CN1167150A - 一种重组人粒细胞集落刺激因子的生产方法 - Google Patents
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Abstract
本发明涉及一种重组人粒细胞集落刺激因子(rhG-CSF)的生产方法。该方法采用反转录后的聚合酶链式(RT-PCR)反应,钓取人粒细胞集落刺激因子基因,转化E.coli菌;蛋白以中空纤维超滤透析方式复性,经离子交换层析、疏水层析和分子筛层析顺序组合,纯化,大规模制得高纯度的药用rhG-CSF蛋白。
Description
本发明涉及基因工程领域,具体地说是人中性粒细胞集落刺激因子(G-CSF)cDNA克隆的获得,表达G-CSF菌种的构建,及G-CSF大规模纯化,制成药品。
人外周血各种成熟血细胞均源自于骨髓造血干细胞的分化增殖,从造血干细胞到外周成熟细胞之间,存在一系列调节因子,形成网络,控制着人体各种血细胞的数量和功能,维持机体白细胞的稳定。
人G-CSF是该网络中存在于正常人体,刺激CFU-GM分化为CFU-G的因子,具有刺激前体细胞分化增殖为成熟外周中性粒细胞并增强其功能的蛋白质调节因子。
人G-CSF的发现是从生物活性研究开始的,早在1980年,Asano等人[Asano,etal.,Br.J.Cancer,41,689-694(1980)]发现一些癌症患者体内有粒细胞增多现象,当把这些人癌细胞移植到裸鼠体内后也发生粒细胞增多现象,表明人癌细胞可产生一种使鼠粒细胞增多的集落刺激因子(CSF)。把人口腔(鳞状)癌细胞株chu-2的条件培养液在体外刺激人和小鼠骨髓细胞时,能形成嗜中性粒细胞集落[Nomura.H,etal.,EMBO,5,871-876(1986)]人膀胱癌5637细胞株的条件培养液,能诱使人骨髓白血病细胞素HL-60和小鼠粒单系白血病细胞系WEHI-3BO+分化,故被称为多向刺激因子。(Sonia.LM.Amgen Patent 1985.08.23US 768,959; Sonia.LM.1986 Science 237:61-65)后来Strife等发现,如果骨髓细胞样品在去除成熟髓系和淋巴系细胞后,再用这种多向刺激因子作用,则其促进嗜中性粒细胞集落形成,其多向刺激活性是由于辅助细胞的间接作用造成的。[Strife.A,etal.,Blood,69,1508-1523(1987)]随后,从上述条件培养基中分离纯化出具有粒细胞集落刺激活性的蛋白组份,它是一种小分子糖蛋白,分子量在18-22KD[Lu Hs,etal.,Arch Biochem Biophys.268 17:81-92(1989)]其糖基化与否并不影响G-CSF刺激嗜中性粒细胞分化增殖的活性。仅影响体内清除速率、稳定性,[Oheda.M,etal.,J.Biol.Chem,265,11432-11435(1990)]并进一步证实其受体细胞主要是嗜中性细胞,是嗜中性粒细胞终端分化和活化因子。在人体正常生理状态下,主要由单核巨噬细胞系产生。由于80年代基因重组技术的飞速发展,已能利用DNA重组技术大量生产可供临床应用的人重组G-CSF。美国柯瑞英-艾格公司就重组生产G-CSF获得专利(优先权号US 768949和US 835548,中国专利号ZL86106234)发明人通过从膀胱癌细胞株5637(A1)中提取总RNA,纯化出PolyA+RNA,制备cDNA库,转化E.coliHB101备用,再把天然极微量的G-CSF纯化,测序,设计并合成一组20bp左右混合探针,利用此探针与cDNA文库杂交,筛选出编码G-CSF的cDNA,扩增这些阳性克隆后,切下G-CSF基因进行序列分析,然后把此基因重组于如PNWI等表达载体,并转化于E.coli中表达G-CSF蛋白。由于人体中含有的天然的G-CSF极其稀少,在如此稀少的情况下把它纯化,用于准确分析N-氨基酸序列是极其困难的,并且混合探针设计合成耗费试剂,设备,且杂交条件严格,工作量大。
利用基因工程手段构建的表达外源目的基因的菌株常采用E.coli表达系统,且多以包含体形式表达于菌体内。包含体是以表达产物为主,聚集形成的蛋白性质的颗粒,目的蛋白具有正确的一级结构顺序,但缺乏正确的构象,因而不具备其生物活性,欲研究或利用其生物活性,必须先经变性剂充分增溶,尔后经复性过程恢复其天然构象,才能显示其活性。到目前为止,复性完全是自发的和随机的,因此复性速度慢,回收率低,目前国内外所用的蛋白质复性方法均是依据排除使蛋白变性的环境,具体采用透析或大量稀释方法,降低变性剂浓度,使目的蛋白复性。
透析法需要时间长,一般要24小时以上,且需要多次更换透析溶液,更主要的是透析袋内部溶液在透析过程中是不均一的,靠近透析膜的溶液变性剂浓度下降快而内部下降慢,只有部分蛋白处于适于复性的变性剂浓度范围,其它蛋白很容易聚集、絮凝、沉淀,使复性回收率下降,而稀释法的大体积稀释试样给后工序的纯化工艺带来不便,而且要增大设备的处理容量。虽有用中空纤维以超滤方式进行复性,但均采用内压式中空纤维,这方式类似于血透原理,虽然可以用于大规模生产,但存在下述缺点:复性蛋白溶液自中空纤维一端流向另一端,变性剂浓度变化剧烈,蛋白被浓缩,部份蛋白来不及复性就超过折叠范围,沉淀析出,此沉淀进一步聚集在中空纤维内,堵塞通路,造成压力上升,中空纤维破损。而且此方式不利于NaOH在位清洗。
本发明的目的是提出一种新的改进的,获得G-CSF cDNA的方法,以及一种新的有效表达系统,使能高效生产G-CSF。
本发明的目的还在于提供一种使变性蛋白复性的简便,快速,高活性回收率,处理量大的新方法。
本发明还有一个目的是提供一种工业化药品生产具有人G-CSF活性多肽的纯化方法。
本发明的目的是这样实现的:利用PCR技术设计一对引物从人血白细胞总RNA中直接钓出G-CSF cDNA(参表I),测序正确后扩增获得较多G-CSF cDNA,并构建于依赖T7 RNA聚合酶的表达载体PGENS1(参图6)成为表达型重组体PGENSG,转化E.coli BL21(DE3)plysS,筛选后获得表达G-CSF的阳性克隆,经发酵、离心收集、匀浆破碎后得到含目的蛋白的包含体。
根据图1,组成复性装置,将待复性蛋白及起始复性液置于复性反应器中,用蠕动泵抽出蛋白液,经中空纤维外侧功能层由下端注入中空纤维柱,变性剂因超滤作用进入纤维中心管路并注入废液缸,而蛋白沿管壁外侧重新回到复性反应器中,同时以同样速度将贮液注入复性反应器,使反应器内体积维持恒定,经不断循环,反应器中变性剂浓度逐步下降,变性蛋白在温和的条件下完成水合表面的脱水过程和折叠过程,直至复性。
复性后蛋白仍含有杂蛋白、热源、核酸,须进一步去除,本发明设计了离子交换层析接疏水层析,后接分子筛层析的顺序组合。离子交换层析填料为DEAE,Q或QAE。rhG-CSF等电点介于5.8-6.2,PH适于7.0-9.0,可直接以复性时稀释液作为上样平衡液,平衡离子交换柱,尔后将复性浓缩好的G-CSF蛋白溶液直接上样,并以上述相应的流动相加0.01-0.5N NaCl梯度进行洗脱,此条件下洗脱的G-CSF蛋白可将复性过程中错配构象及多聚体除去,参图2。疏水柱选用butyl-,phenyl-或octyl基的疏水填料,将离子交换收集的目标峰直接补加0.1-1.0N(NH4)2SO4或0.1-1.0N NaCl,即可上样于疏水柱,疏水柱以离子交换时相应的流动相加上述0.1-1.0N(NH4)2SO4或0.1-1.0N NaCl平衡柱子,上样结束后以20-50mM磷酸缓冲液,PH7.0-9.0洗脱。因磷酸盐具有一定盐析作用,目标蛋白仍吸附在柱上,而在此充分洗涤的过程中核酸及热原被充分洗脱,尔后以Tris盐酸缓冲液或咪唑缓冲液及水顺序地阶段洗脱。G-CSF目标峰在Tris盐酸或咪唑盐酸缓冲液的洗脱峰中。经疏水层析后收集的目标峰,可直接上样于以5-50mM醋酸-醋酸钠为流动相平衡好的Sephacryl或Superdex层析柱,进行精制。
本发明所用此流动相旨在将可能存在的寡聚体进一步去除的同时,可以将前述疏水条件下的体系改换为5-50mM醋酸-醋酸钠,经此分子筛层析得到的目标蛋白G-CSF,可以直接用于制剂的配制,而无须采用透析等其他改换溶液体系的处理,减少处理步骤中可能的污染与损失。
本发明提供一种具有商业价值的,大规模获得重组人粒细胞集落刺激因子的方法,其突出的优点在于:
(1)利用聚合酶链式反应技术代替混合探针钓G-CSF cDNA,所以省去合成大量混合探针的繁琐工作。
(2)采用外压式中空纤维柱复性,可减少中空纤维破损,复性处于可控制变性剂去除速度的过程中,缩短复性时间,使得复性收率高达70%,一次复性规模可达1-10g,所用的聚砜材料便于NaOH进行原位清洗和消毒,可适应药物蛋白开发生产的GMP要求。同时采用此复性方式,还可在复性完成后,直接调整泵的流量差,而将大体积蛋白溶液进行浓缩,以便于后续纯化工艺操作的方便与省时。
(3)离子交换层析、疏水层析和分子筛层析三种不同分离机理层析顺序组合,使复性后的G-CSF得以纯化精制,经反相高效液相层析(HPLC-RP)及毛细管电泳(CE)检测,其纯度均大于95%。并且其生物活性达1×108u/mg。
本发明附图的详细说明都在实施例中:
图1是表达载体PGENS1的组成与特性图;
图2是离子交换层析图谱;
图3是疏水层析图谱;
图4是分子筛层析图谱;
图5是重组工程菌的SDS-PAGE电泳图;
图6是复性装置图:1.代表复性贮液缺罐; 2.代表复性反应器;
3.代表磁力搅拌器; 4.代表中空纤维柱;
5.代表蠕动泵; 6.代表废液罐;
表I是rhG-CSF cDNA的全序列;以下的实例是为了详细说明本发明:
实例1
G-CSF工程菌构建
健康的中国人外周血,用Ficoll作梯度离心,分离出白细胞层。取出白细胞在培养基中作短暂洗涤,直接用异硫氰酸胍/酚氯仿抽提出总RNA。总RNA用作模板。以合成的反义链3′端寡核苷酸为引物进行反转录,反转录液中加入合成的5′端和反义链3′端寡核苷酸引物,用聚合酶链式反应法合成出G-CSF结构基因。经2%琼脂糖凝胶电泳分离出条合成产物,切取λHindIII分子标准564bp处的DNA带,再做第二次聚合酶链式反应,再电泳时可明晰地见到544bp的DNA带。用第二次聚合酶链式反应后的反应液,经纯化后以平末端方法和质粒载体pUC18直接重组连接,并转化到E.coli BL21(DE3)plysS受体菌中得到的转化体按单菌落扩大培养,抽取重组质粒DNA,作DNA测序,证明含有G-CSF的结构基因,其DNA序列与Gene Bank No.X03438 M17706中的G-CSF结构基因完全一致(表2)。
以上有关总RNA抽提,多聚酶循环反应,琼脂糖凝胶电泳,平末端DNA连接反应,重组质粒转化,转化子筛选和DNA测序等一般的DNA克隆技术,都参照J.smbrook等1989,第二版的″分子克隆-实验指南″一书操作。
本方法所用的5′端和3′端DNA寡核苷酸引物分别为:
N端引物:5′GAATCTAGACATGACACCATTAGGCCCTGCCAG
C端引物:5′CATGGAYCCTTAGGGCTGGGCAAGGTGGCGT
本方法所用的聚合酶链式反应条件为:
第一次反应:37℃ 2min→72℃ 2min→90℃ 1min循环5次
再42℃ 2min→72℃ 2min→90℃ 1min循环30次
再72℃保温5min
第二次反应:55℃1min→72℃2min→90℃1min→循环30次
72℃保温5min
本方法所用的表达型重组质粒的构建,是利用引物寡核苷酸引入结构基因两端的NdeI和BamHI酶切点,酶切回收G-CSF基因,插入连接到以pUC为基础,T7噬菌体启动子启动转录的质粒载体PGENS1(图6)上,表达载体各组份来源:复制子(ori)E.coli的coli E1型,选择标记Ampr,启动子T7噬菌体Φ10基因的启动子,终止序列T7噬菌体Φ10基因的终止序列,插入位点NdeI-BamHI。重组体转化E.coliBL21(DE3)plysS,然后筛选而获得rhG-CSF的表达型重组体G-CSF基因的5′端部分编码子作E.coli型的优化,以提高G-CSF的表达量,使之占总蛋白的20%以上(图5)。
实例2
包含体增溶液复性
按图1组配复性装置。将包含体增溶液以一定比例混于复性起始液中。
复性起始液配方:
Urea:1-6M
β-疏基乙醇 5-20mM
Cu2SO4 10-40uM
Tris-HCl 20-50mM
PH7.5-9.5
复性蛋白浓度:0.1mg/ml-1.5mg/ml
EDTA 1-10mM
贮液配方:
Cu2SO4 10uM
Tris-Cl 20-50mM PH 7.5-9.5
EDTA 1-10mM
贮液与复性液的体积比可控制在1∶3-1∶10,复性反应器置于磁力搅拌器上,均匀搅拌,用蠕动泵抽取复性液从中空纤维柱下端注入,从上部注回复性反应器中,透出液被导入废液缸。调节泵流量及进入废液缸的管路口径使之体积维持恒定。
在此循环过程中,水及变性剂不断从中空纤维透出。因超滤膜的分子量截留在3000-10,000,因此蛋白不会漏出,重新回到复性反应器中进行复性。整个复性过程需10小时。中空纤维材质选用聚砜材料或硝酸纤维素酯或醋酸纤维素酯。
实例3
G-CSF纯化制备
用20mM Tris-HCl PH 7.0-9.0平衡DEAE-Sepharose FF柱,以l00cm/h上样,用离子交换流动相I加0.01-0.5N NaCl进行梯度洗脱,收集B峰(图2)。
离子交换目标峰中补加0.1-1.0N(NH4)2SO4,上phenyl-SepharoseFF柱,用20-50mM磷酸缓冲液,在PH7.0-9.0洗脱,而后以20mMTris-HCl pH 7.5洗脱,收集洗脱峰(图3),将疏水层析目标峰中加入(NH4)2SO4 4℃老化过夜,10,000rpm,4℃,10min离心收集沉淀,用5-50mM NaAc-HAc PH4.0溶解沉淀,上Sephacyl S-200柱,洗脱,收集目标峰(图4)。*** SEQUENCE LIST *** (SINGLE)1 605′CCG CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC CAC AAC GGT TTC CCT CTA GCT AGA AAT61 120AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG ACA CCA TTA GGC CCT GCC AGC TCC
Met Thr Pro Leu Gly Pro Ala Ser Ser121 180CTG CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT GGCLeu Pro Gln Ser Phe Leu Leu Cys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly181 240GCA GCG CTC CAG GAG AAG CTG TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG GAG CTG GTGAla Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val241 300CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC AGC TGC CCC AGC CAG GCCLeu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala301 380CTG CAG CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GCC CTT TTC CTC TAC CAG GGG CTCLeu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu381 420CTG CAG GCC CTG GAA GGG ATC TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTGLeu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu421 480GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA ATG GCC CCTAsp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro481 540GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CGG GCAAla Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala541GGA GGG GTC CTG GTT GCC TCC CAT CTC CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTAGly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu601 644CGC CAC CTT GCC CAG CCC TAA GGA TCC GGC TGC TAA CAA AGC CC 3′Arg His Leu Ala Gln Pro
表I rhG-CSF cDNA的全序列
ATG为G-CSF N端第一个氨基酸的编码
TAA为G-CSF C端终止信号
CTG为G-CSF 成熟蛋白的第36位氨基酸(Leu)其后没有
GTGAGTGAG序列(Val-Ser-Glu),表明为β型G-CSF。
Claims (10)
1.一种重组人粒细胞集落刺激因子(rhG-CSF)的生产方法,其特征在于以反转录后的聚合酶链式(RT-PCR)反应方法,钓取人外周血白细胞中粒细胞集落刺激因子(G-CSF)基因的cDNA,构建重组质粒,转化E.coli菌;以外压式或内压式中空纤维超滤透析复性;以离子交换柱层析、疏水柱层析和分子筛柱层析顺序组合纯化,大规模制得高纯度的药用rhG-CSF蛋白。
2.权利要求1所述的生产方法,其特征在于以外压式或内压式中空纤维超滤方式透析变性的rhG-CSF溶液。
3.根据权利要求1所述的生产方法,其特征在于其中复性装置由复性贮液缸、复性反应器、中空纤维超滤器、废液缸、蠕动泵、不锈钢管道和阀门构成。
4.权利要求3所述的复性装置,其特征在于采用并联的中空纤维柱。
5.权利要求3所述中空纤维超滤器,其中中空纤维材料是醋酸纤维素酯、硝酸纤维素酯或聚砜。
6.权利要求3所述复性装置,其中中空纤维超滤器膜中的截留分子量在3000-10000。
7.权利要求1所述的生产方法,其中离子交换柱填料为DEAE、Q、QAE等阴离子交换剂,疏水柱填料为butyl-、phenyl-或octyl基的疏水介质,分子筛柱填料为低吸附的Sephacryl系列或Superdex系列的介质。
8.权利要求1所述的生产方法,其中离子交换柱层析,其流动相I为20-50mM咪唑盐酸、巴比妥盐酸、Tris盐酸、硼砂-硼酸或磷酸缓冲液,PH的范围在7.0-9.0,流动相II为上述相应的流动相I中加0.01-0.5NNaCl梯度洗脱。
9.权利要求1所述的生产方法,其中疏水柱层析,其流动相为20-50mM咪唑盐酸、巴比妥盐酸、Tris盐酸、硼砂-硼酸及磷酸缓冲液,PH范围在7.0-9.0,其中流加0.1-1.0N(NH4)2SO4或0.1-1.0N NaCl,流动相II分别以20-50mM PH范围在7.0-9.0磷酸缓冲液、Tris盐酸缓冲液或咪唑缓冲液及水顺序洗脱。
10.权利要求1所述的生产方法,其中分子筛柱层析,其流动相为5-50mM醋酸-醋酸钠。
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Cited By (4)
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CN1064968C (zh) * | 1996-07-10 | 2001-04-25 | 中国人民解放军第一军医大学 | 重组蛋白提取纯化方法 |
CN1101403C (zh) * | 1998-07-13 | 2003-02-12 | 金磊 | 粒细胞集落刺激因子的制备 |
CN1313612C (zh) * | 2005-12-20 | 2007-05-02 | 山东泉港药业有限公司 | 一种重组人粒细胞集落刺激因子的生产方法 |
CN106222221A (zh) * | 2016-08-05 | 2016-12-14 | 山东科兴生物制品有限公司 | 制备重组人粒细胞刺激因子原液的纯化方法 |
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CN1021461C (zh) * | 1985-09-17 | 1993-06-30 | 中外制药株式会社 | 人粒性白细胞集落刺激因子 |
JPS6293238A (ja) * | 1985-10-18 | 1987-04-28 | Res Dev Corp Of Japan | コロニ−形成刺激因子の製造方法 |
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CN1064968C (zh) * | 1996-07-10 | 2001-04-25 | 中国人民解放军第一军医大学 | 重组蛋白提取纯化方法 |
CN1101403C (zh) * | 1998-07-13 | 2003-02-12 | 金磊 | 粒细胞集落刺激因子的制备 |
CN1313612C (zh) * | 2005-12-20 | 2007-05-02 | 山东泉港药业有限公司 | 一种重组人粒细胞集落刺激因子的生产方法 |
CN106222221A (zh) * | 2016-08-05 | 2016-12-14 | 山东科兴生物制品有限公司 | 制备重组人粒细胞刺激因子原液的纯化方法 |
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