百日咳黏附素的原核表达及其单克隆抗体的制备

目的原核表达百日咳黏附素(Pertactin,Prn),并制备抗Prn单克隆抗体。方法从无细胞百日咳疫苗生产菌株CS株基因组DNA中克隆Prn基因,插入表达载体pQE-30中,构建重组表达质粒pQE-30-Prn,转化E.coli M15,IPTG诱导表达。表达的重组Prn蛋白经阳、阴离子交换层析纯化后,采用Western blot和ELISA法鉴定重组Prn蛋白的反应原性。以纯化的重组Prn蛋白为免疫原,利用杂交瘤技术制备单克隆抗体,并对制备的单抗进行鉴定。结果重组表达质粒经双酶切和测序鉴定构建正确;表达的重组Prn蛋白相对分子质量约为69 000,主要以包涵体形式表达;纯化的重组Prn蛋白的纯度达95%以上,产量可达25 mg/L,能与不同来源的抗Prn-Ab血清特异性结合。获得2株分泌抗Prn单抗的杂交瘤细胞株,分泌的单抗均为IgG1类,轻链类型均为κ,效价均达1∶105以上,并能特异性识别Prn蛋白,而与百日咳杆菌其他抗原蛋白无交叉反应。结论原核表达、纯化了重组Prn蛋白,并制备了2株效价高、特异性强的单抗,为无细胞百日咳疫苗的质量控制及百日咳杆菌致病机制的研究提供了材料。     

幽门螺杆菌CrdR蛋白的原核表达

目的原核表达幽门螺杆菌(Helicobacter pylori,H.pylori)CrdR蛋白(即HP1365),以探讨其在酸适应中的调控机制。方法从H.pylori 26695标准株基因组DNA中PCR扩增hp1365基因,克隆至表达载体pGEX-6P-1中,转化E.coli JM109,IPTG诱导表达,表达产物经SDS-PAGE及Western blot鉴定。结果重组表达质粒pGEX-hp1365经双酶切、PCR和测序鉴定构建正确;表达的重组蛋白相对分子质量约为25 000,主要以包涵体形式表达,可与鼠抗His标签单克隆抗体特异性结合。结论原核表达了H.pylori CrdR蛋白,为进一步探讨其对酸信号的感应机制及其通过阻断酸信号的感应抗H.pylori感染的途径奠定物质基础。     

幽门螺杆菌感应蛋白CrdS的原核表达及生物信息学分析

目的原核表达幽门螺杆菌(Helicobacter pylori,H.pylori)感应蛋白CrdS,并对其进行生物信息学分析,以探讨其酸适应的调控机制。方法从H.pylori 26695标准株全基因组DNA中PCR扩增编码CrdS蛋白的基因hp1364,插入原核表达载体pQE30,构建重组表达质粒pQE30-hp1364,转化大肠埃希菌(E.coli)XL1-blue,IPTG诱导表达重组CrdS蛋白。表达的重组蛋白经Western blot鉴定后,进行生物信息学分析。结果重组表达质粒pQE30-hp1364经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为46 000,表达量占菌体总蛋白的30%,主要以包涵体形式存在,可与Rabbit anti His-Tag Polyclonal Antibody特异性结合;生物信息学分析显示,CrdS的核苷酸序列和氨基酸序列与其他H.pylori菌株(HpG27、F30、B38)具有高度同源性,表面有许多亲水性区域,含多种易被磷酸化的氨基酸。结论成功原核表达了H.pylori CrdS蛋白,并进行了生物信息学分析,为进一步探讨CrdS的感应机制及通过阻断信号感应抗H.pylori感染的途径提供了实验材料。     

重组幽门螺旋杆菌黏附素A的可溶性表达、纯化及其免疫学性质分析

目的 通过分子克隆、蛋白表达及纯化等技术获得重组幽门螺旋杆菌黏附素A(recombinant H. pylori adhesin A,rHpaA),免疫BALB/c小鼠,制备anti-HpaA多克隆抗体后,分析其抗体特异性。方法 利用Phyre2和DNAstar生物信息学软件分析rHpaA的三维结构及抗原性质;通过PCR长片段DNA合成技术获得黏附素HpaA基因,插入质粒pCzn1中,制备重组质粒pCzn1-rHpaA,转化E. coli Arctic Express(DE3),经IPTG诱导表达、Ni-IDA亲和层析纯化后,获得rHpaA蛋白,Western blot鉴定其反应原性。将rHpaA辅以弗氏佐剂免疫雄性BALB/c小鼠,共6只,制备antiHpaA多克隆抗体,ELISA法鉴定其抗体特异性。结果 rHpaA具有较好的三维结构及抗原性质。双酶切鉴定及基因测序证明,重组质粒pCzn1-rHpaA含有完全正确的HpaA基因序列。重组菌株pCzn1-rHpaA/Arctic Express可溶性表达目的蛋白rHpaA,约占上清总蛋白的68.3%,纯度达98.1%。rHpaA可与an...     

幽门螺杆菌双组分系统耐酸相关蛋白ArsS的原核表达及纯化

目的原核表达并纯化幽门螺杆菌双组分系统耐酸相关蛋白ArsS,为深入研究其功能及其在幽门螺杆菌耐酸机制中的作用奠定基础。方法以幽门螺杆菌菌株26695基因组为模板,采用PCR法扩增ArsS基因,插入pET-22b(+)载体,构建重组原核表达质粒pET-22b(+)-ArsS,转化E.coliBL21(DE3),0.5mmol/LIPTG25℃诱导表达,并采用亲和层析与分子筛层析对重组蛋白进行纯化。结果重组原核表达质粒pET-22b(+)-ArsS经双酶切及测序鉴定,证明构建正确;重组蛋白以可溶性形式表达,表达量占菌体总蛋白的30%以上;分子筛层析图谱显示,在150mmol/LNaCl条件下,目的蛋白层析效果较好,纯化后的重组蛋白纯度可达95%以上,浓度约为10mg/ml。结论已成功原核表达并纯化获得了高纯度的ArsS蛋白。     

抗幽门螺杆菌黏附素蛋黄抗体与HP1188蛋黄抗体在小鼠体内防治幽门螺杆菌感染的比较

目的比较抗幽门螺杆菌黏附素(Helicobacter pylori adhesin A,HpaA)蛋黄抗体(immunoglobulin yolk,IgY)(HpaA-IgY)及抗H.pylori HP1188 IgY(HP1188-IgY)在BALB/c小鼠体内预防和治疗H.pylori感染的最低浓度,并评价治疗效果。方法 Western blot鉴定HpaA-IgY及HP1188-IgY抗原特异性。制备1×109 CFU/mL H.pylori菌液,将BALB/c小鼠随机分为阳性对照组(灌喂H.pylori菌液0.5 mL)、阴性对照组(以布氏肉汤替换H.pylori)、预防组(先灌喂1、3、5 mg/mL HpaA-IgY或HP1188-IgY及PBS各1 mL,再灌喂H.pylori菌液0.5 mL)、治疗组(先灌喂H.pylori菌液0.5 mL,再灌喂1、3、5 mg/mL HpaA-IgY或HP1188-IgY及PBS各1 mL)。处理完成后,取各组小鼠胃组织进行H.pylori培养、快速尿素酶试验(rapid urease test,RUT)及组织学检查,观察H.pyl...     

重组幽门螺杆菌粘附素的纯化工艺

目的纯化大肠杆菌表达的重组幽门螺杆菌粘附素。方法将表达HpaA蛋白的工程菌经高压均质机破菌获得包涵体,经洗涤、变性、复性,Q Sepharose High Performance阴离子交换层析和亲和层析分离纯化。采用SDS-PAGE和HPLC检测纯度,用Western blot检测其抗原性。结果纯化后HpaA蛋白纯度高达95％以上,具有良好的抗原性。结论建立了从包涵体中获得高纯度HpaA纯化工艺,为进一步的研究打下了基础。     

幽门螺杆菌VacA-HpaA融合蛋白的表达及其免疫学特性

目的构建H.pylori细胞空泡毒素VacA与黏附素HpaA融合基因的原核表达载体,诱导其表达融合蛋白,并检测表达产物的抗原性与免疫原性。方法用PCR从pQE30-VacA质粒扩增出VacA基因,克隆至pTrc99A-HpaA载体中,与HpaA基因融合后,插入原核表达载体pQE30中,再将pQE30-VacA-HpaA转化入大肠杆菌DH5α,诱导表达并提纯融合蛋白,SDS-PAGE检测融合蛋白的表达,Bradford法检测融合蛋白含量,Western blot鉴定特异性。将融合蛋白免疫家兔,得到多克隆抗血清,用双向免疫扩散和ELISA检测免疫原性。结果SDS-PAGE显示融合蛋白相对分子质量约为65000,表达量在35%以上,主要以包涵体形式表达,蛋白含量为0·72mg/ml,具有良好的VacA和HpaA抗原性与免疫原性。结论VacA-HpaA融合蛋白已成功表达,且具有良好的免疫原性,为进一步研究制备H.pylori疫苗创造了条件。     

单核李斯特菌溶血素蛋白的原核表达及纯化

目的原核表达并纯化单核李斯特菌(Listeria monocytogenes,LM)溶血素(Listeriolysin O,LLO)蛋白。方法用1对LM LLO基因特异性引物从LM基因组DNA中扩增LLO基因,并构建重组原核表达质粒pET-30a(+)/rLLO,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达的rLLO蛋白经镍离子金属螯合柱纯化后,进行SDS-PAGE及质谱分析。结果重组表达质粒pET-30a(+)/rLLO经PCR、双酶切及测序,证明构建正确。表达的rLLO融合蛋白相对分子质量约为78 000,主要以包涵体形式表达。质谱分析显示,两个相对分子质量分别为35 000和45 000的小分子也均为LLO蛋白。结论已成功地在大肠杆菌中表达了rLLO蛋白,并进行了纯化,为进一步研究LLO蛋白的生物学功能及研制基于LLO蛋白的特异性诊断制剂奠定了基础。     

Systematic evaluation and optimization of crystallization conditions for vancomycin purification

This study describes the evaluation and optimization of a crystallizing process capable of efficiently purifying vancomycin in high purity and yield. In particular, we observed how the main process parameters influenced the formation of crystals, determined their morphology, and monitored purity and yield. Acetone was shown to be more effective than alcohol solvents for the crystallization of vancomycin. The optimal distilled water/acetone ratio, storage temperature, storage time, pH, conductivity, initial vancomycin concentration and stirrer velocity were shown to be 1: 3.5 (v/v), 10 °C, 24 h, pH 2.5, 20 ms/cm, 0.1 g/mL, and 640 rpm, respectively. Temperature had a decisive influence on crystal formation; crystals were successfully produced at 10 °C, while at other temperatures, conglomeration, disintegration and cohesion occurred. Crystal growth developed over time and was complete at about 24 h. Vancomycin purity remained at about 97.0% irrespective of storage time while the yield increased over time, reaching a maximum of 95.0% at around 24 h, after which there was no substantial change. Crystallization occurred over a certain range of pH (2.5–3.0), but purity and yield were highest at pH 2.5. When the pH was outside this range, a conglomeration (gelation) phenomenon prevented the efficient production of crystals. Vancomycin crystals were produced irrespective of the stirrer velocity, which had no influence on purity; however, the highest yield of vancomycin was obtained at 640 rpm.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

幽门螺旋杆菌尿素酶多表位疫苗CTB-UE的生物信息学分析及其表达

目的通过生物信息学软件评价幽门螺旋杆菌(Helicobacter pylori,Hp)尿素酶多表位疫苗CTB-UE的抗原结构,并在E.coli中表达、纯化融合蛋白CTB-UE。方法应用生物信息学软件DNAstar、Geno3D、MOE分析Hp尿素酶多表位疫苗CTB-UE的二级结构和三级结构;将人工合成的尿素酶多表位肽基因UE插入含CTB基因的重组质粒p ETC中,构建重组表达质粒p ETCUE,转化E.coli BL21(DE3),经IPTG诱导表达后,通过Ni-NTP镍离子亲和层析纯化重组蛋白CTB-UE,并进行Western blot分析。结果生物信息学软件分析显示,Hp尿素酶多表位疫苗CTB-UE具有合理的结构;重组表达质粒p ETCUE经双酶切和测序鉴定证实,融合基因CTB-UE与设计序列完全一致;表达的融合蛋白CTB-UE相对分子质量约为33 000,主要以包涵体形式表达,表达量占全菌蛋白的22.3%;纯化的融合蛋白CTB-UE纯度达95.6%,可与兔抗Hp多克隆抗体发生特异性结合。结论设计的Hp尿素酶多表位疫苗CTB-UE具有合理的结构,能在E.coli中高效表达,纯化后纯度较高,且具有较强的反应原性,为进一步研究针对尿素酶A、B双亚基的Hp多表位疫苗奠定了基础。     

肠道病毒71型3C基因原核表达质粒的构建表达及纯化

目的构建肠道病毒71型(Entervirus71,EV71)3C基因的原核表达质粒,在大肠杆菌中进行表达并纯化。方法以病毒的反转录产物为模板,PCR扩增EV71 3C基因,克隆至pGEx-4T-1载体中,构建重组表达质粒pGEx-3C,转化大肠杆菌BL21(DE3)中,IPTG诱导表达。表达的重组融合蛋白GSR-3C经GST-Agarose亲和层析和分子筛层析纯化后,Western blot检测其反应原性。结果重组表达质粒pGEx-3C经双酶切及测序证实构建正确;表达的重组融合蛋白相对分子质量约46 000,表达量约占菌体总蛋白的50%,主要以可溶性形式表达;纯化后的重组融合蛋白纯度大于90%,可与小鼠抗EV71单克隆抗体特异性结合。结论成功构建了重组表达质粒pGEx-3C,在大肠杆菌中高效分泌表达并纯化了重组GST-3C融合蛋白,为EV713C蛋白酶结构与功能的研究及3C靶向药物和疫苗的研究奠定了基础。     

DeepHP: A New Gastric Mucosa Histopathology Dataset for Helicobacter pylori Infection Diagnosis

Emerging deep learning-based applications in precision medicine include computational histopathological analysis. However, there is a lack of the required training image datasets to generate classification and detection models. This phenomenon occurs mainly due to human factors that make it difficult to obtain well-annotated data. The present study provides a curated public collection of histopathological images (DeepHP) and a convolutional neural network model for diagnosing gastritis. Images from gastric biopsy histopathological exams were used to investigate the performance of the proposed model in detecting gastric mucosa with Helicobacter pylori infection. The DeepHP database comprises 394,926 histopathological images, of which 111 K were labeled as Helicobacter pylori positive and 283 K were Helicobacter pylori negative. We investigated the classification performance of three Convolutional Neural Network architectures. The models were tested and validated with two distinct image sets of 15% (59K patches) chosen randomly. The VGG16 architecture showed the best results with an Area Under the Curve of 0.998%. The results showed that CNN could be used to classify histopathological images from gastric mucosa with marked precision. Our model evidenced high potential and application in the computational pathology field.     

溶剂结晶法提纯豆甾醇工艺条件的研究

采用溶剂结晶法从混合植物甾醇中提纯豆甾醇,选择了二氯乙烷和正庚烷的二元共沸物作为溶剂,确定了较为合理的豆甾醇结晶工艺条件.溶剂比1∶4,结晶温度30℃,结晶时间2～4 h.     

熔融结晶技术分离纯化有机化合物的研究进展

熔融结晶技术是利用被分离物质各组分间凝固点的差异,通过控制热量的输入和移出,使被分离组分从熔融液中结晶析出,经洗涤、发汗等操作,实现目标组分分离纯化的一种结晶技术。熔融结晶分离纯化技术由于具有不需要使用溶剂、能耗低、设备体积小、能得到高纯产品等优点,在有机化合物的分离纯化领域得到了广泛应用。本文简述了熔融结晶的方式,介绍了熔融结晶器的类型,综述了熔融结晶技术分离纯化有机同分异构体、有机化工原料、日用品、食品和药品的研究进展,分析了熔融结晶技术分离纯化有机化合物过程中存在的问题,展望了熔融结晶技术分离纯化有机化合物的发展方向。文中指出：随着熔融结晶技术的发展,以提高产品质量,减小能耗和降低成本为目的的耦合熔融结晶技术已成为熔融结晶技术发展的方向。以包含熔融结晶设备、工艺、晶体成核和生长动力学、发汗机理以及传热传质模型的系统工程将会成为熔融结晶分离纯化有机化合物的研究热点。     

熔融结晶分离精制2,5-二甲酚

采用熔融结晶分离提纯2,5-二甲酚,考察结晶终温、降温速率、发汗终温对结晶的影响。优化的精制工艺参数为:将物料在熔融状态73℃恒温20 min,按温度控制曲线进行操作,降温速率以3℃/h降温10 h;结晶终温43℃,恒温20 min;然后以3℃/h升温10 h,发汗终温70℃;恒温20 min,最后快速升温至78℃,收集样品,得到含量达99.5%的2,5-二甲酚产品,收率达76.3%。