CN1060521C - 一种人实体肿瘤血管内皮细胞增殖抑制因子的基因克隆方法及在抗肿瘤血管再生治疗中的应用 - Google Patents
一种人实体肿瘤血管内皮细胞增殖抑制因子的基因克隆方法及在抗肿瘤血管再生治疗中的应用 Download PDFInfo
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Abstract
一种人实体肿瘤血管内皮细胞增殖抑制因子的基因克隆方法及在抗肿瘤血管再生治疗中的应用，其特征在于：a.人endostatin基因是从人胶原蛋白十八基因1503至2055cDNA活性片段通过PCR从人肝脏cDNA库中筛选得到；b.人endostatin基因通过内切酶酶切所得到的两种基因片段；c.人endostatin基因及其b项所述2个基因片段通过基因克隆方法在大肠杆菌中表达，具有抑制血管内皮细胞增殖和抗肿瘤血管再生活性而作为生物制品用于治疗实体肿瘤。
Description
本发明是人胶原蛋白十八(human type ⅩⅧ collagen)基因1503至2055cDNA活性片段(Genomics 1994：19，494—499，见文献1)通过PCR方法从人肝脏cDNA库中筛选得到一种人实体肿瘤血管内皮细胞增殖抑制因子(human endostatin)一即人实体肿瘤血管再生抑制因子。该种因子为单一成份可作为生物制品用于抗实体肿瘤血管再生治疗之中。
实体肿瘤临床治疗方法目前是多种多样，但大多是针对不同肿瘤采用不同的方法，并且绝大部分是针对肿瘤细胞进行治疗，用抑制剂或阻断剂来阻断或抑制实体肿瘤血管再生及血液供应方面的疗法较少。用重组人endostatin蛋白进行抗肿瘤血管再生治疗在国内外尚未见报道。
本发明的目的在于提供一种专一性抑制因子，抑制不同的实体肿瘤毛细血管内皮细胞增殖和血管再生，以阻断或专一抑制肿瘤的血液供应及营养来源，使实体肿瘤萎缩，肿瘤细胞凋亡坏死。
本发明的目的可以通过以下措施来达到：
一种人实体肿瘤血管内皮细胞增殖抑制因子(human endostatin)—即人实体肿瘤血管再生抑制因子的基因克隆方法及作为生物制品在治疗实体肿瘤之中的应用，其特征在于：
a．人 endostatin基因的基因克隆方法是从人胶原蛋白十八(human typeⅩⅧ collagen)基因1503至2055cDNA活性片段通过PCR方法从人肝脏cDNA库中筛选得到，首先制备两种引物分别为ATTCATATGCACAGCCACCGCGACTTCCAGCCG和GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT，以人肝脏cDNA库为模板，以PCR条件为94℃1分钟，55℃1分钟和72℃1分钟共计32个循环而得到人endostatin基因(基因核苷酸序列见附表)，通过NdeⅠ和BamHⅠ两种内切酶而克隆入PMAL—c2(NewEngland Biolabs Catalog，见文献2)和PET19b、PET9c(Novagen．Inc Catalog，见文献3)基因载体，然后在大肠杆菌中表达，纯化得到有活性的重组人endostatin蛋白质；
b．采用PstⅠ内切酶从人endostatin基因N末端切去162个核苷酸或采用PstⅠ和SmaⅠ内切酶分别从人endostatin基因N末端和C末端切去162和264个核苷酸后所得基因片段而克隆入PMAL—c2和PET15b、PET9c基因载体，然后在大肠杆菌中表达，纯化得到有活性的重组人endostatin蛋白片段；
c．将纯化得到的重组人endostatin蛋白质及b项所述2个重组蛋白片段加入含有bFGF的牛肾上腺毛细血管内皮细胞培养液中，培养3天后通过细胞计数，测量它们的毛细血管内皮细胞增殖抑制作用活性强度。
本发明的人endostatin基因可通过PCR等基因克隆方法获得。重组人endostatin蛋白可通过基因工程蛋白质纯化方法获得。
实施例：
1、合成两种引物分别为ATTCATATGCACAGCCACCGCGACTTCCAGCCG和GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT。
2、以1微升人肝脏cDNA为模板，加入1项所述两种引物各2微升(浓度为每微升0．1微克)，加入5微升2．5mMdNTP、5微升10 PCR缓冲液和1微升Taq或PwoDNA聚合酶，在PCR仪上94℃1分钟，55℃1分钟和72℃1分钟共计32个循环，然后用1．5％琼脂糖凝胶电泳分离得到人endostatin基因。
3、从2项得到的人endostatin基因采用NdeⅠ和BamHⅠ内切酶酶切，克隆入PMAL—c2和PET15b、PET9c基因载体之中，然后分别在大杆菌中表达重组人endostatin蛋白，分别采用amylose resin beads按照制造者手册(NewEngland Biolabs Catalog，见文献2)纯化重组人endostatin蛋白和采用超滤、高速离心、凝胶过滤等常规生化纯化手段纯化重组人endostatin蛋白(蛋白质氨基酸顺序见附表)。
4、人endostatin基因采用PstⅠ内切酶从人endostatin基因N末端切去162个核苷酸，或采用PstⅠ和SmaⅠ内切酶从人endostatin基因的N末端和C末端分别切去162个和264个核苷酸后所得二种基因片段而克隆入PMAL—c2和PET15b、PET9C基因载体之中，然后按3项所述方法纯化得到重组人endostatin活性蛋白片段。
5、采用牛肾上腺毛细血管内皮细胞(BCE)培养至24孔培养板上，细胞浓度为每毫升25000个细胞，每毫升加入1纳克bFGF和0．008微克至1微克不同浓度的重组人endostatin蛋白片段及两种不同的重组人endostatin蛋白片段，培养3天后通过细胞计数来测量抑制毛细血管内皮细胞增殖的活性。
6、小鼠皮下接种肺癌细胞使肿瘤长至100—200mm3时，将小鼠随机分成三组，一组作对照，另二组分别皮下注射重组人endostatin蛋白及一种人endostatin蛋白片段，每天每公斤体重注射20毫克，连续注射20天，以观察在动物体内抑制实体肿瘤血管再生的作用。
本发明与现有技术相比较具有如下优点：
重组人endostatin蛋白用于抑制人实体肿瘤血管再生理论上优于用鼠重组endostatin蛋白，这是因为采用人的endostatin基因产物用于治疗人实体肿瘤是属于同源性，不被机体所排斥。其中重组鼠endostatin蛋白与重组人endostatin蛋白的氨基酸序列仅有85．33％的同源性(见附表)。重组人endostatin蛋白专一性作用于增殖的毛细血管内皮细胞，对机体许多其它细胞不起作用，而实体肿瘤细胞增殖扩大必须伴随肿瘤内血管不断再生，肿瘤内再生的毛细血管内皮不断增殖，肿瘤内已有的毛细血管内皮细胞也不断增殖，所以重组人endostatin蛋白专一性抑制这些不断增殖的内皮细胞，使肿瘤血管不能再生或抑制血管再生，肿瘤组织得不到足够的营养供应，也得不到血管内皮细胞增殖所带给肿瘤细胞的刺激，从而实体肿瘤产生萎缩，肿瘤细胞凋亡坏死。本发明的另一特点是提供了治疗肿瘤的另一手段，则可从抑制实体肿瘤的血液营养供应来达到治疗的目的，并且这一疗法还可结合已有的许多针对肿瘤细胞的疗法(如放疗、化疗等)结合进行，从而从抑制实体肿瘤血管再生和间接、直接杀死肿瘤细胞两种疗法同时进行治疗，这样能大大提高其疗效。本发明的第三个特点是人endostatin基因在大肠杆菌中表达纯化得到人endostatin蛋白，工艺相对简单，成本相对低廉，利于推广应用。具有活性的两个重组人endostatin蛋白片段分子量小，易于进入体内。尤其是最小的一种重组人endostatin蛋白片段仅42个氨基酸，便于今后人工合成而大规模生产。
附表：一种人实体肿瘤血管内皮细胞增殖抑制因子(human endostatin)
cDNA核苷酸序列The nucleotide sequence of human endostatin cDNA
CATATGCACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGHuman H S H R D F Q P V L H L V A L N S P L S G G MMouse T Q T
CGGGGCATCCGCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCHuman R G I R G A D F Q C F Q Q A R A V G L A G T F R AMouse S
TTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACHuman F L S S R L Q D L Y S I V R R A D R A A V P I V NMouse G S
CTCAAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCAHuman L K D E L L F P S W E A L F S G S E G P L K P G AMouse V S D S Q Q V Q
CGCATCTTCTCCTTTGACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGGCTCGHuman R I F S F D G K D V L R H P T W P Q K S V W H G SMouse R A
GACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGHuman D P N G R R L T E S Y C E T W R T E A P S A T G QMouse S M T T G
GCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACATCGTGCTCTGCHuman A S S L L G G R L L G Q S A A S C H H A Y I V L CMouse S E K N S
ATTGAGAACAGCTTCATGACTGCCTCCAAGTGAHuman I E N S F M T A S KMouse S (F)*Human(人)，Mouse(小鼠)
Claims (1)
1．一种人实体肿瘤血管内皮细胞增殖抑制因子的基因克隆方法，其特征在于：a：人endostatin基因的基因克隆方法是从如附表所示的人胶原蛋白十八(human type ⅩⅧ collagen)基因1503至2055cDNA活性片段通过PCR方法从人肝脏cDNA库中筛选得到，首先制备两种引物分别为ATTCATATGCACAGCCACCGCGACTTCCAGCCG和GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT，以人肝脏cDNA库为模板，以PCR条件为94℃1分钟，55℃1分钟和72℃1分钟共计32个循环而得到人endostatin基因，通过NdeⅠ和BamHⅠ两种内切酶而克隆入PMAL—c2和PET15b、PET9c基因载体，然后在大肠杆菌中表达，纯化得到有活性的重组人endostatin蛋白质；b．采用PstⅠ内切酶从人endostatin基因N末端切去162个核苷酸后得到的基因片段克隆入PMAL—c2基因载体中；采用PstⅠ和SmaⅠ内切酶分别从人endostatin基因N末端和C末端切去162个和264个核苷酸后所得到的基因片段克隆入PET15b和PET9c基因载体中，然后分别在大肠杆菌中表达，纯化得到有活性的两种重组人endostatin蛋白片段。
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| US6346510B1 (en) * | 1995-10-23 | 2002-02-12 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| ES2206602T3 (es) | 1995-10-23 | 2004-05-16 | The Children's Medical Center Corporation | Compuestos y metodos terapeuticos inhibidores de angiogenesis. |
| AU2004202593B2 (en) * | 1995-10-23 | 2008-01-03 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| CN105646701B (zh) * | 2016-03-29 | 2019-03-05 | 徐根兴 | 不同氨基酸结构的重组人内皮抑素蛋白及其制备方法和应用 |
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