HSV-1特异转移因子活性成分的分离和鉴定

取单纯疱疹病毒Ⅰ型(HSV-1)免疫小鼠脾脏制备HSV-1特异转移因子(TF_(HSV-1))。利用亲和色谱法从TF_(HSV-1))分离出能与HSV-1抗原特异结合的成分。TF_(HSV-1)及其成分均可诱导正常鼠淋巴细胞出现抗原依赖的粘附抑制,提纯的TF_(HSV-1)特异成分的免疫活性是TF_(HSV1)的40倍。TF_(HSV-1)及其成分亦可提高HSV-1感染小鼠的存活率,该作用具有剂量依赖性。在保护实验中,TF_(HSV-1)中的特异成分的比活性是_(HSV-1)原有活性的16倍。本文结果表明,我们建立的TF纯化方法实验条件温和,能够迅速有效地分离获得具高免疫活性的TF特异成分。     

抗HSV-1淋巴细胞杂交瘤的建立及应用单克隆抗体对HSV分型

应用HSV-1 SM44株感染的BHK-21细胞及提取的感染细胞膜蛋白抗原免疫Balb/c小鼠。以免疫小鼠脾细胞与小鼠骨髓瘤细胞系Sp_2/0融合,经ELISA筛选仅与HSV-1反应的阳性克隆及克隆化,建立了一株产生抗HSV-1型特异性McAb的杂交瘤细胞系(Mad-2)。经ELISA,IF及抗原吸收试验证明,该细胞系的培养上清及制备的小鼠腹水均与HSV-1呈特异性反应,但不与HSV-2反应。ELISA测定McAb Mad-2的腹水效价为10~(-7)Ig亚类鉴定为IgG-1。应用Mad-2和抗HSV型共同性McAb 1A12及抗HSV-2型特异性McAb CH-A9,对43株临床HSV分离株做了ELISA及IF分型。结果表明,28株口唇、眼部感染分离株和1株宫颈炎分离株均与1A12和Mad-2反应,不与CH-A9反应,为HSV-1。余14株富颈炎分离株均与1A12和CH-A9反应,而不与Mad-2反应,为HSV-2。ELISA和IF分型的结果完全一致。本研究提出作者应用的一套抗HSVMcAb有可能作为HSV型别鉴定的标准试剂。     

氨基酸定性分析技术的一些改进 Ⅰ.快速水解和高温滤纸层析

(1)提供了一种快速水解液,蛋白貭或多肽完全水解仅需20分鉀至2小时,其配制方法是:12NHCl:85%甲酸:水=2∶1∶1。水解温度160—180度。(2)提供了氨基酸高溫层析法及正丁醇:呲啶系統可在4小时內完成单向层析,10小时內完成双向层析,其溶剂系統各成分的比例如下: 酸性溶剂系統:(1)正丁醇:85%甲酸:水=15∶3∶2。(2)正丁醇:冰醋酸:95%乙醇:水=4∶1∶1∶2。溶剂(1)适用于单向层析和双向层析的第一向,溶剂(2)适用于单向层析。硷性溶剂系統:正丁醇:呲啶:60%乙醇:水=5∶1∶1∶1。此溶剂适用于双向层析的第二向。(3)高溫层析的温度有二种:(1)在40—45度进行(2)60度进行一半移室温完成。前者較易操作,重复性也較強。(4)引用了等的吲(口乃木)醌显色法,对氨基酸的分辨极有帮助,并发現一些氨基酸的衍生物或多肽也均显不同顏色。(5)經选择后的新华定性滤紙层析效果頗佳。     

一种女贞叶抗冻蛋白的分离纯化

根据抗冻蛋白与冰结合的特性, 利用碎冰从女贞(Ligustrum lucidum)叶提取液中分离出抗冻蛋白。结果表明, 通过碎冰吸附、凝胶过滤和离子交换层析可以获得4个组分的蛋白质, 其中的1个经鉴定具有热滞活性。在蛋白质浓度为5 mg. mL-1时, 它的热滞活性(thermal hysteresis activity, THA)值为0.678°C, 对其进行全波长扫描(200-1 000 nm)发现在975nm处有吸收峰; 该蛋白亲水性氨基酸含量较高。     

一种女贞叶抗冻蛋白的分离纯化

根据抗冻蛋白与冰结合的特性,利用碎冰从女贞(Ligustrum lucidum)叶提取液中分离出抗冻蛋白。结果表明,通过碎冰吸附、凝胶过滤和离子交换层析可以获得4个组分的蛋白质,其中的1个经鉴定具有热滞活性。在蛋白质浓度为5mg.mL-1时,它的热滞活性(thermal hysteresis activity,THA)值为0.678°C,对其进行全波长扫描(200-1000nm)发现在975nm处有吸收峰;该蛋白亲水性氨基酸含量较高。     

单纯疱疹病毒2型3万道尔顿结构蛋白的纯化及其特性研究

用抗HSV-2型特异性单克隆抗体CH-A9与Sepharose 4B偶联制备免疫吸附柱,以亲和层析的方法,从HSV-2感染的BHK细胞膜上纯化了一种HSV-2特异的结构蛋白(VP30)。实验表明,该结构蛋白的主要特性有:第一,分子量为30,000道尔顿;第二,具有HSV-2型持异性;第三,可在小鼠体内诱发中和抗体。     

单纯疱疹病毒1型感染神经胶质细胞的代谢组学研究

目的研究单纯疱疹病毒1型(HSV-1)对人类少突胶质细胞(OL细胞)代谢的影响,从而探讨HSV-1感染的分子机制。方法将OL细胞随机分为两组,一组为感染组,感染HSV-1,一组为正常对照组,不感染HSV-1,运用核磁共振氢谱(~1H NMR)检测二组细胞培养液水相代谢产物,最后由偏最小二乘法-判别分析(PLS-DA)对数据进行分析。结果感染组与正常对照组细胞培养液水相代谢物的组成有显著差异:被HSV-1感染的OL细胞的培养液中,亮氨酸、谷氨酸、谷氨酰胺增多,丙氨酸、甘氨酸、组氨酸、异亮氨酸、乳酸减少。结论 HSV-1感染改变OL细胞的氨基酸代谢,HSV-1可能通过扰乱宿主细胞的代谢路径,促进自己的繁殖,并影响神经系统的功能活动。     

棕点湍蛙皮肤分泌液中促胰岛素释放肽的分离纯化、基因克隆及活性分析

通过分离纯化棕点湍蛙(Amolops loloensis)皮肤分泌液中的生物活性物质,得到有促胰岛素释放活性的分离峰,并鉴定其结构.采用葡聚糖Sephadex G-50凝胶层析和反相高效液相(RP-HPLC)等手段对棕点湍蛙皮肤分泌液进行分离纯化,利用胰岛素释放实验进行活性检测,Edman降解法测定活性峰的氨基酸序列,反转录法构建cDNA文库并克隆其基因.得到一个具有显著的促胰岛素释放活性的十六肽,测得其氨基酸序列为:FMPIvGKsMSGLSGKL-NH2,命名为amolopin-1.由cDNA(开放阅读框为192bp)推导的氨基酸一级结构显示,其前体由64个氨基酸残基(aa)组成,包括高度保守的信号肽(22aa),酸性肽以及成熟肽.经过数据库序列比对,从棕点湍蛙皮肤中得到一个新的促胰岛素释放肽,进一步分析其作用机理和药代动力学,极有可能得到一个新的治疗糖尿病的降糖药物.     

HSV-1感染对神经胶质瘤细胞神经生长因子及其受体表达的影响

神经生长因子(NGF)主要由神经胶质细胞产生,通过特异的靶细胞表面的神经生长因子受体介导产生生物学效应,与神经细胞的生长发育、分化和凋亡等密切相关。单纯疱疹病毒1型(HSV-1)作为一种嗜神经病毒,易造成神经细胞、神经胶质细胞凋亡或死亡。本实验以U251人神经胶质瘤细胞为研究对象,观察HSV-1感染致U251细胞凋亡的过程中NGF及其受体的变化情况。结果发现U251细胞是HSV-1的容许细胞;HSV-1感染致U251细胞凋亡过程中,NGF及其低亲和力受体p75NTR出现表达强度随时间先增强后减弱的趋势,而高亲和受体Tr-kA持续低表达。推测HSV-1感染致神经细胞凋亡中可能调控了神经营养因子的表达。     

茶树花粉特异蛋白基因CsPSP1的分离及序列分析

利用cDNA-AFLP技术比较了茶树[Camellia sinensis(L.)O.Kuntze cv.Wulong]花蕾发育早期和晚期的基因表达,结果表明存在明显差异。以E12和M20为引物对在晚期发育花蕾中筛选出一条281 bp特异表达的差异条带TDF53(transcipt-derived-fragment,TDF)。RT-PCR分析表明该片段只在晚期发育花蕾中特异表达。用RACE方法延伸其末端序列,克隆并测序获得全长cDNA序列(GenBank登录号:DQ887753)。该基因全长2079 bp,开放阅读框1701 bp,编码567个氨基酸,其分子量为63 kDa。序列和结构的同源性分析表明:该基因编码的氨基酸序列与烟草、油菜的花粉特异蛋白等同源性较高,由此推定,该基因为编码茶树花粉特异蛋白的基因,并将分离到的花粉特异蛋白基因命名为CsPSP1。     

Approach to elaborating immunochemical methods for removing peptides from the blood of kidney failure patients

A homogenic peptide (m. w. 2100 dalton) was isolated from blood serum of patients suffering from renal failure by gel chromatography on a Sephadex G-25 column and by partition chromatography on a column with cellulose MN-300. The peptide was not detectable in the blood of healthy donors. The amino acid composition of the peptide was studied. To obtain an immunosorbent, the peptide was conjugated by the bifunctional reagent, glutaric aldehyde. An optimal conjugation variant promoting the formation of conjugates with a molecular weight of 60000 dalton was developed. The molecular weight of the conjugates was measured by gel chromatography on a Sephadex column G-50 and with the aid of disc electrophoresis in polyacrylamide gel.     

Incorporation of the Ca2+ -binding component (g2) into heavy meromyosin and subfragment-1 of pig cardiac myosin.

A new protein component was found in heavy meromyosin and in subfragment-1 (S-1) prepared by chymotrypsin digestion of pig cardiac myosin in the presence of Ca2+. The molecular weight of this protein was estimated as 15,000 dalton. It was able to bind Ca2+ and showed a similar UV absorption spectrum to that of the g2 light chain. Heavy meromyosin and subfragment-1 which contained the 15,000 dalton component incorporated exogenous g2 and the 15,000 dalton component disappeared after such treatment. We concluded that the 15,000 dalton component was produced from g2 by limitted proteolysis. The subfragment-1 was separated into two protein fractions in equal yield by recycling the gel filtration. One contained the 15,000 dalton component and was able to bind Ca2+ while the other did not contain the component and was unable to bind Ca2+. According to analysis by SDS gel electrophoresis, the large polypeptide chain (the f component) of the first S-1 was approximately 5,000 dalton larger than the f component of the second S-1. The polypeptide corresponding to 5,000 dalton was designated polypeptide-C, because it was released from the C terminal of the f component. It seems to be essential for the attachment of the Ca2+-binding light chain g2. The location of g2 in myosin may thus be at the polypeptide-C which links the head to the tail of myosin.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

The role of histones H3 and H1 in the formation of thymine-lysine cross-links during UV-irradiation of deoxyribonucleoprotein

The effect of UV light (lambda = 254 nm) on calf thymus DNP at low ionic strengths was studied. It was found that at the irradiation doses used the protein in the DNA-protein complex increases as the irradiation dose rises. Thermal treatment and acid hydrolysis resulted in a predominant release of histones H3 and H1 from the complex. Data from liquid high performance chromatography, amino acid analysis, thin-layer chromatography point to the induction by UV-light of a thymine-lysine bond, whose formation involves DNA thymines and histone lysine residues, predominantly H3 and H1 fractions.     

层析聚焦法分离纯化末端脱氧核苷酰转移酶及其抗血清制备和鉴定

 应用层析聚焦和Oligo(dT)-纤维素亲和层析相结合的方法,从小牛胸腺中分离纯化末端脱氧核苷酰转移酶(TdT)。纯化的TdT聚丙烯酰胺凝胶电泳呈一条区带,SDS-聚丙烯酰胺凝胶电泳分子量为24,000及26,000d的两条区带。此纯化TdT径戊二醛交联法,自身交联后免疫家兔,得到兔抗小牛TdT的单价抗血清,并进行了免疫学鉴定。     

常山凝集素的分离纯化及其性质研究

 本文采用分子筛层析和离子交换技术,从植物常山(Dichroa febrifuga Lour)的鲜叶中,分离纯化出一种新的凝集素,定名为常山凝集素(DFL)。并对其理化性质进行了鉴定:测得其亚基分子量为37,000道尔顿;等电点为4.2。DFL是一种糖蛋白,糖含量为2.8％。DNS-CI法测得其肽链的N-末端为L-缬氨酸。本文还对其糖专一性、不同动物红细胞的凝集专一性,以及对猪精子和某些肿瘤细胞的凝集活性等生物学性质进行了研究。     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Primary structure of PDC-109, a major protein constituent of bovine seminal plasma

The two major protein components of bovine seminal plasma, PDC-109 and BSP I, have been purified by gel filtration, partition chromatography and reverse-phase high performance liquid chromatography from an 86% ethanol precipitate of bovine seminal plasma ejaculate. The complete 109-residue amino acid sequence of PDC-109 has been established by automated Edman degradation of the intact peptide as well as its proteolytic digestion and cyanogen bromide cleavage fragments. The 12,774 dalton structure has two structurally similar domains of 38 and 41 amino acids, each containing two disulfide bonds.     

Molecular weight and structural studies on cephalopod rhodopsin.

The protein moiety of squid (Watasenia scintillans) rhodopsin has been shown to have a molecular weight of 46 800 by means of amino acid analysis. This value was comparable to the value (51 000) obtained from SDS-polyacrylamide gel electrophoresis. After the squid eyes were incubated at 10 degrees C for 8 days, the rhodopsin showed a molecular weight of 39 000 on electrophoresis. The smaller molecular weight was ascertained by amino acid analysis of the rhodopsin; and may result from autolysis by the lysosomal enzyme. The rhodopsin in rhabdomeric membranes and in detergent solution was treated with chymotrypsin, papain or subtilisin. These enzymes first produced the 39 000 dalton rhodopsin and then cleaved this into the 25 000 and 14 000 dalton peptides without bleaching. The rhodopsin was attacked by proteases and readily lost an approx. 12 000 dalton peptide portion. This portion included the COOH-terminal and was rich in glutamic acid, proline, glycine, alanine and tyrosine residues.