CN101550181B - 人乳头瘤病毒16型e7蛋白功能拮抗肽及其编码基因与应用 - Google Patents
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Abstract
本发明公开了一种HPV 16型E7蛋白功能拮抗肽及其编码基因与应用。本发明的HPV 16型E7蛋白功能拮抗肽,其氨基酸序列如序列表中序列1所示。本发明以HPV 16型E7蛋白作为靶蛋白,筛选可结合HPV 16型E7蛋白并抑制其与pRb蛋白结合活性的小分子短肽,获得了一组具有一致序列的HPV 16型E7蛋白功能拮抗肽,可以作为治疗HPV感染诱发的宫颈癌的候选药物。本发明的HPV 16型E7蛋白功能拮抗肽在制备HPV感染所致的宫颈癌的药物中具有广泛的应用价值及广阔的市场前景。
Description
技术领域
本发明涉及HPV 16型E7蛋白功能拮抗肽及其编码基因与应用。
背景技术
宫颈癌(cervical cancer,CC)的病死率位于全球妇女癌症死亡率的第二位,近二十多年来有关人乳头瘤病毒(human papillomavirus,HPV)及其在宫颈癌发病中作用的大量研究认为,HPV感染是大多数宫颈癌发生和发展的必要条件(SchiffmanMH,Brinton LA.The epidemiology of cervical carcino genesis.Cancer,1995,76:1888-1901.Cruickshank ME.The role of human papillomavirus in risk management.Gynaecol Practice,2003,3:229-233.)。据报道,来自22个国家的宫颈癌标本中有99.7%可检测到HPV-DNA(Lang J H.To receive the global chanllenge and opportunity forpreventing cervical cancer.Chin JO bstet Gynecol,2002,37:129-131.Walboomers JM.Jacobs MV,Manos MM,et al.Human papillomavirus is a necessary cause of invasivecervical cancer worldwide.J.Pathol,1999,189(1):12-19.),其中与宫颈癌相关的有16、18、31、33、45和58等10余型HPV-DNA(An HJ,Cho NH,Lee SY,et al.Correlationof cervical carcinoma and precancerous lesions with human papillomavirus(HPV)genotypes detected with the HPV DNA chip microarray method.Cancer,2003,97(7):1672-1680.)。目前,宫颈癌传统治疗方法疗效不佳,复发率较高。研究表明,治疗后残余的HPV病毒颗粒DNA是导致其高复发的重要原因,因此利用现代免疫学及分子生物学方法研制针对HPV感染的疫苗、抗体及治疗药物等成为宫颈癌治疗研究的热点。
目前针对HPV的研究主要集中在预防性疫苗和治疗性疫苗的开发上。由于HPV病毒不能在体外增殖,因此生产减毒疫苗不可行。但其衣壳蛋白L1与L2表达后均能自我装配或形成假病毒颗粒(VLP)(Harro CD,Pang YY,Roden RB,et al.Safety andimmunogenicity trial in adult volunteers of a human papillomavirus 16 L 1 virus-likeparticle vaccine.J Natl Cancer Inst,2001,93:284-292.),VLP作为预防性疫苗接种可激发体内的CD4+淋巴细胞介导的体液免疫反应,刺激机体产生保护性中和抗体及产生有效的局部免疫反应(Koutsky LA,Ault KA,Wheeler CM,et al.A controlled trial ofa human papillomavirus type 16 vaccine.N Engl J Med,2002,347(21):1645-1651)。但其成本较高,且HPV是一个多亚型病毒,不同亚型病毒间缺少群共同抗原决定簇,因此多价疫苗可否对不同亚型HPV产生有效的保护作用尚需实验证明。
HPV治疗性疫苗有望清除由HPV感染所引起的肿瘤病灶及生殖器尖锐湿疣,阻断低危型病变向宫颈癌转变的过程。已证明HPV的E6和E7蛋白在细胞发生恶变中发挥重要作用,E6和E7蛋白选择性表达于宫颈癌细胞,可分别使抑癌蛋白p53和pRb失活,导致细胞周期紊乱,还可以激活端粒酶并使细胞永生化,其持续表达是使肿瘤细胞转化和维持恶性特征所必需的。由于正常人体内不存在此病毒蛋白,且其抗原性、特异性均较强,因此E6和E7蛋白已成为HPV治疗性疫苗研究的重要靶标。目前的研究主要包括治疗性蛋白疫苗和治疗性基因疫苗。有报道称,E711-20和E786-93多肽疫苗经动物实验证明能诱发细胞毒性T淋巴细胞反应(DeMarco F,Hallez S,Brulet JM,et al.DNA vaccines against HPV-16 E7 expressing tumour cells.AnticancerRes,2003,23:1449-1454);L1-E7嵌合蛋白疫苗在动物实验中可分别激发针对L1的体液免疫和E7的细胞免疫(程浩,叶俊,含HPV16E7的病毒样颗粒诱导小鼠产生特异性CTL反应。中华皮肤科杂志2005年5月第38卷第5期:291-293。Wakabayashi MT,DaSilva DM,Potkul RK,et al.Comparison of human papillomavirustype 16 L1 chimeric virus2like particles versus L1/L2chimeric virus-like particles intumor revention.Intervirology,2002,45:300-307.);HSP65-E7融合蛋白疫苗可诱导针对E7蛋白的细胞免疫(Chu NR,Wu HB,Wu T,et al.Immunotherapy ofa humanpapillomavirus(HPV)type 16 E7-expressing tumor by administration of fusion proteinconprising Mycobacterium bovis bacilli Calmette Guerin(BCG)hsp65 and HPV 16 E7.Clin Exp Immunol,2000,121(2):216-225.);利用重组牛痘病毒构建的TA-HPV疫苗融合了E6和E7蛋白的基因,在临床实验中证明可诱发体内特异性的细胞毒性T淋巴细胞反应和特异性的血清反应(Kaufmann AM,Stem PL,Rankin EM,et al.Safety andimmunogenicity of TA-HPV,A recombinant vaccinia virus expressing modified humanpapillomavirus(HPV)16 and HPV18 E6 and E7 genes in women with progressivecervical cancer.Clin Cancer Res,2002,8(12):3676-3685.)。但多肽疫苗免疫原性较弱,且存在MHC-I类分子限制性,使其应用具有一定的局限性;而基因疫苗的安全性和诱导有效免疫反应的作用还有待进一步的改善。
预防性疫苗和治疗性疫苗需通过诱发体内产生有效的细胞免疫及体液免疫来发挥作用,具有一定的时效性,且多数疫苗免疫原性较弱,需合适的佐剂辅助才可诱发机体产生有效的免疫应答;同时由于HPV病毒亚型的多样性,尚未有广谱性的针对不同亚型HPV的有效疫苗的报道。随着对HPV病毒研究的深入,研究可直接作用于HPV E6和E7蛋白并阻断其活性的疫苗成为了一个新的热点(Veldman T,Horikawa I,Barrett Carl J,et al.Transcriptional activation of the telomerase hTERTgene by human papillomavirus type 16E6 oncoprotein.J Virol,2001,75(9):4467-4472.Nishimura A,Nakahara T,Ueno T,et al.Requirement of E7 oncoprotein for viability ofHeLa cells.Microbes Infect.2006 Jan 17.)。研究发现,E7蛋白致癌作用的关键是干扰视网膜母细胞瘤蛋白pRb与转录因子E2F的结合,从而导致细胞增殖周期紊乱;E7蛋白还可以通过与pRb相关蛋白p107或p130的结合,使蛋白质磷酸化而灭活,并释放后续转录因子E2F使细胞过度增殖,在宫颈癌发生、演进中起到重要作用(GammohN,Grm HS,Massimi P,et al.Regulation of human papillomavirus type 16 E7 activitythrough direct protein interaction with the E2 transcriptional activator.J Virol.200680(4):1787-97.Zhang B,Chen W,Roman A.The E7 proteins of low-and high-riskhuman papillomaviruses share the ability to target the pRB family member p 130 fordegradation.Proc Natl Acad Sci U S A.2006 10;103(2):437-42.Caldeira S,Dong W,Tommasino M.Analysis of E7/Rb associations.Methods Mol Med.2005;119:363-79.)。因此,研究可以阻断或抑制E7蛋白的活性作用,从而恢复pRb蛋白抑癌活性的拮抗剂将成为宫颈癌治疗的新途径。Accardi L等通过筛选噬菌体抗体库获得了抗HPV16-E7的单链抗体,利用真核表达载体构建重组表达质粒后转染HPV-16阳性肿瘤细胞SiHa,实验证明,该单链抗体可以有效地抑制E7蛋白的活性,对细胞转化起到逆转作用(Accardi L,Dona MG,Di Bonito P,et al.Intracellular anti-E7 humanantibodies in single-chain format inhibit proliferation of HPV 16-positive cervicalcarcinoma cells.Int J Cancer.2005Sep 10;116(4):564-70.Griffin H,Elston R,JacksonD,et al.Inhibition of papillomavirus protein function in cervical cancer cells byintrabody targeting.J Mol Biol.2006 Jan 20;355(3):360-78.Epub 2005Nov 14.),提示E7蛋白可作为HPV相关恶性肿瘤或宫颈癌前期病变治疗的重要靶标。
通过对E7蛋白的结构分析发现,该蛋白2区中的LXCXE位点(25-29aa)以及3区的锌指结构区是其结合pRb的关键位点,锌指结构区的突变可完全破坏E7蛋白的转化和永生化能力。Liu X等利用X-射线晶体衍射对E7蛋白CR3锌指结构进行分析后发现,E7-CR3包含了两个保守性的片段,其中一个片段为结合pRb所必需,而另一片段为结合E2F所必需(Liu X,Clements A,Zhao K,et al.Structure ofthe humanPapillomavirus E7 oncoprotein and its mechanism for inactivation of the retinoblastomatumor suppressor.J Biol Chem.2006Jan 6;281(1):578-86.)。研究证明,不同亚型HPV病毒E7蛋白均具有保守性LXCXE位点以及CR3锌指结构区(Alonso LG,Smal C,Garcia-Alai MM,et al.Chaperone holdase activity of human papillomavirus E7oncoprotein.Biochemistry.2006Jan 24;45(3):657-67.Fiedler M,Campo-Femandez B,Laich A,et,al.Purification and characterisation of the E7 oncoproteins of the high-riskhuman papillomavirus types 16 and 18.J Virol Methods.2005Dec 26.),且HPV E7蛋白与人体蛋白之间无任何同源性,因此以E7蛋白保守性LXCXE位点以及CR3锌指结构区为靶标,找寻作用于该位点的小分子化合物,可望获得广谱性且可与不同亚型HPV E7蛋白结合并抑制其与pRb结合的小分子抑制剂。
目前噬菌体肽库技术已成功应用于新型多肽药物的筛选中,该技术的优势在于,可以获得与靶分子特异性结合的高亲和力的短肽,且短肽的DNA序列可知,可同时为蛋白表位分析及空间构象分析提供有效的依据(McLaurin J,Cecal R,Kierstead ME et al:Therapeutically effective antibodies against amyloid-beta peptidetarget amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis.Nat Med.2002;8(11):1263-9.Casey JL,Coley AM,Anders RF et al:Antibodies to malariapeptide mimics inhibit Plasmodium falciparum invasion of erythrocytes.InfectImmun.2004 Feb;72(2):1126-34.)。在针对HPV蛋白功能的研究中,Fujii T等利用噬菌体肽库技术,以HPV E2蛋白为筛选靶分子获得了E2蛋白拮抗肽,实验证明,筛选获得的短肽在细胞内可以有效的抑制E2蛋白的活性(Fujii T,Austin D,Guo D,et al.Peptides inhibitory for the transcriptional regulatory function of human papillomavirusE2.Clin Cancer Res.2003 Nov 1;9(14):5423-8.),该结果为利用噬菌体肽库技术筛选有效的针对HPV E7蛋白活性的小分子短肽抑制剂提供了有利的证据。
发明内容
本发明的一个目的是提供一种HPV 16型E7蛋白功能拮抗肽。
本发明所提供的HPV 16型E7蛋白功能拮抗肽,其氨基酸序列如序列表中序列1所示。
本发明的另一个目的是提供一种HPV 16型E7蛋白功能拮抗肽的编码基因。
所述HPV 16型E7蛋白功能拮抗肽的编码基因,其脱氧核糖核苷酸序列如序列表中序列2所示。
含有上述HPV 16型E7蛋白功能拮抗肽编码基因的重组表达载体、转基因细胞系和重组菌也属于本发明的保护范围。
所述重组表达载体具体可为重组噬菌体。
本发明的七肽可用于制备预防和/或治疗由HPV16感染所致疾病的药物。
本发明以HPV 16型E7蛋白作为靶蛋白筛选噬菌体展示七肽库,采用靶蛋白分子竞争性洗脱的方式,通过筛选获得了能够与HPV 16型E7蛋白特异性结合并可抑制其功能活性的七肽。该七肽具有以下优点:
①能够阻断HPV 16型E7蛋白的生物学活性,抑制HPV 16型E7蛋白与其配体蛋白pRb的结合,达到恢复pRb生物学活性的目的,该七肽可用于制备预防和/或治疗由HPV感染所致疾病(如宫颈癌)的药物,亦可为利用基因治疗手段进行宫颈癌治疗提供可用的药物前体。
②渗透力强、易于到达病变部位,且该短肽针对的靶蛋白为HPV特异性表达蛋白,与人体内正常蛋白无同源性,因此无毒副作用,使用较为安全。短肽的免疫原性较弱,不易诱发机体产生抗短肽的免疫应答。
③分子特异性强,易于人工合成,纯化工序简便,容易实现规模化生产,并可结合计算机辅助分子模拟技术对筛选获得的短肽分子进行相关的改造以提高其亲和力及活性。
本发明的HPV 16型E7蛋白功能拮抗肽的获得为短肽型HPV 16型E7蛋白功能抑制剂的研发奠定了基础,可结合腺病毒载体或其他基因工程载体进行宫颈癌基因治疗药物的研发。本发明的HPV 16型E7蛋白功能拮抗肽在制备HPV感染所致的宫颈癌的药物中具有广泛的应用价值及广阔的市场前景。
附图说明
图1为噬菌体克隆的ELISA鉴定结果
图2为阳性噬菌体克隆的竞争性ELISA鉴定结果
图3为阳性噬菌体克隆对HPV 16型E7蛋白与其配体蛋白pRb结合的抑制实验结果
图4为阳性噬菌体克隆测序图
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。
实施例1、以HPV 16型E7蛋白为靶蛋白筛选得到本发明的重组噬菌体七肽
本发明利用商品化噬菌体展示七肽库(New England BioLabs公司)来筛选获得HPV 16型E7蛋白功能拮抗肽。
一、以HPV 16型E7蛋白为靶蛋白进行筛选
1、将HPV 16型E7蛋白(Santa Cruz公司)用pH8.6的碳酸盐缓冲液稀释为100mg/L,取100uL上述稀释过的HPV 16型E7蛋白包被聚苯乙烯酶联板,4℃孵育过夜;
2、倒去酶联板中的蛋白溶液,将酶联板用TBS-Tween(10mmol/L Tris.HCl,pH7.5,500mmol/L NaCl,1mL/L Tween 20)洗涤5次后,加入5g/L BSA于37℃封闭2h;
3、酶联板再经TBS-Tween洗涤5次后,加入噬菌体展示七肽库(New EnglandBioLabs公司)10uL(含噬菌体颗粒1.7X1011个)和90uL TBS,于室温条件下摇床轻摇孵育1h;
4、用TBS-Tween洗涤酶联板10-15次后,加入浓度为800mg/L的HPV 16型E7蛋白进行竞争性洗脱,室温条件下摇床轻微振荡孵育1h;
5、收集洗脱液,测定噬菌体滴度,扩增后用于下一轮筛选;
6、重复上述1-5的步骤,共进行三轮筛选,将第三轮筛选获得的洗脱液经梯度稀释后与大肠杆菌ER2738(New England BioLabs)侵染后铺板,24h后挑取培养平板上的单个蓝斑加至2mL大肠杆菌ER2738培养液内,37℃空气摇床250rpm振荡孵育4.5h,以扩增单克隆噬菌体;其中,第二轮和第三轮筛选除TBS-Tween的浓度与第一轮不同外,其它方法均与第一轮筛选相同。第二轮和第三轮筛选的TBS-Tween组成如下:10mmol/L Tris·HCl,pH 7.5,500mmol/L NaCl,5mL/L Tween 20。
7、将上述步骤6获得的噬菌体培养液10,000rpm离心后取上清,加入160uLPEG8000-NaCl沉淀过夜,次日离心弃上清,将沉淀溶于200uL PBS缓冲液中,测定单个噬菌体克隆的滴度。
为得到亲和力较强的特异性噬菌体克隆,在上述三轮筛选中,逐渐提高洗涤液TBS-Tween中Tween的含量(第一轮筛选时,洗涤液中Tween的含量为1mL/L;第二轮和第三轮筛选时,洗涤液中Tween的含量均为5mL/L),以去除非特异性结合的噬菌体克隆。同时用靶蛋白竞争性洗脱的方式替代传统的酸性缓冲液洗脱,以提高特异性阳性噬菌体克隆的获得率。
二、阳性噬菌体克隆的ELISA鉴定
亲和性筛选的目的在于获得特异性结合的噬菌体克隆,但不能完全去除非特异性结合的噬菌体克隆,因此对筛选获得的噬菌体单克隆要进行必要的鉴定,以确定其与靶蛋白的特异性结合。
1、将HPV 16型E7蛋白用pH8.6的碳酸盐缓冲液稀释为100mg/L,取100uL上述稀释过的HPV 16型E7蛋白包被聚苯乙烯酶联板,4℃孵育过夜;
2、倒去酶联板中的蛋白溶液,经PBST洗涤5次后,每孔内加入200uL 10g/L的脱脂奶粉,37℃封闭2h;
3、PBST洗涤5次后,每孔内加入100uL上述步骤一筛选得到的单克隆噬菌体的培养上清(约2.5X1011pfu),37℃水浴箱孵育1h;
4、PBST洗涤5次后,每孔内加入100uL HRP-抗M13抗体(GE Healthcare公司),37℃水浴箱呼育30min;
5、PBST洗涤5次后,每孔内加入100uL TMB显色液作用2min,并以100uL 2MH2SO4终止反应,测定反应液的A450值。
每个单克隆噬菌体均设未包被HPV 16型E7蛋白的酶联孔作为对照,以排除非特异性吸附的阴性噬菌体克隆对实验结果的影响。
噬菌体克隆的ELISA鉴定结果如图1所示,其中,图1a为第1-20个单克隆噬菌体的ELISA鉴定结果,图1b为第21-40个单克隆噬菌体的ELISA鉴定结果。结果表明,挑取的40个单克隆噬菌体中,有8个单克隆噬菌体可与HPV 16型E7蛋白特异性结合,分别为:A-5、A-9、A-15、A-17;B-3、B-6、B-15、B-17(以A450值高于1且蛋白包被孔A450值高于未包被蛋白孔A450值3倍为阳性标准),此8个噬菌体克隆为阳性噬菌体克隆。
三、阳性噬菌体克隆的竞争性ELISA鉴定
为鉴定阳性噬菌体克隆与HPV 16型E7蛋白的特异性结合,以上述步骤二获得的阳性噬菌体克隆作为阻断剂,抑制HPV 16型E7蛋白与其相应单克隆抗体的结合,以检验阳性噬菌体克隆的拮抗活性。
1、HPV 16型E7蛋白的包被及封闭过程同步骤二的1和2;
2、将上述步骤二获得的阳性噬菌体克隆倍比稀释为1X1013、1X1011、1X109、1X107和1X105pfu/mL,取50uL上述各浓度的阳性噬菌体克隆分别与50uL抗HPV16-E7单克隆抗体(NeoMarkers公司)(1∶1000)混匀,37℃孵育30min;
3、将上述步骤1的酶标板用PBST洗涤5次后,加入100uL上述步骤2的混合液,37℃孵育30min,同时以同样稀释倍数的原库噬菌体(New England BioLabs公司)作为对照;
4、酶标板用PBST洗涤5次后,加入HRP-兔抗鼠多克隆抗体(深圳晶美)(1∶3000),37℃孵育30min;
5、酶标板用PBST洗涤5次后,加入TMB显色液显色2min,2M H2SO4终止反应,测定反应液的A450值,计算抑制率。
以阳性噬菌体克隆竞争抑制抗HPV 16-E7单克隆抗体与HPV 16型E7蛋白的结合,来判断阳性噬菌体克隆与HPV 16型E7蛋白的特异性结合,阳性噬菌体克隆的竞争性ELISA鉴定结果如图2所示。其中,横坐标A、B、C、D和E分别表示阳性噬菌体克隆和对照噬菌体的滴度分别为1X1013、1X1011、1X109、1X107和1X105pfu/ml。结果表明,阳性噬菌体克隆能够阻断HPV 16型E7蛋白与其相应抗体的结合,且抑制效果随着阳性噬菌体克隆滴度的升高而增大。而对照的噬菌体克隆不能够阻断HPV16型E7蛋白与其相应抗体的结合。
四、阳性噬菌体克隆对HPV 16型E7蛋白与其配体蛋白pRb结合的抑制实验
为了获得可与HPV 16型E7蛋白结合,并能够阻断HPV 16型E7蛋白与其配体蛋白pRb结合的拮抗肽,要通过竞争性抑制实验来证明所获得的阳性噬菌体克隆的拮抗活性。
1、HPV 16型E7蛋白的包被及封闭过程同步骤二的1和2;
2、将上述步骤1的酶标板用PBST洗涤5次后,每孔加入上述步骤二获得的阳性噬菌体克隆上清50uL(阳性噬菌体克隆的浓度为1X1012pfu/ml),37℃孵育30min,同时以浓度为1.0×1012pfu/ml的原库噬菌体作为对照;
3、将pRb蛋白(购自Active Motif公司)梯度稀释为5、10、20、40和80mg/L,分别取50uL不同浓度的pRb蛋白加入到上述步骤2的孔内,37℃孵育30min;
4、PBST洗涤5次后,每孔内加入100uL HRP-抗M13抗体(GE Healthcare公司),37℃水浴箱孵育30min;
5、PBST洗涤5次后,每孔内加入100uL TMB显色液作用2min,并以100uL 2MH2SO4终止反应,测定反应液的A450值。
阳性噬菌体克隆对HPV 16型E7蛋白与其配体蛋白pRb结合的抑制实验结果如图3所示。结果表明,阳性噬菌体克隆可与pRb蛋白竞争,与HPV 16型E7蛋白结合,表明阳性噬菌体克隆具有特异地与HPV 16型E7蛋白结合的活性,并可有效的抑制HPV 16型E7蛋白与其配体蛋白pRb的结合,是HPV 16型E7蛋白功能拮抗肽。而对照的噬菌体克隆不能抑制HPV 16型E7蛋白与其配体蛋白pRb的结合。
六、阳性噬菌体克隆的测序
经上述实验鉴定,共获得5个具有拮抗HPV 16型E7蛋白结合活性的阳性噬菌体克隆,将这5个阳性噬菌体克隆进行DNA测序,测序图如图4所示。结果发现这5个阳性噬菌体克隆中插入的七肽的编码基因序列一致,具体脱氧核糖核苷酸序列如序列表中序列2所示,其编码的七肽的氨基酸序列如序列表中序列1所示。
序列表
<160>2
<210>1
<211>7
<212>PRT
<213>人乳头瘤病毒(Human papillomavirus)
<400>1
Gln Pro Lys Gly Leu Asp Trp
1 5
<210>2
<211>21
<212>DNA
<213>人乳头瘤病毒(Human papillomavirus)
<400>2
cagccgaagg gtcttgattg g 21
Claims (9)
1.一种七肽,其氨基酸序列如序列表中序列1所示。
2.权利要求1所述七肽的编码基因。
3.含有权利要求2所述基因的重组表达载体。
4.根据权利要求3所述的重组表达载体,其特征在于:所述重组表达载体为重组噬菌体。
5.含有权利要求2所述基因的转基因细胞系。
6.根据权利要求5所述的转基因细胞系,其特征在于:所述细胞系为导入权利要求3或4所述的重组表达载体的转基因细胞系。
7.含有权利要求2所述基因的重组菌。
8.根据权利要求7所述的重组菌,其特征在于:所述重组菌为导入权利要求3或4所述的重组表达载体的重组菌。
9.权利要求1所述的七肽在制备预防和/或治疗由HPV16感染所致疾病的药物中的应用。
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CN1445237A (zh) * | 2002-03-19 | 2003-10-01 | 中国人民解放军军事医学科学院基础医学研究所 | 肿瘤坏死因子拮抗肽及其应用 |
CN1683395A (zh) * | 2005-02-22 | 2005-10-19 | 徐祥 | 核因子-κB p65亚基拮抗肽及其应用 |
CN1756845A (zh) * | 2002-11-12 | 2006-04-05 | A·B·德塞罗斯 | 腺病毒载体疫苗 |
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CN1445237A (zh) * | 2002-03-19 | 2003-10-01 | 中国人民解放军军事医学科学院基础医学研究所 | 肿瘤坏死因子拮抗肽及其应用 |
CN1756845A (zh) * | 2002-11-12 | 2006-04-05 | A·B·德塞罗斯 | 腺病毒载体疫苗 |
CN1683395A (zh) * | 2005-02-22 | 2005-10-19 | 徐祥 | 核因子-κB p65亚基拮抗肽及其应用 |
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