大黄鱼肝脏免疫保护蛋白的鉴定

本研究以大黄鱼(Larimichthys crocea)肝脏为研究对象,采用比较蛋白质组学技术分析大黄鱼免疫保护后蛋白表达水平的变化。结果发现,对照组和免疫保护组中共有20个表达水平差异显著的蛋白点,经MALDI-TOF-MS分析,获得18个差异蛋白点肽质量指纹图谱(peptide mass fingerprint,PMF),通过数据库查询,与免疫型受体、新型抗原受体等蛋白具有高度同源性。在此基础之上,进一步选取4个蛋白差异点(1、10、14和18号)进行Westen blot验证。结果表明,该4个蛋白差异点可以与所制备的抗体发生特异性的结合,且变化趋势与蛋白质组学分析结果一致。由此推测,免疫型受体等18个蛋白应该与大黄鱼免疫保护密切相关,所获研究结果为揭示大黄鱼免疫保护机理奠定了良好的基础。教育部新世纪优秀人才支持计划项目(NCET-11-0922)~

Modification of Energy Metabolism-related Proteins is Responsible for Ceftriaxone-resistance

随着抗生素药物的开发与利用,细菌在对抗药物过程中逐渐发展出各种不同的耐药机制.近年来高通量的蛋白质组学技术已逐渐用于细菌耐药性机理研究,但主要集中在对细菌外膜蛋白的作用进行分析.本文采用2-d nATIVE/SdS PAgE方法从蛋白质复合物角度分析接近生理条件的胞浆蛋白在头孢曲松耐药性中的作用.结果发现8个耐药性相关蛋白质,通过对蛋白质功能分析,揭示了细菌通过调整能量代谢相关蛋白产生耐药性的新机制.进一步对相关菌株的次抑菌浓度和生存率分析,提示MAlP和SuCC等关键蛋白质可作为设计和开发新型抗菌分子的作用靶点.Outer membrane proteins of bacteria have been investigated systematically in response to antibiotic resistance by high-throughput proteomic techniques in recent years,but the data of the cytoplasmic proteins analyzed on a proteome scale are not available yet.In this study,2-D native/SDS-PAGE was first applied for characterization in cytoplasmic proteins responding to ceftriaxone-resistance in Escherichia coli.Eight proteins involved in modification of energy production and conversion were identified responsible for ceftriaxone-resistance.Among these proteins,down-regulated MalP and up-regulated SucC were found to play critical roles in response to resistance according to the MIC and survival ability analysis,and the two proteins may be candidate targets for development of novel antimicrobial drugs.浙江省自然科学基金项目(No.Y2090396);浙江理工大学科研启动基金项目(No.11615432610862)---

Study on Quantitative Method and Immune Regulation of Ig/Ig two-component-Determined Circulating Immune Complexes

目的　建立Ig/Ig -TCIC的定量方法 ,研究Ig/Ig -TCIC是否具有免疫调节作用。 方法　以Ig的免疫生物学性质和数学计算相结合进行定量。通过研究Ig/Ig-TCIC与C3/Ig -TCIC、全血清和复合Ig与补体等体液免疫指标的关系来探讨其免疫调节作用。结果　成功地建立了Ig/Ig -TCIC的定量方法 ,并发现Ig/Ig -TCIC与有关体液免疫指标的相互关系具有明显规律性。结论　本定量法简便实用。Ig/Ig -TCIC作为免疫网络的中间产物在免疫调节中起重要作用Objective To establish a quantitative method to Ig/Ig two component determined circulating imune complexes(Ig/Ig-TCIC) and study whether Ig/Ig-TCIC was related to the function of immune regulation.Methods Quantity of Ig/Ig-TCIC was carried out by the combination of Ig immune biology with mathematics.The immune regulation of Ig/Ig-TCIC was characterized by the correlation of Ig/Ig-TCIC with C3/Ig-TCIC,serum Ig and C3,and complexed Ig and C3.Results The quantitative method was established and significant regularity was found from the association of Ig/Ig-TCIC with these humoral immune markers.Conclusion This quantitative method was simple and useful.Ig/Ig-TCIC plays an important role in immune regulation as a middle product of immune network.教育部优秀骨干教师基金;; 福建省自然科学基金资助项目(C970 16

Association of TolC expression with resistance to tetracycline in Escherichia coli K-12

随着抗生素的开发与使用,细菌在对多种抗生素的适应过程中逐渐发展出对抗生素耐药的反应机制。TolC是药物排出转运体系的外膜成分,与AcrAB一起形成主要的药物排出泵,有关其表达量与耐药间的关系目前尚不清楚。试验将tolC克隆到pET-32a载体上进行诱导表达,镍柱纯化,免疫新西兰大白兔,获得1:4000的抗血清。Western blotting分析表明,TolC的表达量在耐四环素的大肠埃希菌K-12中比对照组提高50%。细菌TolC高表达试验发现,其MIC从100μg/ml提高到200μg/ml。结果说明TolC的表达量与四环素耐药性直接相关,提示细菌可以通过调节外膜蛋白的表达实现对抗生素的耐受。Association of TolC expression with resistance to tetracycline in Escherichia coli K-12 was studid.TolC was an outer membrane protein,which was bound with AcrAB to form a critical multidrug efflux pump.However,association of TolC amount with drug resistance was ill-defined.In the study,tolC was cloned into pET-32a vector induced expression and its recombinant proteins were purified by Ni-NTA.The purified recombinant proteins were used for preparing of anti-TolC.The prepared antiserum with titer 1:4000 was utilized as the primary antibody in Western blotting.It showed that TolC amount was increased 50% in tetracycline-resistant E.coli K-12 compared to its original control strain.When TolC was overexpressed,the strain showed strong resistance to tetracycline with its MIC from 100μg/ml to 200μg/ml.These findings indicated that TolC amount was directly correlated to bacterial resistance to tetracycline,suggesting that bacteria mounted an adaptive feedback to antibiotics by regulating outer membrane protein expression

虾类免疫学的基础和应用研究

国家海洋局第三海洋研究所海洋生物工程重点实验室开放课题基金资助项

Universal primer PCR with SSCP and RFLP for identification of fish disease pathogens

采用通用引物PCR(UPPCR)、PCR RFLP、PCR SSCP技术 ,研究快速鉴别鱼病病原菌的分子生物学诊断技术。结果发现 ,采用细菌 16SrRNA基因保守区特异性引物 ,以嗜水气单胞菌、鲁克氏耶尔森菌、鳗弧菌、柱状曲挠杆菌、乙型链球菌、荧光假单胞菌等部分常见鱼病病原菌为对象 ,可以建立一种UPPCR技术。该技术能在保证实验条件不变的基础上 ,检出上述所有细菌 ,并还可检出大肠杆菌和双歧杆菌等非鱼病病原菌。并且认为 ,该法与SSCP配合即采用UPPCR SSCP技术能较好地鉴别被检菌而用于鱼病病原菌的快速诊断。A universal primer PCR(UPPCR), PCR RFLP and PCR SSCP had been screened for a molecular biological method that permitted the rapid identification of fish pathogens among them. The results showed that a universal primer PCR technology was available with specific primers from conserved regions of bacterial 16S ribosomal RNA genes. The bacteria tested included some common causative agents of fish diseases such as Aeromonas hydrophila, Yersinia ruckeri, Vibrio anguillarum, Flexibacter columnaris, beta Streptococcus, Pseudomonas fluorescens. The approach allowed the bacteria above and Escherichia coli and Bifidobacterium catenulatum detectable without any alterations of the experiment conditions. However, this study also found that the identification of species of bacteria tested depended on the combination of UPPCR and SSCP(PPCR SSCP), which was better than that of UPPCR and RFLP(UPPCR RFLP) and made rapid diagnosis of fish disease pathogens possible.国家自然科学基金!资助项目 (39770 5 85 );; IFS基金!(ImmunecomplexesinfreshfishinfectedFlexibactercolumnaris)资助项

In┐depthStudyofSerumImmunoglobulininCancerPatientsHuangXueFangWangSanyingPengXuanxianetalTheStateLaboratoryForTumorCelEnginering,XiamenUniversity,XiamenAbstractInordertostudythechangesinimmunoglobulinIgpaternsincancerpa-tients,serumIg(totalIg)wascategori

目的：评价恶性肿瘤患者的免疫球蛋白反应类型。方法：将血清免疫球蛋白（Ig）剖解为游离和复合两部分进行研究。以食管癌、喉癌、胃癌、乳腺癌、子宫癌、鼻咽癌、卵巢癌和非霍奇金淋巴瘤（nHl）与健康者分别为实验组和对照组。结果：患者游离Ig的变化与总Ig一致，而复合Ig则与之不同，对其的检测更有利于对患者免疫状况的评价。从而说明，既往有关血清Ig含量的测定，主要反映游离Ig部分的免疫反应状况。结论：免疫反应产物的剖解分析可以更进一步认识免疫反应本质。在对肿瘤患者Ig比例的研究中，发现肿瘤患者的复合Igg普遍严重下降。从而认为特异性Igg水平的明显下降是患者免疫功能降低的重要原因，并提出深入研究其机理，将可能为探索肿瘤发病机理和治疗找到一条新路In order to study the changes in immunoglobulin Ig patterns in cancer patients, serum Ig (total Ig) was categoried into Free Ig and complex Ig For analysis in patient with cancers of esophagus,larynx,stomach, breast, nasopharynx, ovary and non Hodgkin's lymphomas as well as in healthy individuals as control.It was Found that the variations of Free Ig were in good mimicry with those of the total Ig,whereas complex Ig had its characteristic Features paralleling the immune status of the patients.It was thought, thereFore, previous reports on serum Ig mainly concentrated in the immune responses of Free Ig,and that in depth analysis of immune elements could only enable unveiling the truth .Furthermore, we Found that complex IgG was markedly decreased in almost all of these malignancies in terms of Ig ratios.It is suggested that the marked decrease of speciFic IgG levels might be the key reason For declining immune ability of cancer patients and that Further study of its mechanism would be possible to Find a new approach For exploring tumor pathogenesis and therapy.福建省自然科学基

HBVDNA/补体双特异性免疫复合物的检测

以羊抗人C1Q和b因子Igg为捕捉抗体，分别用以捕捉通过经典和旁路途径激活补体的dAnE颗粒抗原抗体免疫复合物中的C1Q和b因子，再利用引物的特异性和PCr的强大扩增能力对捕捉物中的HbVdnA进行检测，成功地建立了检测HbVdnA/补体双特异性循环...国家自然科学基