CN1174097C - 由克隆的维生素d结合蛋白衍生的巨噬细胞激活因子 - Google Patents
由克隆的维生素d结合蛋白衍生的巨噬细胞激活因子 Download PDFInfo
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Abstract
借助于杆状病毒载体克隆了维生素D结合蛋白(Gc蛋白质)和其小区域(约为Gc肽的1/5,已知称作为区域III)。用固定化的β-半乳糖苷酶和唾液酸酶处理克隆的Gc蛋白质和克隆的区域(Cd)肽分别产生巨噬细胞激活因子GcMAFc和CdMAF。这些克隆的巨噬细胞激活因子和GcMAF可用于治疗癌症,HIV感染和骨质疏松症,也用作为免疫和接种的佐剂。研制了在癌症和HIV感染的患者中检测血清或血浆α-N-乙酰氨基半乳糖苷酶的方法和抗体-夹心ELISA试剂盒。
Description
发明领域
本发明涉及利用克隆的维生素D结合蛋白(Gc蛋白)和克隆的Gc蛋白区域III的低聚糖消化作用,产生有效的巨噬细胞激活因子,以及这些巨噬细胞激活因子在各种癌症、HIV感染和骨质疏松症方面的应用,以及作为免疫接种和接种疫苗的一种佐剂的应用,还研制了利用癌症和HIV感染的患者血液中检测到的特殊酶,α-N-乙酰氨基半乳糖苷酶的诊断和预后方法。
术语
Gc蛋白质 维生素D3结合蛋白
MAF 巨噬细胞激活因子
GcMAF Gc蛋白衍生的巨噬细胞激活蛋白
GcMAFc 克隆的Gc蛋白衍生的巨噬细胞激活因子
Gc domain III Gc区域III
CdMAF 克隆的区域III衍生的巨噬细胞激活因子
NagAg 作为抗原的α-N-乙酰氨基半乳糖苷酶
发明概述
借助于杆状病毒载体克隆了维生素D结合蛋白(Gc蛋白)及其小区域(约占Gc肽长度的18%,也称区域III)。用固定的β-半乳糖苷酶和唾液酸酶处理克隆的Gc蛋白和克隆的区域(Cd)肽以分别产生巨噬细胞激活因子,GcMAFc和Cd MAF。这些克隆的巨噬细胞激活因子和GcMAF将用于癌症治疗,HIV感染和骨质疏松症,也能作为免疫接种和接种疫苗的一种佐剂。研制了用于检测癌症和HIV感染患者血清或血浆的α-N-乙酰氨基半乳糖苷酶的方法及抗体夹心ELISA试剂盒,并将它们作为诊断及预后标志。
附图描述
参考下面详细的说明及附图能更好地理解该发明,那么本发明的其它目的及一些伴随的特征将是显而易见的。
图1a是巨噬细胞激活因子(MAF)形成图。
图1b是癌症或者HIV感染患者血液中的Gc蛋白的去糖基作用图解。
图2表示克隆的GcMAF的氨基酸序列,是SEQ ID NO:1,它是完整的克隆的Gc蛋白质。
图3表示编码EcoRI片段E1的前导序列和Gc蛋白区域III的DNA片段的构建;A,编码Gc蛋白质的整个cDNA;B,插入到非融合载体的构建体;阴影区域表明约1000个碱基对(bp)的压缩区。
图4表示用非融合载体,克隆的区域III(CdMAF1)的89个氨基酸序列,SEQ ID NO:2。
图5表示用于克隆Gc蛋白区域III的杆状病毒融合载体。
图6表示用融合载体克隆的区域III(CdMAF2)的94个氨基酸序列SEQ ID NO:3。
图7表示血浆的α-N-乙酰氨基半乳糖苷酶的活性与埃利希腹水肿瘤的腹腔中肿瘤积存量(全部细胞数)的关系。
图8A表示本发明的GcMAF对患有前列腺癌的成年患者的疗效。
图8B表示本发明的GcMAF对患有乳腺癌的成年患者的疗效。
图8C表示本发明的GcMAF对患有结肠癌的成年患者的疗效。
发明背景
A.炎性应答导致巨噬细胞的激活
炎症导致巨噬细胞的激活。炎症损伤释放溶血磷脂酰胆碱。把小剂量(5-20微克/鼠)的溶血磷脂酰胆碱(Lyso-Pc)和其它溶血磷脂给小鼠给药能大大增强巨噬细胞的吞噬作用和分泌超氧化物的能力(Ngwenya和Yamamoto,Proc.Soc.Exp.Biol.Med.193:118-124(1990);Yamamoto,等人,Inf.Imm.61:5388-5391(1993);Yamamoto,等人,Inflammation.18:311-322(1994)。
这种巨噬细胞的激活需要B淋巴细胞和T淋巴细胞和血清的维生素D结合蛋白(DBP;人DBP被称为Gc蛋白)的参与。由Lyso-Pc体外激活小鼠的腹腔巨噬细胞需要通过Lyso-Pc处理的B细胞的β-半乳糖苷酶和T细胞的唾液酸酶对Gc蛋白逐步修饰,可产生巨噬细胞激活因子(MAF),即携带N-乙酰氨基半乳糖的蛋白质为保留的糖部分的蛋白质(图1a)(Yamamoto,等人,Proc.Natl.Acad.Sci.USA.88:8539-8543(1991);Yamamoto,等人,免疫学杂志。151:2794-2902(1993);Naraparaju,和Yamamoto等人Immunol.Lett.43:143-148(1994))。因此Gc蛋白是MAF的一种前体物。
用固定的β-半乳糖苷酶和唾液酸酶保温Gc蛋白,产生非常高效价的MAF(GcMAF)(Yamamoto,等人,Proc.Natl.Acad.Sci.USA.88:8539-8543(1991);Yamamoto,等人,免疫学杂志。151:2794-2902(1993);美国专利5177002;美国专利5326749)。以极少量(10pg/鼠,100纳克/人)的GcMAF给药导致巨噬细胞的吞噬作用增强和超氧化物分泌能力提高。
用100pg GcMAF/毫升体外处理326个各种癌症病人的外周血液的单核细胞/巨噬细胞(下文称为巨噬细胞),各种癌症患者的巨噬细胞被激活而具有吞噬作用和分泌超氧化物的能力。该结果表明:癌症患者的巨噬细胞能被激活。然而约70%癌症患者的血浆Gc蛋白的MAF前体活性丧失或降低。发现Gc蛋白前体活性丧失或者降低的原因是癌症患者血液里的α-N-乙酰氨基半乳糖苷酶引导血浆Gc蛋白的去糖基作用。去糖基的Gc蛋白不能转变成MAF(图1b)。这样,Gc蛋白MAF前体活性的丧失阻止了MAF的生成。因此,在一些癌症患者中巨噬细胞并不能被激活。由于巨噬细胞的激活是发生免疫级联过程的第一步。这样的癌症患者就成为免疫抑制者。这至少能部分解释癌症患者死于严重感染的原因。
类似地,在体外用100pg的GcMAF/毫升处理了196名HIV感染/AIDS患者的外周血液巨噬细胞,全部患者的巨噬细胞都被激活而具有吞噬作用和超氧化物生产能力。然而约35%的HIV-感染患者的血浆Gc蛋白的MAF前体活性降低。在癌症患者中,这些患者的血浆Gc蛋白被HIV感染患者的α-N-乙酰氨基半乳糖苷酶进行去糖基作用。这种机制解释了后期HIV感染患者(AIDS)严重免疫抑制的原因。
已严重降低了血浆Gc蛋白前体的活性的癌症和HIV感染患者,携带大量的α-N-乙酰氨基半乳糖苷酶。而中等降低前体活性的患者具有中等水平的血浆α-N-乙酰氨基半乳糖苷酶活性。具有高前体活性的患者,包括无症状的HIV-感染患者,具有的α-N-乙酰氨基半乳糖酶活性较低,但却显著的水平。既然大量的(260微克/毫升)Gc蛋白存在于血液中,那么低水平酶不能影响前体活性。此外,在各种癌症和HIV-感染患者的血浆中发现α-N-乙酰氨基半乳糖苷酶并且与其血浆Gc蛋白的前体物活性成倒相关。因此,患者血清或血浆α-N-乙酰氨基半乳糖苷酶活性的增加会导致血浆Gc蛋白质的前体活性的降低。这些观察使本发明人作出如下推断:即血清或血浆α-N-乙酰氨基半乳糖苷酶在对癌症患者和HIV感染的患者/AIDS病患者的免疫抑制中起作用。因此,患者血清或血浆的α-N-乙酰氨基半乳糖苷酶的活性能作为一种诊断和预后标志。
B.巨噬细胞激活级联的缺损表现为骨质疏松症
激发炎症的巨噬细胞活化过程似乎是主要的巨噬细胞激活级联。这个过程由其它吞噬细胞如破骨细胞和单核细胞共同分担。在巨噬细胞激活级联中,B淋巴细胞可诱发的β-半乳糖苷酶的缺损引起破骨细胞的功能异常导致表现为骨质疏松症,(Yamamoto等人,免疫学杂志,152:5100,1994),新生儿缺乏或延迟骨髓的形成。
常染色体隐性骨质疏松症以大量骨质积累为特征,由于破骨细胞的功能异常,从而导致骨质再吸收的减少(Marks,Clin.Orthop.189:239,1984)。在骨质疏松症的动物模型中,根据破骨细胞功能异常的程度,骨髓腔的发育和牙齿的生长要么延迟,要么是常见的缺失(Marks,Am.J.Med.Genet.34:43,1989)。在人类早期的骨质疏松症,通常覆盖感染的生命的第一级联出现死亡,表现为免疫抑制(Reeves,儿科学,64:202,1979)。累积的证据表明:骨硬化的哺乳动物(包括人类)的破骨细胞的缺失或者功能异常经常伴随着巨噬细胞的缺失或功能异常。本发明关于在骨硬化变异中破骨细胞和巨噬细胞被激活的研究表明;一种普通的信号因子巨噬细胞激活因子能激活破骨细胞和巨噬细胞,并且缺乏β-半乳糖苷酶的B细胞则不能产生巨噬细胞激活因子(Yamamoto等人免疫学杂志,152:5100,1994)。既然GcMAF及其克隆的衍生物能绕过Gc蛋白和淋巴细胞的作用而直接作用于巨噬细胞和破骨细胞上,那么,把这些因子给骨硬化宿主给药,应该能修饰骨质病变。事实上,本发明人最近发现把4种GcMAF或者克隆的巨噬细胞激活因子(GcMAFC)(100pg/周)给出生4周的骨质疏松症突变小鼠给药,结果导致巨噬细胞和破骨细胞的激活以及随后吸收过量骨骼母质,并增大了骨髓腔的体积。
C.利用GcMAF及其克隆的衍生物治疗癌症的效果
尽管癌症,HIV-感染和骨硬化患者的巨噬细胞激活级联缺失,GcMAF能绕过Gc蛋白和淋巴细胞的作用而直接作用于巨噬细胞(或破骨细胞)进行激活。当被活化时,巨噬细胞有消除癌细胞和HIV-感染细胞的潜力。当每个癌症患者每周用100纳克GcMAF治疗几个月后,GcMAF对各种人类癌症都显示了显著的疗效。
除了从人类血库中获取GcMAF,也可从克隆的Gc蛋白或能激活巨噬细胞的小区域中获取。克隆的Gc蛋白需要能使克隆产物糖基化的真核细胞的载体/宿主。分子量约52000的Gc蛋白(458个氨基酸残基)是一种多功能蛋白且携带3个独立的区域(Cooke和Haddad,Endocrine Rev.,10:294,1989)。
区域I与维生素D能进行相互作用,而区域III与肌动蛋能相互作用(Haddad等人,生物化学,31:7174,1992)。化学裂解或者蛋白质水解得到的Gc蛋白片段表明:最小的区域--区域III包含80个氨基酸残基,其中包括糖基化位点和一个激活巨噬细胞的必需肽。因此,利用一种杆状病毒载体和一种昆虫宿主克隆了Gc蛋白和全部区域III肽,用固定的β-半乳糖苷酶和唾液酸酶处理产生有效的巨噬细胞激活因子。象GcMAF一样,这些克隆的GcMAFc和CdMAF对多种癌症显出很大的疗效。
D.用于抗原或疫苗免疫接种时GcMAF的有效的佐剂活性
巨噬细胞是提供抗原的细胞。由GcMAF激活的巨噬细胞迅速吞噬靶抗原或靶细胞,并且提供处理过的抗原到生成抗体的细胞。接种少量GcMAF(100pg/鼠)和绵羊红细胞(SRBC)后,立即(1-4天)就能观察到大量抗体分泌细胞迅速发育。该发现表明:GcMAF及其克隆的衍生物,GcMAFc和CdMAF应该能作为免疫接种和接种疫苗的一种有效的佐剂。
编码巨噬细胞激活因子的基因克隆的方法描述
A.把Gc蛋白的cDNA克隆到一种昆虫病毒中
利用Stratagene of La Jolla,CA的pico BlueTm免疫筛选试剂盒,能从噬菌体λgtll(Clontech,Palo Alto,CA)的人类肝脏cDNA库分离出bm人类Gc蛋白的全长cDNA。具有几种事实说明昆虫细胞中的杆状病毒的表达系统的多角体蛋白的优点:(a)在感染周期后期中,该系统包含总细胞蛋白一半以上,在感染细胞中以非常高的水平表达;(b)对病毒感染或复制不是必要的,是指重组病毒不需要任何辅助机能;(c)缺少多角体基因的病毒具有来源于包含该克隆基因的病毒特殊的噬斑形态;(d)与细菌细胞不同,昆虫细胞能有效地使克隆的基因产物糖基化。
这个表达系统的优点之一是一种可视筛选以允许辨别重组病毒和对其定性。在病毒感染周期后期,感染细胞的细胞核以很高的水平产生多角体蛋白。累积的多角体蛋白组成了含有内嵌病毒质粒的包含体。这些包含体大小达15um是很强的折射体,给它们以明亮的外观后在光学显微镜下很容易看到。用重组病毒感染的细胞缺少包含体。为了从野生型病毒中分辩出重组病毒,在昆虫细胞的单细胞层形成转染上清液(含有病毒裂解液的重组体)。在光学显微镜下筛选存在(表示野生型病毒)或者不存在(表示重组病毒)包含体的噬斑。
此外,杆状病毒表达系统利用了不依赖于辅助病毒的病毒,这种病毒在适于昆虫细胞生长的悬浮培养条件下,能繁殖出高效价的病毒,相对容易地可获得大量的重组蛋白,与从细菌里获得的不溶性蛋白不同,在昆虫细胞里,大部分过量生产的蛋白仍然可溶。而且,病毒基因组大(130kbp),这样就能接纳大的外源DNA片段。
1)杆状病毒载体的选择
所有有效的杆状病毒载体是基于pUC的并且授予氨苄青霉素抗性。各含有多角体基因启动子,可变长度的多角体编码序列,和用于将所需外源基因克隆到病毒序列两侧的插入位点,该病毒序列位于启动子的5’和外源基因插入物的3’。这些两侧序列有助于载体和野生型杆状病毒DNA之间进行同源重组(Ausubel等人,现代分子生物学技术,1990)。在选择合适的杆状病毒表达载体时,主要考虑是否以融合或非融合蛋白形式表达重组体。由于Gc多肽的糖基化需要用于将多肽转移到内质网的引导信号序列,应该将含有从起始密码子穿过引导序列到天然Gc蛋白质的+1氨基酸(leu)的cDNA导入到具有携带多接头的EcoRI位点的非融合载体,pLV393(Invitrogen,San Diego,CA)。
在用EcoRI酶对λgtll的编码Gc蛋白质的cDNA部分消化期间,通过电泳分离具有EcoRI末端的全长Gc cDNA,与EcoRI切割的pVL1393混合,并且用T4连接酶连接。该正确定位的构建体应该表达整个Gc肽,总共458个氨基酸(图2)。为了获得正确的构建体,将感受态的大肠杆菌HB101细胞转化到pVL载体并且在含有氨苄青霉素Luria肉汤琼脂平板(LB/氨苄青霉素平板)选择转化体。制备DNA用于测序方案,通过首先寻找3’多聚A延伸端,以测出含有具有正确的阅读方向的插入物或基因的菌落。然后从5’末端开始对具有3’多聚A(来自mRNA的多聚A尾)的克隆测序以证实编码Gc肽的全长DNA的正确方向。
2)用克隆的质粒DNA和野生型病毒DNA共转染昆虫细胞
用克隆的质粒(载体)DNA(2微克)和野生型(AcMNPV)杆状病毒DNA(10微克)在950微升转染缓冲液中共转染秋粘虫细胞的单细胞层(Sf9)(2.5×106个细胞/25平方厘米的烧瓶)(Ausubel等人,现代分子生物学技术,1990)。当细胞培养4或5天时,转染上清液含有重组病毒。
3)重组杆状病毒的鉴定
将共转染裂解液稀释104,105或106,并且于Sf9细胞平板培养4-6天。在噬斑充分形成之后,以1.3%的频率鉴定闭合现象阴性的细胞的噬斑。分离几个假设的重组病毒噬斑,两次再形成噬斑以便于纯化。分离纯化的重组病毒噬斑。
B.从重组杆状病毒中分析所需的蛋白质
1)重组病毒溶解产物的制备
将昆虫细胞秋粘虫(Spodoptera frugiperda)单细胞层(Sf9)(2.5×106个细胞/25平方厘米的烧瓶)用重组病毒克隆感染并且培养于5毫升的GIBCO无血清培养基里(来自于GIBCO生化公司,Rockville,MD)或补加0.1%卵清蛋白的培养基中以避免牛血清维生素D结合的蛋白的污染。培养瓶在27℃培养及每天检查感染的征状。4-5天后,轻轻地从烧瓶里收集细胞并将细胞和培养基转移到离心管里,在1000xg,4℃的条件下离心10分钟。为了感染最大化以便生产重组蛋白质,秋粘虫(Sf9)细胞培养于具有50毫升的完全培养基的100毫升旋转悬浮培养瓶,达到2×106个细胞/毫升。在1000xg的条件下离心10分钟收获细胞,重悬浮于10到20毫升含有感染系数(MOI)为10的重组病毒的无血清培养基中。在室温下培养1小时后,将感染的细胞转移到一个200毫升容积的有100毫升无血清培养基的旋转烧瓶并且培养40小时。分泌的多于40%的蛋白质是所需蛋白质,分离上清液的蛋白质。
2)所需蛋白质的定性测定
对十二烷基硫酸钠一聚丙烯酰胺凝胶用考马斯蓝染色,每个泳道装载20到40微克总细胞蛋白,用于估计表达蛋白质的数量。因为这些样品含有细胞蛋白质,同未侵染的细胞蛋白质比较易于测定该重组蛋白质。
3)克隆的Gc蛋白质经酶转化为巨噬细胞激活因子(GcMAFc)
采用维生素D亲和柱分离分子量为52000和458个氨基酸残基的克隆的Gc蛋白质(2微克)(图2)(Link.等人,分析生物化学157:262,1986)和用固定化的β-半乳糖苷酶和唾液酸酶处理,获得的克隆的巨噬细胞激活因子(GcMAFc)加入到小鼠和人类的巨噬细胞,分析其吞噬细胞和产生超氧化物的能力。用每毫升10pg的巨噬细胞激活因子培养巨噬细胞3小时,导致巨噬细胞吞噬细胞的活性增加5倍和巨噬细胞产生超氧化物的能力增加15倍。
C.巨噬细胞激活所需区域的亚克隆
I.克隆步骤/非融合载体
1)负责巨噬细胞激活区域(CdMAF)的克隆
通过EcoRI消化,将包括76个碱基对的上游5’侧翼区片段和204个碱基对的3’侧翼区片段的λgtll中编码Gc蛋白质的完整的cDNA序列片段化,以产生4个限制性片段,各片段分别命名为E1,120;E2,314;E3,482和E4,748碱基对。用Cooke和David的方法将每个限制片段克隆到来自Promega的质粒pSP65(Madison,WI)的EcoRI位点。尽管发现一个小于区域III的1/2的区域对巨噬细胞的激活有作用,但是小于40个氨基酸残基的小的片段不能在昆虫细胞里表达。加之短肽很快被人类血浆蛋白酶降解,所以不能应用于临床。因此,这一完整区域III(近80个氨基酸残基)被亚克隆进入一个昆虫病毒,其中预料侵染细胞里能有效地生产多肽和使其糖基化。
2)cDNA片段亚克隆进入杆状病毒多角体基因
由于肽糖基化作用需要一个前导信号序列,以便转移肽进入内质网,应该将包括起始密码子(-16Met),整个前导序列到天然Gc蛋白质+1氨基酸(Leu)的E1的DNA片段插入到该载体。因为这一片段携带Gc蛋白质的起始密码子,因此,可以使用非融合载体,pVL1393(Invitrogen,San Diego,CA)。含有cDNA克隆E1的起始密码子-前导序列的片段和编码克隆E4的85个C-末端氨基酸(整个区域III)+3’非编码延伸端的cDNA片段连接在一起,并且克隆到昆虫病毒pVL载体的EcoRI位点。为了获得这一构建体,用HaeIII将E1和E4 DNA裂解成片段以各产生两个片段,分别为E1hl(87个碱基对),E1hs(33个碱基对)和E4hs(298个碱基对)和E4hl(450个碱基对)。将两个E1hl和E4hl大的片段电泳分离,与EcoRI消化的pVL混合和用T4连接酶连接,正如图3所示。这一正确定位的构建体应表达整个区域III,总数为89个的氨基酸,包括4个E1hl氨基酸,也是指如图4所显示的CdMAF1。为了获得正确的构建体,感受态大肠杆菌HB101细胞转化到pVL载体和在LB/氨苄青霉素平板上筛选转化体。制备DNA用于测序步骤,通过首先寻找3’多聚A延伸端,以测出含有具有正确的阅读方向的构建体的菌落。然后从5’末端开始对具有3’多聚A(来自mRNA的多聚A尾)的克隆测序以证实E1hl片段的正确方向。发现载体含有正确定位的整个构建体(区域III)。
3)重组杆状病毒的分离,克隆的区域肽(Cd)的纯化和克隆的巨噬细胞激活因子的酶促产生
用克隆的质粒(载体)DNA(2微克)和野生型(AcMNPV)杆状病毒DNA(10微克)在950微升转染缓冲液中共转染秋粘虫细胞的单细胞层(Sf9)(2.5×106个细胞/25平方厘米的烧瓶)。分离重组杆状病毒噬斑和用于在昆虫细胞里生产Gc区域III肽。这一克隆的区域具有分子量(MW)为10000和89氨基酸,正如图4所显示,采用肌动蛋白亲和柱纯化该区域(Haddad等人,生物化学31:7174,1992)。2微克的克隆的区域(Cd1)肽用固定化的β-半乳糖苷酶和唾液酸酶处理产生一个克隆的巨噬细胞激活因子,命名为CdMAF1。
II.克隆步骤II:融合载体
1)克隆对巨噬细胞激活有作用的区域(CdMAF)
杆状病毒融合载体,pPbac载体(Stratagene,La Jolla,CA)含有人类胎盘碱性磷酸酶分泌的信号序列,它指导新生克隆肽趋向于细胞的分泌途径,导致分泌进入培养基。这一信号肽被信号序列肽酶裂解,而新生克隆肽被进入寄主昆虫细胞的分泌途径,导致克隆区域(Cd)肽的分泌。图5显示载体携带为基因替代的填充片段和用于鉴别基因插入的LacZ基因。
用限制性酶SmaI和BamHI载体消化载体DNA将pPbac载体的填充片段切开,通过电洗脱去除。Gc蛋白质的E4cDNA片段用HaeIII和BamHI消化,产生一个实际上同E4h1(图3)一样的片段。该片段与上述pPbac载体混合和用T4连接酶连接。这一方案不仅固定了连接的定位,而且也将该片段与读码框架融合。大肠杆菌DH5aF’细胞用反应混合液转化。用HaeIII和BamHI消化后,从大量菌落中分离这一克隆的DNA插入片段。这一插入片段由测序鉴定。序列显示正确的定位。
2)由用野生型杆状病毒和克隆的DNA插入片段转染的Sf9昆虫细胞分离重组杆状病毒
为了转染昆虫细胞(秋粘虫,Sf9),使用线性的野生型(AcMNPV)杆状病毒DNA和昆虫杀菌素脂质体(Invitrogen,SanDiego,CA)。脂质体介导的昆虫细胞的转染是最有效的适用的转染方法。为了在一个60mm培养皿里转染Sf9细胞的单细胞(2×106),轻轻加入下列混合物:
3微克克隆的质粒DNA,
10微升线性野生型杆状病毒(AcMNPV)DNA(0.1微克/微升)
1毫升培养基,
29微升昆虫杀菌素脂质体,
轻轻摇动该培养皿,在室温下培养4小时。转染后,加1毫升培养基和在27℃下培养在一个湿润的环境条件48小时。获得的转染溶菌产物用于噬斑测定。用克隆的步骤1里描述的方法,纯化重组病毒,分离克隆的区域肽(Cd2)和酶促产生命名为CdMAF2的克隆的巨噬细胞激活因子。该CdMAF是由94个氨基酸残基组成的,如图6所示,且包括融合载体的9个氨基酸,本文命名为CdMAF2,尽管CdMAF2比来自于非融合载体的CdMAF1肽多5个氨基酸,但他们显示相同的生物活性。
癌和艾滋病毒感染的患者的血液中α-N-乙酰氨基半乳糖苷酶活性测定方法的描述
1.用于检测癌和艾滋病毒感染的患者的血液中α-N-乙酰氨基半乳糖苷酶的测试方案(图示说明)
在癌和艾滋病毒感染的患者的血液中α-N-乙酰氨基半乳糖苷酶及血清Gc蛋白质去糖基化的检测步骤。
步骤1:血浆或血清中30和70%的硫酸铵逐步沉淀:
血浆/血清(1毫升)+30%和然后70%的饱和硫酸铵
70%的沉淀→在50毫摩尔的柠檬酸盐-磷酸盐缓冲液(pH6.0)中溶解→于同样缓冲液在4℃的情况下透析一夜。
步骤2:α-N-乙酰氨基半乳糖苷酶酶促测定
反应混合液:透析过的样品100微升+1毫升的50毫摩尔柠檬酸盐-磷酸盐缓冲液(pH6.0),这一缓冲液含有作为底物的5微摩尔的对硝基苯N-乙酰-α-D-氨基半乳糖苷。
培养时间:60分钟,加入200微升的0.5MNa2CO3终止。
活性测量:释放的对硝基酚在420nm的光吸收和以纳摩尔/毫克/分钟表示。
2.α-N-乙酰氨基半乳糖苷酶酶促测定步骤:
健康人和病人的血浆/血清(1毫升)用70%饱和硫酸铵沉淀。该硫酸铵沉淀溶解于50mM柠檬酸盐-磷酸盐缓冲液(pH6.0)和在相同的缓冲液4℃下透析,这一透析液的体积被增加到1毫升。硫酸铵沉淀将酶与抑制剂分离。该酶活性是在37℃500微升反应混合物中测定的,该混合液含有50mM柠檬酸盐-磷酸盐缓冲液(pH6.0)和作为底物的5微摩尔的对硝基苯N-乙酰-α-D-氨基半乳糖苷。这一反应最初是加入250微升透析样品开始的,然后60分钟后加入250微升0.5M Na2CO3溶液终止。将反应混合物用离心机离心,将上清液在420nm吸收以纳摩尔/克/分钟表示来决定释放出对硝基苯酚的量。
3.用酶联免疫吸附测定法(ELISA)来测定作为抗原的α-N-乙酰氨基半乳糖苷酶。
在免疫测定步骤中,为测定作为抗原(NagAg)的血清或血浆α-N-乙酰氨基半乳糖苷酶(Nag),制备抗体夹心ELISA试剂盒。
1)抗体的制备:制备抗α-N-乙酰氨基半乳糖苷酶多克隆抗体(山羊和兔子)和单克隆抗体。用硫酸铵(50%饱和液)分级分离(分馏)和DE52离子交换或者A蛋白质柱从抗血清或单克隆的腹水液中纯化免疫球蛋白分馏物。
2)碱性磷酸酶与抗体缀合。在0.1M的磷酸盐缓冲液中,在pH6.8(PBC)在4℃透析5毫克/毫升抗体溶液过夜,将100微升透析抗体溶液(3毫克/毫升)加入到一个1.5毫升微量离心管中的90微升10毫克/毫升碱性磷酸酶(免疫测定级;Boehringer Mannheim,Indianapolis,IN)中。加入5微升25%戊二醛且轻轻混合。放入温室下。在时间0,5,10,15,30,60和120分钟时去掉25微升样品。然后放在另一1.5毫升微量离心管,加入125微升PBS到每个样品中,然后加1.1毫升Tris/卵清蛋白溶液。在4℃下在PBS透析样品过夜。用直接的ELISA测试每一样品的碱性磷酸酶活性来决定哪一种缀合时间产生最有效的酶缀合。30分钟缀合时间产生最好的酶的活性。这一最佳缀合时间被用来制备大量的碱性磷酸酶抗体缀合物。
3)抗体包被的微滴板的制备:用多头移液管和尖头,将溶于PBSN(PBS包括0.05%叠氮钠)中的50微升的免疫球蛋白溶液2到10微克/毫升分配进入微滴板的每个孔(微孔)内,用塑料盖将平板盖住,然后在37℃下培养2小时或在室温下过夜。用蒸馏水漂洗包被的微滴板三次以上。用封闭剂(含有0.05%吐温20,1mM乙二胺四乙酸,0.25%BSA和0.05%NaN3的PBS)填满孔,所述封闭剂从瓶中流加分配到各个孔,和在室温下培养30分钟。用蒸馏水漂洗平板3次和轻轻地用纸巾覆盖去除残留的液体。
4)抗原(NagAg)的准备
(a)用于标准曲线的标准抗原。用封闭缓冲液以连续的1∶3稀释抗原原液,制备一个标准抗原稀释系列,稀释液的范围从0.1到1000纳克/毫升。
(b)测试抗原:稀释癌症病人或HIV感染病人的血浆/血清10到100倍,因为他们含有每毫升20到1000纳克的脱辅基蛋白质的病人血浆或血清。
5)测定:
步骤1:将50微升的抗原测试液等分试样和标准抗原稀释溶液加入到抗体包被的孔中,然后在室温下保温2小时,在蒸馏水中漂洗3次。用封闭缓冲液填满每个孔,然后在室温下保温10分钟,用水漂洗三次,然后去掉残留液体。
步骤II:加入50微升特异性抗体碱性磷酸酶缀合物(通常25到400纳克/毫升),然后在室温下保温2小时。如步骤I洗涤平板。
步骤III:每个孔加入75微升对硝基苯磷酸盐(NPP)底物溶液。然后在室温下保温1小时,酶活性引起溶液改变颜色,颜色的强度与NagAg的存在与否有关。置于有405nm滤器的微量滴定板读数器平板培养。
6)数据分析:通过标准抗原的系列稀释产生的数据来制备一个标准曲线,x轴为抗原浓度,是对数尺度,吸收率在y轴上,是线性尺度,在测试溶液中插入抗原(NagAg)浓度。计算α-N-乙酰氨基半乳糖苷酶的活性。由于抗癌和艾滋病毒的酶的抗体是特异于各自的酶(NagAg),这一抗体夹心ELISA将对照的抗原(α-半乳糖苷酶)与各个酶区分。酶活性也通过在另一标准曲线插入抗原(NagAg)浓度而决定血浆/血清α-N-乙酰氨基半乳糖苷酶活性,所述曲线是α-N-乙酰氨基半乳糖苷酶活性对抗原浓度的曲线。该ELISA测定法对于酶的活性测定不仅精确而且经济。
证据观察
A.克隆的巨噬细胞激活因子GcMAFc和CdMAF对培养的吞噬细胞(巨噬细胞和破骨细胞)的影响
用pg量的克隆的巨噬细胞激活因子GcMAFc和CdMAF处理人巨噬细胞和破骨细胞3小时,导致这些吞噬细胞的超氧化物分泌能力增强,正如表1所示。这一吞噬细胞激活水平是与GcMAF的巨噬细胞激活相似的(Yamamoto等人,AIDS Res.Human Ret.11:1373,1995)。
表1用GcMAF及其克隆的衍生物体外处理激活吞噬细胞
产生的超氧化物,纳摩尔/分钟/106吞噬细胞
浓度pg/ml 人巨噬细胞* 鼠腹腔巨噬细胞 人破骨细胞
GcMAFc 0 0.07 0.06 0.03
10 3.20 3.46 2.56
100 5.18 5.08 4.22
CdMAF 0 0.01 0.02 0.08
10 2.96 2.87 2.43
100 4.26 4.53 4.09
*癌病人外周血单核细胞/巨噬细胞,当使用艾滋病毒感染的病人时也会观察到相似的结果。
B.以克隆的巨噬细胞激活因子,GcMAFc和CdMAF给药,激活鼠腹腔的巨噬细胞
以pg量(10和100pg/鼠)的GcMAFc或CdMAF给药到BALB/C鼠1天后,分离鼠腹腔巨噬细胞,然后测定超氧化物的产生能力,正如表2所示,巨噬细胞被有效地激活,这一结果类似于用GcMAF激活巨噬细胞(Naraparaju和Yamamoto,Immunol.Lett.43:143,1994)。
表2以克隆的GcMAF衍生物给药激活小鼠的腹膜巨噬细胞
剂量 产生的超氧化物,纳摩尔/分钟/106吞噬细胞
pg/小鼠 小鼠腹腔巨噬细胞
GcMAFc 0 0.05
10 3.18
100 5.23
CdMAF 0 0.03
10 2.54
100 4.23
C.GcMAF,GcMAFc,或CdMAF对携带肿瘤的小鼠和骨质疏松症小鼠的治疗效果
1)GcMAF,GcMAFc,或CdMAF对携带埃利希腹水肿瘤的小鼠的治疗效果
当以GcMAF,GcMAFc,或CdMAF(100pg/小鼠)给药BALB/c小鼠并且接收105埃利希腹水肿瘤细胞/小鼠,它们至少存活5星期。所有对照小鼠仅接收腹水肿瘤并且在约2星期时死亡。当以额外量的GcMAF,GcMAFc,或CdMAF(100pg/小鼠)在移植后4天后给小鼠给药,肿瘤细胞被完全去除(表3)。
表3GcMAF和克隆的衍生物对携带埃利希腹水肿瘤的小鼠的治疗效果
组别 小鼠数量 移植后治疗a 存活小鼠的数量/时间
组1
6只小鼠 未治疗对照 6只小鼠/13±3天
10只小鼠 0天100pg GcMAF/小鼠 10只小鼠/36±7天
组2
6只小鼠 未治疗对照 6只小鼠/14±4天
10只小鼠 0天100pgGcMAF/小鼠 10只小鼠/35±6天
组3
6只小鼠 未治疗对照 6只小鼠/14±5天
10只小鼠 0天100pgCdMAF/小鼠 10只小鼠/34±3天
组4
8只小鼠 未治疗对照 8只小鼠/15±5天
12只小鼠 0天100pgCdMAF/小鼠
4天100pgCdMAF/小鼠 12只小鼠/>65天
组5
8只小鼠 未治疗对照 8只小鼠/14±5天
12只小鼠 0天100pgCdMAFc/小鼠
4天100pgCdMAFc/小鼠 12只小鼠/>65天
组6
8只小鼠 未治疗对照 8只小鼠/14±5天
12只小鼠 0天100pgCdMAFc/小鼠
4天100pgCdMAFc/小鼠 12只小鼠/>65天
aGcMAF,GcMAFc,或CdMAF肌内(全身的)给药,并且所有组小鼠接收105肿瘤细胞/小鼠
2)GcMAF和克隆GcMAF衍生物(GcMAFc和CdMAF)对骨质疏松症小鼠的治疗效果
通过从出生1天后每周注射100pg,共4星期进行以GcMAFc或CdMAF给新生的同窝仔给药。在28天杀死小鼠。从治疗的和未治疗的对照小鼠取出胫骨,纵向二等分,在解剖显微镜下检查以检测骨髓腔的大小。骨髓腔的大小以胫骨的骺平板之间的距离百分数表示。未治疗的小鼠组形成具有30%总长的胫骨的骨髓。在经过未治疗的小鼠组相同时期后治疗的小鼠组骨髓的形成增加20%。骨髓腔形成的增加是破骨细胞激活和破骨吸收增加的指标。
D.癌症和HIV感染患者的免疫抑制起源
癌症患者的α-N-乙酰氨基半乳糖苷酶的来源似乎是癌细胞。在各种器官的肿瘤组织匀浆液包括11个不同肿瘤组织包括4个肺,3个乳腺,3个结肠和1个宫颈肿瘤中检测到高α-N-乙酰氨基半乳糖苷酶活性,虽然α-N-乙酰氨基半乳糖苷酶活性从15.9-50.8纳摩尔/毫克/分钟。外科手术去除人癌产生的恶性损伤导致血浆α-N-乙酰氨基半乳糖苷酶缓慢降低,同时前体物活性增加,特别是如果恶性细胞被定位。
在临床前的小鼠肿瘤模型中,用5×105埃利希腹水肿瘤细胞/小鼠移植到BALB/c小鼠的腹腔,并且分析血清α-N-乙酰氨基半乳糖苷酶活性。当由于移植的埃利希腹水肿瘤在小鼠腹腔生长而检测血清酶水平时,酶活性与肿瘤负载(腹腔总细胞数)直接成比例,如图7所示。这也可以用移植了KB细胞的裸鼠(人口腔鳞状细胞癌细胞系)证实。随着固态肿瘤的肿瘤大小(由重量测定)增加,血清α-N-乙酰氨基半乳糖苷酶活性增加。因此,我们利用血浆α-N-乙酰氨基半乳糖苷酶活性作为预后性症状指数监测治疗进展。
人癌症放射性治疗降低了血浆α-N-乙酰氨基半乳糖苷酶活性,同时前体物活性增加。这表明放射性治疗降低了能够分泌α-N-乙酰氨基半乳糖苷酶的癌细胞数量。这些结果也证实血浆α-N-乙酰氨基半乳糖苷酶活性与Gc蛋白质的MAF活性具有相反的关系。甚至在外科手术去除人癌产生的恶性损伤之后,大多数手术后的患者在其血液中携带大量的α-N-乙酰氨基半乳糖苷酶活性。采用其它方法例如X-照射,闪烁法等等可检测到残留于手术后患者的癌损伤。我们利用大多数的敏感酶检测方法作为GcMAF治疗期间预后性症状指数。
HIV感染的细胞似乎分泌α-N-乙酰氨基半乳糖苷酶到血液中。当将HIV感染的患者的外周血单核细胞(PBMC)培养并且用作为前病毒诱导剂的丝裂霉素处理时(Sato等人,Arch.Virol.54:333,1977),α-N-乙酰氨基半乳糖苷酶分泌到培养液中。这些结果使我们建议α-N-乙酰氨基半乳糖苷酶是病毒编码的产物。事实上,HIV被膜蛋白gp120似乎是携带α-N-乙酰氨基半乳糖苷酶活性的。
E.GcMAF,GcMAFc,或CdMAF对人癌症和病毒感染患者的治疗效果
1.癌症患者:GcMAF对前列腺,乳腺和结肠癌和成人白血病患者的治疗效果
以GcMAF(100和500纳克/人)给健康志愿者给药结果导致大大地增加巨噬细胞的激活,如测定出巨噬细胞的吞噬能力增加7倍和产生超氧化物的能力增加15倍。以GcMAF给药证明接受者没有产生副作用的迹象。以各种剂量给许多小鼠给药,在各种器官包括肝,肺,肾,脾等等没有产生病症也没有组织学变化。当以GcMAF(100纳克/星期)治疗各种类型的癌症患者时,观察到对各种类型的癌症的显著的治愈效果。根据肿瘤特异性血清α-N-乙酰氨基半乳糖苷酶活性对GcMAF对携带各种类型的癌的患者的治疗效果进行评价,因为血清酶水平与癌细胞总数(肿瘤负载)成比例。图8a-8c描述了GcMAF对前列腺,乳腺和结肠癌的治愈效果。在以100纳克GcMAF给药25星期之后,大多数(>90%)的前列腺,乳腺癌患者明显地显示低水平的血清酶。在以GcMAF给结肠癌患者给药35次之后,也观察到类似的结果。观察到GcMAF对肺,肝,胃,脑,膀胱,肾,子宫,卵巢,喉,食管,口腔和皮肤癌具有类似的愈合效果。因此,GcMAF似乎对各种各样的癌的治愈效果没有差别。但是,在治疗6星期以上之后GcMAF显示患者没有产生任何副作用的证据。这也可通过血细胞数概括,肝和肾功能等等进行证实。
当以GcMAFc(100纳克/星期)和CdMAF(100纳克/星期)各给两个前列腺癌患者给药,观察到愈合效果类似于GcMAF。
2.病毒感染的患者
用100pgGcMAF/毫升对HIV感染/AIDS患者的外周血巨噬细胞进行治疗导致巨噬细胞的激活大大增加(Yamamoto等人,AIDS Res.Human Ret.11:1373,1995)。HIV感染的患者携带抗HIV的抗体。HIV感染的细胞在细胞表面表达病毒抗原。因此,当激活时,巨噬细胞具有借助于介导杀死/吞噬Fc受体的细胞去除感染细胞的潜力。
类似地,用100纳克GcMAF/毫升对慢性感染了EB病毒(EBV)和感染了疱疹的患者的外周血巨噬细胞进行治疗导致巨噬细胞的激活大大增加。类似于HIV,EBV感染的淋巴细胞(B细胞)。由于这些被膜病毒编码α-N-乙酰氨基半乳糖苷酶并且感染的细胞分泌所述酶到血流中,患者血清中该酶的活性可用作为治疗期间预后性症状的指数。在以GcMAFc(100纳克/星期)给慢性感染EBV和疱疹的患者25次给药之后,酶活性降低到健康的水平。当以GcMAFc(100纳克/星期)和CdMAF(100纳克/星期)给EBV感染的患者给药时,观察到愈合效果类似于GcMAF。
F.GcMAF,GcMAFc和CdMAF用作为免疫和接种的佐剂的活性:在GcMAF和绵羊红细胞给药之后小鼠中细胞分泌抗体的数量快速增加(PFC)
在腹膜内给药50pgGcMAF/小鼠之后6小时用SRBC接种BALB/c小鼠。在接种之后各个间歇(1-5天),利用Jerne噬斑试验测定脾中分泌IgM抗体的细胞(Jerne等人,细胞结合的抗体,Wistar研究院出版,1963)。GcMAF和SRBC给药1天之后产生1.35×104PFC/脾。GcMAF和SRBC给药2天之后,细胞分泌抗体的数量已经增加到8.2×104PFC/脾。在第4天,细胞分泌抗体的数量达到最大水平(约23.6×104PFC/脾),如表4所示。相反,在SRBC注射后4天仅接收SRBC注射的小鼠产生约3.8×104PFC/脾。
为了了解剂量应答,在以从1-50pg/小鼠范围的各种剂量的GcMAF给药后6小时,用SRBC注射小鼠。在以GcMAF和SRBC给药后第4天,利用Jerne噬斑试验测定每个脾中分泌抗体的细胞数量。给药4天之后,形成噬斑的细胞的数量同样地增加,而GcMAF的浓度增加约1pg/小鼠。在GcMAF的剂量为5,10和50pg/小鼠时,分别检测到12.6×104,20.2×104,24.3×104PFC/脾。因此,GcMAF是免疫接种的有效的佐剂。
表4在GcMAF和SRBCa给药后产生抗绵羊红细胞(SRBC)的分泌抗体的细胞的时间过程的研究
SRBC接种 分泌抗体的细胞/脾(×104)
之后天数 仅SRBC GcMAF+SRBC
1 0.01±0.002 1.35±0.21
2 0.08±0.02 8.28±1.26
3 1.18±0.42 14.42±2.32
4 3.86±0.95 23.68±6.05
5 2.15±0.63 18.63±3.43
a在GcMAF(50pg/小鼠)给药之后6小时用SRBC接种小鼠。在SRBC注射后每天用显微镜计算噬斑(分泌IgM细胞)的数量。形成噬斑的细胞的数量/脾以三个重复试验的平均值±SEM表示。
不需要进一步的阐述,前面的描述足于描述本发明,通过应用现在或将来的知识,在各种服务条件下同样可以使用其它情况。
参考文献
描述了下面的参考文献,其整个内容全部引入本文作为参考,如上述说明书中记载的参考文献一样。
美国专利文献
美国专利No.5177001,5177002和5326749(Yamamoto)。
其它出版物
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2.Sato.M.,Tanaka,H.,Yamada,T.和Yamamoto,N.,风疹病毒对BHK/WI-2细胞的持久感染和对风疹变异体的定性。Arch.Virology 54:333-343,(1977)。
3.Reeve,J.D.,August,C.S.,Humbert,J.R.,Weston,W.L.,对婴儿骨质疏松症的寄主防御。儿科学,64:202(1979)。
4.Marks,S.C.,Jr.,破骨细胞的来源,结构和功能的探针的先天的骨质疏松症突变。Clin.Prthop.189:239(1984)。
5.Link,R.P.,Perlman,K.L.,Pierce,E.A.,Schnoes,H.K.,Deluca,H.F.通过25-羟基维生素D3-Sepharose层析纯化人血清维生素D结合蛋白。Anal.Biochem.157:262-269(1986)。
6.Cook,N.E.和Haddad,J.G.,维生素D结合蛋白(Gc蛋白质)。Endocrine Rev.10:294-307(1989)。
7.Marks,S.C.,Jr.,破骨细胞生物学:哺乳动物突变课,Am.J.Med.Genet.34:43-54(1989)。
8.Ngwenya,B.Z.和Yamamoto,N.,溶血磷脂酰胆碱处理的非吸附细胞对巨噬细胞的刺激作用。Proc.Soc.Exp.Biol.Med.193:118124(1990)。
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10.Yamamoto,N.,和Homma,S.,维生素D3结合蛋白(组特异性成份,Gc)是来自溶血磷脂酰胆碱处理的淋巴细胞的巨噬细胞激活信号的前体物。Proc.Natl.Acad.Sci.USA.88:8539-8543(1991)。
11.Cooke,N.E.,和David,E.V.,血清维生素D3结合蛋白是白蛋白和α-甲胎蛋白基因簇。J.Clin.Invest.76:2420-2424(1985)。
12.Haddad,J.G.,Hu,Y.Z.,Kowalski,M.A.,Laramore,C.,Ray,K.,Robzyk,P.和Cooke,N.E.,血浆维生素D结合蛋白的固醇和肌蛋白结合的区域(Gc-球蛋白)的定性。生物化学31:7174-7181(1992)。
13.Yamamoto,N.,和Kumashiro,R.,通过逐步的B细胞的β-半乳糖苷酶作用和T细胞的唾液酸酶的作用将维生素D结合蛋白(组特异性成份)转变为巨噬细胞激活因子。免疫学杂志。151:2794-2902(1993)。
14.Yamamoto,N.,和Kumashiro,R.,Yamamoto,M.Willett,N.P.和Lindsay,D.D.,由两种血清因子,维生素结合蛋白和白蛋白调节巨噬细胞对炎症的激活。Inf.Imm.61:5388-5391(1993)。
15.Yamamoto,N.,Lindsay,D.D.,Naraparaju,V.R.,Irelalnd,R.A.,和Popoff,S.M.,在骨质疏松症大鼠中缺失激活炎症的巨噬细胞串联体,免疫学杂志152:5100-5107(1994)。
16.Yamamoto,N.,Willett,N.P.和Lindsay,D.D.,沉淀激活炎症的巨噬细胞的血清蛋白质。Inflammation.18:311-322(1994)。
17.Naraparaju,V.R.,和Yamamoto,N.,激活炎症的巨噬细胞中β-半乳糖苷酶对B淋巴细胞和唾液酸酶对T淋巴细胞的作用。Immunol.Lett.43:143-148(1994)。
18.Yamamoto,N.,Naraparaju,V.R.,和Srinivasula,S.M.,血清维生素D3结合蛋白的结构修饰和对HIV感染的患者的免疫抑制。AIDS Res.Human Ret.11:1373-1378(1995)。
Claims (10)
1.用于将维生素D3结合蛋白即Gc蛋白质或维生素D3结合蛋白区域III即Gc区域III克隆到杆状病毒的方法,包括选择和利用杆状病毒载体克隆Gc蛋白质或Gc区域III的步骤:
(a)利用免疫筛选方法从人肝λgt11分离全长Gc蛋白质DNA文库并且插入到非融合的杆状病毒载体中;或通过用EcoRI和HaeIII酶消化Gc蛋白质DNA而分离Gc蛋白质的起始前导序列加5’末端4个氨基酸的DNA和Gc蛋白质区域IIIDNA,并利用连接酶将其插入到非融合杆状病毒载体pVL1393的EcoRI位点;或利用HaeIII和BamHI酶分离Gc蛋白质区域IIIcDNA并且利用连接酶将其插入到融合的杆状病毒载体pPbac中以取代信号序列下游的SmaI-BamHI填充片段;
(b)通过将昆虫细胞用载体DNA和野生型杆状病毒DNA共转染以分离携带全长Gc蛋白质cDNA或Gc蛋白质区域IIIcDNA的重组杆状病毒;和
(c)在昆虫细胞中繁殖重组杆状病毒并且纯化克隆的Gc蛋白质或克隆的区域蛋白质Cd。
2.用于生产克隆的Gc蛋白衍生的巨噬细胞激活因子GcMAFc或克隆的区域III衍生的巨噬细胞激活因子CdMAF的方法,包括将克隆的Gc蛋白质或克隆的Gc蛋白质区域III与固定化的β-半乳糖苷酶和唾液酸酶体外接触并且获得克隆的Gc蛋白衍生的巨噬细胞激活因子GcMAFc或克隆的区域III衍生的巨噬细胞激活因子CdMAF。
3.一种克隆的Gc蛋白衍生的巨噬细胞激活因子GcMAFc,其是权利要求2所述方法的产物,具有SEQ.ID.NO:1的氨基酸序列。
4.一种克隆的区域III衍生的巨噬细胞激活因子CdMAF,其是权利要求2所述方法的产物,具有SEQ.ID.NO:2或SEQ.ID.NO:3的氨基酸序列。
5.一种巨噬细胞激活因子在制备用于治疗癌症或人免疫缺陷病毒的药物中的应用,所述巨噬细胞激活因子是权利要求2所述方法的产物,具有SEQ.ID.NO:1、SEQ.ID.NO:2或SEQ.ID.NO:3的氨基酸序列。
6.一种佐剂,其含有根据权利要求2所述方法得到的克隆的Gc蛋白衍生的巨噬细胞激活因子GcMAFc。
7.一种佐剂,其含有根据权利要求2所述方法得到的克隆的区域III衍生的巨噬细胞激活因子CdMAF。
8.一种克隆的维生素D3结合蛋白,其具有图2的氨基酸序列SEQ.ID.NO:1,其是克隆的Gc蛋白衍生的巨噬细胞激活因子GcMAFc。
9.一种克隆的维生素D3结合蛋白区域III,其具有图4的氨基酸序列SEQ.ID.NO:2,其是克隆的区域III衍生的巨噬细胞激活因子CdMAF1。
10.一种克隆的维生素D3结合蛋白区域III,其具有图6的氨基酸序列SEQ.ID.NO:3,其是克隆的区域III衍生的巨噬细胞激活因子CdMAF2。
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