CN114671956A - 一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体 - Google Patents
一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体 Download PDFInfo
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Abstract
本发明属于分子生物学领域,具体公开了一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体。本发明在TNHH的基础上,对易导致翻译表达错配的半胱氨酸进行突变,并且对与凝血酶结合部位的部分非极性氨基酸突变为其他氨基酸得到的TNHH突变体。TNHH是一种具有双功能的嵌合蛋白,我们通过基因定点突变技术同时解决了两个难题,不仅仅降低了其异构体含量,提高了蛋白纯度,还使其生物学活性大大提高,具有很高的实践意义。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体。
背景技术
在我国,脑血管疾病一种致死率仅次于恶性肿瘤疾病,其中缺血性脑损伤导致的死亡率占了其中的2/3。缺血性脑卒中主要有两种类型:一种是由于脑动脉粥样硬化或脑血管栓塞引起的卒中,一种是由于心脏骤停等引起的脑血流低灌注引起的卒中。目前主要通过溶栓治疗,应用阿司匹林、氯吡格雷等抗血小板聚集药物等手段来进行治疗。虽然能够改善脑卒中的情况,但是治疗周期长且存在严重的后遗症,例如偏瘫,认知障碍等。而且治疗费用昂贵,副作用多。
研究表明缺血性脑损伤能够导致人体凝血酶活性升高,白细胞粘附聚集。而一种生物创新药TNHH能够有效抑制这两种现象。TNHH(Targeted Neutrophil InhibitoryFactor and Hirugen Hybrid,即重组白细胞抑制因子和水蛭肽嵌合蛋白)是由重庆富进生物医药公司合成研究,在白细胞抑制因子(NIF)和水蛭肽(Hirulog)中间加入了一段交联肽连接而成,是一种双功能的重组嵌合蛋白,具有两者的功能,既能够抑制白细胞的转移聚集又能够抑制凝血酶的活性。
TNHH作为一种用于治疗急性脑血管疾病的新型蛋白药物,主要依据脑出血和脑血栓形成过程中局部凝血酶原被激活,成凝血酶后引起的血小板所致微血管阻塞和脑水肿,白细胞浸润和激活后所致脑组织受损的病理机理而设计的针对白细胞活化和凝血酶活性的新型抗脑卒中双功能蛋白的药物。在急性发病后24小时内给予治疗7天左右,该药物可达到恢复受损脑组织,减轻脑水肿和改善局部微循环的多重目的。由于TNHH具有较好的安全性和显著的有效性,双靶点融合蛋白的设计实现了降低NIF和Hirulog片段的风险性,增加有效性的目的。因此,TNHH成为临床上对急性脑卒中(特别是急性脑栓塞)有显著治疗和预防效果(加速康复和减少后遗症)的一种全新药物。这将为逐渐增多的老年性急性脑血管以外患者带来福音,同时节约大量的医疗和社会成本。
TNHH由282个氨基酸组成,N末端含257个氨基酸的NIF,C末端含20肽水蛭肽(Hirulog),中间由5个甘氨酸铰链区组成,其中N末端第一个氨基酸为甲硫氨酸。TNHH中NIF区域与天然的NIF除第1位增加了Met外完全一致,而其中水蛭肽(Hirulog区域)与国外上市的文献报道的Hirulog有两个氨基酸差异,即在结合肽FPRPGGGG中将第一位D型脯氨酸变成L型,第6位的甘氨酸变成了丝氨酸,其核苷酸序列如SEQ.ID NO:1,氨基酸序列如SEQ.IDNO:2所示。TNHH的具体结构示意为:Met-NIF(257)-(Gly)5-FPRPGSGG-Hirugen(53-64)。TNHH的分子量为31.5KD,pI4.5,含有10个半胱氨酸,5对二硫键,无糖基化。由于半胱氨酸侧链较长,因此在翻译表达时容易产生错配,从而产生异构体降低了蛋白纯度,同时也使其生物活性有所降低。因此需要提供一种TNHH突变体来减少翻译表达时错配机会,减少异构体的产生,提高了蛋白纯度,进一步提高了生物学活性。
发明内容
基于上述现有技术的不足,本发明的目的在于提供一种重组白细胞抑制因子和水蛭肽嵌合蛋白的突变体。该突变体翻译表达时不易产生异构体,产品纯度更高,且具有能够抑制白细胞的转移聚集又能够抑制凝血酶的生物活性更高。
本发明在重组白细胞抑制因子和水蛭肽嵌合蛋白TNHH(氨基酸序列如SEQ.ID NO:2所示)的基础上,对易导致翻译表达错配的半胱氨酸进行突变,得到纯度更高的TNHH突变体。并将与凝血酶结合部位的部分非极性氨基酸突变为其他氨基酸,进一步提高TNHH突变体的生物活性。
本发明的第一方面,提供一种重组白细胞抑制因子和水蛭肽嵌合蛋白的突变体,与原始TNHH相比,所述的TNHH突变体的氨基酸发生突变,得到一类高纯度,高生物活性,抑制白细胞粘附力的TNHH突变体。其中所述的突变一方面是将含有二硫键易配错的半胱氨酸突变为侧链较短的氨基酸;另一方面将与凝血酶结合部位的非极性氨基酸突变为其他氨基酸。
一种重组白细胞抑制因子和水蛭肽嵌合蛋白的突变体,其氨基酸序列如下所示;
包含:
Asn-Glu-His-Asn-Leu-Arg-Cys-Pro-Gln-Asn-Gly-Thr-Glu-Met-Pro-Gly-Phe-Asn-Asp-Ser-Ile-Arg-Leu-Gln-Phe-Leu-Ala-Met-His-Asn-Gly-Tyr-Arg-Ser-Lys-Leu-Ala-Leu-Gly-His-Ile-Ser-Ile-Thr-Glu-Glu-Ser-Glu-Ser-Asp-Asp-Asp-Asp-Asp-Phe-Gly-Phe-Leu-Pro-Asp-Phe-Ala-Pro-Arg-Ala-Ser-Lys-Met-Arg-Tyr-Leu-Glu-Tyr-Asp-Cys-Glu-Ala-Glu-Lys-Ser-Ala-Tyr-Met-Ser-Ala-Arg-Asn-Cys-Ser-Asp-Ser-Ser-Ser-Pro-Pro-Glu-Gly-Tyr-Asp-Glu-Asn-Lys-Tyr-Ile-Phe-Glu-Asn-Ser-Asn-Asn-Ile-Ser-Glu-Ala-Ala-Leu-Lys-Ala-Met-Ile-Ser-Trp-Ala-Lys-Glu-Ala-Phe-Asn-Leu-Asn-Lys-Thr-Lys-Glu-Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser-Asn-His-Asp-Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-Ala-Arg-Glu-Lys-Phe-Gly-Xaa162-Ala-Val-Val-Asn-Cys-Pro-Leu-Gly-Glu-Ile-Asp-Asp-Glu-Thr-Asn-His-Asp-Gly-Glu-Thr-Tyr-Ala-Thr-Thr-Ile-His-Val-Val-Cys-His-Tyr-Pro-Lys-Ile-Asn-Lys-Thr-Glu-Gly-Gln-Pro-Ile-Tyr-Lys-Val-Gly-Thr-Pro-Xaa211-Asp-Asp-Xaa214-Ser-Glu-Tyr-Thr-Lys-Lys-Ala-Asp-Asn-Thr-Thr-Ser-Ala-Asp-Pro-Val-Cys-Ile-Pro-Asp-Asp-Gly-Val-Cys-Phe-Ile-Gly-Ser-Lys-Ala-Asp-Tyr-Asp-Ser-Lys-Glu-Phe-Tyr-Arg-Phe-Arg-Glu-Leu-Gly-Gly-Gly-Gly-Gly-Phe-Pro-Arg-Pro-Gly-Ser-Gly-Gly-Asn-Gly-Asp-Xaa274-Glu-Glu-Xaa277-Xaa278-Glu-Glu-Tyr-Xaa282。
其中,
Xaa162为Cys,Ala,Leu,Gly,Ser或Val中的一种;
Xaa211为Cys,Ala,Leu,Gly,Ser或Val中的一种;
Xaa214为Ala,Leu,Gly,Ser,或Val中的一种;
Xaa274为Phe,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa277为Ile,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa278为Pro,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa282为Leu,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种。
优选地,
Xaa162为Cys,Ala,Ser或Val中的一种;
Xaa211为Cys,Ala,Gly或Ser中的一种;
Xaa214为Ala或Gly中的一种;
Xaa274为Phe,Asn,Glu,Thr,Gly或Ser中的一种;
Xaa277为Ile,Cys,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa278为Pro,Cys,Thr,Tyr,Gly或Ser中的一种;
Xaa282为Leu,Glu,Gln,Thr,Tyr,Gly中的一种。
更进一步优选,
Xaa162为Cys或Ala;
Xaa211为Cys或Ala;
Xaa214为Ala;
Xaa274为Phe或Ser;
Xaa277为Ile或Gly;
Xaa278为Pro或Thr;
Xaa282为Leu或Glu。
在一个实施例中,所述的TNHH突变体
Xaa162为Ala;
Xaa211为Cys;
Xaa214为Ala;
Xaa274为Phe;
Xaa277为Ile;
Xaa278为Thr;
Xaa282为Glu;所述的TNHH突变体的氨基酸序列如SEQ.ID NO:3所示。
在另一个实施例中,所述的TNHH突变体;
Xaa162为Ala;
Xaa211为Cys;
Xaa214为Ala;
Xaa274为Ser;
Xaa277为Gly;
Xaa278为Pro;
Xaa282为Leu;所述的TNHH突变体的氨基酸序列如SEQ.ID NO:4所示。
在另一个实施例中,
Xaa162为Cys;
Xaa211为Ala;
Xaa214为Ala;
Xaa274为Phe;
Xaa277为Ile;
Xaa278为Thr;
Xaa282为Glu;所述的TNHH突变体的氨基酸序列如SEQ.ID NO:5所示。
在另一个实施例中,
Xaa162为Cys;
Xaa211为Ala;
Xaa214为Ala;
Xaa274为Ser;
Xaa277为Gly;
Xaa278为Pro;
Xaa282为Leu;所述的TNHH突变体的氨基酸序列如SEQ.ID NO:6所示。
本发明的第二方面,提供一种多核苷酸,所述多核苷酸编码本发明所述的TNHH突变体。
本发明的第三方面,提供一种表达载体,所述表达载体包含第二方面所述的多核苷酸。在一个实施例中,所述的表达载体为pET-3c。
本发明的第四方面,提供一种宿主细胞,所述宿主细胞包含第三方面所述的表达载体,或者所述宿主细胞的基因组整合有第二方面所述的多核苷酸。
在优选的实施方式中,所述的宿主细胞为原核细胞,优选大肠杆菌。
本发明的第五方面,提供TNHH突变体的制备方法。
为了获得TNHH突变体,本发明采取如下步骤:①参考TNHH原序列SEQ.ID NO:2,找出容易产生错配的氨基酸以及具有白细胞抑制作用的活性部位中没有发挥功能的氨基酸,确定突变位点,设计引物。②准备好模板引物后通过PCR扩增技术得到完整的质粒。由全自动测序仪测序确定人工合成的cDNA片段的碱基序列与所设计一致。③制备大肠杆菌感受态细胞DH5α,并将质粒转化至DH5α中,甘油菌送样测序,确定质粒与所设计一致。④将重组质粒转化至大肠杆菌BL21(DE3)pLysS中,涂平板过夜,生长单菌落,即为转化成功。⑤转化成功的大肠杆菌pET-3c-BL21(DE3)pLysS经测序分析,目的基因正确嵌入载体并且目的基因序列与所设计的相同。使TNHH突变体蛋白在宿主细胞中能够表达。最后从菌体中提取纯化得到TNHH突变体蛋白。
本发明的第六方面,提供TNHH突变体在制备治疗心脑血管疾病药物中的应用,尤其是脑缺血、脑损伤或相关并发症中的应用。
本发明的第七方面,提供一种药物组合物,所述组合物包含第一方面所述的TNHH突变体与药学上可接受的辅料。
本发明所述的药物组合物可为任何剂型,如注射剂型,其包括注射液和冻干粉等形式。制备注射剂型时,可将本发明TNHH加入一定量的无机盐或氨基酸缓冲,如磷酸盐、乙酸盐、碳酸盐、柠檬酸盐、甘氨酸、组氨酸,其中盐主要是指钠盐,其离子强度为5-100mmol/L。药物组合物的pH维持5.0-9.0。该组合物中还可以加入蛋白保护剂如白蛋白、明胶、多糖、淀粉、甘油等。其中多糖优选为甘露醇、蔗糖、海藻糖中的一种或多种。
本发明与现有技术相比,具有如下优势:
(1)将TNHH中部分Cys突变为Ala,氨基酸侧链变短,能有效降低出现错配的几率,降低了异构体的含量,提高蛋白纯度。
(2)突变TNHH基因序列中水蛭肽段的非极性氨基酸为其他极性氨基酸,能够提高其生物学活性,增强抑制白细胞粘附的能力。
(3)TNHH是一种具有双功能的嵌合蛋白,我们通过基因定点突变技术同时解决了两个难题,不仅仅降低了其异构体含量,提高了蛋白纯度,还使其生物学活性大大提高,具有很高的实践意义。
附图说明
图1是TNHH及其突变体诱导表达SDS-PAGE电泳图;
其中M:蛋白质Marker;
泳道1:TNHH使用IPTG诱导前;
泳道2:TNHH;
泳道3:突变体TNHH-M1;
泳道4:突变体TNHH-M2;
泳道5:突变体TNHH-M3;
泳道6:突变体TNHH-M4。
图2是纯化后TNHH突变体SDS-PAGE电泳图。
其中M:蛋白质Marker;
泳道1:纯化后的TNHH;
泳道2:纯化后的突变体TNHH-M1;
泳道3:纯化后的突变体TNHH-M2;
泳道4:纯化后的突变体TNHH-M3;
泳道5:纯化后的突变体TNHH-M4。
图3是TNHH的HPLC色谱图。
图4是突变体TNHH-M1的HPLC色谱图。
图5是突变体TNHH-M2的HPLC色谱图。
图6是突变体TNHH-M3的HPLC色谱图。
图7是突变体TNHH-M4的HPLC色谱图。
具体实施方式
定点突变技术
定位突变是在已知蛋白质结构与功能的基础上,在已知DNA序列中取代、插入或删除特定的氨基酸,从而产生具有新性状的突变蛋白分子的一种蛋白质工程技术。
定位突变技术可以改变蛋白的理化性质,例如,提高蛋白药物的稳定性;增强蛋白与药物的溶解性能;改善生物学性质,包括但不限于改变酶对底物的专一性、提高酶的活性,改善亲和性、特异性等等。
定点突变技术可以通过突变结合域的氨基酸达到提高或消除配体与受体,酶与底物之间的结合活性,这种突变改变的蛋白质特定造成二级结构或高级结构和所带电荷等特定的变化。如果突变的氨基酸正好处于关键的抗原-抗体作用位点,那么该氨基酸变化很有可能导致该位点的电荷和二级结构或高级结构发生改变,配体与受体,酶与底物之间不能结合从而达到突变的目的。同时也导致原来的抗体不能识别这个位点,即形成了新的抗原。这是这种传统突变方法必然可能出现的。
下面结合具体实施例来对本发明做进一步的说明,应当理解的是以下实施例仅用于说明本发明的技术方案,并不用于限制本发明的保护范围。实施例中所用到的原料、材料等如非特殊说明均可通过商业途径获得,实施例中未注明具体条件的实验方法,可通过本领域的常规技术或厂商建议的方法实施。
实施例1TNHH基因突变体的获得
一、引物合成
根据表1所示的TNHH的突变位点,使用Primer软件设计突变所需要的引物,委托上海生工设计合成,TNHH突变体所需引物见2。
表1 TNHH不同突变体突变位点
突变体 | 突变位点 |
TNHH-M1 | 第278位Pro→Thr,第282位Leu→Glu,第162、214位Cys→Ala |
TNHH-M2 | 第274位Phe→Ser,第277位Ile→Gly,第162、214位Cys→Ala |
TNHH-M3 | 第278位Pro→Thr,第282位Leu→Glu,第211、214位Cys→Ala |
TNHH-M4 | 第274位Phe→Ser,第277位Ile→Gly,第211、214位Cys→Ala |
表2 TNHH突变体所需引物
二、PCR反应
将突变TNHH原序列第162位氨基酸所需的引物,模板DNA以及缓冲体系等按如下反应体系进行混合。
混合完成后,进行PCR扩增,扩增程序如下:
得到pET-3c-TNHH-M1第一个位点突变完成质粒,然后根据表2中所需要的引物,重复以上步骤,直至pET-3c-TNHH-M1突变体四个位点突变完成,得到完整的pET-3c-TNHH-M1突变质粒,标记为pET-3c-TNHH-M1。送样至上海生工进行测序分析,确认得到的突变质粒中氨基酸序列与所设计一致。其中本发明中TNHH突变体氨基酸序列如SEQ.ID NO:3~6所示,核苷酸序列如SEQ.ID NO:7~10所示。
三、突变质粒转化DH5α
DH5α感受态细胞的制备:
1、将DH5α甘油菌划线LB平板,37℃培养过夜;
2、挑取单菌落接种至5ml LB液体培养基中,37℃,150rpm培养过夜,按1%接种量转接5ml LB培养基中,37℃,150rpm培养2-3h,测定OD600为0.4~0.6;
3、将培养菌液置于冰浴或放入4℃冰箱预冷,取1ml菌液4℃,6000rpm,离心5min收集菌体;
4、菌体中加入1ml预冷无菌的0.1mol/L的CaCl2溶液洗2次;
5、菌体中加入100μL预冷的0.1mol/L的CaCl2溶液重悬细胞,4℃保存备用;
质粒转化感受态细胞:
1、将突变产物M1分别转入感受态细胞中,轻轻混匀,冰浴30min;
2、将EP管放入预热至42℃的水浴锅中热击90秒,勿动EP管;
3、快速取出EP管,冰浴冷却3-5min;
4、每管加入800μL SOC培养基,37℃,100rpm温育45min;
5、取100μL培养液涂布LB氨苄平板,37℃培养过夜。
从平板上挑取单菌落接种至含100μg/ml氨苄青霉素的5ml LB液体培养基,37℃,150rpm培养过夜。
取4ml菌液离心收集菌体,用大连宝生物质粒提取试剂盒提取质粒,加60μL灭菌水洗脱,做好标记,-20℃保存。
四、重组质粒转化至大肠杆菌BL219(DE3)pLysS内
BL21(DE3)pLysS感受态细胞的制备:
1、将BL21(DE3)pLysS甘油菌划线LB平板,37℃培养过夜;
2、挑取单菌落接种至5ml LB液体培养基中,37℃,150rpm培养过夜,按1%接种量转接5ml LB培养基中,37℃,150rpm培养2-3h,测定OD600为0.4~0.6;
3、将培养菌液置于冰浴或放入4℃冰箱预冷,取1ml菌液4℃,6000rpm,离心5min收菌菌体;
4、菌体中加入1ml预冷灭菌的0.1mol/L的CaCl2溶液洗涤2次;
5、菌体中加入100μL预冷的0.1mol/L的CaCl2溶液重悬细胞,4℃保存备用;
质粒转化感受态细胞:
1、将突变质粒M1分别转入感受态细胞中,轻轻混匀,冰浴30min;
2、将EP管放入预热至42℃的水浴锅中热击90秒,勿动EP管;
3、快速取出EP管,冰浴冷却3-5min;
4、每管加入800μL SOC培养基,37℃,100rpm温育45min;
5、取100μL培养液涂布LB氨苄平板,37℃培养过夜。
平板上生长单菌落,即转化成功,为TNHH突变菌株pET-3c-TNHH/BL21(DE3)pLysS-M1。
同样方法制备pET-3c-TNHH/BL21(DE3)pLysS-M2、pET-3c-TNHH/BL21(DE3)pLysS-M3和pET-3c-TNHH/BL21(DE3)pLysS-M4菌株。
实施例2突变体pET-3c-TNHH/BL21(DE3)pLysS-M1~pET-3c-TNHH/BL21(DE3)pLysS-M4菌株摇瓶表达
将4种突变菌株及原始大肠菌株pET-3c-TNHH/BL21(DE3)pLysS在摇瓶上进行初步表达研究。首先从菌株相应平板上挑取单克隆接种至LB氨苄培养基(0.5%酵母粉,1%蛋白胨,1%氯化钠,100mg/L氨苄青霉素),每株接种2瓶,37℃培养10h,OD600在0.8~1.2之间,使用终浓度为0.4mol/LIPTG进行诱导表达,诱导4h培养结束。菌液12000rpm离心3min弃上清,通过SDS-PADE对其表达量进行分析,结果见图1,从图1可知基因突变后蛋白表达量与初始TNHH表达量相比有所提高。
实施例3对TNHH及突变体TNHHM1~M4进行发酵纯化
一、原始菌株pET-3c-TNHH/BL21(DE3)pLysS及突变体菌株pET-3c-TNHH/BL21(DE3)pLysS-M1~pET-3c-TNHH/BL21(DE3)pLysS-M4发酵罐发酵诱导表达
1、种子培养:
挑取原始菌株pET-3c-TNHH/BL21(DE3)pLysS、及突变体菌株pET-3c-TNHH/BL21(DE3)pLysS-M1~pET-3c-TNHH/BL21(DE3)pLysS-M4单菌落分别接种至含100mg/L氨苄青霉素的LB培养基中,37℃,150rpm培养10h,得到发酵用种子液。
2、30L发酵罐发酵:
30L发酵罐中加入10L发酵基础培养基(0.3%酵母粉,0.5%蛋白胨,3.5%十二水和磷酸氢二钠,0.5%磷酸二氢钾,0.1%氯化铵,0.1%氯化钠,0.1%硫酸镁,1%葡萄糖),高压灭菌,待培养基冷却至37℃用氨水调节pH至7.0,接种,调整转速和通气量控制溶氧量保持在30~70%进行培养。培养6h后,到溶氧回升到90%开始补料(0.3%酵母粉,0.5%蛋白胨,2%葡萄糖),补料2h后使用IPTG进行诱导表达,诱导12h结束发酵。发酵培养液,12000rpm离心10min,弃上清,收集菌体,4℃保存备用。
二、TNHH及突变体TNHH-M1-M4纯化
1、压力匀浆破菌
将pET-3c-TNHH/BL21(DE3)pLysS-M1每100g菌体加入1L破菌缓冲液,40MPa压力匀浆2-3次。4℃离心,12000rpm,15min,保留沉淀。
2、洗涤包涵体
同上体积洗涤缓冲液,充分搅拌溶解,4℃离心,12000rpm,15min,保留沉淀。然后使用同上体积溶解缓冲液充分搅拌溶解。
3、复性浓缩
使用同上体积复性液复性,10℃左右,3KD截流分子量超滤浓度20-30倍。
4、Butyl Sepharose疏水层析柱纯化
将疏水层析柱连接到AKTA层析仪,用pH8.0的平衡缓冲液(25mmol/L Tris,0.3M(NH4)2SO4 0.5mol/LNaCl)平衡,开始上样,完毕后用平衡缓冲液洗脱收集透过峰蛋白,pH8.0 25mmol/L Tris洗脱杂蛋白。用盐酸调节pH3.0~4.0沉淀目的蛋白,4℃离心,12000rpm,15min,保留沉淀。
5、Source 30Q离子交换层析柱纯化
沉淀用20mmol/L PB溶解并用0.45μm滤膜过滤。将离子交换层析连接到AKTA层析仪,用pH8.0的平衡缓冲液(20mmol/L PB,0.2mol/LNaCl)平衡,开始上样,完毕后用pH8.0的洗脱液(20mmol/L PB,0.5mol/LNaCl)收集透过峰蛋白,用磷酸调节pH3.0~4.0沉淀目的蛋白,4℃离心,12000rpm,15min,保留沉淀。
6、Superdex75层析柱纯化
将凝胶层析连接到AKTA层析仪,用pH 6.5 10mmol/L PB平衡,开始上样,完毕后用pH 6.5 10mmol/L PB洗脱,收集所得目的蛋白溶液即为突变体TNHH-M1。
同样方法得到TNHH蛋白溶液及突变体TNHH-M2~M4蛋白溶液。
对纯化后的TNHH及突变体TNHH-M1~M4蛋白溶液进行SDS-PAGE电泳,电泳图见图2,结果显示各泳道内只有单一目的条带;HPLC法测定各蛋白溶液纯度,HPLC色谱图见图3~7,根据面积归一化法计算,原始TNHH蛋白溶液纯度为95.8%;在TNHH色谱峰前有一保留时间为8.760min的杂质。从图4-7可知,突变体TNHH-M1的纯度为98.3%。突变体TNHH-M2的纯度为98.77%。突变体TNHH-M3的纯度为99.01%。突变体TNHH-M4的纯度为98.94%。突变体TNHH-M1~M4与原始TNHH蛋白溶液95.8%相比较,异构体含量明显降低,HPLC纯度显著升高。
实施例5体内生物活性测定
选取wistar大鼠90只,喂养一周后,选取体重在200~300g之间的大鼠50只,随机分为5组,即TNHH组,M1组,M2组,M3组,M4组。制备成为缺血性脑损伤模型,造模成功后,用尾静脉注射方式进行用药治疗。TNHH组给药6.5mg/kg,0.5ml/只;M1~M4组分别给药突变体TNHH-M1~M4为6.0mg/kg,0.5ml/只。每12h给药一次,连续给药14天后,处死,检测其脑梗死面积,脑组织IL-10含量,血清中TNF-α含量及MPO活性。测定结果见表3。
表3 TNHH及突变体体内生物活性测定
组别 | 脑梗死面积(%) | IL-10 | TNF-α(ng/ml) | MPO活性(U/g) |
TNHH组 | 24.51±6.61 | 23.11±3.18 | 1.035±0.131 | 0.168±0.019 |
TNHH-M1组 | 22.85±5.77 | 20.74±3.14 | 1.003±0.075 | 0.155±0.017 |
TNHH-M2组 | 21.63±3.50 | 22.97±3.41 | 0.903±0.115 | 0.161±0.014 |
TNHH-M3组 | 19.88±4.79 | 20.57±2.33 | 0.871±0.092 | 0.147±0.012 |
TNHH-M4组 | 21.34±3.27 | 21.52±2.47 | 0.984±0.147 | 0.154±0008 |
从表3可知,与TNHH相比较,突变体TNHH-M1~M4能够更有效的降低大鼠脑梗死面积,血清中TNF-α的含量也有所下降,还能够提高MPO活性,说明抗炎能力更强。
实施例6抗白细胞粘附测定
一、细胞传代
人原髓细胞白血病细胞HL-60细胞,使用完全培养基(含有20%胎牛血清的RPMI1640培养基)培养传代。
二、抑制白细胞粘附实验
1、将上述细胞离心弃上清,用完全培养基重悬计数,以1.0×106个/ml接种于培养瓶中,并加入终浓度为10ng/ml的PMA诱导剂,37℃,5%CO2培养箱培养24-36h,细胞贴壁视为诱导成功。
2、弃细胞培养液,用完全培养基洗涤三次,使用细胞刮刀轻轻刮下,并加入2ml完全培养基混匀,转移至4ml离心管中,1000rpm离心5min,弃上清,用细胞维持液(含有5%胎牛血清的RPMI1640培养基)重悬稀释至1.0~1.5×106个/ml,加入终浓度为1μM的PMA诱导剂诱导。
3、将TNHH及突变体TNHH-M1~M4待测品以50μL/孔加入96孔高效吸附细胞培养板,每个样品3个复孔。并加入100μL/孔的HL-60细胞,置于37℃,5%CO2培养箱中培养3.5h。
4、吸出上层未贴壁细胞,弃掉。96孔板用细胞维持液洗涤三次。
5、加入100μL/孔的CCK-8显色液,避光于37℃,5%CO2培养箱中培养3-5h后,酶标仪450nm测定OD值。测定结果见表4。
表4TNHH及突变体活性测定
产品名称 | 抗白细胞粘附活性(U/mg) |
TNHH | 6.0 |
TNHH-M1 | 8.0 |
TNHH-M2 | 8.2 |
TNHH-M3 | 9.4 |
TNHH-M4 | 9.1 |
从表4结果可知,突变体TNHH-M1~M4抗白细胞粘附能力比TNHH要好,活性均高于TNHH,高达8.0~9.4×104U/mg。
序列表
<110> 鲁南制药集团股份有限公司
<120> 一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体
<130> 2020
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60
atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120
atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180
ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240
gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300
aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360
tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420
agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480
ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540
gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600
ggtcaaccaa tctacaaggt tggtactcca tgtgacgact gttccgaata cactaagaag 660
gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720
ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780
ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cccagaagaa 840
tacttg 846
<210> 2
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly
1 5 10 15
Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr
20 25 30
Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu
35 40 45
Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg
50 55 60
Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser
65 70 75 80
Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu
85 90 95
Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser
100 105 110
Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn
115 120 125
Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His
130 135 140
Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe
145 150 155 160
Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr
165 170 175
Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His
180 185 190
Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly
195 200 205
Thr Pro Cys Asp Asp Cys Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr
210 215 220
Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile
225 230 235 240
Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu
245 250 255
Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly
260 265 270
Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu
275 280
<210> 3
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly
1 5 10 15
Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr
20 25 30
Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu
35 40 45
Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg
50 55 60
Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser
65 70 75 80
Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu
85 90 95
Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser
100 105 110
Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn
115 120 125
Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His
130 135 140
Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe
145 150 155 160
Gly Ala Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr
165 170 175
Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His
180 185 190
Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly
195 200 205
Thr Pro Ala Asp Asp Cys Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr
210 215 220
Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile
225 230 235 240
Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu
245 250 255
Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly
260 265 270
Asp Phe Glu Glu Ile Thr Glu Glu Tyr Glu
275 280
<210> 4
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly
1 5 10 15
Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr
20 25 30
Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu
35 40 45
Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg
50 55 60
Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser
65 70 75 80
Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu
85 90 95
Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser
100 105 110
Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn
115 120 125
Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His
130 135 140
Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe
145 150 155 160
Gly Ala Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr
165 170 175
Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His
180 185 190
Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly
195 200 205
Thr Pro Ala Asp Asp Cys Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr
210 215 220
Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile
225 230 235 240
Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu
245 250 255
Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Ser Gly
260 265 270
Asp Gly Glu Glu Ile Pro Glu Glu Tyr Leu
275 280
<210> 5
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly
1 5 10 15
Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr
20 25 30
Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu
35 40 45
Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg
50 55 60
Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser
65 70 75 80
Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu
85 90 95
Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser
100 105 110
Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn
115 120 125
Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His
130 135 140
Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe
145 150 155 160
Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr
165 170 175
Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His
180 185 190
Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly
195 200 205
Thr Pro Ala Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr
210 215 220
Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile
225 230 235 240
Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu
245 250 255
Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Asn Gly
260 265 270
Asp Phe Glu Glu Ile Thr Glu Glu Tyr Glu
275 280
<210> 6
<211> 282
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asn Glu His Asn Leu Arg Cys Pro Gln Asn Gly Thr Glu Met Pro Gly
1 5 10 15
Phe Asn Asp Ser Ile Arg Leu Gln Phe Leu Ala Met His Asn Gly Tyr
20 25 30
Arg Ser Lys Leu Ala Leu Gly His Ile Ser Ile Thr Glu Glu Ser Glu
35 40 45
Ser Asp Asp Asp Asp Asp Phe Gly Phe Leu Pro Asp Phe Ala Pro Arg
50 55 60
Ala Ser Lys Met Arg Tyr Leu Glu Tyr Asp Cys Glu Ala Glu Lys Ser
65 70 75 80
Ala Tyr Met Ser Ala Arg Asn Cys Ser Asp Ser Ser Ser Pro Pro Glu
85 90 95
Gly Tyr Asp Glu Asn Lys Tyr Ile Phe Glu Asn Ser Asn Asn Ile Ser
100 105 110
Glu Ala Ala Leu Lys Ala Met Ile Ser Trp Ala Lys Glu Ala Phe Asn
115 120 125
Leu Asn Lys Thr Lys Glu Gly Glu Gly Val Leu Tyr Arg Ser Asn His
130 135 140
Asp Ile Ser Asn Phe Ala Asn Leu Ala Trp Asp Ala Arg Glu Lys Phe
145 150 155 160
Gly Cys Ala Val Val Asn Cys Pro Leu Gly Glu Ile Asp Asp Glu Thr
165 170 175
Asn His Asp Gly Glu Thr Tyr Ala Thr Thr Ile His Val Val Cys His
180 185 190
Tyr Pro Lys Ile Asn Lys Thr Glu Gly Gln Pro Ile Tyr Lys Val Gly
195 200 205
Thr Pro Ala Asp Asp Ala Ser Glu Tyr Thr Lys Lys Ala Asp Asn Thr
210 215 220
Thr Ser Ala Asp Pro Val Cys Ile Pro Asp Asp Gly Val Cys Phe Ile
225 230 235 240
Gly Ser Lys Ala Asp Tyr Asp Ser Lys Glu Phe Tyr Arg Phe Arg Glu
245 250 255
Leu Gly Gly Gly Gly Gly Phe Pro Arg Pro Gly Ser Gly Gly Ser Gly
260 265 270
Asp Gly Glu Glu Ile Pro Glu Glu Tyr Leu
275 280
<210> 7
<211> 846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60
atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120
atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180
ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240
gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300
aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360
tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420
agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480
ggtgctgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540
gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600
ggtcaaccaa tctacaaggt tggtactcca tgtgacgacg cttccgaata cactaagaag 660
gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720
ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780
ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cactgaagaa 840
tacgaa 846
<210> 8
<211> 846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60
atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120
atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180
ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240
gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300
aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360
tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420
agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480
ggtgctgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540
gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600
ggtcaaccaa tctacaaggt tggtactcca tgtgacgacg cttccgaata cactaagaag 660
gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720
ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780
ggtggcttcc caagaccagg tagcggtggc aacggtgact ccgaagaagg tccagaagaa 840
tacttg 846
<210> 9
<211> 846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60
atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120
atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180
ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240
gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300
aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360
tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420
agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480
ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540
gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600
ggtcaaccaa tctacaaggt tggtactcca gctgacgacg cttccgaata cactaagaag 660
gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720
ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780
ggtggcttcc caagaccagg tagcggtggc aacggtgact tcgaagaaat cactgaagaa 840
tacgaa 846
<210> 10
<211> 846
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aacgaacaca acttgagatg tccacaaaac ggtactgaaa tgccaggttt caacgactcc 60
atcagattgc aattcttggc tatgcacaac ggttacagat ccaagttggc tttgggtcac 120
atctccatca ctgaagaatc cgaatccgac gacgacgacg acttcggttt cttgccagac 180
ttcgctccaa gagcttccaa gatgagatac ttggaatacg actgtgaagc tgaaaagtcc 240
gcttacatgt ccgctagaaa ctgttccgac tcctcctccc caccagaagg ttacgacgaa 300
aacaagtaca tcttcgaaaa ctccaacaac atctccgaag ctgctttgaa ggctatgatc 360
tcctgggcta aggaagcttt caacttgaac aagactaagg aaggtgaagg tgttttgtac 420
agatccaacc acgacatctc caacttcgct aacttggctt gggacgctag agaaaagttc 480
ggttgtgctg ttgttaactg tccattgggt gaaatcgacg acgaaactaa ccacgacggt 540
gaaacttacg ctactactat ccacgttgtt tgtcactacc caaagatcaa caagactgaa 600
ggtcaaccaa tctacaaggt tggtactcca gctgacgacg cttccgaata cactaagaag 660
gctgacaaca ctacttccgc tgacccagtt tgtatcccag acgacggtgt ttgtttcatc 720
ggttccaagg ctgactacga ctccaaggag ttctacagat tcagagaatt gggcggtggc 780
ggtggcttcc caagaccagg tagcggtggc aacggtgact ccgaagaagg tccagaagaa 840
tacttg 846
Claims (9)
1.一种重组白细胞抑制因子和水蛭肽嵌合蛋白突变体,其特征在于,其氨基酸序列如下所示;Asn-Glu-His-Asn-Leu-Arg-Cys-Pro-Gln-Asn-Gly-Thr-Glu-Met-Pro-Gly-Phe-Asn-Asp-Ser-Ile-Arg-Leu-Gln-Phe-Leu-Ala-Met-His-Asn-Gly-Tyr-Arg-Ser-Lys-Leu-Ala-Leu-Gly-His-Ile-Ser-Ile-Thr-Glu-Glu-Ser-Glu-Ser-Asp-Asp-Asp-Asp-Asp-Phe-Gly-Phe-Leu-Pro-Asp-Phe-Ala-Pro-Arg-Ala-Ser-Lys-Met-Arg-Tyr-Leu-Glu-Tyr-Asp-Cys-Glu-Ala-Glu-Lys-Ser-Ala-Tyr-Met-Ser-Ala-Arg-Asn-Cys-Ser-Asp-Ser-Ser-Ser-Pro-Pro-Glu-Gly-Tyr-Asp-Glu-Asn-Lys-Tyr-Ile-Phe-Glu-Asn-Ser-Asn-Asn-Ile-Ser-Glu-Ala-Ala-Leu-Lys-Ala-Met-Ile-Ser-Trp-Ala-Lys-Glu-Ala-Phe-Asn-Leu-Asn-Lys-Thr-Lys-Glu-Gly-Glu-Gly-Val-Leu-Tyr-Arg-Ser-Asn-His-Asp-Ile-Ser-Asn-Phe-Ala-Asn-Leu-Ala-Trp-Asp-Ala-Arg-Glu-Lys-Phe-Gly-Xaa162-Ala-Val-Val-Asn-Cys-Pro-Leu-Gly-Glu-Ile-Asp-Asp-Glu-Thr-Asn-His-Asp-Gly-Glu-Thr-Tyr-Ala-Thr-Thr-Ile-His-Val-Val-Cys-His-Tyr-Pro-Lys-Ile-Asn-Lys-Thr-Glu-Gly-Gln-Pro-Ile-Tyr-Lys-Val-Gly-Thr-Pro-Xaa211-Asp-Asp-Xaa214-Ser-Glu-Tyr-Thr-Lys-Lys-Ala-Asp-Asn-Thr-Thr-Ser-Ala-Asp-Pro-Val-Cys-Ile-Pro-Asp-Asp-Gly-Val-Cys-Phe-Ile-Gly-Ser-Lys-Ala-Asp-Tyr-Asp-Ser-Lys-Glu-Phe-Tyr-Arg-Phe-Arg-Glu-Leu-Gly-Gly-Gly-Gly-Gly-Phe-Pro-Arg-Pro-Gly-Ser-Gly-Gly-Asn-Gly-Asp-Xaa274-Glu-Glu-Xaa277-Xaa278-Glu-Glu-Tyr-Xaa282。
其中,
Xaa162为Cys,Ala,Leu,Gly,Ser或Val中的一种;
Xaa211为Cys,Ala,Leu,Gly,Ser或Val中的一种;
Xaa214为Ala,Leu,Gly,Ser,或Val中的一种;
Xaa274为Phe,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa277为Ile,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa278为Pro,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa282为Leu,Asn,Cys,Glu,Gln,Thr,Tyr,Gly或Ser中的一种。
2.根据权利要求1所述的突变体,其特征在于,
Xaa162为Cys,Ala,Ser或Val中的一种;
Xaa211为Cys,Ala,Gly或Ser中的一种;
Xaa214为Ala或Gly中的一种;
Xaa274为Phe,Asn,Glu,Thr,Gly或Ser中的一种;
Xaa277为Ile,Cys,Gln,Thr,Tyr,Gly或Ser中的一种;
Xaa278为Pro,Cys,Thr,Tyr,Gly或Ser中的一种;
Xaa282为Leu,Glu,Gln,Thr,Tyr,Gly中的一种。
3.根据权利要求1所述的突变体,其特征在于,
Xaa162为Cys或Ala;
Xaa211为Cys或Ala;
Xaa214为Ala;
Xaa274为Phe或Ser;
Xaa277为Ile或Gly;
Xaa278为Pro或Thr;
Xaa282为Leu或Glu。
4.根据权利要求1~3任一项所述的突变体,其特征在于,
Xaa162为Ala;
Xaa211为Cys;
Xaa214为Ala;
Xaa274为Phe;
Xaa277为Ile;
Xaa278为Thr;
Xaa282为Glu;
或Xaa162为Ala;
Xaa211为Cys;
Xaa214为Ala;
Xaa274为Ser;
Xaa277为Gly;
Xaa278为Pro;
Xaa282为Leu;
或Xaa162为Cys;
Xaa211为Ala;
Xaa214为Ala;
Xaa274为Phe;
Xaa277为Ile;
Xaa278为Thr;
Xaa282为Glu;
或Xaa162为Cys;
Xaa211为Ala;
Xaa214为Ala;
Xaa274为Ser;
Xaa277为Gly;
Xaa278为Pro;
Xaa282为Leu。
5.一种多核苷酸,所述多核苷酸编码本发明所述的TNHH突变体。
6.一种表达载体,包含权利要求5所述的多核苷酸。
7.一种宿主细胞,包含权利要求6所述的表达载体,或者所述宿主细胞的基因组整合有权利要求5所述的多核苷酸。
8.一种权利要求1所述突变体在制备治疗心脑血管疾病药物中的应用。
9.一种药物组合物,所述组合物包含权利要求1所述的突变体及药学上可接受的辅料。
Priority Applications (1)
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