CN106279406A - 猪皮中分离纯化的皮抑素g1的活性成分四连接素及其应用 - Google Patents
猪皮中分离纯化的皮抑素g1的活性成分四连接素及其应用 Download PDFInfo
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Abstract
本发明属于生化分离技术领域,具体涉及一种猪皮中分离纯化的皮抑素G1的活性成分四连接素及其应用。本发明采用生化分离纯化技术,结合分子生物学技术,首次证明表皮细胞抑裂素中对表皮细胞有丝分裂有抑制作用的主要活性成分为四连接素(Tetranectin,TN),所述猪四连接素由181个氨基酸组成,分子量19792.9,pI为5.75,TN为从猪皮制备的皮抑素G1的主要活性物质,可以制备成治疗银屑病的药物。
Description
技术领域
本发明属于生化分离技术领域,具体涉及一种猪皮中分离纯化的皮抑素G1的活性成分四连接素及其应用。
背景技术
皮抑素G1具有明显的抑制表皮细胞有丝分裂的活性,因此可以作为缓解银屑病症状的药物,但半个多世纪来皮抑素G1既未用于治疗银屑病,亦未得到纯化,因此人们对其主要活性成分的化学性质知之甚少。目前有关皮抑素G1的研究除发明专利ZL201410036118.X外,未见其他报。
发明内容:
本发明需要解决的问题是探索皮抑素G1的主要活性成分,了解其化学结构和性质及其在制备治疗银屑病药物中的应用。本发明从猪皮经部分纯化得到的皮抑素G1开始,进一步纯化,通过阳离子交换凝胶层析,制备性SDS-PAGE,分离后将活性成分抽提物送质谱鉴定,发现在各蛋白成分中含量最高的成份为猪四连接素,和磷脂酰乙醇胺结合蛋白(PEBP)。通过DNA重组技术,在大肠杆菌中成功表达了四连接素(TN)和PEBP两种蛋白,并证实了TN具有很高的抑制活性,TN的单体活性更高,而PEBP虽然也有肯定的活性;但与TN比较则活性较低;显示皮抑素G1中主要的具有抑制活性的成分为TN。
本发明所述猪四连接素由181个氨基酸组成,分子量19792.9,pI为5.75,其氨基酸序列为:
本发明以皮抑素G1为起始材料,通过阳离子交换凝胶SP-sepharos FF柱层析,层析柱用pH 6.8 0.02mol/L,PBS平衡后,将皮抑素G1调节pH至6.8上柱分离,最早出现一个极大的穿过峰,改变洗脱缓冲液PBS的pH值为8.0,出现一个很小的尖峰,称为洗脱峰1,再在此碱性缓冲液中加入0.5m NaCl,又得到洗脱峰2,层析图见图1.将三部分蛋白,分别对水透析、脱盐、冻干,加入pH 6.8的0.02mol/L PBS溶解,测定活性.
1、皮抑素G1活性测定方法
(1)采用待试样品对人表皮永生细胞株(HaCat)细胞生长的抑制百分率表示活性。HaCat细胞由上海复祥生物技术有限公司提供。
(2)测定方法:将HaCat细胞在含10%胎牛血清的RPMI1640培养液中,在5%CO2培养箱中37℃培养至对数生长期,用0.25%胰酶从培养瓶壁上消化10分钟左右,振动培养瓶,收集细胞,离心洗去酶液,用培养液配成3×104/ml细胞悬液,加入96孔培养板中,每孔加160μL细胞悬液,在CO2培养箱中培养24小时后,加入待测样品40μL,待测样品的配制:样品除菌后测定蛋白浓度,用培养液配成2.5μg/ml浓度(100ng/40μL)并作17次倍比稀释,每种浓度在每孔中加入40μL,共三个平行孔,对照组加40μL培养液,设6个平行孔,96孔板四周36个孔,因误差较大,一般只加200μL Hank’s液,不参与实验,加样后96孔板在CO2培养箱中继续培养72小时,每孔中加入20μL 5mg/ml的MTT,继续培养4小时后,倾去上清,每孔加入100μL二甲基亚砜,溶液显蓝紫色,在酶标仪OD 492nm处读数,以3个平行样品孔的平均OD值与6个对照孔的平均OD值,计算抑制百分比。
(3)活性单位,中值(ED50值)及比活性的计算:活性单位:以蛋白质倍比稀释的次数为横坐标,以抑制百分率为纵坐标作图可得一S型曲线,画出上下平台,取上下平台的中值(ED50)查出中值时的抑制百分数,每抑制1%规定为1个毫单位(1mU),从中值处的蛋白含量可计算出该蛋白的比活性(U/mg)
比活性=活性单位(U)/mg蛋白
从倍比稀释数换算成为蛋白量:
如果中值时的稀释次数为6.95。起始蛋白浓度为100ng/孔。
中值为6.95的蛋白量为2-6.95×100ng=0.8088ng
如中值ED50为27.7mU
则比活性为27.7mU/0.8088ng=34248.2U/mg
(4)活性测定结果,发现仅洗脱峰1表现高活性,穿过峰无活性,洗脱峰2的比活性极低。峰1的活性测定结果见图2.
(5)将洗脱峰1进行制备性SDS-PAGE分离,按考玛斯蓝染色显示有四群蛋白,见图3,将四群相应位置的蛋白胶条切下,置于玻璃瓶中,用平头玻璃棒尽量压碎,用0.02mol/L的pH6.8PBS 4℃浸提过夜,次日吸出上清液,重复此过程三次,合并四次抽提液,透析,冻干,溶解,测蛋白,测活性,结果显示仅分子量2万左右的第3蛋白群表现高活性,见图4.
(6)将带3蛋白再次进行SDS-PAGE,并在前沿溴酚蓝色带出胶后再继续电泳一小时,凝胶染色后带3出现分子量很接近的二条蛋白,将此二块凝胶送苏州普泰公司进行质谱分析,结果发现第一条带含7种成分,第二条带含6种成分,分析结果见下表。
表1.质谱分析3号蛋白群中带1和带2中各成分的性质(带1含7个成分)
编号 | 含量% | 分子量MW | 性质 |
1 | 10.4 | 96262.41 | 猪未知蛋白 |
2 | 1.6 | 41522.89 | 猪未知蛋白 |
3 | 43.9 | 22575.29 | 猪四连接素 |
4 | 0 | 34388.22 | 猪未知蛋白 |
5 | 1.3 | 63287.39 | 猪G-6-P isomerarc |
6 | 37.9 | 21072.67 | 猪磷脂酰乙醇胺结合蛋白 |
7 | 4.9 | 15086.84 | 猪血红蛋白亚基α |
(带2含6个成分)
编号 | 含量% | 分子量MW | 性质 |
1 | 76.7 | 22575.29 | 猪四连接素 |
2 | 1.7 | 122636.09 | 猪未知蛋白 |
3 | 3.1 | 18966.95 | 猪磷酸丙酮酸水合酶 |
4 | 14.8 | 16212.48 | 猪血红蛋白亚基β |
5 | 3.7 | 15086.84 | 猪血红蛋白亚基α |
6 | 0.1 | 96262.41 | 猪未知蛋白 |
其中含量最高的为猪四连接素(TN),其次为磷脂酰乙醇胺(PEBP)结合蛋白,其余含量较低的蛋白有的是参与新陈代谢中已知的酶和血红蛋白亚基,还有几种猪未知蛋白,显然其中TN和PEBP是皮抑素G1的主要活性成分。
2为了辨明皮抑素G1的活性成分是TN还是PEBP,或者二者都没有活性,而是含量极低的猪未知蛋白,为此委托南京德泰公司进行TN和PEBP的DNA重组,并在大肠杆菌中成功表达,南京德泰生物技术公司对二种蛋白通过DNA重组技术分别在大肠杆菌中成功表达了四连接素TN和PEBP(分离方法相同)。
工程细菌在含50μg/ml卡那霉素的LB试管中摇床(37℃,15rpm)中生长过夜,次日接种到含50μg/ml卡那霉素的1LTB培养基中在摇床中生长至OD600nm=0.6-0.8时,将温度降至15℃,1小时后加入终浓度0.5mol/L,IPTG诱导生长16小时,离心收集细菌,分离纯化步骤如下。
(1)菌泥破碎和初步纯化
用Buffer A:50mM NaCl,2mM DTT,1%TritonX-100,1μg/ml pepstatin A,1μg/mlLeupeptin pH 8.0裂解菌泥。
(2)超声裂解超声3s间歇8s,总计20min,离心13000rpm,20分钟,4℃离心取沉淀,重复此步骤2次。
(3)用Buffer B:50mM Tris,150mM NaCl,2mM DTT,2mM EDTA,1%TritonX-100,pH8.0洗涤包涵体,离心取沉淀,重复两次。
(4)用Buffer C:50mM Tris,150mM NaCl,2mM DTT,2mM EDTA,2M尿素,pH8.0洗涤包涵体,离心取沉淀。
(5)用Buffer D:50mM Tris,150mM NaCl,10mM DTT,6M GdmCl,pH8.0洗涤包涵体,离心取上清。
(6)分子筛层析纯化
Superdex TM200(prep级),层析柱25/100,流速:2ml/min
Buffer E:50mM Tris,150mM NaCl,8M尿素,Ph8.0
分离结果见图5.
合并纯度较高的13-17管,用考马斯亮蓝G250测定浓度,然后进行稀释复性
(7)稀释复性
将纯化后的目标蛋白缓慢加入到200ml复性液Buffer F:50mM Tris-HCl,150mMNaCl,2mM GSH,0.4Mm GSSG,0.4M L-精氨酸,2mM CaCl2,pH8.0中,使其终浓度约为0.3mg/ml,用磁力搅拌器4℃旋转,总计复性24小时。
(8)复性后纯化
稀释复性液用0.45um滤膜过滤,去除不溶物,用浓缩管浓缩至3ml.
(9)分子筛层析,纯化。
填料类型SuperdexTM200,prep gradc.
层析柱型号16/100
流速1ml/min
纯化buffer G 50mM Tris,150mM Nacl,Ph8.0.
分子筛层析纯化结果见图6
(10)合并收集,测蛋白后分装,冻干。
SDS-PAGE分析,稀释复性样品的纯度,结果见图7.
得率:高密度培养70-80g湿菌/L
3用人表皮永生细胞Hacat测定样品对细胞有丝分裂的抑制活性。
结果显示TN具有很高的抑制活性,因TN为一同质三聚体。在分离纯化过程中有保护剂DTT存在下单体以非共价结合方式聚合为三聚体,纯化过程如不加保护剂则以单体形式存在,无论聚体或单体均有很高活性,但单体的比活性,达到150430μ/mg.而聚体为主的蛋白比活性为34248.3μ/mg.显然单体表现更高的比活性。而PEBP也有一定活性但与TN比较则比活性相对低很多。仅4834μ/mg.结果见图8、9、10.
本发明的有益效果:
本发明通过以上结果显示TN显然是皮抑素的主要抑制活性的贡献者,皮抑素G1的主要活性成分为猪四连接素(TN),PEBP的比活性虽比TN低,但不能排除在银屑病的治疗中PEBP也起着一定作用,因PEBP本身除了有抑制表皮细胞有丝分裂的活性,它还具有促进细胞凋亡的作用。
TN是1986年Clemmensen等人从人血浆中发现的,2002年已通过DNA重组技术得到纯品,但至今仍处在研究阶段。已知TN与很多疾病相关。如心血管病,免疫,骨质疏松,肥胖,癌症如乳腺癌、宫颈癌,肺癌,但无人报导TN与银屑病有关。本发明采用TN的单克隆抗体检测试剂盒测定了16例正常人(体检人员)。16例肺癌病人和22例银屑病人血浆中TN含量。结果肺癌病人血浆中TN的含量和报导的结果一致,与正常人水平比较十分低下。而银屑病人血浆中TN的含量竟然和肺癌病人一样绝大多数都十分低下,接近于零。
正常人(体检人员) 425pg/ml-1740pg/ml
肺癌病人 <5pg/ml
银屑病人 <5pg/ml
根据以上结果判断TN与银屑病之间应该有紧密的关系,目前虽然还不清楚TN的降低与银屑病的因果关系,但是为皮抑素G1可以缓解银屑病的症状提供了有力的依据。TN为从猪皮制备的皮抑素G1的主要活性物质,TN可以制备成治疗银屑病的药物。
四.附图说明
图1皮抑素G1的SP-sepharose FF凝胶层析图
图2 SP-sepharose FF柱层析分离峰1(PI)的活性测定
图3洗脱峰1(P1)的SDS-PAGE
图4 SDS-PAGE分离第3号和第4号蛋白质的生物活性
图5 SEC分离图谱及SDS-PAGE纯度检查
图6 SDS-PAGE分析分子筛层析纯化结果
图7 SDS-PAGE分析TN的纯度(5为加保护剂,6未加保护剂)
图8 TN(三聚体为主)的活性测定
图9 rhu-TN单体的活性测定
图10 PEBP的活性测定
五、具体实施方式
1、皮抑素G1的制备
7.5kg去脂猪皮,绞肉机绞成肉皮糜,加水44kg,胶体磨磨成匀浆,纱布粗滤后,渣滓再次加水匀浆。最后将滤渣和滤液合并,总体积达80L,10℃以下搅拌3小时,细布过滤,滤渣再加水搅拌2小时,合并二次滤液,以截止分子量1万膜超滤,以去除小分子杂质,体积浓缩至1/10左右,以1:0.46比例加入95%医用乙醇,使乙醇终浓度为30%,冰箱过夜,次日离心取上清,再按1:5.5比例加入95%医用乙醇,使乙醇终浓度达到85%,4℃冰箱过夜。次日吸去上清,离心取沉淀。沉淀溶解在PH7.4,0.02mol/L PBS中离心取上清,沉淀再次悬浮,离心取上清,合并二次上清液得4.1L上清液除菌测蛋白,测活性,比活性为3520U/mg,经试验对皮肤无刺激等毒性反应后,即为皮抑素G1,蛋白浓度为36.3mg/ml。
2、皮抑素软膏的制备
皮抑素软膏配方采用中国医科院皮研所药房配方略作修改,3kg软膏中含白蜡419g,白凡士林2374g。加热至65℃融化,冷却至55℃加入防腐剂2,6-二叔丁基对甲酚3g,皮抑素207ml(7.5g蛋白)混合均匀后在无菌室中加入已经消毒的药盒中,每盒30g,每克含皮抑素2.5mg,共100盒,存放冰箱中,供病人使用。
3、治疗效果
第一例志愿病人为南京大学教师,男性70多岁,患银屑病50年,有家族史。其外祖父患银屑病,但母代无患者,病人为全身性严重寻常型银屑病。患处皮肤为死皮屑覆盖,弯折处常出血,影响饮食、睡眠,十分痛苦,曾经中西药治疗,都是暂时缓解,不久又复发。在英国治疗用钙泊三醇药膏煤焦油油膏,煤焦油药浴,也是缓解又复发。
试用本发明研制的皮抑素软膏,为慎重起见,建议先用一条腿治疗,每日早晚擦一次药,试用三星期后病人兴奋地告知治疗的腿皮屑完全脱落,皮肤恢复柔软平滑,只残留色素沉淀。另一条未治疗的腿仍是白色死皮屑覆盖,形成鲜明对照。病人反映,皮抑素是他所有用过的药中效果最好,无色无味无副作用,是最容易被接受的一种治疗药物。
在这一病例的鼓舞下,在志愿者的要求和医生配合下,共进行了10多例斑块型银屑病人的治疗。绝大多数都取得明显改善。有的医生再三询问药物中是否加有激素,经告知未加任何激素完全是天然分离的蛋白,医生反映说效果比加激素的进口药更好,而且从未有副作用发生,是一种安全有效的外用药物。
Claims (2)
1.一种猪皮中分离纯化的皮抑素G1的活性成分四连接素,其特征是由181个氨基酸组成,分子量19792.9,pI为5.75,其氨基酸序列为:
2.权利要求1所述猪皮中分离纯化的皮抑素G1的活性成分四连接素在制备治疗银屑病药物中的应用。
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