CN100351377C - 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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- CN100351377C CN100351377C CNB2005100870265A CN200510087026A CN100351377C CN 100351377 C CN100351377 C CN 100351377C CN B2005100870265 A CNB2005100870265 A CN B2005100870265A CN 200510087026 A CN200510087026 A CN 200510087026A CN 100351377 C CN100351377 C CN 100351377C
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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,将其插入到原核表达载体pBV220中,转化入大肠杆菌DH5α中,建立了重组全长人肝再生增强因子原核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,具体涉及一种通过大肠杆菌表达重组全长人肝再生增强因子的DNA,以及利用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)。
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CAB87993),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大肠杆菌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,其等电点为7.2、分子量23kDa,根据密码子图表分析其对应的DNA核苷酸序列,并根据大肠杆菌偏爱密码子原则,重新设计全长人肝再生增强因子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,得到如SEQ ID NO.2所述的核苷酸序列,以下步骤既是制备重组全长人肝再生增强因子的过程也是对SEQ ID NO.2所述的核苷酸序列的鉴定过程;
2、将步骤1所得的核苷酸序列经EcoR I和BamH I酶酶切,连接入pBV220,得到表达载体pBV220-whALR;
3、将表达载体pBV220-whALR转化入大肠杆菌DH5α中,通过含有氨苄青霉素的选择平皿培养基上进行含表达载体pBV220-whALR的大肠杆菌DH5α的挑选;
4、将步骤3中挑选的细菌单菌落接种于培养基上进行发酵表达,获得含包涵体的菌体;
5、对步骤4中获得的菌体进行破碎、离心、弃上清液,得包涵体;
6、对步骤5中的包涵体进行变性和复性后,用阴离子交换柱Sepharose DEAE F.F进行层析,收集获得目的蛋白;
7、将步骤6中获得的目的蛋白进行透析后,用阳离子交换柱Sepharose CM F.F进行层析,收集目的蛋白;
8、将步骤7中获得的目的蛋白进行凝胶过滤柱Superdex 75层析,收集最终纯化产物,用12%聚丙烯酰氨凝胶电泳进行鉴定。
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增强因子的工艺流程图。
图2为重组全长人肝再生增强因子DNA表达产物的聚丙烯酰胺凝胶电泳图。
图3为重组全长人肝再生增强因子纯化产物的聚丙烯酰胺凝胶电泳图。
具体实施方式
实施例1 重组全长人肝再生增强因子原核表达的制备工艺
1、DNA的制备 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所示,根据密码子图表分析其对应的DNA核苷酸序列,并根据大肠杆菌偏爱密码子原则,重新设计全长人肝再生增强因子DNA序列,将所得序列交由“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所述的核苷酸序列;
2、表达载体pBV220-whALR的构建 将步骤1所得的DNA序列经限制性内切酶EcoRI和限制性内切酶BamHI酶切后以粘性末端克隆入质粒pBV220。由“上海申友生物技术有限责任公司”进行酶切和测序鉴定;
3、转化DH5α大肠杆菌 自LB琼脂平板上挑取大肠杆菌DH5α接种入LB液体培养基中,37℃ 200g摇床振摇8小时。取100μl菌液接种入另一LB液体培养管中,37℃ 200g摇床振摇1.5小时,将菌液分装入微离心管中,冰上15分钟,4℃ 4000g离心10分钟,弃去上清液。加入0.1M冰预冷的氯化钙500μl,将沉淀的菌体吹起,并将2管合为1管,冰上静置15分钟,4℃ 4000g离心10分钟,弃去上清。加入0.1M冰预冷的氯化钙200μl,将沉淀的菌体吹起。加入pBV220-whALR质粒,混匀后置冰水浴中30分钟,转入42℃水浴2分钟,再入冰浴1分钟,加入LB液800μl,37℃振摇60分钟。均匀地铺在氨苄青霉素选择平皿上,待平皿液体吸收后,倒置平皿,于37℃放置16小时后出现菌落;
4、重组全长人肝再生增强因子的表达从细菌平皿中挑取2-3个细菌单菌落,接种于5ml LB/Amp(100μg/ml)培养基中,30℃ 200rpm振荡培养12h;后取200μl菌液接种于50ml LB中,30℃ 200rpm振荡培养12h,后取4ml菌液接种于1000mlLB中,30℃ 200rpm振荡培养12h,作为种子液。配20L培养基加入发酵罐,发酵罐原位高压,加入Amp 2g。将1L种子液接种于发酵罐内,进行发酵。发酵参数为通气量5L/min,溶氧量50%,搅拌速度250rpm,温度为30℃,补充50%葡萄糖42ml,4h后测OD600=0.5,5h后测OD600=1,进行诱导,发酵罐升温至42℃,补充50%葡萄糖40ml,搅拌速度300rpm,3h后测OD600=2,停止诱导,收获菌体。4℃下5000rpm离心30min,收集菌体,1L TE洗涤一次,弃上清,4℃下5000rpm离心30min,共收获菌体共150g,-70℃保存;
5、裂菌 称取菌体30g,加入TE(pH 7.5)160ml磨碎,加入溶菌酶160mg,冰浴30分钟,超声破碎:功率35%、脉冲9sec/9sec、时间20min,10000rpm 4℃离心20分钟,弃上清,加入4M尿素60ml磨碎,10000rpm 4℃离心20分钟,弃上清,加入TE(pH 7.5)160ml磨碎,10000rpm 4℃离心20分钟,弃上清,得到包涵体共7.2g,经12%聚丙烯酰胺凝胶(SDS-PAGE)电泳鉴定,符合预期蛋白分子量,ALR已成功表达;
6、包涵体的变复性 包涵体中加入8M尿素60ml,仔细研磨,加入β-巯基乙醇600μl,转入100ml三角瓶中,37℃ 80rpm振荡2h,室温10000g离心20分钟,取上清滴入2L的复性液(50mM Tris、2M尿素、0.75g GSH、0.153g GSSG)中,摇动,置于4℃复性24h;
7、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2 5ml/min;上样2L,流速为25ml/min;洗去未结合蛋白:3CV,流速为10ml/min;样品洗脱:0-100% B液(1M NaCl)5CV,流速为10ml/min,收集目的蛋白;
8、透析 将收集的目的蛋白装入透析袋中,以20mMNaH2PO4(pH 4.7)10L进行透析,4℃透析24h;
9、阳离子交换柱Sepharose CM F.F层析 将步骤8中的透析产物进行层析,选用XK 16进行装柱。柱平衡:20mM NaH2PO4(pH4.7)5CV,流速为10ml/min;上样110ml,流速为10ml/min;洗去未结合蛋白:2CV,流速为10ml/min;样品洗脱:0-100%B液(1M NaCl)5CV,流速为10ml/min,收集目的蛋白;
10、凝胶过滤柱Superdex 75层析 对目的蛋白进行层析,选用XK 16进行装柱。柱平衡:50mM PBS(pH 7.5)2CV,流速为1.5ml/min;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,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:人肝细胞系HepG2购自上海细胞生物研究所,HepG2细胞生长于高糖9MEM+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100ul,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
组别 | 20ng/ml(cpm/105cells) | 50ng/ml(cpm/105cells) | 100ng/ml(cpm/105cells) |
重组全长人肝再生增强因子生理盐水对照组空白对照组 | 1346±133794±9741±20 | 1473±274 | 1469±325 |
4、结果:实验证明,通过大肠杆菌表达的重组全长人肝再生增强因子能够在体外促进肝癌细胞系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
组别 | X±SD(cpm/mgDNA) |
重组全长人肝再生增强因子生理盐水对照组空白对照组 | 7005±31204893±102136±10 |
4、结果:实验证明,通过大肠杆菌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、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
组别 | 24h(%) | 48h(%) |
重组全长人肝再生增强因子生理盐水对照组 | 45.084.1 | 77.499.7 |
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4 MTT法细胞存活率的测定数据
组别 | 吸光度(10μl) | 吸光度(1μl) | 吸光度(0.1μl) | 吸光度(0.01μl) |
重组全长人肝再生增生因子生理盐水对照组 | 2.1360.312 | 1.2550.441 | 1.3610.163 | 0.680.795 |
4、结果:实验证明,通过大肠杆菌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20ug,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
组别 | 1天 | 2天 | 3天 |
注射重组全长人肝再生增强因子生理盐水对照组正常组 | 40%100%0 | 80%100%0 | 80%100%0 |
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20ug,对照组注射生理盐水。48h后测定DNA增值率,同时取外周血测定ALT、LDH水平(参见表6)。
表6 外周血ALT、LDH水平测定数据
组别 | 血清ALT(IU/L×103) | 血清LDH(IU/L×103) |
重组全长人肝再生增强因子生理盐水对照组 | 2.70±0.333.12±0.45 | 2.55±0.583.27±0.24 |
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7 DNA增值测定中阳性细胞数
组别 | 阳性细胞数/视野 | 刺激指数 |
重组全长人肝再生增强因子生理盐水对照组 | 88.0±26.832.6±14.4 | 2.7 |
4、结果:实验证明,通过大肠杆菌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
<160>2
<210>1
<211>204
<212>PRT
<213>人
<220>
<400>1
Met Ala Ala Pro Gly Glu Arg Gly Arg Phe His Gly Gly Asn Leu Phe
1 5 10 15
Phe Leu Pro Gly Gly Ala Arg Ser Glu Met Met Asp Asp Leu Ala Thr
20 25 30
Asp Ala Gly Pro Gly Arg Gly Ala Glu Arg Arg Gly Arg Leu Gly Leu
35 40 45
Asp Ala Ser Pro Gly Ala Asp Leu Arg Phe Ser Cys Arg Arg Gly Arg
50 55 60
Leu Pro Glu Ala Ala Cys Arg Ala Cys Val Asp Phe Lys Thr Trp Met
65 70 75 80
Arg Thr Gln Gln Lys Arg Asp Thr Lys Phe Arg Glu Asp Cys Pro Pro
85 90 95
Asp Arg Glu Glu Leu Gly Arg His Ser Trp Ala Val Leu His Thr Leu
100 105 110
Ala Ala Tyr Tyr Pro Asp Leu Pro Thr Pro Glu Gln Gln Gln Asp Met
115 120 125
Ala Gln Phe Ile His Leu Phe Ser Lys Phe Tyr Pro Cys Glu Glu Cys
130 135 140
Ala Glu Asp Leu Arg Lys Arg Leu Cys Arg Asn His Pro Asp Thr Arg
145 150 155 160
Thr Arg Ala Cys Phe Thr Gln Trp Leu Cys His Leu His Asn Glu Val
165 170 175
Asn Arg Lys Leu Gly Lys Pro Asp Phe Asp Cys Ser Lys Val Asp Glu
180 185 190
Arg Trp Arg Asp Gly Trp Lys Asp Gly Ser Cys Asp
195 200
<210>2
<211>615
<212>DNA
<213>人工序列
<220>
<223>根据大肠杆菌偏爱密码子原则而设计
<400>2
atggcggcgc ccggcgagcg tggccgcttc cacggcggga acctcttctt cctgccgggg 60
ggcgcgcgct ccgagatgat ggacgacctg gcgaccgacg cggggccggg gcgcggggcg 120
gagagacgcg gccgcctcgg cctcgacgcc agcccaggcg ccgacctccg attctcctgt 180
cgccgaggac gcctcccgga ggcggcgtgc cgggcctgcg ttgacttcaa gacgtggatg 240
cgtactcaac agaaacgtga tactaaattt cgtgaagact gcccgccgga tcgcgaggaa 300
ctgggccgcc acagctgggc tgttctgcac accctggccg cctactaccc ggacctgccg 360
accccagaac agcagcaaga catggcccag ttcatccatc tgttttctaa gttttacccg 420
tgtgaggagt gtgctgaaga cctgcgtaaa cgtctgtgcc gtaaccaccc agacacccgc 480
acccgcgcat gcttcaccca gtggctgtgc cacctgcaca atgaagtgaa ccgcaagctg 540
ggcaagccgg acttcgactg ctctaaagtg gatgagcgct ggcgcgacgg ctggaaggat 600
ggctcctgtg actaa 615
Claims (7)
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pBV220-whALR。
4、制备权利要求1所述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寄主细胞,培养转化体,发酵表达;
2)裂菌,包涵体的变性和复性;
3)阴离子交换柱纯化;
4)阳离子交换柱纯化;
5)凝胶过滤柱纯化。
5、权利要求4所述的制备方法,其中步骤1中所述的表达载体是pBV220-whALR。
6、权利要求4或5所述的制备方法,其中寄主细胞是大肠杆菌细胞。
7、权利要求4或5所述的制备方法,其中寄主细胞是大肠杆菌DH5α细胞。
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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,李克,成军.中华肝脏病杂志,第10卷第5期 2002 * |
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