用单克隆抗体和蛋白水解酶研究人绒毛膜促性腺激素的抗原决定簇

 本文报道,在不同酶解条件下,用嗜热菌蛋白酶、糜蛋白酶、胰蛋白酶,酶解人绒毛膜促性腺激素(hCG)得四个片段,而用胃蛋白酶则得三个片段;这些酶解片段在聚丙烯酰胺凝胶电泳中的条带位置不同,说明为非均一的酶解产物。应用蛋白质转移电泳技术和在硝酸纤维素膜上进行免疫酶标染色,检测出抗hCG的单克隆抗体9C_2和8B_5可与完整hCG及所有hCG酶解片段反应;而7F_3、BAH和10E_8只与完整hCG反应。表明hCG分子中可能存在序列型和结构型的两种不同的抗原决定簇。     

人绒毛膜促性腺激素酶解片段抗原性的研究

 本文应用制备电泳从人绒毛膜促性腺激素(hCG)的嗜热菌蛋白酶酶解混合物中,分离制备出了三个hCG酶解片段。经聚丙烯酰胺凝胶电泳和酶标免疫染色鉴定,表明它们具有较高纯度。用SDS-聚丙烯酰胺凝胶电泳法测定了这三个hCG酶解片段的表观分子量分别为4.35×10~4,4.20×10~4和4.00×10~4;而完整hCG分子的表观分子量为4.75×10~4。用DNS-Cl法测知这三个酶解片段各含有两个N末端氨基酸,分别为Leu/Gly,Ile/Gly和Val/Gly,这些末端与完整hCG的N末端氨基酸不同。用ELISA法测定了这三个酶解片段对抗hCG单克隆抗体9C_2的反应性,发现它们的抗原活性分别是完整hCG分子的1.44倍、1.50倍和1.63倍。表明经酶解后,片段的抗原活性有所提高,这可能是其抗原决定区经酶解后向外暴露,使单克隆抗体更加可及之故。     

人绒毛膜促性腺激素β亚基酶解片段特性的研究

以嗜热菌蛋白酶水解人绒毛膜促性腺激素,亚基(hCG_β),用HPLC分离制备酶解产物。酶解片段经竞爭性放射分析,不能与LH/hCG受体结合,但保留近乎全部hCG_β的免疫反应性.园二色谱分析,与hCG_β比较,酶解片段所含α螺旋减少,β折叠与无规卷曲含量增多。表明hCG_β可能有几个肽段和/或糖链参与hCG受体相互作用;酶解片段中仍含有hCG_β的主要决定簇,该决定簇与hCG_β结合受体的结构有区别。     

萤火虫荧光素酶单克隆抗体的制备与抗原决定簇的研究

以大肠杆菌表达的萤火虫荧光素酶 (fireflyluciferase)为抗原 ,免疫小鼠并进一步筛选与克隆 ,共得到 6株单克隆抗体 .制备腹水并纯化获得抗体后 ,对这 6株抗体与天然态和热变性态蛋白质以及蛋白酶解片段的结合性质进行了鉴定 .认为这 6株抗体的抗原决定簇都是顺序决定簇 .发现其中有 2株单抗与热变性态蛋白质及酶解片段的结合能力较强 ,而不与天然态蛋白质结合 ,其抗原决定簇可能是位于蛋白质内部的肽段 .另外 4株抗体既可与热变性态蛋白质以及酶解片段结合 ,也可与天然态蛋白质结合 ,其抗原决定簇可能位于蛋白质分子表面 .     

从免疫及天然噬菌体抗体库中筛选抗rP27Kip1单链抗体的研究

用重组p27Kip1蛋白(rP27Kip1) 免疫小鼠,从免疫和未经免疫的小鼠脾脏抽提mRNA并扩增小鼠H链(H链)及L链(L链)基因,分别组装成单链可变区片段(ScFv)基因,构建噬菌体免疫抗体库及天然抗体库. 文库仅经一轮抗原-抗体亲和筛选后,用TaqⅠ/HinfⅠ酶切分析转化子. 获自免疫抗体库的64个克隆中,有11个克隆的酶切片段相同,而天然抗体库的64个克隆的片段则都彼此不同,但有1个克隆的酶切片段与免疫抗体库的11个克隆酶切片段相同. 将这些酶切图谱相同的重组片段分别克隆入原核表达载体pET28b(+),并在大肠杆菌(E. coli)中表达,表达产物经ELISA分析,证实可特异结合rP27Kip1抗原,一方面说明在抗体筛选过程中辅以酶切图谱分析,可以有效提高筛选效率,另一方面,也说明从噬菌体抗体库筛选特异性抗体是制备单克隆抗体(McAb)的理想途径之一.     

长叶车前花叶病毒上海分离株(HRVsh)外壳蛋白免疫多肽的研究

长叶车前花叶病毒上海分离株(HRVsh)的外壳蛋白中含有4个甲硫氨酸残基,本文采用溴化氰裂解,并结合葡聚糖凝胶G-100柱层析、高压纸电泳及纸层析等方法,分离纯化了5个多肽片段,经~(125)I标记抗体对免疫多肽的鉴定,表明其中二段多肽与~(125)-IgG的结合能力接近完整病毒的水平,说明这二段多肽具有HRVsh的抗原专一性,决定HRVsh抗原性的抗原决定簇主要分布于这二个肽段中。 外壳蛋白的胰蛋白酶酶解肽谱及多肽氨基酸序列分析的结果,表明HRVsh和HRV标准株系间在氨基酸序列上有很大相似性,这就决定了两者密切的血清学亲缘关系。     

通过乙型肝炎病毒核心基因5''''端的修饰使核心抗原在大肠杆菌中高效表达

为了使乙型肝炎病毒核心抗原(HBcAg)在大肠杆菌中获得高效表达,本文首次采用一种新方法对核心基因前区进行改造与修饰,即用限制性内切酶Taq I从核心基因内部5′端切开,去除核心基因起始信号ATG及ATG 5′端上游的全部前核心区(Precore),再化学合成一段既包含核心基因起始信号又具有多种功能的DNA片段。将两者拼接重组到表达载体pUC9上,转化受体菌,成功地获得高效表达HBcAg的菌株。用ELISA法检测,表达滴度为1:80000。表达产量占菌体总蛋白的16％。其菌体裂解液经免疫电镜观察,可见到成堆聚集的典型HBcAg颗粒。抗原单体分子量约为22000道尔顿,双体为44000。与目前国内外所普遍采用的方法比较,本文的方法有许多明显的优点,可用于其它基因改造。     

λ拟操纵基因的克隆

以HindtTI和艮o Rl双重酶解p1)G3|质粒DNA制备了29个硷基对DNA片段。该DNA片段含有l‘个硷基对A拟操纵基因(pseud~perator),其一端为HindLTI接头,另一端为EcoRI接头。29个玲基对片段与pBR325质粒连接、转化、筛选获得克隆pQHl00(Ap‘Tc‘。M’)。FQHl00DNA分别以Hznd[1l,EcoRl单独酶解或双重酶解,经凝胶电泳分析,其结果表明,pQH1~)0DNA含有两个A拟操纵基因,这两个DNA片段在HtnI川位点上,以头对头的方式相连。     

胰岛素依赖型糖尿病HLAⅡ类抗原DNA多态性研究

 用Southern DNA分析法,对正常人和胰岛素依赖型糖尿病(IDDM)病人外周血白细胞DNA进行限制性片段长度多态性(RFLP)研究。HLA抗原与IDDM相关,我们用HLA-DQβcDNA探针和EcoR Ⅰ、BamH Ⅰ内切酶,测得EcoR Ⅰ 2.2kb片段与正常人DR2抗原相关联(r=0.78,P=1×10~(-6),与IDDM DR2抗原无关联;此片段在DR2的正常组和IDDM组中的频率有显著差异(P=0.02)。EcoR Ⅰ 3.0kb和BamH Ⅰ 3.3kb片段在IDDM组中的频率均降低,与正常组比较其频率有显著差异(P=3.2×10~(-3)和P=1.8×10~(-3),这二片段的差异还未见报道。DNA的RFLP研究提示,IDDM病人中可能是由于基因片段的缺失或是基因结构的改变,导致经酶切后与探针杂交的结果与正常者有差异。     

酵母丙氨酸转移核糖核酸5′-端GpGpGpCpGPUpGpUp~(m~1)GpGpCpGpU十三核苷十二磷酸片段(1～13)的合成

本文介绍用T_4 RNA连接酶酶促合成方法合成酵母丙氨酸转移核糖核酸5′端十三核苷十二磷酸片段(1～13)GpGpGpCpGpUpGpUp~(m~1)GpGpCpGpU的工作。我们将这一片段分为二段来合成,先合成5′-端6Nt和3′-端7Nt(A合成路线)或5′端7Nt和3′-端6Nt(B合成路线),再将它们连接起来。并对由于片段含鸟便嘌呤核苷酸较多而在合成和分离上出现的一些特性进行了探讨。     

Proteolytic and chemical dissection of the human erythrocyte glucose transporter.

Treatment of the purified, reconstituted, human erythrocyte glucose transporter with trypsin lowered its affinity for cytochalasin B more than 2-fold, and produced two large, membrane-bound fragments. The smaller fragment (apparent Mr 18000) ran as a sharp band on sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis. When the transporter was photoaffinity labelled with [4-3H]cytochalasin B before tryptic digestion, this fragment became radiolabelled and so probably comprises a part of the cytochalasin B binding site, which is known to lie on the cytoplasmic face of the erythrocyte membrane. In contrast, the larger fragment was not radiolabelled, and ran as a diffuse band on electrophoresis (apparent Mr 23000-42000). It could be converted to a sharper band (apparent Mr 23000) by treatment with endo-beta-galactosidase from Bacteroides fragilis and so probably contains one or more sites at which an oligosaccharide of the poly(N-acetyl-lactosamine) type is attached. Since the transporter bears oligosaccharides only on its extracellular domain, whereas trypsin is known to cleave the protein only at the cytoplasmic surface, this fragment must span the membrane. Cleavage of the intact, endo-beta-galactosidase-treated, photoaffinity-labelled protein at its cysteine residues with 2-nitro-5-thiocyanobenzoic acid yielded a prominent, unlabelled fragment of apparent Mr 38000 and several smaller fragments which stained less intensely on SDS/polyacrylamide gels. Radioactivity was found predominantly in a fragment of apparent Mr 15500. Therefore it appears that the site(s) labelled by [4-3H]cytochalasin B lies within the N-terminal or C-terminal third of the intact polypeptide chain.     

An amphiphilic structure of the ninth component of human complement. Evidence from analysis of fragments produced by alpha-thrombin

Purified human C9 was treated separately with three proteolytic enzymes: trypsin, plasmin, and alpha-thrombin, and the digestion products were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Trypsin initially cleaved the Mr = 71,000 C9 to produce a Mr = 47,000 fragment plus numerous smaller fragments and prolonged digestion reduced the molecule to small polypeptides. Plasmin produced a Mr = 37,000 fragment which was stable to further digestion, plus fragments smaller than Mr = 10,000. Human alpha-thrombin cleaved C9 (7.8% carbohydrate) at a single internal site to produce a Mr = 37,000 fragment (11.3% carbohydrate) and a Mr = 34,000 fragment (3.9% carbohydrate). Statistical analysis of the amino acid compositions of the fragments and alkaline polyacrylamide gel electrophoresis showed that C9 is highly amphiphilic; the Mr = 34,000 fragment contains a majority of the acidic amino acids and migrates rapidly on alkaline gels; the Mr = 37,000 fragment is hydrophobic with a slow electrophoretic mobility. The two fragments remain noncovalently associated, but were separated by sodium dodecyl sulfate-hydroxylapatite chromatography. The NH2-terminal sequence analysis of native C9, of alpha-thrombin-cleaved C9, and for the isolated fragments showed that the acidic Mr = 34,000 fragment is the NH2-terminal C9a domain and the more hydrophobic Mr = 37,000 fragment is the carboxyl-terminal C9b domain. Hemolytic activity of C9 was unaffected by alpha-thrombin cleavage.     

Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane

Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.     

Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1 M sucrose results in degradation of the Mr = 102,000 ATPase enzyme to two fragments of Mr = 55,000 and 45,000 with subsequent appearance of fragments of Mr = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the Mr = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the Mr = 55,000 and 45,000 fragments, indicating that they were derived from a single linear cleavage of the larger enzyme. There was antigenic cross-reactivity between the Mr = 20,000 and 55,000 fragments, indicating an origin of the Mr = 20,000 fragment in the Mr = 55,000 fragment. None of the antibodies inhibited (Ca2+ + Mg2+)-dependent ATPase or Ca2+ transport. The Mr = 20,000 fragment and the Mr = 55,000 fragment were active in Ca2+ ionophore assays. The active site of ATP hydrolysis was labeled with [gamma-32P]ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the Mr = 55,000 and 30,000 fragments, indicating that the Mr = 30,000 fragment was also derived from the Mr = 55,000 fragment. Amino acid composition data showed that the Mr = 45,000 fragment contained about 60% nonpolar and 40% polar amino acids, while the Mr = 55,000 fragment and the Mr = 20,0000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca2+-binding protein were not. The use of antibodies against the various fragments indicated that the Mr = 55,000 fragment was in large part exposed, whereas the Mr = 20,000 and the 45,000 fragments were only poorly exposed. It is probable that the site of ATP hydrolysis in the Mr = 55,000 fragment is external, whereas the ionophore site is only partially exposed and the Mr = 45,000 fragment is largely buried within the membrane.     

The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half.

Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.