幽门螺杆菌Catalas/GST融合蛋白的可溶性表达及凝血酶柱上切割GST标签

目的 利用GST融合基因表达系统表达幽门螺杆菌Catalase/GST融合蛋白,并利用凝血酶柱上切割GST标签.方法 将重组表达质粒Catalase/pGEX-4T-1转化大肠杆菌BL21 (DE3)感受态中并用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌进行裂解.采用谷胱甘肽琼脂糖树脂Glutathione Sepharose 4B对Catalase/GST融合蛋白可溶性表达上清进行纯化,并同时利用凝血酶柱上切割GST标签,用鼠抗Catalase抗体对纯化产物进行Western blot鉴定.结果 高效表达出相对分子质量约85 kDa的Catalase/GST融合蛋白,以部分可溶性的形式表达,凝血酶柱上成功地切割了GST标签,Catalase蛋白能被鼠抗Catalase单克隆抗体识别.结论 成功表达了Catalase/GST融合蛋白并柱上切割了GST标签,为深入研究Catalase的功能奠定了基础.     

刚地弓形虫活化蛋白激酶C受体1基因的克隆和原核表达及纯化和抗原性分析

目的 克隆刚地弓形虫活化蛋白激酶C受体1(TgRACK1)基因,原核表达、纯化TgRACK1,并分析其抗原性.方法 制备弓形虫RH株速殖子总RNA,根据TgRACK1基因全长编码序列(GenBank登录号:AY547291)的开放阅读框设计引物并进行逆转录-聚合酶链式反应(RT-PCR)扩增,扩增产物经双酶切后连接入pGEX-6p-1载体,重组质粒转化大肠埃希菌DH5α,阳性菌落经菌液PCR、双酶切及DNA测序进行鉴定.将重组质粒pGEX-6p-1-TgRACK1转化至大肠埃希菌BL21(DE3)并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物;分别以抗GST标签抗体和兔抗弓形虫血清为一抗,免疫印迹(Western blotting)分析鉴定重组蛋白及其抗原性.重组融合蛋白经GST琼脂糖凝胶亲和纯化,纯化蛋白经SDS-PAGE分离,考马斯亮蓝染色结合凝胶成像系统分析其纯度.结果 RT-PCR扩增产物约为970 bp,重组质粒pGEX-6p-1-TgRACK1构建成功.经IPTG诱导获得相对分子质量约63 kDa的可溶性重组蛋白.诱导表达的蛋白质为带GST标签的重组融合蛋白,且能被兔抗弓形虫血清识别.亲和纯化后获得纯度大于95％的重组TgRACK1蛋白质.结论 克隆得到弓形虫活化蛋白激酶C受体1基因,原核表达并纯化出该基因编码的蛋白质,且该重组蛋白具有抗原性.     

结核分枝杆菌ESAT-6的原核表达与纯化及免疫原性检测

目的构建及鉴定含有GST标签的结核分枝杆菌esat-6基因的原核表达载体,并在大肠埃希菌中高效表达融合蛋白。方法 PCR扩增结核分枝杆菌esat-6基因,并构建到pGEX4T-1上,重组质粒经测序鉴定后进行诱导表达,采用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白。结果扩增出了结核分枝杆菌esat-6基因,构建了具有正确基因序列的质粒载体pGEX4T-1-ESAT-6,转化大肠埃希菌BL21后经诱导产生了高水平的表达产物,蛋白纯化后可被结核病患者血清特异性识别。结论构建了质粒载体pGEX4T-1-ESAT-6,并经诱导后高效表达了ESAT-6融合蛋白,为进一步研究结核菌的免疫机制奠定了基础。     

单纯疱疹病毒2型糖蛋白G的表达、纯化及鉴定

目的 对pGEX-4T- 1/gG-2重组质粒进行诱导表达,并纯化、鉴定表达的目的蛋白.方法 用最佳诱导条件对重组质粒进行诱导表达,表达产物经SDS-PAGE电泳检测验证后,对存在于超声离心上清液中的融合蛋白用亲和层析技术进行纯化,并用ELISA间接法对纯化的GST/gG-2融合蛋白的灵敏性和特异性等生物活性进行鉴定.结果 目的蛋白经诱导后表达于上清中,经鉴定纯化的目的蛋白保留了天然蛋白原有的生物活性.结论 GST/gG-2融合蛋白的获得,为研制以重组蛋白代替传统的全病毒作为检测抗原的新型HSV-2型特异性免疫学检测试剂盒奠定了基础.     

HSV-1重组糖蛋白G的表达、纯化及活性鉴定

目的对构建的pGEX-4T-1/gG-1重组质粒进行诱导表达,并纯化、鉴定表达的目的蛋白。方法用最佳诱导条件对重组质粒进行诱导表达,表达产物经SDS-PAGE电泳检测验证后,对存在于超声离心后上清液中的融合蛋白用亲和层析技术进行纯化,并用ELISA间接法对纯化的GST/gG-1融合蛋白进行灵敏性和特异性等生物活性的初步鉴定。结果目的蛋白经诱导后表达于上清中,初步鉴定纯化的目的蛋白保留了天然蛋白原有的生物活性。结论 GST/gG-1融合蛋白的获得,为研制以重组蛋白代替传统的全病毒作为检测抗原的新型HSV-1型特异性免疫学检测试剂盒奠定了基础。     

肠出血型大肠埃希菌O104:H4转运蛋白AatA单克隆抗体制备及鉴定

目的 表达肠出血型大肠埃希菌O104 :H4的融合蛋白GST-AatA,获得单克隆抗体。方法 人工合成肠出血型大肠埃希菌O104 :H4的转运蛋白基因aatA,连接至pMDl8-T载体,转化E.coli DH5α,双酶切回收,构建原核表达质粒PGEX-6P-1-GST-AatA,测序鉴定后,转化E.Coli BL21,IPTG诱导表达,SDS-PAGE和Western Blot分析目的蛋白的表达并纯化该重组蛋白。将纯化后的融合蛋白GST-AatA免疫Balb/c小鼠,制备相应单克隆抗体;并对该单克隆抗体的纯度、亚型、效价、特异性进行鉴定和分析。结果 构建的原核表达载体PGEX-6P-1-GST-AatA能表达融合蛋白GST-AatA;亲和层析纯化后融合蛋白GST-AatA纯度达到95 %,免疫小鼠后得到相应特异性单克隆抗体。结论 成功制备出一株抗转运蛋白AatA的单克隆抗体,该抗体为IgG1亚型,特异性好,效价高,纯度可达97%。     

大鼠Clara细胞分泌蛋白CC16基因的克隆和原核表达及纯化

目的 克隆大鼠Clara细胞分泌蛋白(CC16)基因,原核表达并纯化重组的CC16蛋白,为进一步研究CC16蛋白的功能及对炎症性气道疾病的治疗作用奠定基础.方法 制备大鼠肺组织总RNA,根据大鼠CC16基因全长编码序列(GenBank登录号:NM_013051)的开放阅读框设计引物并进行逆转录PCR(RT-PCR)扩增,扩增产物经双酶切后连接入pET30a(+)载体,重组质粒转化大肠埃希菌(E.coli DH5α),阳性菌落经PCR和双酶切鉴定并测序.将重组质粒pET30a(+)-rCC16转化至E coli Rosetta(DE3)并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物,重组融合蛋白经Ni2+琼脂糖凝胶亲和纯化.分别以抗His标签抗体和山羊抗CC16抗体进行免疫印迹(Western blotting)分析并鉴定重组蛋白.结果 RT-PCR扩增产物约为290 bp.菌落PCR、双酶切及测序结果显示,重组质粒pET30a(+)-rCC16构建成功.SDS-PAGE结果显示,经IPTG诱导获得相对分子质量约16 kDa的可溶性重组蛋白.Western blotting结果表明,诱导表达的蛋白质为带His标签的重组融合蛋白,且能被山羊抗CC16抗体识别.结论 成功构建基因重组体pET30a(+)-rCC16,并制备出可溶性大鼠重组CC16蛋白.     

结核分枝杆菌85B基因原核表达及间接ELISA方法的建立

目的原核表达结核分枝杆菌85B融合蛋白,建立该抗原对结核病患者诊断的ELISA方法。方法制备结核分枝杆菌基因组DNA,从结核分枝杆菌H37Rv基因组中进行PCR扩增85B基因并构建至表达载体p GEX4T-1上。原核表达GST-85B融合蛋白,并通过包涵体对其进行变性和透析复性后,进行GST亲和层析纯化得到可溶性的目的蛋白。用聚丙烯酰胺凝胶电泳和免疫印迹分析重组蛋白,ELISA检测表达融合蛋白的抗原性。结果扩增出了结核分枝杆菌85B基因,构建了具有正确基因序列的质粒载体p GEX4T-85B,转化大肠杆菌BL21后经诱导产生了高水平的表达产物。超声裂菌法分离的结果显示,目的蛋白在菌体沉淀中以包涵体形式居多。将包涵体变性、复性后用GST亲和层析纯化,蛋白纯化后可被结核病患者血清特异性识别。结论构建了质粒载体,并诱导表达了GST-85B融合蛋白,为进一步研究结核菌的免疫机制奠定了基础。     

大肠埃希菌中疟原虫MSP1-17编码基因蛋白重组及表达

目的为免疫原性及疟疾特异性早期诊断研究,以及高效抗红内期疟原虫复合多价基因疫苗的研究提供基础。方法(1)以鼠约氏疟原虫MSP1编码基因为模板,片段自4855到5271共416 bp,扩增出pyMSP1 DNA片段,将该片段克隆入质粒pPGEX-2T,构建含有GST编码的质粒pGEX-2T-PyMSP1-17。(2)用上述构建的质粒植入大肠埃希菌DH-5α表达重组蛋白。结果(1)构建出约氏疟原虫裂殖子表面蛋白1融合基因片段真核重组表达质粒。(2)获得MSP1-GST融合蛋白,在大肠埃希菌DH-5α收获到重组的约氏疟原虫裂殖子表面蛋白浓度为46 mg/L。(3)原核表达的蛋白产物免疫小鼠后分离血清,采用间接免疫荧光法(IFA)检测疟原虫,滴度在1∶400~1∶1000之间均可识别IFA血片中的疟原虫。结论设计重组MSP1-17基因在大肠埃希菌中真核系统内能够得到充分表达,并具有免疫原性和抗原性。无论是作为DNA疫苗研究的候选者抑或是制备特异性抗体进行疟疾早期诊断都具有重要的意义;由于该抗原的表达量高、免疫原性强,是目前很有希望的疟疾疫苗候选抗原之一。     

亚洲牛带绦虫26kDa GST基因表达及免疫学分析

目的 对亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移酶基因(glutathione S-transferase,GST)进行克隆、表达和免疫学初步分析研究,为深入研究其生物学功能提供依据.方法 将亚洲牛带绦虫成虫26kDa GST克隆到原核表达质粒pET-30a( )中,在大肠埃希菌BL-21/DE3中用异丙硫代-β-D半乳糖苷(IPTG)诱导表达,表达产物通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进行鉴定,用镍离子金属螯合剂亲和层析柱进行纯化,纯化的重组蛋白用蛋白印迹(Western Blotting)进行免疫学分析.结果 PCR、双酶切及DNA测序结果均表明重组质粒pET-30a( )-Ta GST构建成功.SDS-PAGE结果表明,目的基因在大肠埃希菌BL-21/DE3中获得高效表达,经亲和层析获得了高纯度蛋白.重组蛋白可被其免疫的SD大鼠血清识别,表明其具有免疫原性;并且能识别感染了亚洲牛带绦虫的猪血清和亚洲牛带绦虫患者血清,表明其具有免疫反应性.结论 亚洲牛带绦虫成虫26kDa谷胱甘肽S-转移酶基因可在原核表达系统中获得具有免疫学活性的高效表达.     

小鼠截短型CDC25B蛋白PKA体外磷酸化分析

目的通过体外磷酸化研究和放射自显影技术证实小鼠CDC25B蛋白是蛋白激酶A(PKA)的直接作用底物。方法构建小鼠截短型pGEX-4T-2-CDC25B201原核表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,glutathione Sepharose 4B亲和层析纯化CDC25B蛋白,与PKA进行体外磷酸化反应,分别采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白免疫印迹(western blot)和放射自显影鉴定CDC25B是否为PKA直接磷酸化底物。结果在E.coli BL21细胞内,采用0.1 mmol/L的IPTG在27℃条件下诱导表达3 h即可达到较高表达水平;SDS-PAGE显示在约50 kD处有明显GST-CDC25B201融合蛋白特异条带;放射自显影显示GST-CDC25B201融合蛋白的磷酸化条带大约位于55 kD处;Western blot分析,在重组质粒表达的总蛋白、载体表达的GST蛋白、纯化蛋白及磷酸化蛋白的相应位置均出现GST抗体的杂交条带。结论小鼠CDC25B蛋白是PKA的直接作用底物。     

脆性X智力低下蛋白在大肠杆菌中的表达及纯化

目的:在大肠杆菌中对脆性X智力低下蛋白(FMRP)进行重组表达,获得高产量及高纯度的可溶性重组蛋白。方法:以人脑cDNA文库为模板,PCR扩增FMR1基因后将其克隆至融合表达载体pGEX-6P-1中,重组载体转化进入大肠杆菌BL21(DE3)pLysS,并用IPTG进行诱导表达。应用谷胱甘肽-琼脂糖凝胶4B进行亲和层析纯化GST-FMRP融合蛋白,并以SDS-PAGE电泳和Westernblot对融合蛋白的表达进行检测。结果:亲和纯化后的融合蛋白经FMRP及GST单抗分别进行Westernblot鉴定为GST-FMRP融合蛋白。结论:大肠杆菌中FMRPISO10的重组表达产量高,纯化得到可溶性重组蛋白,为FMRP抗体的制备及FXS检测试剂盒的开发奠定基础。     

幽门螺杆菌Lpp20真核表达重组体的构建及其表达鉴定

目的构建含幽门螺杆菌（Hp）Lpp20基因的真核表达重组体,并鉴定其在Hela细胞中能否有效表达,为进一步开展Lpp20核酸疫苗与该蛋白的生物学功能的研究打下基础。方法抽提Hp标准菌株26695基因组DNA,应用聚合酶链式反应（PCR）技术从基因组DNA扩增Lpp20基因,经过一系列酶切、连接反应将其克隆入真核表达载体pcD-NA3.1（＋）,转入大肠杆菌,筛选阳性克隆,通过PCR和酶切反应鉴定;用脂质体法将构建好的重组载体pcDNA3.1（＋）-Lpp20转染Hela细胞,通过免疫细胞化学法及Western印迹法检测其在Hela细胞中表达的Lpp20蛋白。结果成功扩增出长约528 bp的Lpp20基因,测序结果表明扩增出的Lpp20基因与Hp Lpp20序列一致,PCR和酶切鉴定结果证实Lpp20基因正确克隆入真核表达载体pcDNA3.1（＋）,并经免疫细胞化学法和Western印迹法检测到重组体可在Hela细胞中有效表达。结论成功构建了Lpp20基因的Hp真核表达重组体,并检测到其在Hela细胞中可表达出免疫反应性良好的蛋白,为进一步探索其免疫作用及生物学功能奠定了基础。     

幽门螺杆菌Lpp20真核表达重组载体的构建及鉴定

目的以幽门螺杆菌外膜脂蛋白Lpp20为目的基因,构建H.pylori Lpp20基因的真核表达重组载体pcD-NA3.1( )-Lpp20,为H.pylori核酸疫苗的研制奠定实验基础。方法用Pri mer5.0软件分析GenBank中H.pylori外膜脂蛋白Lpp20基因序列,设计合成相应特异性引物,引入BamHⅠ和XhoⅠ酶切位点,以H.pylori26695株基因组为模板,PCR扩增Lpp20目的基因片段,定向插入真核表达载体pcDNA3.1( )多克隆酶切位点中,构建重组体pcDNA3.1( )-Lpp20;通过酶切分析,PCR鉴定及测序鉴定,筛选阳性重组体。结果以H.pylori26695株基因组DNA为模板扩增出特异的Lpp20基因片段,大小约为528bp;双酶切及测序鉴定证明成功构建了H.pylori真核表达重组载体pcD-NA3.1( )-Lpp20。结论PCR扩增得到了大小约为528bp的H.pylori Lpp20目的基因片段;并成功构建了pcD-NA3.1( )-Lpp20真核表达重组载体。     

Monoclonal antibody,mAb 4C13, an effective detoxicant antibody against ricin poisoning

Ricin is a glycoprotein produced in castor seeds and consists of two polypeptide chains named Ricin Toxin A Chain (RTA) and Ricin Toxin B Chain (RTB), linked via a disulfide bridge. Due to its high toxicity, ricin is regarded as a high terrorist risk for the public. However, antibodies can play a pivotal role in neutralizing the toxin. In this research, the anti-toxicant effect of mAb 4C13, a monoclonal antibody (mAb) established using detoxicated ricin as the immunized antigen, was evaluated. Compared with mAb 4F2 and mAb 5G6, the effective mechanism of mAb 4C13 was analyzed by experiments relating to its cytotoxicity, epitope on ricin, binding kinetics with the toxin, its blockage on the protein synthesis inhibition induced by ricin and the intracelluar tracing of its complex with ricin. Our result indicated that mAb 4C13 could recognize and bind to RTA, RTB and exert its high affinity to the holotoxin. Both cytotoxicity and animal toxicity of ricin were well blocked by pre-incubating the toxin with mAb 4C13. By intravenous injection, mAb 4C13 could rescue the mouse intraperitoneally (ip) injected with a lethal dose of ricin (20 μg/kg) even at 6 h after the intoxication and its efficacy was dependent on its dosage. This research indicated that mAb 4C13 could be an excellent candidate for therapeutic antibodies. Its potent antitoxic efficiency was related to its recognition on the specific epitope with very high affinity and its blockage of protein synthesis inhibition in cytoplasm followed by cellular internalization with ricin.     

Long-lasting specific antibodies against P277 induced by mucosal administration of P277 repeat sequences carried by Hsp65 in the absence of adjuvants

To improve the weak immunogenicity of peptide P277, the recombinant expression plasmid pET28-Hsp65-6 x P277 was constructed by inserting 5 x P277 which was amplified by PCR into the vector pET28-Hsp65-P277. It was transformed into Escherichia coli BL21 (DE3) and the fusion protein (Hsp65-6 x P277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified by means of cell disruption, ammonium sulfate precipitation, double-distilled H2O dialysis, DEAE52-cellulose column chromatography, and then used to immunize female NOD mice with three i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at 3 weeks interval. Antibodies against P277 and HSP65 were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and Western blot. Specific antibodies were successfully induced and lasted for more than 20 weeks in animals immunized with the fusion protein via intranasal route even in the absence of adjuvants.     

Inhibition of sperm-egg binding and fertilisation in mice by a monoclonal antibody reactive to 57-kDa human sperm surface antigen

Monoclonal antibodies (mAbs) against spermatozoa are a popular approach to define sperm antigens involved in the process of fertilisation. The identification and characterisation of a 57-kDa fertility asssociated sperm antigen (FASA-57) from human spermatozoa was reported in an earlier paper by the authors. In the present report, studies to develop mAbs against partially purified FASA-57 are extended. From a panel of mAbs raised, one clone designated as 3H(4)B(9) was selected and characterised because it recognised native FASA-57. Indirect immunofluorescence studies revealed that FASA-57 localised on the acrosome of non-acrosome-reacted human spermatozoa and on the equatorial region after the acrosome reaction. Spermatozoa from several other mammalian species were also found to express this antigen, suggesting its evolutionary conservation across the species. The antigen localised specifically in spermatogonial cells and luminal spermatozoa of the testis and epididymis. Western blot studies showed the presence of a FASA-57-like protein in the mouse brain also, indicating that testis and brain share antigenic similarities. Further, the role of FASA-57 in sperm-egg interaction was investigated using a mouse model. The mAb 3H(4)B(9) inhibited sperm-egg binding and fusion in a dose-dependent manner with half-maximal inhibition at 2 microg mL(-1). In conclusion, FASA-57 appears to play an important role in sperm-egg recognition, fusion and fertilisation. Therefore, FASA-57 could be used as a diagnostic marker in the evaluation of male infertility.     

恙虫病东方体蛋白基因的原核表达及活性鉴定

目的 对恙虫病东方体Gilliam株56kDa外膜蛋白基因进行原核表达，并对表达产物进行纯化，以获得有生物活性的重组蛋白，用于恙虫病诊断试剂的研制。方法 根据GiUiam株东方体56kDa蛋白基因序列设计特定引物。用PCR法扩增出编码56kDa抗原长约1320bp的DNA，克隆至原核载体PET28a，构建了表达载体PET-OTG，并获表达。表达产物经镍(Ni2 )柱亲和层析纯化，聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白印迹(Western—blot)分析鉴定。并用间接ELISA进行抗原性分析。结果SDS—PAGE检测表明重组蛋白分别以可溶性蛋白和包涵体两种方式表达，在相对分子质量约52kDa处有表达目的条带。经 Ni^2 层析柱纯化得到目的蛋白，包涵体蛋白纯化后纯度大于可溶性蛋白，达电泳纯。Western-blot证实该蛋白能被患者阳性血清所识别，用阴性血清则未出现相应印迹。间接ELISA结果表明重组蛋白具有良好的抗原性，能有效区分恙虫病阳性和阴性血清。结论 重组蛋白具有免疫反应活性。有望作为诊断抗原用于恙虫病的诊断。     

重组结核分枝杆菌PPE17蛋白原核可溶性表达纯化和血清学诊断研究

目的利用原核系统可溶性表达结核分枝杆菌PPE17蛋白并进行纯化,研究其免疫学特性,并评价该重组蛋白在结核病血清学诊断方面的价值。方法从结核分枝杆菌H37Rv基因组中利用PCR扩增PPE17核酸序列然后克隆至融合表达载体pET-DsbC中,转入大肠埃希菌BL21(DE3)进行诱导、表达和纯化,用Western Blot和EL ISA方法进行抗原性初步评价。结果融合蛋白Ds-bC-PPE17在原核系统内经IPTG诱导表达后,主要以可溶性形式表达存在,经镍柱层析获得了纯的重组蛋白,纯度达95%以上。Western Blot和EL ISA方法结果证明重组DsbC-PPE17蛋白具有较强的抗原活性。用纯化的PPE17蛋白做抗原,临床诊断结核病人血清,阳性率达60%。结论融合蛋白DsbC-PPE17在大肠埃希菌中以可溶性表达形式存在,高纯度的重组融合蛋白有可能成为结核病的血清学诊断抗原。     

Diagnosis of cystic echinococcosis, central Peruvian Highlands

We evaluated prevalence of cystic echinococcosis (CE) in a central Peruvian Highland district by using 4 diagnostic methods: ultrasonography for 949 persons, radiography for 829, and 2 serologic tests for 929 (2 immunoblot formats using bovine hydatid cyst fluid [IBCF] and recombinant EpC1 glutathione S-transferase [rEpC1-GST] antigens). For the IBCF and rEpC1-GST testing, prevalence of liver and pulmonary CE was 4.7% and 1.1% and seropositivity was 8.9% and 19.7%, respectively. Frequency of seropositive results for IBCF and rEpC1-GST testing was 35.7% and 16.7% (all hepatic cysts), 47.1% and 29.4% (hepatic calcifications excluded), and 22.2% and 33.3% (lung cysts), respectively. Weak immune response against lung cysts, calcified cysts, small cysts, and cysts in sites other than lung and liver might explain the poor performance of the serodiagnostic tests. We confirm that CE is highly endemic to Peru and emphasize the limited performance of available serologic assays in the field.