国产蛇毒激活血浆蛋白C的研究

目的:从9种国产蛇毒中筛选具有激活血浆蛋白C作用的蛇毒。方法:运用活化部分凝血活酶时间(APTT)和发色底物实验分别观察抗凝活性和酰胺酶活性,综合抗凝活性和酰胺酶活性确定蛋白C蛇毒激活作用。结果:在9种国产蛇毒中烙铁头蛇毒及蝮蛇毒在1.5mg/L浓度下即使人血浆纯蛋白C产生酰胺酶活性,并使APTT显著延长。结论:9种国产蛇毒中烙铁头蛇毒及蝮蛇毒具有激活人体血浆蛋白C成为活化蛋白C(APC)的作用。     

中国强毒赖型钩端螺旋体新基因OmpL17的表达及其免疫原性

将Omp L17基因克隆于p GEX- 1λT载体,在大肠杆菌JM10 9中表达分子量约为5 4 KDa的GST-Omp L17融合蛋白,凝血酶切割后得到了大小约2 8KDa的Omp L17蛋白。用纯化的Omp L17蛋白免疫大白兔,制备了高滴度的特异性抗体(1∶4 896 )。本研究获得了纯化的Omp L17及其特异性抗体,为该外膜蛋白致病作用及免疫保护力研究奠定基础。     

醛酮还原酶AKR7A5蛋白的纯化及其对萘醌类化合物的底物特异性研究

目的:探讨小鼠醛酮还原酶AKR7A5蛋白对萘醌及其衍生物的底物特异性。方法:IPTG(异丙基硫代半乳糖糖苷)诱导BL21pLysS大肠杆菌中His标签的AKR7A5融合蛋白大量表达,利用FPLC系统通过HiTrap亲和柱纯化His-AKR7A5融合蛋白,经SDS-PAGE和Western blot法鉴定纯化的AKR7A5蛋白。使用AKR酶活性实验检测纯化的重组AKR7A5蛋白对萘醌类化合物的底物特异性。结果:经SDS-PAGE和Western blot验证,成功纯化了His标签的AKR7A5融合蛋白;AKR酶活性实验结果显示,重组AKR7A5蛋白对散沫花醌有中等亲和力,对胡桃醌和维生素K3有较低的亲和力,对1,4-萘醌无亲和力。结论:成功纯化了重组AKR7A5蛋白,并检测了其对萘醌类化合物的底物特异性,结果表明醛酮还原酶很可能选择性地参与萘醌类化合物的代谢。     

抗人补体C4α链单克隆抗体的制备与鉴定

人补体C4属β-1E球蛋白。血清平均浓度为430μg/ml。分子量为202,000。由α、β和γ三条肽链组成。已证实C4被C_1s激活的部位,纯化C4被胰蛋白酶作用而断裂的部位,胺、甲胺等化学物质的作用部位,以及Ig、SRBC的结合部位和该分子的α链有关。此外,C4补体型的遗传特征和某些自身免疫性疾病的发生关系十分密切。C4遗传的多态性很可能位于其     

蛋白C、蛋白S、抗凝血酶、凝血因子Ⅷ在不同Child-Pugh肝功能分级的慢性肝硬化患者中的应用研究

目的 探讨血浆蛋白C(protein C,PC)、血浆蛋白S(protein S,PS)、血浆抗凝血酶(antithrombin, AT)、血浆凝血因子Ⅷ(coagulation factor Ⅷ,F Ⅷ)在不同Child-Pugh肝功能分级的慢性肝硬化患者中的应用意义。方法 选取2020年12月至2021年12月首都医科大学附属北京佑安医院进行诊治的96例慢性肝硬化患者作为研究对象，另选择同期体检正常的15例正常人作为对照组，分别采用发色底物法和凝固法测定不同Child-Pugh肝功能分级患者的PC、PS、AT、FⅧ等水平并进行比较，并分析相关性。结果 蛋白C、蛋白S、AT、FⅧ在不同Child-Pugh肝功能分级的分组间有显著差异。随着Child-Pugh肝功能分级变差，患者的蛋白C、蛋白S和AT的活性明显降低，FⅧ活性增加。结论慢性肝硬化有高凝风险，建议检测和评估血栓形成的可能。     

应用SELDI技术检测反义寡核苷酸作用前后胃癌细胞培养液中的差异蛋白

目的：利用针对人端粒酶RNA（hTR）的反义寡核苷酸（ASODN）和正义寡核苷酸（NODN）作用于人胃癌细胞SGC7901,比较ASODN和NODN作用后细胞培养液蛋白的变化。方法：利用SELDI技术检测差异蛋白的表达变化。对照组为未加任何处理因素的SGC7901细胞。结果：SELDI技术检测发现,ASODN作用组有31个差异蛋白分子低表达,10个差异蛋白分子高表达。NODN作用组有25个差异蛋白分子低表达,16个差异蛋白分子高表达。所有差异蛋白分子量均〈10000KDa。ASODN作用组中有6个低表达蛋白在NODN作用组高表达,分子量分别为4180.7KDa、4825KDa、7925.2KDa、8138KDa、8605.1KDa、8935.3KDa。结论：ASODN和NODN作用SGC7901细胞后细胞培养液中所表达的差异蛋白十分相似。     

淋巴细胞脉络丛脑膜炎病毒GP2单克隆抗体的制备和鉴定

目的制备淋巴细胞脉络丛脑膜炎病毒(LCMV)单克隆抗体(m Ab)。方法采用杂交技术获得并筛选出杂交瘤细胞,收集含单克隆抗体的培养上清行免疫荧光(IFA)、dot-blot、ELISA法鉴定抗体重链类型和测定单克隆抗体的免疫效价。用蛋白G Sepharose亲和层析填料纯化抗体,纯化后的抗体采用SDS-PAGE检测纯化效果,并进一步用Western blot检查抗体对LCMV的作用部位。结果成功获得能稳定分泌抗LCMV的特异性杂交瘤细胞株,顺利获得纯化的单克隆抗体,命名为anti-GP2。免疫荧光、ELISA结果均证实获得的单克隆抗体能识别LCMV抗原。用ELISA法鉴定出该单克隆抗体重链类型为Ig G2b,培养液免疫效价大约为1.35×10~3。蛋白G Sepharose亲和层析填料纯化抗体后经SDS-PAGE检测证实纯化抗体成功。Western blot提示该单克隆抗体对LCMV的作用部位为病毒颗粒表面糖蛋白抗原GP2。结论成功制备出了针对LCMV病毒颗粒表面糖蛋白抗原GP2的单克隆抗体(anti-GP2 m Ab)。     

副溶血弧菌TDH单克隆抗体的研制与性质鉴定

目的:制备副溶血弧菌耐热直接溶血毒素TDH单克隆抗体,并分析其免疫学特性。方法:用纯化的TDH毒素蛋白作为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与骨髓瘤细胞融合,用间接ELISA方法进行阳性细胞筛选,有限稀释法进行亚克隆。并对该单克隆抗体进行了分子量大小、效价、亚类、特异性和亲和力分析。结果:筛选出一株能够稳定分泌TDH单克隆抗体的杂交瘤细胞株命名为T9H4,其免疫球蛋白亚型鉴定为IgG1,亲和力常数可达2.19×109L/mol,并表现出较强的特异性。抗体纯化后效价达1∶1 024 000,SDS-PAGE电泳结果显示单抗蛋白重链分子量约为50 kD和轻链23 kD。结论:成功获得一株能稳定分泌单克隆抗体的杂交瘤细胞,为开发免疫学快速检测方法提供重要实验基础。     

Western印迹分析嗜人按蚊中肠膜蛋白免疫原性

采用Western-blot免疫学方法对嗜人按蚊中肠粗蛋白和膜蛋白(即纯化后的粗蛋白)分子量及免疫原性进行分析。结果,经SDS-PAGE电泳,嗜人按蚊中肠粗蛋白显示8条主带,分子量范围在(66200~15850Da);而膜蛋白显示4条主带,分子量范围在(66200~35480Da)。利用中肠粗蛋白免疫小鼠所获得的抗血清,可特异性识别粗蛋白中的3个条带;而特异性识别膜蛋白的仅1条,分子量为38·9kDa。可见,利用蔗糖不连续梯度离心可获得膜蛋白,该蛋白含有与抗蚊中肠粗蛋白抗体相结合的膜蛋白表面抗原,这对今后制备嗜人按蚊中肠单克隆抗体具有参考价值。     

亲和层析一步纯化IgM类单克隆抗体

本文介绍采用兔抗鼠IgM血清制备的亲和层析柱从小鼠腹水中一步提纯单克隆抗体IgM的方法。提纯的IgM应用琼脂糖电泳检测,为一个区带;应用还原条件下的SDS—PAGE检测,分离为分子量～80K的重链和～25K的轻链两条区带,几未见其他杂带;应用免疫印染法鉴别,进一步确证为IgM,不含常见的杂质IgG。实验过程还发现提纯的IgM不够稳定,在贮存过程有降解现象。另外应用同一亲和柱从吸附IgG后的小鼠血清中提取多克隆抗体IgM的效果也是良好的,经用还原条件下的SDS-PAGE检测,同样也只分离为轻、重链两条区带,未见混有其他杂蛋白。     

Construction,expression and biochemical characterisation of a novel triskringle plasminogen activator gene

A DNA sequence coding for the second kringle of tissue-type plasminogen activator (t-PA) was synthesised chemically and inserted just before the double-kringle region of the t-PA gene. This tris-kringle PA or hybrid F gene was expressed in a BPV-based expression vector and the expression product was purified to homogeneity. The specific activity of the protein on fibrin-agar plate was about 250 000 iu/mg compared to ∼ 500 000 iu/mg for rt-PA. Analysis of the protein by SDS-PAGE showed that it was present mainly in the single chain form and displayed doublet bands of Mr 68 000 and 73 000 Da, probably due to differential glycosylation. Although hybrid F showed tighter binding to lysine-Sepharose, its binding to a fibrin clot was comparable to that of t-PA. Hybrid F and t-PA were found to have comparable amidolytic activities toward a synthetic chromogenic substrate, S-2288, suggesting that the extra kringle in hybrid F did not affect its catalytic function. However, hybrid F exhibited considerably lower activity in fibrin-dependent plasminogen activation characterised by higher Km and lower kcat values than the native t-PA. This shows that addition of an extra kringle in t-PA somehow interferes in the ternary complex formation with fibrin and plasminogen and thereby adversely affects its fibrin-stimulated activity.     

Lack of isotype exclusion and expression of aberrant lambda light chain on secreted MOPC-315 myeloma proteins

The presence of aberrant lambda 1 light (L) chain fragment (lambda 1 F) on the secreted myeloma protein of MOPC-315 has been demonstrated by serological and immunochemical methods. We developed a highly sensitive radioimmunoassay that utilizes exquisitely specific xenogeneic anti-lambda 1 antibodies to detect the minute amounts of lambda 1 F on lambda 2-bearing MOPC-315 myeloma proteins. In addition, structural evidence that lambda 1 F is present on MOPC-315 myeloma protein was demonstrated by subjecting 125I-labeled MOPC-315 myeloma protein to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions followed by autoradiography. The relative amounts of lambda 1 F and lambda 2-chain on MOPC-315 myeloma were measured by two independent methods. The molar ratio of lambda 1 F to lambda 2 was calculated to be 1:68 by radioimmunoassay and 1:80 by analytical SDS-PAGE. This represents the first demonstration that an aberrant L-chain fragment combines with a heavy chain and is secreted in association with antigen-binding myeloma proteins. The implications of these results on L-chain isotype exclusion are discussed.     

补体终末阶段同源限制因子的分离纯化

为了研究补体终末阶段同源限制的作用机制，本文首先分析了正常人红细胞膜不同分子量蛋白组份对反应性溶血的抑制作用。五次重复试验结果均发现１８ＫＤａ６５ＫＤａ的组份具有抑制活性，提示：红细胞膜上同时存在两种具有同源限制功能的蛋白分子。将红细胞膜蛋白抽提物经ＤＥＡＥ－纤维素ＤＥ－３２．Ｓｅｐｈａｒｏｓｅ ６Ｂ层析纯化分离到一个活性峰。进一步用ＳＤＳ－ＰＡＧＥ制备电泳纯化出ｍｐ１８具有较高的抑制活性。本文还     

HLA-A*0203-BSP的表达和复性及其四聚体的鉴定

目的：优化诱导条件大批量表达生物素化酶BirA底物肽（BSP）与HLA-A＊0203重链胞外域的融合蛋白（HLA—A＊0203、BSP），并制备负载HLA-A＊0203限制性EB病毒抗原肽EBNA3 596-604的四聚体（HIA—A}0203／SVR）。方法：以HLA—A＊0203-BSP原核表达载体转化E．coli BL21（DE3）菌株，优化诱导条件进行大批量重组蛋白的表达。通过稀释法复性可溶性HLA-A＊0203／SVR单体，然后以BirA对其进行生物素化，并以阴离子交换树脂纯化。将纯化的HLA-A＊0203／SVR单体与荧光素标记的链亲和素按4：1的比例混合形成四聚体，通过对特异性CTL进行染色验证其结合活性。结果：当IPTG的浓度为0．4mmol／L，于37℃诱导过夜后，融合蛋白的表达最多。该重组蛋白相对分子质量（Mr）为34003，与HLA—A＊0203-BSP的理论Mr相一致。该重组蛋白以包涵体形式存在于沉淀部分，约占菌体总蛋白的30％。负载抗原肽的可溶性HLA-A＊0203／SVR单体是在同时存在HLA-A＊0203，BSP、β2微球蛋白及HLA-A＊0203限制性抗原肽SVR的情况下通过稀释法复性而获得。该单体生物素化并纯化后与荧光素标记的链亲和素按4：1的比例混合后即形成四聚体。流式细胞术（FCM）分析证实，该四聚体具有与HLA—A2^＋供者特异性CTL结合的活性。结论：HLA—A＊0203-BSP融合蛋白在优化条件下获得高效表达。以此蛋白制备的HLA-A＊0203／SVR四聚体具有与HLA-A2^＋供者特异性CTL结合的活性，为研究HLA—A＊0203个体EB病毒特异性CTL的免疫应答打下了基础。     

Purification of Chicken C3 and a Structural and Functional Characterization

A major plasma protein from chicken, analogous to mammalian complement component C3, was purified by the removal of plasminogen, precipitation with polyethyleneglycol, and ion-exchange chromatography. Purification was guided by a rabbit antiserum specific to chicken C3. The yield of native C3 was 27%, and purity and functional activity was assessed by SDS-PAGE, immunoprecipitation techniques, and the ability of the purified C3 to restore the haemolytic activity of C3-depleted chicken serum. Monoclonal antibodies were raised against purified chicken C3. These antibodies were characterized and used to prepare an immunosorbent column to deplete chicken plasma specifically of C3. Chicken C3 has a mol.wt of 185,000-195,000 and a two-chain structure with an alpha chain (118,000) and beta chain (68,000). Complement activation leads to changes in the electrophoretic mobility of chicken C3 and to a decrease in mol.wt to 144,000 corresponding to the release of a 15,000 C3a and a 34,000 C3d/C3dg fragment. Chicken C3 exists in multiple molecular forms with pI values of 6.4-6.6. A genetic polymorphism of chicken C3 based on electrophoretic mobility has not yet been detected after analysis of more than 500 individuals. The function of chicken C3 is dependent on a reactive thioester because treatment of purified chicken C3 with methylamine causes functional inactivation of C3.     

Purification and characterization of neurotoxin produced by Clostridium botulinum type C 6813.

The toxin produced by Clostridium botulinum type C 6813 (C-6813) was purified 1,009-fold from the culture supernatant in an overall yield of 30%. The specific toxicity was 1.1 X 10(7) mouse minimum lethal doses per mg of protein. The toxin had a molecular weight of 144,000, composed of the light and heavy chains with molecular weights of 52,000 and 92,000, respectively, linked by one or two disulfide bond(s). The purified C-6813 toxin heavy and light chains reacted strongly with anti-type D heavy chain immunoglobulin G and anti-type C1 light chain immunoglobulin G, respectively. The amino acid compositions of C-6813 toxin heavy and light chains were more similar to those of type D heavy chain and type C1 light chain than to those of type C1 heavy chain and type D light chain, respectively. These results suggest that in the toxin produced by the type C strain at least two subtypes exist.     

Isolation and biological characterization of boar sperm capacitation-related antigen

PROBLEM: Monoclonal anti-capacitated sperm antibody has been used as a probe to identify, isolate, and characterize specific, fertilization-related antigen with some characteristic features that point to its possible significance in immunocontraception. METHOD OF STUDY: Fast protein liquid chromatography (FPLC), enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectrofocusing were used for isolation, immunochemical and physicochemical characterization of a monoclonal antibody (mAb) 1F10 cognate antigen. Sperm-zona pellucida binding and hemizona assay were used for testing the biological roles of mAb 1F10 and Ag 1F10 in boar and human fertilization processes. RESULTS: The Ag 1F10 was found to be eluted in the eighth protein peak of FPLC-fractionated Nonidet P40 (NP40) extracts of capacitated boar spermatozoa. The SDS-PAGE and immunoelectrofocusing experiments showed that Ag 1F10 is a protein composed of a single peptide chain with a relative molecular mass of 68/70 kDa and an isoelectric point of 3.5. It was demonstrated that the zona binding activity of spermatozoa preincubated in the presence of mAb 1F10 was significantly inhibited both in porcine and human in vitro fertilization (IVF) systems. A dose-dependent manner of inhibition of the sperm/ligand activities of porcine and human zona pellucida was observed when the effect of purified Ag 1F10 was investigated by its preincubation with zona pellucida. CONCLUSIONS: It is assumed that the protein bearing the epitope recognized by mAb 1F10 may be accepted as one of the molecules with receptor function in sperm-zona pellucida interaction during fertilization.     

F10蛋白的原核表达及多克隆抗体的制备

目的：表达F10蛋白，并制备兔抗F10多克隆抗体。方法：利用PCR方法扩增F10基因片段，经BamHⅠ和EcoRⅠ酶切后连接人pET-GST原核表达载体，构建的pET-GST／F10融合重组表达质粒转化大肠杆菌B121，经IPTG诱导表达蛋白，并以亲和层析的方法进行纯化。表达产物用SDS—PAGE电泳和Western blot进行分析鉴定。以纯化的F10蛋白免疫新西兰大白兔，制备兔抗F10的多克隆抗体，并以ELISA法检测抗体效价。结果：经酶切和核酸序列分析证实重组质粒包含有正确编码的F10读码框。SDS—PAGE电泳分析显示pET—GST／F10诱导后表达一相对分子质量（Mr）约为61000的融合蛋白，与预期结果相符。目的蛋白纯化后的纯度达90％以上，Western blot证实该蛋白是GST／F10的融合蛋白。将纯化的GST／F10融合蛋白免疫家兔，得到的兔抗F10抗体效价达1：20000。结论：成功构建了人F10基因原核表达载体，并获得了高纯度的F10重组蛋白及兔抗F10抗体，为下一步研究F10基因功能奠定了实验基础。