CN1096541A - 家蚕基因工程生产重组人巨噬细胞集落刺激因子 - Google Patents
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Abstract
本发明属于生物技术制药工业中的基因工程生
产多肽药物的技术领域,其目的是利用我国资源十分
丰富的家蚕为原料,从家蚕幼虫中的血淋巴中大量表
达rhM-CSF,表达水平可达250毫克/升,
rhM-CSF基因为含有完整的天然信号肽和基因第
1—149位氨基酸密码子及终止密码的DNA片段。
并从家蚕血淋巴中分离纯化出比活大于107单位/
毫克,纯度98%的rhM-CSF精品,用于生产抗真
菌,抗病毒感染,抗肿瘤及升白血球等用途的药物。
Description
本发明属于生物技术制药工业中的基因工程生产多肽药物技术领域。
人巨噬细胞集落刺激因子(hM-CSF)是一种重要的造血细胞生长因子。它对巨噬细胞的分化、增殖及成熟后的功能具有特异的刺激作用。同时它也是一种免疫效应剂,能促进和诱导效应细胞分泌干扰素、肿瘤坏死因子、白介素-1、粒细胞集落刺激因子(G-CSF)、前列腺素及纤溶酶原激活剂等,有效地增强巨噬细胞在炎症反应中的抗炎症作用。临床上主要应用于促进造血功能的恢复。如放疗化疗后的白细胞减少或骨髓移植造成的骨髓发育不良;增强机体抗感染、抗肿瘤的防御能力,对治疗某些致死性真菌病和某些病毒病特别有效;此外,还能选择性地降低血清中的低密度脂蛋白,用于抗动脉硬化。国外基因工程生产的rhM-CSF已进入Ⅲ期临床。1985年美国Cetus公司首先分离到hM-CSF的短链cDNA基因(Science(1985)230:291),并在COS细胞中表达出有活性的rhM-CSF,但表达水平仅为2×103单位/毫升,随后不同实验室相继从大肠杆菌、酵母或昆虫细胞中表达获得rhM-CSF。但表达水平一般在10毫克/升。1988年Cetus公司采用大规模高密度培养的草地贪夜蛾(S.F)细胞,使rhM-CSF在培养细胞中的表达量达到40毫克/升。(Biotechnology(1988)6:1406)。但成本偏高。不利于投入生产。
本发明的目的是利用我国资源来源十分广泛便宜的家蚕为原料,代替大肠杆菌或酵母发酵,或昆虫SF培养细胞,从家蚕幼虫的血淋巴中大量表达rhM-CSF,表达水平可达250毫克/升,并创造一条简便可行的工艺,从家蚕血淋巴中分离纯化出比活大于107单位/毫克,纯度98%的rhM-CSF精品。使整个工艺成本大为降低,成为一条更适于大规模生产可供临床应用的rhM-CSF生产工艺。
本发明的技术内容包括:1.家蚕基因工程表达载体-带hM-CSF(1-149aa)的重组家蚕核型多角体病毒BmNPV-M-CSF的构建;2。重组病毒对家蚕幼虫的感染及rhM-CSF在家蚕血淋巴中的高效表达和分泌;3.从家蚕血淋巴中分离纯化rhM-CSF的精品。
1.天然的hM-CSF具有长中短三种型式,它们的cDNA基因具有相同的信号肽(32个氨基酸)和N端第1-149位氨基酸密码子.据此,我们设计的rhM-CSF基因为含有完整的天然信号肽(32个氨基酸包括起始密码ATG)和基因第1-149位氨基酸密码子及终止密码的DNA片段。具体合成过程与重组病毒的构建过程如下:hM-CSF信号肽质粒含有hM-CSF信号肽32个氨基酸及基因第1-6位氨基酸密码子,经化学合成后克隆于pUC18的EcoRI、PstI位点中,由中科院上海生物工程研究中心合成提供。另以hM-CSF的长链cDNA基因片段为模板,用PCR法合成5′端带EcoRI位点3′端带终止密码及Hind Ⅲ位点的含hM-CSF基因第3-149位密码子的基因片段,克隆于M13mp18之EcoRI、Hind Ⅲ位点中。利用hM-CSF基因第6密码子中的ScaI位点,将信号肽与基因片段拼接成5′端有EcoRI位点,含信号肽32个密码子、基因第1-149位密码子及终止密码,3′端有Hind Ⅲ位点的分泌型短基因,克隆于pUC18的EcoRI、Hind Ⅲ位点中。经序列分析证明序列正确,ScaI位点恢复。上述质粒经扩增纯化后用EcoRI、Hind Ⅲ双酶解切出带信号肽及N端1-149位密码子的rhM-CSF基因片段,用klenow酶补平后插入至家蚕转移质粒pBE284(S.Maeda提供)之补平的XbaI位点中.限制酶分析筛选出重组方向正确-即多角体基因启动子与基因编码方向一致的表达转移质粒pBE-M-CSF。扩增纯化后与纯化的野生型家蚕核型多角体病毒Bm NPV(C6株)的基因组DNA混合,经脂质体(Lipofectin)共转染培养的单层家蚕细胞Bm N.27℃,培养7天后在琼脂板上用原位杂交筛选出阳性重组病毒空斑。再经4次以上96孔板有限稀释法纯化病毒。并结合测定集落刺激活性选出高水平表达rhM-CSF的不含野生型病毒的纯净重组病毒BmNPV-M-CSF。
2.重组病毒感染家蚕幼虫及表达rhM-CSF的家蚕血淋巴的收获:纯化后的重组病毒以感染指数(MOI)=0.1空斑形成单位(PFU)/细胞感染单层培养的BmN细胞,27℃培养4-5天后,4000转/分离心弃细胞收集上清液作为毒种。毒种液滴度为107CFU/ml。用天然桑叶或人工饲料饲养的家蚕,5龄起眠后第1天,以10微升(105PFU)/虫毒种腹部注射或穿刺感染重组病毒。继续饲养4-5天后,剪腹足加一倍体积抗氧剂保护液(1mMDTT,10mM抗坏血酸)防止血淋巴黑化,离心8,000-10,000转/分,4℃,20分钟或低温压滤收集血淋巴。血淋巴可在-20℃存放。测定血淋巴的集落刺激活性可达2-3×106集落刺激单位(CFU)/ml。
3.从家蚕血淋巴中分离纯化rhM-CSF:上述方法收集的家蚕血淋巴,在70℃水浴保温15分钟,8,000转/分离心除去热处理沉淀的杂蛋白。上清液对pH6.0,0.05M NaAc缓冲液透析,上DEAE 52柱,柱子预先用样品缓冲液平衡.先用0.05M NaAc,0.05M NaCl,pH6.0缓冲液充分洗涤,然后用pH6.0,0.05M NaAc,0.2M NaCl缓冲液洗脱rhM-CSF。收集洗脱液对pH6.5,3mM磷酸钠缓冲液透析,上预先平衡的羟基磷灰石柱子。先用10mM,pH6.5磷酸钠缓冲液充分洗涤,再用50mM,pH6.5,磷酸钠缓冲液洗脱rhM-CSF。收集洗脱液透析浓缩后,于低压液相层析系统上经SP柱离子交换层析。用pH4.5,0.02M NaAc缓冲液盐浓度梯度0-0.5M NaCl进行梯度洗脱,梯度时间30分钟,流速1ml/分,rhM-CSF洗脱峰位于20-25分钟区段.收集该区段rhM-CSF洗脱液,透析脱盐后冻干保存。
本发明构建的重组家蚕核型多角体病毒BmNPV-M-CSF,其中带完整信号肽的hM-CSF基因(1-149)位于BmNPV的强大的多角体基因启动子控制下,多角体基因被hM-CSF基因所取代。在家蚕的培养细胞、幼虫或蛹体内,随着重组病毒的感染和复制,rhM-CSF能高效表达并分泌至细胞外。表达产物经免疫印迹法(western-blotting)检定,产生的rhM-CSF为糖基化程度不同分子量分别为18、20、22KD三种免疫特异性及生物活性相似的蛋白。精品经N末端分析N端十个氨基酸序列为Glu-Glu-Val-Ser-Glu-Tyr-Ser-His-Met,表明rhM-CSF的天然信号肽在表达分泌过程中已被正确地切除。
采用本发明构建的重组病毒按本发明所述方法感染家蚕幼虫和收集血淋巴,家蚕血淋巴中表达积累的rhM-CSF可达3×106CFU/ml,按精品比活1.2×107CFU/mg折算,表达量为250mg/L。实现了rhM-CSF的高效表达。
按本发明的技术路线,热处理可简便有效地灭活家蚕血淋巴中存在的大量酪氨酸氧化酶,防止了黑化并除去一部分易热变性的杂蛋白。按本发明提供的分离纯化方法可以从家蚕感染重组病毒后采集的血淋巴中获得纯化程度基本符合基因工程制药要求的rhM-CSF精品。rhM-CSF精品比活达1.2×107CFU/mg以上。在高压液相层析(HPLC)系统TSK2000SW分子筛柱上分析呈一个主峰,纯度>98%。用SDS-PAGE银染法检测,无明显的杂蛋白电泳区带。rhM-CSF精品临床可用于生产抗真菌、抗病毒感染、抗肿瘤及升白血球等药物。
下面是本发明的实施例:1.rhM-CSF在不同家蚕幼虫中的表达。以5龄家蚕幼虫,品种苏5×苏6,每批5,000条为材料,以M.O.I=105PFU/虫的剂量注射感染重组病毒,第4天剪去一对腹足,加一倍体积抗氧剂溶液(1mMDTT,10mM抗坏血酸),离心8,000转/分,4℃,20分钟,收集血淋巴。第5天家蚕幼虫开始大批死亡。将收集的血淋巴存放于-20℃。血淋巴测定集落刺激活性,1993年春蚕二批的平均表达水平为2×106CFU/ml血淋巴原液。93年秋蚕二批平均表达水平为3×106CFU/ml血淋巴原液,约合250微克/毫升。
单层培养的家蚕细胞BmN,在接近汇合时,以MOI=10PFU/细胞剂量感染重组病毒BmNPV-M-CSF,感染后27℃继续培养,第6天培养液中rhM-CSF的表达量达10-14×104CFU/ml(3×105细胞)。
2.取家蚕血淋巴表达液100ml,加抗氧剂液稀释1倍后,70℃保温15分钟,8,000转/分离心20分钟,弃杂蛋白沉淀取上清液。对pH6.0,0.05M NaAc缓冲液透析后,上DEAE52柱(16mm×200mm),柱子预先用样品缓冲液平衡。先用三倍床体积之pH6.0,0.05M,NaAc,洗涤,然后用pH6.0,0.05M NaAc。0.2M NaCl缓冲液洗出rhM-CSF,收集洗出液。柱子用pH6.0,0.05M NaAc,0.05M NaCl缓冲液洗除杂蛋白,再生平衡后再使用。rhM-CSF洗出液对pH6.5,3mM磷酸钠缓冲液透析后,上预先平衡的羟基磷灰石柱。先用三倍床体积pH6.5,10mm磷酸钠缓冲液洗涤,而后用pH6.5,50mM磷酸钠缓冲液洗出rhM-CSF组分。留存的杂蛋白可用0.5M磷酸缓冲液洗净后再生平衡待用。收集的rhM-CSF洗出液经透析脱盐浓缩后,于Waters 650E蛋白纯化系统上经SP离子交换柱层析(FPLC)。选择梯度缓冲液为pH4.5,0.02M NaAc,0-0.5M Nacl,梯度时间30分钟,流速1ml/分.收集20-25分钟区段rhM-CSF洗出峰。上述rhM-CSF洗出液经透析脱盐后,冷冻干燥分装成rhM-CSF精品。分别测定蛋白质含量和集落刺激活性,算出精品比活为1.2×107CFU/mg。HPLC测定纯度>98%。整个分离纯化过程纯化倍数为200倍以上。活性得率大于30%。
按本发明所述方法每一万条秋蚕可得血淋巴表达液约6升,可提取rhM-CSF精品约500毫克。
Claims (4)
1、一种基因工程人巨噬细胞集落刺激因子(rhM-CSF)的生产方法,其特征是以家蚕为原料,通过带人rhM-CSF基因的重组家蚕核型多角体病毒(BmNPV-M-CSF)为载体,在感染重组病毒的家蚕血淋巴中大量表达rhM-CSF,并从家蚕血淋巴中分离纯化rhM-CSF精品。
2、根据权利要求1.所述的方法其所用的重组病毒载体的构建特征是,将化学法合成的hM-CSF信号肽片段与PCR法合成的人M-CSF基因片段,经体外重组拼接成含人M-CSF信号肽32个密码子和基因第1-149位密码子及终止密码、两端有适当的限制酶切位点的分泌型rhM-CSF基因片段,通过平头连接插入家蚕转移质粒pBE284中补平的XbaI位点,选出重组方向正确即多角体基因启动子与rhM-CSF基因编码方向一致的表达转移质粒pBE-M-CSF,扩增纯化后再与纯净的野生型家蚕核型多角体病毒(BmNPV)C6株的基因组DNA混合,经脂质体共转染培养的单层家蚕细胞BmN,再经多次病毒的纯化筛选出高水平表达rhM-CSF的纯净重组病毒BmNPV-M-CSF。
3、根据权利要求1.,2.所述的方法,从感染重组病毒的家蚕血淋巴中表达和分离纯化rhM-CSF的特征是,用重组病毒10微升毒种(105PFU/虫)腹部注射或穿刺感染5龄家蚕幼虫,剪腹足加抗氧剂保护后,离心收集血淋巴,然后经热处理→DEAE52柱→羟基磷灰石柱→FPLC(SP柱)逐步纯化。获得比活大于1.2×107集落刺激单位(CFU)/毫克,纯度达98%以上(HPLC分析)的rhM-CSF精品,活性得率为30%以上。
4、本方法生产的rhM-CSF产品的结构特征是其氨基酸序列与天然人M-CSF第1-149位氨基酸相同,N端第1位是谷氨酸残基,产物为三种糖基化程度不同分子量分别是18、20、22KD的蛋白质混合物。临床可用于生产抗真菌、抗病毒感染、抗肿瘤及升白血球等药物。
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WO2001029204A3 (en) * | 1999-10-19 | 2001-11-01 | Roger Craig | Protein production system |
CN1110563C (zh) * | 1997-11-05 | 2003-06-04 | 浙江农业大学 | 家蚕生产基因工程生白细胞药物的方法 |
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CN1110563C (zh) * | 1997-11-05 | 2003-06-04 | 浙江农业大学 | 家蚕生产基因工程生白细胞药物的方法 |
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