鸡(Gallus gallus)IFNγ基因重组蛋白复性条件优化及生物活性鉴定研究

为提高鸡IFNγ基因重组蛋白的复性效率,用18种不同的复性缓冲液对在大肠杆菌中表达的cIFNγ进行复性处理,通过SDS-Page分析、考马斯亮蓝定量、对鸡胚成纤维细胞以及对鸡胚的保护实验检测复性产物的得率和生物学活性。研究结果表明:去污剂和疏水剂能保证蛋白的复性得率和复性效率,重组表达产物得率可高达94.7%;氧化还原电势对是cIFNγ成功复性的关键,有生物学活性的复性产物的复性液中均存在氧化还原对GSSG/GSH;经去污剂处理后再复性的产物对细胞保护的有效浓度显著低于直接复性的蛋白。     

马铃薯钾离子通道蛋白SKT1的原核表达与纯化

克隆马铃薯(Solanum tuberosum L.)钾离子通道蛋白SKT1基因片段后原核表达并纯化SKT1蛋白,为进一步研究SKT1的晶体结构与功能,以及与其它钾离子通道蛋白的相互作用奠定基础.以马铃薯试管苗总RNA反转录出的cDNA为模板,PCR扩增SKT1蛋白的胞内区,基因片段为1758bp,构建原核表达载体pET30-SKT1,转化大肠杆菌BL21表达株菌,IPTG诱导表达.经SDS-PAGE检测发现SKT1蛋白主要是以包涵体的形式存在.利用6M盐酸胍对SKT1包涵体进行溶解,利用Ni柱亲和纯化,最后在复性缓冲液中复性.获得了SKT1蛋白,复性得率大于40%,纯度达到95%以上,Western Blotting鉴定正确.     

葡激酶突变体Y1-Sak的体外变复性研究

具有低免疫原性的双功能葡激酶突变体Y1-Sak(△15,S16K,K74A,E75A,R77A,K109R,F111D)在大肠杆菌中的表达产物为包涵体,必须进行体外变复性才能得到活性.详细研究了复性方式、pH、温度、复性的初始蛋白浓度和添加L-精氨酸对Y1-Sak的体外变复性的影响,发现复性温度和添加的L-精氨酸对Y1-Sak的复性有重要的影响.经过工艺优化后,Y1-Sak的纯化活性回收率达到40%左右,蛋白比活性由20 kU/mg提高到53 kU/mg.用动态光散射分析发现,工艺优化后复性的高比活性Y1-Sak的水合动力学半径与野生型葡激酶相近,分子基本处于单体状态.     

重组人胰岛素的体外复性纯化

对大肠杆菌表达的重组人胰岛素原包涵体蛋白的变性复性条件进行了优化。考察了变性液中DTT的浓度,复性液的pH值,还原型谷胱甘肽(GSH)与氧化型谷胱甘肽(GSSG)的物质的量比,甘氨酸(Gly)浓度对复性的影响。结果表明,在变性液中加入60mmol/L DTT,复性液pH9.5,GSH与GSSG物质的量比为5∶1,Gly浓度为50mmol/L条件下复性率最高。复性后的重组人胰岛素原过DEAE-Sepharose FF柱,经过TPCK-胰蛋白酶和羧肽酶B酶切后,再过Sephadex G-25柱,得到纯度较高的重组人胰岛素。目的蛋白收率为96mg/g干菌体。     

H3N2SIV河南株非结构蛋白1(NS1)重组的纯化及Dot-ELISA的建立

为建立鉴别猪流感病毒感染猪和灭活疫苗免疫猪的诊断方法,利用pET-28a载体在大肠杆菌BL21中表达H3N2 SIV河南株NS1,并通过Ni2+-NTA亲和色谱法纯化His-NS1融合蛋白.用SDS-PAGE,Western blotting和Dot-ELISA分析纯化蛋白,表明重组NS1能与猪流感病毒感染猪血清进行特异性反应,而不与灭活疫苗免疫猪血清反应.结果表明重组蛋白能作为区别猪流感病毒感染猪和灭活疫苗免疫猪的诊断抗原,为控制猪群中的流感病毒奠定了基础.     

重组人TRAIL胞外片段的原核表达系统选择及蛋白复性研究

采用PCR技术从人肝脏cDNA文库扩增出编码114～281位氨基酸的TRAIL基因片段,将之克隆至原核表达载体pET-30a、pQE-30和pJLA503中,上述重组载体经IPTG诱导表达后,SDS-PAGE鉴定显示pET-30a系统表达效率最高,目的蛋白占菌体总蛋白含量的40%左右;采用透析法或梯度稀释法对TRAIL胞外区片段包涵体进行复性,非还原SDS-PAGE的结果显示梯度稀释法明显好于透析法;纯化后的TRAIL胞外区片段蛋白具有明显的生物学活性,效果与标准品相似;通过添加不同的折叠促进剂对复性效率影响的研究,确立了TRAIL胞外区片段适宜的复性方法,复性效率达75%～80%.     

人P53的原核表达及包涵体复性研究

通过检测血清中p53蛋白及其抗体变化诊断肿瘤的ELISA试剂盒的研发需要大量制备p53蛋白.本研究通过构建人野生型p53基因的原核表达质粒pET-32a-p53,在大肠杆菌中诱导表达得到p53包涵体蛋白,经包涵体变性、亲和纯化、复性得到可溶p53蛋白.发现利用8M尿素对包涵体进行变性溶解,经镍柱亲和纯化得到纯度超过95%的变性蛋白.尝试透析复性、稀释复性和柱上复性3种方法分别对p53变性蛋白进行复性.结果表明透析复性的复性率最高,这为原核表达制备p53提供了一种参考.本研究为ELISA法检测血清中p53蛋白及其抗体诊断肿瘤相关试剂盒研发奠定了基础.     

小鼠PD-1胞外段重组蛋白表达及其包涵体复性

旨在利用原核表达系统制备可溶性的PD-1胞外段(以下简称exPD-1)蛋白.以C57BL/6小鼠脾细胞cDNA为模板,扩增小鼠exPD-1基因,成功构建重组质粒pET-28a-exPD-1,并在E.coliBL21(DE3)中诱导表达.结果显示,小鼠exPD-1重组蛋白大小约为17kD,以包涵体形式存在.exPD-1包涵体蛋白经8mol/L尿素变性、Ni-NTA亲和层析后,采用尿素梯度透析法对蛋白进行复性,复性效果不佳,添加氧化/还原型谷胱甘肽复性后,复性率达到50%以上,提高了复性效率.小鼠exPD-1蛋白的成功制备,为进一步研究PD-1的免疫学功能奠定了材料基础.     

人胰蛋白酶原-2在大肠杆菌中的表达、纯化与活性测定

胰蛋白酶原-2是急性胰腺炎相关的敏感而特异的标志物,并且与多种肿瘤转移的过程密切相关．对胰蛋白酶原-2基因5’端4个氨基酸的密码子碱基进行简并突变,并在其3’端连接6个组氨酸编码序列构建含重组质粒pBVTg2的大肠杆菌工程菌-pBvTg2／DH5α．重组大肠杆菌经温度诱导后获得了高效表达,重组蛋白占总菌体蛋白的20％,并以包涵体形式存在．包涵体经尿素缓冲液裂解后,通过Ni-NTA亲和树脂一步分离纯化．纯化蛋白经SDS-PAGE分析及利用anti—histidine mAb进行Western blotting分析,以及蛋白N末端测序分析,结果表明与目的蛋白特征相符合．纯化的重组人胰蛋白酶原-2经复性后能与天然人胰蛋白酶原-2的单抗antihTrypsinogen2 mAb特异结合,复性后的蛋白在肠激酶作用后测得具有74BAEEU／mg的胰蛋白酶比活力,为进行胰蛋白酶原-2单克隆抗体的制备及其检测和应用奠定了基础．     

传染性法氏囊病病毒分子生物学研究的一些进展

对IBDV近年来的分子生物学方面的研究进展进行概况，主要包括IBDv的基因组结构及理化特性、病毒蛋白、不同型IBDV变异的分子基础和分子生物学诊断技术等，以探讨利用病毒重要基因核酸序列的差异进行IBDV分子流行病学研究及病毒各致病型快速鉴别诊断的可能性．     

Dot—ELISA及其在植物病毒单克隆抗体筛选中的应用

确立了以硝酸纤维素膜(NCM)作固相载体的Dot-ELISA的最适试验程序,并用此技术检测了284个杂交瘤培养上清,筛选出80个分泌抗甜菜花叶病毒(Beet mo(>)ic virus)抗体的上清样品,阳性率达28.17％。用Dot-ELISA测定小鼠抗甜菜花叶病毒多克隆抗体,与常规ELISA比较,结果表明该技术灵敏度较常規ELISA为高,而且具有操作简便、快速、经济以及适于大量样品检测等优点,是杂交瘤筛选的一种比较理想的有效途经。引入亲和素-生物素系统(ABS)后,该技术灵敏度增高了10倍。此外,还在消除非特异性反应方面进行了某些探索。     

抗伊氏锥虫单克隆抗体的研究——二 、用McAb直接斑点法检测伊氏锥虫抗原的实验研究

抗伊氏锥虫糖蛋白的单克隆抗体ⅡG_6标记过氧化物酶后,用于直接法斑点试验检测血清中伊氏锥虫(T.e.)抗原,36份感染2—4d的鼠血清全部呈阳性反应;12份正常鼠血清全部呈阴性反应.上述结果表明本方法具有较好的敏感性和特异性.ⅡGMcAb Dot ELISA检测结果显示血中伊氏锥虫密度与抗原滴度呈正相关.该法操作简便易行,不需特殊设备.肉眼即可观察结果.本次实验研究将为现场应用提供快速诊断的重要依据.     

烟草花叶病毒单克隆抗体的制备和鉴定

经工程菌表达与纯化,得到了纯度95%以上的TMV-CP-F重组蛋白,配合提取的TMV天然病毒颗粒作为免疫原.通过杂交瘤技术获得了14株能分泌特异针对TMV外壳蛋白的单克隆抗体杂交瘤细胞株.经鉴定14株细胞所分泌的抗体亚类为IgG1型,抗体轻链均为κ型.经ProteinA一步法亲和层析纯化所得抗体经鉴定相对分子质量在149.36～157.23 ku之间,抗体纯度在80%以上.经间接ELISA测定,14株抗体均与TMV-CP重组蛋白和TMV病毒有良好特异性反应.所制备的抗TMV抗体的特异性高,可用于与其相关的免疫检测研究和应用.     

抗Der p 2单克隆抗体细胞株的建立与鉴定

目的:建立Der p 2(屋尘螨Ⅱ类变应原)杂交瘤细胞株,并对其分泌的Der p 2单克隆抗体进行鉴定.方法:用重组Der p 2蛋白作为免疫原,免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,经筛选和3次克隆化,制备出单克隆抗体.采用间接ELISA法、Western blot法确定单克隆抗体的免疫球蛋白的类型和亚类、相对亲和力、特异性,对单克隆抗体进行鉴定.结果:获得3株能稳定分泌高效价Der p 2单克隆抗体的杂交瘤细胞株,1B6为IgG1类,1G11、4B1为IgG2a类.间接ELISA法检测细胞上清效价为104~105,腹水效价为105~106.Westernblot试验证明3株单抗与Der p 2蛋白均有特异性反应.结论:成功制备了3株抗Der p 2的单克隆抗体,均具有良好的特异性和亲和力,为进一步建立Der p 2蛋白检测和纯化的方法奠定了基础.     

Smurf2单克隆抗体的制备与鉴定

Smad泛素调节因子家族的两个E3泛素连接酶成员Smurf1和Smurf2 (Smad ubiquitin regulatory factor 1 and 2)具有至少80％的同源性.为了更好地研究它们的功能,需要获得一个能区分Smurf1和Smurf2的单克隆抗体.选取Smurf2与Smurf1蛋白序列中仅有的非同源区域序列作为抗原免疫Bal b/C小鼠,成功制备若干株能稳定分泌抗Smurf2抗体的杂交瘤细胞株.对所获得的单克隆抗体的效价和特异性等进行一系列检测,结果表明该抗体能特异性地识别过表达及细胞内源Smurf2蛋白.此外该抗体能被应用于Western blot和免疫荧光实验,从而为深入研究Smurf2的细胞生物学功能提供了良好的实验基础.     

改进双抗夹心ELISA用于筛选单抗杂交瘤细胞株

本文利用改进双抗夹心ELISA筛选一种可测定血清样品中与血浆蛋白结合的蛋白质药物的单抗。用兔抗K102多抗经亲和层析纯化并包被后,加入用10%猴血浆PBS还原后的抗原K102,再加入杂交瘤细胞株上清液和HRP-羊抗鼠IgG、底物反应液筛选阳性单抗杂交瘤细胞株。结果用改进后的双抗夹心ELISA筛成功地选出6株能稳定分泌针对K102的单抗杂交瘤细胞株,分别命名为1C9、2D7、3E2、5F4、5F6、6B9;细胞株产生的单抗用ELISA法能检测出血清样品中的K102的浓度。提示改进的双抗夹心ELISA可用于针对与血浆蛋白结合的蛋白质药物的单抗杂交瘤细胞株的筛选。     

抗人骨桥蛋白单克隆抗体制备及其特异性研究

利用RT-PCR技术克隆人骨桥蛋白(hOPN)基因,构建OPN原核表达质粒pET-32a(+)-hOPN,转化BL21菌株,经IPTG诱导表达重组人骨桥蛋白(rhOPN).以纯化的rhOPN为免疫原,免疫BABL/c小鼠,取其脾细胞与小鼠骨髓瘤细胞NS1融合.通过有限稀释法进行克隆和间接ELISA筛选,获得抗人OPN蛋白单克隆抗体杂交瘤细胞株,以ELISA、Western blot对抗体特异性进行鉴定.通过竞争抑制试验对单克隆抗体识别抗原位点进行分析.结果共获得2株抗人OPN单克隆抗体,分别命名为8F1和2B5,亚型测定皆为IgG1.通过细胞侵袭抑制试验检测,2株抗人OPN mAb皆能很好地抑制细胞迁移.本研究成功获得了抗人骨桥蛋白的特异性单克隆抗体,为进一步研究OPN蛋白在自身免疫病和肿瘤中的功能提供了重要的工具.     

Exon shuffling: mapping polymorphic determinants on hybrid mouse transplantation antigens

The mouse major transplantation antigens H-2K, H-2D and H-2L are highly polymorphic cell-surface glycoproteins which may serve as recognition elements in cell-cell interactions. Each antigen possesses a number of alloantigenic determinants defined by antisera of various specificities. Recently, monoclonal antibodies have been produced which redefine and extend our knowledge of these determinants2,3, but structural information has not yet been correlated with the serological definition of the antigens. We have previously reported the molecular cloning of genes for H-2Ld and H-2Dd transplantation antigens from the BALB/c mouse and the expression of these genes in mouse L cells4,5. To localize the serological determinants to discrete regions of the H-2 protein, we have now constructed new H-2 antigen genes by joining together fragments of the H-2Ld and H-2Dd genes. In L cells, these genes direct the synthesis of hybrid H-2 proteins and by using monoclonal antibodies of defined specificities, we have mapped classically defined serological specificities to structurally defined domains of the transplantation antigen protein. We conclude that polymorphic determinants recognized by monoclonal antibodies are located in functionally distinct portions of the protein.     

An anti-idiotype vaccine against experimental schistosomiasis

Schistosomiasis is a parasitic infection of man which is widespread in tropical countries, and which so far has resisted attempts at control. We have been approaching the problem from an immunological angle. We have previously reported the production of a rat monoclonal IgG2a antibody against Schistosoma mansoni which exhibits marked cytoxicity for schistosomula in the presence of eosinophils and a high degree of protection by passive transfer in naive rats. This antibody, IPLSm1, was shown to bind specifically to a schistosomulum membrane target antigen defined as a glycoprotein of relative molecular mass 38,000 (38K), which is strongly immunogenic in schistosome infection of various animal species including man. Although theoretically the 38K protein represents an excellent candidate for a potential vaccine against schistosomiasis, the glycanic nature of the epitope recognized by IPLSm1 limits its production by DNA recombinant technology. It was, moreover, shown that, together with protective antibodies, the 38K molecule was able to induce the production of blocking IgG2c antibodies that inhibit the functional properties of IPLSm1 both in vitro and in vivo. Therefore, following Jerne's network theory, we considered an alternative approach, the possibility of immunization using anti-idiotype antibodies. In the present study, rat monoclonal anti-idiotype antibodies were produced against IPLSm1 (AB1). Anti-idiotype antibodies (AB2) were selected by their capacity to inhibit the binding of radioiodinated AB1 to its 38K target antigen. Sera from naive LOU rats immunized with a purified AB2 preparation contained specific anti-schistosome antibodies (AB3) which bound to 38K. AB3 antibodies were strongly cytotoxic for schistosomula in the presence of rat eosinophils and conferred highly significant protection by passive transfer. Most importantly, rats immunized with AB2 demonstrated marked protection (50-80%) to a challenge infection.     

Co-localization of GABA receptors and benzodiazepine receptors in the brain shown by monoclonal antibodies

The most abundant inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid (GABA), exerts its main effects via a GABAA receptor that gates a chloride channel in the subsynaptic membrane. These receptors can contain a modulatory unit, the benzodiazepine receptor, through which ligands of different chemical classes can increase or decrease GABAA receptor function. We have now visualized a GABAA receptor in mammalian brain using monoclonal antibodies. The protein complex recognized by the antibodies contained high- and low-affinity binding sites for GABA as well as binding sites for benzodiazepines, indicative of a GABAA receptor functionally associated with benzodiazepine receptors. As the pattern of brain immunoreactivity corresponds to the autoradiographical distribution of benzodiazepine binding sites, most benzodiazepine receptors seem to be part of GABAA receptors. Two constituent proteins were identified immunologically. Because the monoclonal antibodies cross-react with human brain, they provide a means for elucidating those CNS disorders which may be linked to a dysfunction of a GABAA receptor.