CN1141142C - 人乳腺癌muc-1抗原基因工程疫苗 - Google Patents
人乳腺癌muc-1抗原基因工程疫苗 Download PDFInfo
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Abstract
本发明提供了一种对人乳腺癌有明确的预防和治疗作用的基因工程疫苗。这种疫苗是将结核杆菌(卡介苗)热休克蛋白65(BCG HSP65)的编码基因和人乳腺癌细胞表达的MUC1抗原细胞毒性T淋巴细胞(CTL)表位基因融合在一起，在大肠杆菌表达的HSP-MUC1抗原CTL表位融合蛋白。本发明的疫苗可有效地预防和治疗乳腺癌。
Description
技术领域
本发明涉及一种基因工程重组蛋白疫苗，特别是涉及一种预防和治疗人乳腺癌的基因工程疫苗。
背景技术
粘蛋白(mucins)是表达在乳腺癌细胞表面的一种蛋白质，和肿瘤细胞的侵袭力相关的粘蛋白被称为MUC1。过度表达的MUC1可以减弱细胞和细胞间的相互作用，促进细胞间的解离和瘤细胞发生转移。乳腺癌细胞表达的MUC1分子具有很强的免疫原性(Segal-Eiras A，et al.Breast cancer associated mucin：a review.Allergol Immunopathol(Madr)1997 Jul-Aug；25)，被认为是免疫细胞攻击的靶点。因此，采用MUC1分子制成的疫苗可能对乳腺癌有预防和治疗作用。在这方面，已有人做了一些工作：
Brossart P等用加载MUC1细胞毒性T淋巴细胞(CTL)表位(PDTRPAPGSTAPPAHGVTSA)多肽的人树突状细胞体外免疫可诱导出乳腺癌细胞特异性的CTL(Brossart P，et al，Iddentification of HLA-A2-rrestricted T cell epitopes derived from theMUC1 tumor antigen for broadly applicable vaccine therapies.Blood 1999 Jun 15；93(12)：4309-17)。Lees CJ等用MUC-1 mannan(一种自酵母细胞壁分离出来的多聚甘露糖)偶联物免疫小鼠诱导了MHC I类抗原限制的CTL，此CTL可杀伤乳腺癌细胞(Lees CJ，et al.Theeffect of T1 and T2 cytokines on the cytotoxic T cell response to mannan-MUC 1.CancerImmunol Immunother 2000 Feb；48(11)：644-52)。Reddish M等将由16个氨基酸组成的MUC1多肽(GVTSAPDTRPAPGSTA)和KLH(匙孔槭血蓝素)的偶联物免疫16位乳腺癌转移的病人，在7位病人诱导出了对表达MUC1肿瘤细胞有杀伤作用的MHC I限制的CTL(Reddish M，et al.Anti-MUC-1class I restricted CTLs in metastatic breast cancer patientsimmunized with a synthetic MUC1 peptide，Int J Cancer 1998 Jun 10；76(6)：817-23)。Apostolopoulous V等用MUC1多肽(20氨基酸残基)-Mannan融合蛋白免疫动物可诱导特异性CTL，使受免疫动物产生对接种肿瘤细胞的保护性免疫力(Apostolopoulous V，et al.Cyclosphosphamide enhances the CTL precursor frequency in mice immunized with MUC1-mannan fusion protein(M-FP)，J Immunotherapy，1998 Mar；21(2)：109-13)。Karanikas V等采用MUC1-mannan融合蛋白免疫25位乳腺癌、结肠癌、胃癌和直肠癌患者，在2人诱导了T淋巴细胞的增生(Karanikas V.et al.Antibody and T cell responses of patients withadenocarcinoma immunized with mannan-MUC1 fusion protein.J Clin Invest 1997 Dec1；100(11)：2783-92)。Goydos JS等以BCG为佐剂，用150个氨基酸残基的MUC1合成肽免疫63个乳腺腺癌患者，诱导出MUC1特异性CTL(Goydos JS，et al.A phase I trial of asynthetic mucin peptide vaccine.Induction of specific immune reactivity in patients withadenocarcinoma)。
细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)是人体杀伤肿瘤细胞的最高效的免疫细胞。任何一种激发机体抗肿瘤免疫的疫苗是否有效，决定于这种疫苗是否能激发肿瘤特异性CTL。外源性的蛋白类肿瘤抗原在免疫人体后，通常被抗原提呈细胞摄取、加工，进入MHC II类提呈途径，激发体液免疫反应(Heikema A，Agsteribbe E，Wilschut J，Huckriede A.Generation of heat shock protein-based vaccines by intracellular loading of gp96with antigenic peptides.Immunol Lett 1997 Jun 1；57(1-3)：69-74)，但不能有效激活肿瘤特异性的CTL的产生，因而不能产生有效的肿瘤预防和治疗作用。因此，赋予MUC1特异性激活CTL的性质就成为研究以MUC-1为抗原的、预防和治疗人乳腺癌疫苗的一个技术关键。
热休克蛋白(heat shock protein，HSP)是存在于多种生物体内的一个分子伴侣蛋白质家族。在免疫应答的过程中，热休克蛋白可表现如下三种基本的功能：1、协助抗原性物质进入包括树突状细胞在内的抗原提呈细胞；2、在抗原提呈细胞中和加工处理的抗原物质相互作用使之进入MHC I类抗原提呈途径，进而激活抗原特异性CTL；3、刺激树突状细胞表达协同刺激分子(如B7等)和分泌细胞因子。
近年的研究表明，HSP可使非共键结合的肿瘤抗原在抗原提呈细胞内加工后和MHC I类分子结合并提呈在其表面，有效地激活CTL去高效杀伤表达特异性肿瘤抗原的肿瘤细胞。注射从自体肿瘤提取的热休克蛋白-抗原肽复合物可抑制荷瘤小鼠原发肿瘤的生长、降低肿瘤的转移率，延长荷瘤小鼠的生存时间(Tamura Y.Peng P.Liu K.Daou M，SrivastavaPK.Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations.Science1997 Oct 3；278(5335)：117-20)。
发明内容
本发明的目的是将结核杆菌(BCG)热休克蛋白65(HSP65)基因和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因融合在一起，研制一种对人乳腺癌有预防和治疗作用的基因工程重组疫苗。
本发明将结核杆菌(BCG)热休克蛋白65(BCG HSP65)的编码基因和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因相连接，连接方式是结核杆菌HSP65位于5’端，MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因连接于BCG HSP65的3’端。在BCGHSP65-MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的3’端连接6聚组氨酸编码序列，得到重组融合蛋白疫苗。编码所述融合蛋白的基因序列是SEQ ID NO：1所示的核苷酸序列；所述融合蛋白的结构是SEQ ID NO：2所示的氨基酸序列。所述的人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因序列是SEQ ID NO：3所示的核苷酸序列。
本发明的疫苗的制备方法是：1、获取结核杆菌(BCG)65KD热休克蛋白(HSP65)的编码基因
1)培养结核杆菌(BCG)
2)提取结核杆菌基因组DNA
3)用PCR方法自结核杆菌分离热休克蛋白65(HSP65)结构基因
4)PCR产物的克隆
5)PCR产物的序列分析2、构建HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因3、克隆HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因
1)用NcoI和HindIII消化PCR产物。
2)消化产物经脂糖凝胶电泳分离。
3)切下琼脂糖凝胶上的DNA电泳带。
4)将回收的PCR产物以NcoI和HindIII切点克隆入原核细胞表达载体(pET-28a(+)质粒)。
5)将含HSP-65基因的重组pET-28a(+)质粒转化大肠杆菌。4、测定HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的序列。5、表达HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因。6、纯化HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位融合蛋白。7、分离HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因融合蛋白。8、鉴定HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因融合蛋白的纯度。
此融合蛋白即为结核杆菌热休克蛋白65-人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因融合蛋白基因工程疫苗。
具体实施方式
实施例1 获取卡介苗结核杆菌(BCG)65KD热休克蛋白(HSP65)的编码基因
1、卡介苗结核杆菌来源于长春生物制品研究所。
2、采用苏通马铃薯培养基培养卡介苗结核杆菌，培养的温度为37-39℃，生长出的卡介苗结核杆菌呈现干皱浅黄色的菌膜。收集菌膜，从中提取卡介苗结核杆菌基因组DNA。(提取结核杆菌基因组DNA的方法参照MolecularCloning一书(J.Sambrook，Isolation of high-molecular-weight DNA from mammalian cells，9.16-9.22，ColdSpring Harbor Laboratory Press，Molecular cloning，1989)。)
3、采用PCR方法自结核杆菌分离热休克蛋白65(HSP65)结构基因。采用的5’端引物序列为5’CCATG GCC AAG ACA ATT GCG3’，3’端引物序列为5’ACC GAA TTC GCTAGC CAT ATG GAA ATC CAT GCC ACC CAT 3’。
所述PCR操作程序是：在一500μl微量离心管中加入下列试剂：模板cDNA5μl(mmol/L)10×PCR缓冲液(含氯化镁)5μldNTPs(10mmol/L)1μl5’端和3’端引物(0.01mmol/L)各0.5μlTaq DNA聚合酶(5u/μl)0.25μl加去离子水至终体积50μl混合后加入矿物油3滴反应条件：94℃，30″；55℃，1’；72℃，2’，30个循环周期后，72℃延伸10分钟。
4、PCR产物的克隆
采用TA克隆方法克隆PCR产物，方法见文献(于永利，麻彤辉，杨贵贞：TA克隆及双链DNA测序，介绍一种快速克隆及分析PCR产物的方法，中国免疫学杂志，1994，10(1)：5)。
5、PCR产物的序列分析
按常规方法(J.Sambrook，Polyacrylamide gel electrophoresis 1.21-1.32，Cold Spring Harbor Laboratory Press，Molecular cloning，1989)提取质粒，采用双脱氧末端终止法，对插入片段进行序列测定。
实施例2构建HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因
采用PCR方法合成HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因，模板为结核杆菌65KD热休克蛋白(HSP)的编码基因(浓度为0.01pmol/L)，PCR引物的为，5’端引物序列为：5’ttc gcc atg gcc aag aca att gcg 3′，3′端引物的序列为：5’ggc cgc aag ctt cag agc cgg acg gtt gtc cgg agc aga ggt aac acc gtg agc cgg cgg agcggt aga acc cgg agc cgg acg ggt gtc cgg agc aga ggt aac acc gtg agc cgg cgg agc ggt aga acc gaattc gct agc cat atg caa atc 3′
反应条件为：94℃，30″；55℃，1’；72℃，4’，30个循环周期后，72℃延伸10分钟。
实施例3 HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因融合
基因的克隆
1、用NcoI和HindIII在37℃消化PCR产物，时间为2小时。
2、消化产物琼经脂糖凝胶电泳分离。电泳的条件是：1％琼脂糖凝胶，1×TAE缓冲液，150-200mA，电泳0.5-1小时。20×TAE缓冲液：0.8mol/L Tris base0.4mol/L NaOAc0.04mol/L Na2 EDTA用冰醋酸调pH8.3。
3、在紫外灯下观察并切下琼脂糖凝胶上的DNA电泳带。将含DNA区带的琼脂糖凝胶切下，置-70℃冷冻15分钟，然后在65℃水浴中使胶融化；加入等倍体积TE-饱合酚，剧烈振荡30秒钟，然后-70℃冷冻15分钟；室温融化后，12,000rpm离心5分钟，将上层液相移至另一管中，用2-2.5倍乙醇沉淀、洗涤和干燥DNA。
4、将回收的PCR产物
克隆入经限制性内切酶NcoI和HindIII消化的原核细 胞表达载体pET-28a(+)质粒(美国Novagen公司)的6聚组氨酸(histidine)密码子的上游。
质粒DNA的消化反应：1μg质粒DNA1μl10×缓冲液(见Promega Corporation产品说明书)。1μl限制性内切酶NcoI(10单位/μl)1μl限制性内切酶HindIII(10单位/μl)用双蒸水补齐至10μl混合后37℃温育30-120分钟。
连接反应：质粒DNA(0.5μg/μl)2μlDNA插入片段(300ng/μl)5μl10×连接缓冲液(见Protocols and Applications Guide，p57，Promega Corporation，Second Edition.1991)1μlT4 DNA连接酶1μl用双蒸水补齐至10μl混合后置14-16℃水浴6-12小时。
5、将含HSP-65基因的重组pET-28a(+)质粒转化大肠杆菌。感受态细胞的制备：
a、将大肠杆菌在LB琼脂培养基上划线，37℃培养12-16小时；
b、次日从琼脂平板上取一单菌落于2ml LB培养基中，37℃以225rpm速度震荡培养12-16小时；
c、取1ml上述培养物接种于100ml LB培养基中，37℃以225rpm的速度震荡培养直至OD值为0.5左右(大约3小时)；
d、将菌液冰浴2小时，然后2,500Xg，4℃离心20分钟收集菌体；
e、加入100ml冰冷的Trituration缓冲液(100mmol/LCaCl2，70mmol/L MgCl2，40mmol/L醋酸钠，pH5.5)，混匀，置冰上45分钟；
f、1,800Xg，4℃离心10分钟，弃上清，加入10ml冰冷的Trituration缓冲液悬浮细胞；
g.按每份200ul分装，4℃可保存1-2周。若需长期保存，可加甘油至终浓度为15％，置-70℃备用。
转化方法：
a、将200ul感受态细胞置冰上融化，然后加入3ul DMSO或β-巯基乙醇，混合后，加入3-5μl连接反应液(含重组质粒)，温和混匀，置冰上30分钟；
b、42℃ 45秒，然后迅速放回冰中1-2分钟；
c、加入2ml LB培养液，37℃以225rpm的速度摇荡培养1小时；
d、4,000Xg离心10秒钟，弃上清，用200ul LB培养液重悬菌体；
e、将菌液铺于含有适当抗生素的LB琼脂培养板上，涂匀，室温放置20-30分钟，倒置于37℃孵箱中培养12-16小时。用限制性内切酶消化的方法鉴定重组克隆。
f、质粒和菌株的保存：质粒冰冻保存于-20℃。菌株在含20-50％甘油培养液中保存于-20℃或-70℃。
HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因的测序(采用双脱氧末端终止法，具体方法见文献：于永利，麻彤辉，杨贵贞.TA克隆及双链DNA测序：介绍一种快速克隆及分析PCR产物的方法.中国免疫学杂志，1994，10(1)：5)结果表明，所得到的HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因和设计的完全一致。
实施例4 HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位基因的融合基因的表达
1、将单菌落的细菌接种于50ml LB培养基中，于250ml三角烧瓶中，37℃水浴震荡培养至OD600为0.6。
2、入IPTG使其终浓度为0.4mM，37℃水浴震荡培养2-3小时。
3、三角烧瓶于冰上5分钟，4℃离心5分钟(5000xg)。
4、吸弃上清，收集细菌，立即使用或冻存。
实施例5 HSP-65和人MUC-1抗原细胞毒性T淋巴细胞(CTL)表位融合蛋白的纯化
1、将大肠杆菌细胞(3g湿重)于室温下解冻。组织经组织破碎器短暂匀浆后，重悬于20ml含0.2％脱氧胆酸钠(DOC)的缓冲液A。
缓冲液A(1000ml)的配制：终浓度1mol/L Tris.HCL(pH7.9)50ml 50mmol/L0.5mol/EDTA 1ml 0.5mmol/L5mol/L NaCL 10ml 50mmol/L100％甘油50ml 5％
2、用台式超声细胞破碎仪(SANYO，MSE SONIPREP 150)，设8个循环和50％的时间间隔，在冰浴中超声90秒破菌，搅拌10-20min。
3、加DOC，使终浓度为0.2％(即，加约240ul的20％脱氧胆酸钠(DOC)。混匀，静置10min。20％脱氧胆酸钠(DOC)贮存液应提前1天配制，配方为10g无水DOC，溶于水中，调体积至50ml。得粗制裂解物(A)
4、13000rpm，4℃离心10min。轻轻倾出上清。
5、加18ml缓冲液A和2ml 20％DOC贮存液于包涵体沉淀。用组织破碎器充分重悬沉淀，室温下静置至少10分钟。将悬液于4℃离心10min，13000rpm。
6、重复步骤5。
7、加39.4ml缓冲液A和0.6ml 20％十二烷基肌氨酸钠(SKL)储液(SKL净浓度为0.3％)。剧烈搅动使沉淀慢慢溶解。静置至少30分钟。
十二烷基肌氨酸钠(SKL)储液：10g无水十二烷基肌氨酸钠(SKL)，溶于H2O中，调体积至50ml。
8、将悬液于4℃离心10min，13000rpm。
9、用缓冲液A+0.3％SKL稀释溶解的蛋白质使其浓度为1mg/ml。用缓冲液A将溶解的蛋白质稀释10倍，使其浓度为0.1mg/ml，SKL的终浓度为0.03％。
10、在4℃溶解的蛋白质制备物(体积约为400ml)对缓冲液A(体积约为4000ml)透析8小时，同时充分搅拌。然后换新鲜缓冲液并重复。
11、从透析袋(口径1cm)中移出经透析的蛋白质溶液，4℃、8000rpm离心20min，去除所有的的聚集物。留取上清，做镊-Saphrose-4-B层析分离HSP-65和前列腺特异性抗原(PSA)的T细胞表位的融合基因融合蛋白。
12、采用常规的SDS-PAGE方法(J.Sambrook，Polyacrylamide gelelectrophoresis 6.36-6.49，Cold Spring Harbor Laboratory Press，Molecularcloning，1989)。鉴定HSP-65和前列腺特异性抗原(PSA)的T细胞表位的基因融合蛋白的纯度，纯度为：98％。
实施例6体内抑制MCF-7细胞生长实验
一、材料
1、MCF-7细胞：MCF7是人乳腺癌细胞系细胞(American Type Culture Collection，ATCC)，表达MUC-1分子，可在小鼠形成原位实体肿瘤。
2、实验动物：6周的雌性BALB/c，疫苗组和对照组各20只。
3、实验方法
1)采用DMEM培养基培养MCF7细胞。在DMEM培养基中含10％的灭活胎牛血清，2mML-谷胱甘肽，100单位/ml青霉素，100μg/ml链霉素。在注射之前，用无血清DMEM将细胞洗三次。每只小鼠注射1×107个MCF7细胞，注射部位是小鼠两肩胛骨间皮下。在6周以后，测量小鼠肿瘤块基底部的面积。
2)在接种肿瘤30天前和15天前，疫苗组分别腹腔注射100μg人乳腺癌基因工程疫苗；对照组分别腹腔注射同疫苗组同体积生理盐水。
4、实验结果及结论
动物肿瘤块基底部面积的平均值：疫苗组为61mm2，对照组为300mm2。
结论：该人乳腺癌基因工程疫苗能明显抑制人乳腺癌细胞在小鼠体内生长。
序列表SEQ ID NO：1信息(i)序列特征(A)长度：1800个碱基(B)类型：核酸(C)链性：单链(D)拓扑结构：线性(ii)分子类型：DNA(iii)序列描述：SEQ ID NO：1
1 ATGGCCAAGA CAATTGCGTA CGACGAAGAG GCCCGTCGCG GCCTCGAGCG GGGCTTGAAC 6061 GCCCTCGCCG ATGCGGTAAA GGTGACATTG GGCCCCAAGG GCCGCAACGT CGTCCTGGAA 120121 AAGAAGTGGG GTGCCCCCAC GATCACCAAC GATGGTGTGT CCATCGCCAA GGAGATCGAG 180181 CTGGAGGATC CGTACGAGAA GATCGGCGCC GAGCTGGTCA AAGAGGTAGC CAAGAAGACC 240241 GATGACGTCG CCGGTGACGG CACCACGACG GCCACCGTGC TGGCCCAGGC GTTGGTTCGC 300301 GAGGGCCTGC GCAACGTCGC GGCCGGGGCC AACCCGCTCG GTCTCAAACG CGGCATCGAA 360361 AAGGCCGTGG AGAAGGTCAC CGAGACCCTG CTCAAGGGCG CCAAGGAGGT CGAGACCAAG 420421 GAGCAGATTG CGGCCACCGC AGCGATTTCG GCGGGTGACC AGTCCATCGG TGACCTGATC 480481 GCCGAGGCGA TGGACAAGGT GGGCAACGAG GGCGTCATCA CCGTCGAGGA GTCCAACACC 540541 TTTGGGCTGC AGCTCGAGCT CACCGAGGGT ATGCGGTTCG ACAAGGGCTA CATCTCGGGG 600601 TACTTCGTGA CCGACCCGGA GCGTCAGGAG GCGGTCCTGG AGGACCCCTA CATCCTGCTG 660661 GTCAGCTCCA AGGTGTCCAC TGTCAAGGAT CTGCTGCCGC TGCTCGAGAA GGTCATCGGA 720721 GCCGGTAAGC CGCTGCTGAT CATCGCCGAG GACGTCGAGG GCGAGGCGCT GTCCACCCTG 780781 GTCGTCAACA AGATCCGCGG CACCTTCAAG TCGGTGGCGG TCAAGGCTCC CGGCTTCGGC 840841 GACCGCCGCA AGGCGATGCT GCAGGATATG GCCATTCTCA CCGGTGGTCA GGTGATCAGC 900901 GAAGAGGTCG GCCTGACGCT GGAGAACGCC GACCTGTCGC TGCTAGGCAA GGCCCGCAAG 960961 GTCGTGGTCA CCAAGGACGA GACCACCATC GTCGAGGGCG CCGGTGACAC CGACGCCATC 10201021 GCCGGACGAG TGGCCCAGAT CCGCCAGGAG ATCGAGAACA GCGACTCCGA CTACGACCGT 10801081 GAGAAGCTGC AGGAGCGGCT GGCCAAGCTG GCCGGTGGTG TCGCGGTCAT CAAGGCCGGT 11401141 GCCGCCACCG ACGTCGAACT CAAGGAGCGC AAGCACCGCA TCGAGGATGC GGTTCGCAAT 12001201 GCCAAGGCCG CCGTCGAGGA GGGCATCGTC GCCGGTGGGG GTGTGACGCT GTTGCAAGCG 12601261 GCCCCGACCC TGGACGAGCT GAAGCTCGAA GGCGACGAGG CGACCGGCGC CAACATCGTG 13201321 AAGGTGGCGC TGGAGGCCCC GCTGAAGCAG ATCGCCTTCA ACTCCGGGCT GGAGCCGGGC 13801381 GTGGTGGCCG AGAAGGTGCG CAACCTGCCG GCTGGCCACG GACTGAACGC TCAGACCGGT 14401441 GTCTACGAGG ATCTGCTCGC TGCCGGCGTT GCTGACCCGG TCAAGGTGAC CCGTTCGGCG 15001501 CTGCAGAATG CGGCGTCCAT CGCGGGGCTG TTCCTGACCA CCGAGGCCGT CGTTGCCGAC 15601561 AAGCCGGAAA AGGAGAAGGC TTCCGTTCCC GGTGGCGGCG ACATGGGTGG CATGGATTTC 16201621
GOCAATTCGG TTCTACCGCT
CTCTGCTCCG 16801682
CGGCTCCGGG TTCTACCGCT
CTCTGCTCCG 17411742
CGGCTCTGAA GCTTGCGGCC
CCACCTCTGA 1800SEQ ID NO：2信息(i)序列特征(A)长度：599个氨基酸残基(B)类型：氨基酸(C)链性：单链(D)拓扑结构：线性(ii)分子类型：蛋白质(iii)序列描述：SEQ ID NO：21 Met Ala Lys Thr Ihr Ala Tyr Asp Glu Glu Ala Arg Arg Gly Leu 1516 Glu Arg Gly Leu Asn Ala Leu Ala Asp Ala Val Lys Val Thr Leu 3031 Gly Pro Lys Gly Arg Asn Val Val Leu Glu Lys Lys Trp Gly Ala 4546 Pro Thr Ihr Thr Asn Asp Gly Val Ser Ile Ala Lys Glu Ile Glu 6061 Leu Glu Asp Pro Tyr Glu Lys lle Gly Ala Glu Leu Val Lys Glu 7576 Val Ala Lys Lys Thr Asp Asp Val Ala Gly Asp Gly Thr Thr Thr 9091 Ala Thr Val Leu Ala Gln Ala Leu Val Arg Glu Gly Leu Arg Asn 105106 Val Ala Ala Gly Ala Asn Pro Leu Gly Leu Lys Arg Gly Ile Glu 120121 Lys Ala Val Glu Lys Val Thr Glu Thr Leu Leu Lys Gly Ala Lys 135136 Glu Val Glu Thr Lys Glu Gln lle Ala Ala Thr Ala Ala Ile Ser 150151 Ala Gly Asp Gln Ser Ile Gly Asp Leu Ile Ala Glu Ala Met Asp 165166 Lys Val Gly Asn Glu Gly Val Ile Thr Val Glu Glu Ser Asn Thr 180181 Phe Gly Leu Gln Leu Glu Leu Thr Glu Gly Met Arg Phe Asp Lys 195196 Gly Tyr Ile Ser Gly Tyr Phe Val Thr Asp Pro Glu Arg Gln Glu 210211 Ala Val Leu Glu Asp Pro Tyr Ile Leu Leu Val Ser Ser Lys Val 225226 Ser Thr Val Lys Asp Leu Leu Pro Leu Leu Glu Lys Val Ile Gly 240241 Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly Glu 255256 Ala Leu Ser Thr Leu Val Val Asn Lys Ile Arg Gly Thr Phe Lys 270271 Ser Val Ala Val Lys Ala Pro Gly Phe Gly Asp Arg Arg Lys Ala 285286 Met Leu Gln Asp Met Ala Ile Leu Thr Gly Gly Gln Val Ile Ser 300301 Glu Glu Val Gly Leu Thr Leu Glu Asn Ala Asp Leu Ser Leu Leu 315316 Gly Lys Ala Arg Lys Val Val Val Thr Lys Asp Glu Thr Thr Ile 330331 Val Glu Gly Ala Gly Asp Thr Asp Ala Ile Ala Gly Arg Val Ala 345346 Gln Ile Arg Gln Glu Ile Glu Asn Ser Asp Ser Asp Tyr Asp Arg 360361 Glu Lys Leu Gln Glu Arg Leu Ala Lys Leu Ala Gly Gly Val Ala 375376 Val Ile Lys Ala Gly Ala Ala Thr Glu Val Glu Leu Lys Glu Arg 390391 Lys His Arg Ile Glu Asp Ala Val Arg Asn Ala Lys Ala Ala Val 405406 Glu Glu Gly Ile Val Ala Gly Gly Gly Val Thr Leu Leu Gln Ala 420421 Ala Pro Thr Leu Asp Glu Leu Lys Leu Glu Gly Asp Glu Ala Thr 435436 Gly Ala Asn Ile Val Lys Val Ala Leu Glu Ala Pro Leu Lys Gln 450451 Ile Ala Phe Asn Ser Gly Leu Glu Pro Gly Val Val Ala Glu Lys 465466 Val Arg Asn Leu Pro Ala Gly His Gly Leu Asn Ala Gln Thr Gly 480481 Val Tyr Glu Asp Leu Leu Ala Ala Gly Val Ala Asp Pro Val Lys 495496 Val Thr Arg Ser Ala Leu Gln Asn Ala Ala Ser Ile Ala Gly Leu 510511 Phe Leu Thr Thr Glu Ala Val Val Ala Asp Lys Pro Glu Lys Glu 525526 Lys Ala Ser Val Pro Gly Gly Gly Asp Met Gly Gly Met Asp Phe 540541 His Met Ala Ser Glu Phe Gly Ser Thr Ala Pro Pro Ala His Gly 555556 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala 570571 Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro Ala 585586 Leu Lys Leu Ala Ala Ala Leu Glu His His His His His His 600SEQ ID NO：3信息(i)序列特征(A)长度：120个碱基(B)类型：核酸(C)链性：单链(D)拓扑结构：线性(ii)分子类型：DNA(iii)序列描述：SEQ ID NO：3CTCTGCTCGG 1680CTCTGCTCCG 17411742 GACAACCGTC CGGCTCTG
Claims (2)
1、一种预防和治疗人乳腺癌的基因工程重组蛋白疫苗，其特征是该疫苗含有结构是SEQ ID NO：2所示的氨基酸序列的基因工程重组融合蛋白。
2.权利要求1所述的基因工程重组蛋白疫苗，其中编码所述融合蛋白的基因是SEQ IDNO：1所示的核苷酸序列。
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| Application Number | Priority Date | Filing Date | Title |
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| CNB011026146A CN1141142C (zh) | 2001-02-08 | 2001-02-08 | 人乳腺癌muc-1抗原基因工程疫苗 |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB011026146A CN1141142C (zh) | 2001-02-08 | 2001-02-08 | 人乳腺癌muc-1抗原基因工程疫苗 |
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| CN1368384A CN1368384A (zh) | 2002-09-11 |
| CN1141142C true CN1141142C (zh) | 2004-03-10 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1727362B (zh) * | 2004-07-30 | 2010-12-01 | 中国人民解放军第二军医大学 | 癌胚抗原阳性肿瘤治疗性疫苗的制备及应用 |
| CN1762491A (zh) * | 2004-10-19 | 2006-04-26 | 北京迪威华宇生物技术有限公司 | Bcg作为激活细胞毒性t淋巴细胞活性蛋白的佐剂 |
| CN101429251B (zh) * | 2007-11-06 | 2012-05-23 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种抗肿瘤融合蛋白,其制备方法及用途 |
| CN101967193B (zh) * | 2010-03-19 | 2016-03-30 | 上海医学生命科学研究中心有限公司 | 能与LOX-1结合的Hsp60片段、其衍生物及应用 |
| CN102174555A (zh) * | 2010-12-29 | 2011-09-07 | 中国人民解放军第四军医大学 | 一种Hsp65-hIL-2融合表达的重组表达载体及重组菌株 |
| CN103146734B (zh) * | 2013-03-12 | 2014-10-01 | 中国人民解放军军事医学科学院军事兽医研究所 | 抗烧烫伤感染多器官衰竭绿脓杆菌毒素疫苗 |
| CN106279435B (zh) * | 2016-08-16 | 2019-06-07 | 新乡医学院 | 靶向VEGF与mucin1的抗肿瘤疫苗、编码基因、表达载体、表达工程菌及应用 |
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