIC9K-whALR转化入毕赤酵母GS115中，得到转化体，将转化体涂布于RDB平板进行含表达载体pPIC9K-whALR的毕赤酵母GS115的挑选；
4、取转化后的菌落进行培养发酵，收集上清液；
5、对收集的上清液进行透析，置换缓冲液；
6、对步骤5中的透析产物，用阴离子交换柱Sepha rose DEAEF.F进行层析；
7、对步骤6中的层析产物，用凝胶过滤柱Superdex 75进行层析纯化；
经以上步骤获得的重组全长人肝再生增强因子，可促进肝部分切除术后大鼠肝脏的再生，在临床上，重组全长再生增强因子对肝损伤、急性肝功能衰竭、重症肝炎和肝硬化患者的肝细胞损伤具有保护作用，并促进肝细胞的再生和修复。
附图说明
图1为制备重组全长人肝再生因子的工艺流程图。
图2为重组全长人肝再生因子DNA表达产物的聚丙烯酰胺电泳图。
图3为重组全长人肝再生因子纯化产物的聚丙烯酰胺电泳图。
具体实施方式
实施例1重组全长人肝再生增强因子毕赤酵母表达的制备工艺
1、DNA的制备从Genebank里选取Genebank号为CAB87993的全长人肝再生增强因子氨基酸序列如SEQ ID NO.1，其等电点为7.2，分子量为23kDa，根据密码子图表分析其对应的DNA核苷酸序列，并根据毕赤酵母偏爱密码子原则重新设计适合毕赤酵母表达的全长人肝再生增强因子的DNA序列，将所得序列表交由“上海博亚生物技术公司”进行DNA序列合成，将合成后的DNA序列交由“上海申友生物技术有限责任公司”进行DNA序列测序、鉴定，得到如SEQ ID NO.2所述的DNA核苷酸序列；
2、将步骤1所得的DNA核苷酸序列以EcoRI酶切位点连接入pPIC9K载体，得到表达载体pPIC9K-whALR，经酶切和测序鉴定(由上海申友生物技术有限责任公司进行测序)正确。
3、电转化毕赤酵母 挑取酵母单菌落，接种至含有5ml YPD培养基的50ml三角瓶中，30℃300g培养过夜。取500μl的培养物接种至含有500ml新鲜培养基的2L三角摇瓶中，30℃300g培养过夜，将细胞培养物于4℃1500g离心5分钟，用500ml的冰预冷的无菌水将菌体沉淀重悬；4℃1500g离心5分钟，用250ml的冰预冷的无菌水将菌体沉淀重悬；4℃1500g离心5分钟，用1ml的冰预冷的1M的山梨醇溶液将菌体沉淀重悬，其终体积约为1.5ml。将10μl Sal I线性化的pPIC9K-whALR加入80μl毕赤酵母GS115感受态细胞，混匀，加入电击杯底部，电压1500V，电阻400Ω，电容25uf电击。加入1ml预冷的山梨醇，混匀后涂布于RDB平板，30℃培养至菌落出现。
4、重组全长人肝再生增强因子的表达从RDB平板中挑取10个单菌落，接种于5ml MD培养基中，30℃250rpm振荡培养24h，后取500μl菌液接种于1L BMGY培养基中，30℃250rpm振荡培养18h，作为种子液。配10L含葡萄糖的基本盐培养基加入发酵罐，发酵罐原位高压，加入43ml PTM1微量盐。将1L种子液接种于发酵罐内，进行发酵。发酵参数为通气量7L/min，溶氧量30％，搅拌速度250rpm，温度为30℃，pH值维持于5.0，补充25％葡萄糖30ml，4h后测OD600＝1，进行诱导，以80ml/h的速度加入甲醇，搅拌速度300rpm，溶氧维持于20％-30％，35h后停止发酵。4℃下10000rpm离心30min，收集上清，用100g/L三氯醋酸(TCA)沉淀，进行12％聚丙烯酰胺凝胶电泳鉴定(图3)，表达产物条带符合预期蛋白分子量，ALR已成功表达。
5、透析将收集的上清装入透析袋中，以20mM Tris-HCl(pH 7.6)100L进行透析，4℃透析48h。
6、阴离子交换柱Sepharose DEAE F.F层析选用XK 26进行装柱，柱平衡：20mM Tris-HCl(pH 7.6)5CV，流速为25ml/min；上样的表达上清为2L，流速为25ml/min；洗去未结合蛋白为3CV，流速为10ml/min；样品洗脱为0-100％B液(1MNaCl)5CV，流速为10ml/min。收集目的蛋白峰。
7、凝胶过滤柱Superdex 75层析选用XK 16进行装柱。柱平衡：50mM PBS(pH 7.5)2CV，流速为1.5ml/min；以DEAE层析后的收集液上样，每次15ml，流速为1.5ml/min；样品洗脱：50mM PBS，流速为2ml/min。收集最终纯化的表达产物，进行12％聚丙烯酰胺凝胶电泳鉴定(图4)，ALR纯化成功。
实施例2重组全长人肝再生增强因子体外促进肝癌细胞系HepG2的DNA合成
1、HepG2细胞培养：HepG2细胞生长于高糖DMEM+10％胎牛血清中，37℃5％CO2培养。待细胞生长至对数期，胰酶消化，调整细胞密度至4×105/ml，在96孔培养板中每孔接种100μl，继续培养6h。
2、加样：在96孔培养板中加入待测样品，共3个浓度(20ng/ml，50ng/ml，100ng/ml)，每个浓度设置3个复孔，继续培养24h。加入2.0×104Bq 3H-TdR，继续培养3h。
3、检测：利用细胞收集器将细胞收集到滤纸片上，上液闪计数器计数(参见表1)。
表1体外促进肝癌细胞系HepG2DNA合成
4、结果：通过实验证明，毕赤酵母表达的重组全长人肝再生增强因子能够在体外促进肝细胞系HepG2的DNA合成。
实施例3重组全长人肝再生增强因子体内促进肝部分切除术后大鼠肝脏细胞DNA合成
1、大鼠肝部分切除术模型的建立：大鼠单笼饲养1周，实验前禁食过夜，术前腹腔注射戊巴比妥钠(40mg/kg)麻醉，上腹部脱毛后以750mL/L酒精消毒皮肤，以上腹部正中切口，显露肝脏，按Hoggin法充分游离左叶及中叶肝脏的韧带后，于各肝叶的根部结扎牢固，迅速于结扎线的远端切除左肝叶及肝中叶，即相当于2/3肝切除。保留余肝叶，缝合腹部切口。
2、加药：术后腹腔注射重组全长人肝再生增强因子1ml(0.1mg/ml)，同时设生理盐水对照组。20h后按照100uCi/kg腹腔注射3H-TdR，2h后处死大鼠。
3、检测：开腹，取肝组织，用Promega试剂盒提取组织DNA，进行3H-TdR计数(参见表2)。
表2体内促进肝部分切除术后大鼠肝脏细胞DNA合成
4、结果：通过实验证明，毕赤酵母表达的重组全长人肝再生增强因子能够在体内促进肝部分切除术后大鼠肝脏细胞DNA合成。
实施例4重组全长人肝再生增强因子体外促进CCl4损伤肝细胞的修复
1、体外CCl4损伤肝细胞模型的建立：按照原位灌注法分离BalB/C小鼠肝细胞，将分离的细胞用含10％小牛血清，10-9mol/L胰岛素的RPMI1640培养液悬浮，按每孔3×104细胞/100ul的数量接种于96孔板，37℃5％CO2培养10h。换用含5mM CCl4，10％小牛血清及不同浓度重组全长人肝再生增强因子的RPMI1640培养液。
2、乳酸脱氢酶(LDH)释放实验：于换液后不同时间收集细胞培养上清，利用Cytox 96非放射性细胞毒检测试剂盒测定细胞LDH释放率(参见表3)。
表3细胞LDH释放率数据
3、细胞存活率的测定：使用MTT法评价肝细胞的存活率，用酶联仪检测OD490值(参见表4)。
表4MTT法细胞存活率的测定
4、结果：通过实验证明，毕赤酵母表达的重组全长人肝再生增强因子能够明显抑制CCl4损伤肝细胞LDH释放，并显著增加CCl4损伤后肝细胞的存活率。
实施例5重组全长人肝再生增强因子体内促进CCl4损伤肝细胞的修复
1、体内CCl4肝中毒模型的建立：BalB/C小鼠腹腔注射10％CCl4(4ml/kg)，4h后随机分组，治疗组每12h腹腔注射重组全长人肝再生增强因子20μg，对照组注射生理盐水。记录动物死亡时间，存活超过96h即为存活(参见表5)。
表5体内CCl4肝中毒小鼠致死率
2、体内CCl4肝损伤模型的建立：BalB/C小鼠腹腔注射10％CCl4(2ml/kg)，4h后随机分组，治疗组每12h静脉注射重组全长人肝再生增强因子20μg，对照组注射生理盐水。48h后测定DNA增值率，同时取外周血ALT、LDH水平测定(参见表6)。
表6外周血ALT、LDH水平测定数据
3、DNA增殖测定：在最后一次注射重组全长人肝再生增强因子后12h，按照1ml/kg的量腹腔注射5-FU标记液，2h后处死动物，开腹取肝组织，甲醛固定，石蜡包埋。常规石蜡切片，二甲苯脱蜡，酒精脱水后PBS洗涤三次，抗5-FU单抗室温孵育60min，PBS洗涤二次，切片以过氧化物酶标记的鼠抗兔IgG室温孵育30min，洗涤三次后显色10min，脱水、透明、封片，显微镜下随即挑选五个视野，计数阳性细胞(参见表7)。
表7DNA增值测定中阳性细胞数
4、结果：通过实验证明，毕赤酵母表达的重组全长人肝再生增强因子在体内能够显著提高CCl4肝中毒小鼠的存活率，显著降低CCl4肝损伤小鼠的外周血ALT、LDH水平。光镜下可见重组全长人肝再生增强因子能够显著减轻肝细胞变性坏死的比率，并能够促进CCl4诱导肝损伤模型小鼠肝细胞DNA合成。
序列表
<110>北京地坛医院
<120>表达重组全长人肝再生增强因子的基因及重组全长人肝再生增强因子的制备方法和用途
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Asp Ala Gly Pro Gly Arg Gly Ala Glu Arg Arg Gly Arg Leu Gly Leu
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Asp Ala Ser Pro Gly Ala Asp Leu Arg Phe Ser Cys Arg Arg Gly Arg
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Leu Pro Glu Ala Ala Cys Arg Ala Cys Val Asp Phe Lys Thr Trp Met
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Arg Thr Gln Gln Lys Arg Asp Thr Lys Phe Arg Glu Asp Cys Pro Pro
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Asp Arg Glu Glu Leu Gly Arg His Ser Trp Ala Val Leu His Thr Leu
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Ala Ala Tyr Tyr Pro Asp Leu Pro Thr Pro Glu Gln Gln Gln Asp Met
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Ala Gln Phe Ile His Leu Phe Ser Lys Phe Tyr Pro Cys Glu Glu Cys
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Ala Glu Asp Leu Arg Lys Arg Leu Cys Arg Asn His Pro Asp Thr Arg
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Thr Arg Ala Cys Phe Thr Gln Trp Leu Cys His Leu His Asn Glu Val
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ggcgcgcgct ccgagatgat ggacgacctg gcgaccgacg cggggccggg gcgcggggcg 120
gagaggcgcg gccgcctcgg cctcgacgcc agcccaggcg ccgacctccg attctcctgt 180
cgccgaggac gcctcccgga ggcggcgtgc cgggcctgcg ttgacttcaa gacttggatg 240
cggactcagc agaagcggga caccaagttt agggaggact gcccgccgga tcgcgaggaa 300
ctgggccgcc acagctgggc tgttcttcac accctggccg cctactaccc cgacctgccc 360
accccagaac agcagcagga catggcccag ttcatacatt tattttctaa gttttacccc 420
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acccgggcat gcttcacgca gtggctgtgc cacctgcaca atgaagtgaa ccgcaagctg 540
ggcaagcctg acttcgactg ctcaaaggtg gatgagcgct ggcgcgacgg ctggaaggat 600
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Claims (6)
1.一种表达重组全长人肝再生增强因子的DNA，其核苷酸序列如SEQ ID NO.2所述。
2.包含权利要求1所述DNA的表达载体。
3.包含权利要求1所述DNA的表达载体pPIC9K-whALR，所述表达载体pPIC9K-whALR是通过将SEQ ID N0.2所述的核苷酸序列经EcoR I酶酶切，连接入质粒pPIC9K而制得。
4.制备重组全长人肝再生增强因子的方法，包括以下步骤：
1)将权利要求2所述的表达载体转化入宿主细胞，培养转化体，发酵表达，其中宿主细胞是毕赤酵母细胞；
2)对发酵表达的产物进行离心，收集上清液；
3)对收集的上清液进行透析，置换缓冲液；
4)对步骤3的产物进行阴离子交换柱层析纯化；
5)对步骤4的产物进行凝胶过滤柱层析纯化；
6)对步骤5所得的纯化产物分装后冻干。
5.权利要求4所述的方法，其中步骤1中所述的表达载体是pPIC9K-whALR，所述表达载体pPIC9K-whALR是通过将SEQIDNO.2所述的核苷酸序列经EcoR I酶酶切，连接入质粒pPIC9K而制得。
6.权利要求4或5所述的方法，其中宿主细胞是毕赤酵母GS115细胞。
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| CN101234191B (zh) * | 2007-01-29 | 2011-04-27 | 首都医科大学 | 人重组肝刺激因子增强肝癌细胞抗凋亡的作用及其应用 |
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| CN1418964A (zh) * | 2002-08-16 | 2003-05-21 | 中国人民解放军传染病研究所 | 重组全长人肝再生增强因子的生产方法及其治疗严重肝病的用途 |
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| CN1418964A (zh) * | 2002-08-16 | 2003-05-21 | 中国人民解放军传染病研究所 | 重组全长人肝再生增强因子的生产方法及其治疗严重肝病的用途 |
Non-Patent Citations (2)
| Title |
|---|
| 人肝再生增强因子表达载体的构建及其在酵母中的表达. 王琳等.中华肝脏病杂志，第10卷第5期. 2002 |
| 人肝再生增强因子表达载体的构建及其在酵母中的表达. 王琳等.中华肝脏病杂志，第10卷第5期. 2002 * |
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