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第十四届全国免疫学学术大会

作者： Chuanbao Li Haizhi Tao Weijun Li Yubin Lin Jun Yu Bio-Rad (Shanghai) Life Science Research and Development Co. Ltd.

western blotting is an important technique used in life science research and clinical *** is widely used to detect specific proteins in lysates from whole cells,tissues,or fractionated *** are key reagents for western blotting,however,not all antibodies produced via immunization can be used for western blotting and using poor-quality antibodies can lead to inconsistent *** address this issue,we established a rapid and efficient method to generate high-quality monoclonal antibodies for western ***,immunogens are designed based on target immunogenicity analysis,then expressed in *** or HEK293 ***,mice are immunized with these immunogens using a fasttrack immunization method to stimulate iliac lymph nodes for two ***,the lymphocytes from enlarged iliac lymph nodes are fused with SP2/0 via ***,the hybridomas are screened with a western blotting-specific screening method that can identify hybridomas that secrete antibodies that can be used for western ***,stable hybridoma clones are obtained via multiple rounds of screening and subcloning and used to produce ***,antibodies are tested against multiple biologically relevant cell lysates and tissues that express the endogenous *** each antibody,we carefully reviewed the literature to identify lysates expected to contain the target and used these to evaluate the *** specificity of the antibodies was further validated by IP/MS,genetic knockout using CRISPR/Cas9,and siRNA-mediated *** western blotting-specific screening and validation was performed,all the antibodies produced with this approach were high-quality western blotting antibodies,and all researchers can generate reliable and reproducible western blots using these *** have produced high-quality western blotting monoclonal antibodies for 313 targets based on this approach,and these antibodies are launched as Precisio

关键词： Monoclonal antibody western blotting Hybridoma western blotting-specific screening

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猪肺表面活性蛋白A western blotting检测方法的建立

猪肺表面活性蛋白A western blotting检测方法的建立

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第四届中国畜牧科技论坛

作者：乔莉娟王立贤颜华中国农业科学院北京畜牧兽医研究所邯郸学院生物科学系

对肺表面活性蛋白A（surfactant associated protein A,SP-A）的检测已是人类临床医学中研究某些肺部疾病的标志物,为了在兽医临床上建立准确、简便的检测SP-A含量的方法,为兽医临床上通过检测SP-A诊治猪呼吸系统疾病奠定基础。本文探... 详细信息

对肺表面活性蛋白A（surfactant associated protein A,SP-A）的检测已是人类临床医学中研究某些肺部疾病的标志物,为了在兽医临床上建立准确、简便的检测SP-A含量的方法,为兽医临床上通过检测SP-A诊治猪呼吸系统疾病奠定基础。本文探讨了用westernblotting技术检测猪的支气管肺泡灌洗液（branchoalveolar lavage fluid,BALF）和血清中SP-A含量的方法。结果成功建立了用western blotting技术检测猪的BALF中SP-A含量的方法,但遗憾的是未能得到用western blotting技术检测猪血清中SP-A含量的结果,认为失败的主要原因是由于本实验中没有使用特异性抗猪SP-A的一抗。

关键词：猪肺表面活性蛋白A 支气管肺泡灌洗液血清 western blotting

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Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

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Acta Pharmacologica Sinica 2004年第6期25卷 19-24页

作者： Lu-yang YU, Bo LIN, Zhen-lin ZHANG2, Li-he GUO3 Instituteof Biochemistryand Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy ofSciences, Shanghai 200031 Center for Preventing and Treating Osteoporosis, Osteoporosis Research Unit, 2 Shanghai JiaoTong UniversityAffiliated Sixth People’s Hospital, Shanghai 200233, China Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Center for Preventing and Treating Osteoporosis Osteoporosis Research Unit Shanghai JiaoTong University Affiliated Sixth People’s Hospi

AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS: PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and western blots. Urine amylase, NO generation, and histological examination were examined. RESULTS: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen, duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.

关键词： adenoviridae streptozocin insulin-dependent diabetes mellitus gene therapy nitric oxide glucose amylases reversed transcriptase polymerase chain reaction western blotting

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Anti-epitope antibody, a novel site-directed antibody against human acetylcholinesterase

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Acta Pharmacologica Sinica 2004年第4期25卷 33-37页

作者： Xing-mei ZHANG, GangLIU, Man-ji SUN2 Institute of Pharmacology and Toxicology, Academyof MilitaryMedical Sciences, Beijing 100850, China Institute of Pharmacology and Toxicology Academy of Military Medical Sciences

AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induc- tion and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody. METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChEwas chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE) polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

关键词： acetylcholinesterase butyrylcholinesterase epitopes antibodies enzyme-linked immunosorbent assay western blotting

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Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2/Bax and activating caspase-3/ICAD pathway

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Acta Pharmacologica Sinica 2004年第5期25卷 149-156页

作者： Shin-ichi TASHIRO,Satoshi ONODERA,Takashi IKEJIMA Department of Clinical and Biomedical Sciences Showa College of Pharmaceutical Sciences Tokyo 194-8543 Japan Department of Clinical and Biomedical Sciences Showa College of Pharmaceutical Sciences Tokyo 194-8543 Japan China-Japan Research Institute of Medical and Pharmaceutical Sciences Shenyang Pharmaceutical University Shenyang 110016 China prof

AIM: To study the mechanisms by which oridonin inhibited HeLa cell growth in vitro. METHODS: Viability oforidonin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualizedby phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel *** activity was assayed using fluorometric protease assay. ICAD, Bcl-2, and Bax proteins expression weredetected by western blot analysis. RESULTS: Oridonin induced oligonucleosomal fragmentation of DNA andincreased caspase-3 activity, on the other hand, reduced the expression of inhibitor of caspase-3-activated DNase(ICAD), a caspase-3 substrate, at 12 h in HeLa cells. Oridonin-induced DNA fragmentation, caspase-3 activationand down-regulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). However, pretreatment with an inhibitor of poly (ADP-ribose) polymerase (PARP), 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), did not suppress oridonin-induced HeLa cell death. Inaddition, oridonin-induced apoptosis was associated with an increase in the expression of the apoptosis inducerBax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. CONCLUSION:Oridonin induces HeLa cells apoptosis by altering balance of Bcl-2 and Bax protein expression and activation ofcaspase-3/ICAD pathway.

关键词： oridonin HeLa cells apoptosis caspase 3 caspases bcl-2 genes bax genes western blotting

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Cloning, expression, and polymorphism of the ECI1 gene in various pig breeds

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Journal of Integrative Agriculture 2017年第8期16卷 1789-1799页

作者： LU Yun-feng CHEN Ji-bao ZHANG Bo LI Qing-gang WANG Zhi-xiu ZHANG Hao WU Ke-liang College of Animal Science and Technology China Agricultural University School of Life Science and Technology Nanyang Normal University Institute of Animal Sciences and Veterinary Medicine Anhui Academy of Agricultural Sciences

The enzyme △3,△2-dienoyl-CoA isomerase （ECI1） plays a crucial role in the mitochondria113-oxidation of fatty acids with a double-bond in odd and even positions. The ECll gene might be a qualified candidate for studies pertaining to lipid deposition and meat quality in swine. In the present study, ECI1 cDNA of the Tibetan pig was obtained by in silico cloning and verified by PCR analysis. Single-nucleotide polymorphisms （SNPs） of ECll were screened by PCR-sequencing and genotypes of those SNPs were tested by PCR-restriction fragment length polymorphism （PCR-RFLP） in Diannan small-ear pigs （DSP, n=40）, Tibetan pigs （TP, n=60） and Yorkshire pigs （YP, n=30）. The expression levels of ECll were analyzed by real-time quantitative PCR and western blotting in tissues of the liver, backfat, and Iongissimus dorsi （LD） muscle of DSP （n=8）, TP （n=8） and YP （n=8）. Single factor linear correlation analysis was applied separately for each breed to evaluate correlations between ECll gene expression in the LD muscle and intramuscular fat （IMF） content. We obtained an ECll gene length of 1 401 bp from the cDNA that contained a full coding region of 909 bp. Three novel SNPs （g.42425337G〉A; g.42424666A〉G; and g.42422755A〉G） were detected, and only g.42424666A〉G exhibited three genotypes among the three breeds. The ECll expression levels in the LD muscle of DSP and TP were significantly higher than that of YP （P〈0.05）. Moreover, TP had the highest ECI1 expression in backfat （P〈O.01）, and a positive correlation was observed between gene expression and IMF content. The results suggest that differences in ECI1 gene expression might be related to lipid depo- sition and meat quality in pig.

关键词： ECll gene lipid deposition mRNA expression pig single-nucleotide polymorphism western blotting

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Treatment with NADPH oxidase inhibitor apocynin alleviates diabetic neuropathic pain in rats

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Neural Regeneration Research 2018年第9期13卷 1657-1664页

作者： Murat Olukman Aytül onal Fatma Gül celenk Yigit Uyankgil Türker cavusoglu Neslihan Düzenli Sibel ülker Department of Pharmacology Faculty of Medicine Ege University Bornova IzmirTurkey Department of Medical Genetics Faculty of Medicine Ege University Bornova Izmir Turkey Department of Histology and Embryology Faculty of medicine cord Blood Cell and Tissue Research and Application Center Ege University Bornova Izmir Turkey

Increased reactive oxygen species by the activation of NADPH oxidase（NOX） contributes to the development of diabetic ***,a NOX inhibitor,increases sciatic nerve conductance and blood flow in diabetic *** investigated potential protective effect of apocynin in rat diabetic neuropathy and its precise mechanism of action at molecular *** models of streptozotocin-induced diabetes were treated with apocynin（30 and 100 mg/kg per day,intragastrically） for 4 *** hyperalgesia and allodynia were determined weekly using analgesimeter and dynamic plantar *** blot analysis and histochemistry/immunohistochemistry were performed in the lumbar spinal cord and sciatic nerve *** injection reduced pain threshold in analgesimeter,but not in *** treatment increased pain threshold *** blot analysis showed an increase in catalase and NOX-p47 phox protein expression in the spinal ***,protein expressions of neuronal and inducible nitric oxide synthase（n NOS,i NOS）,superoxide dismutase,glutathion peroxidase,nitrotyrosine,tumor necrosis factor-α,interleukin-6,interleukin-1β,aldose reductase,cyclooxygenase-2 or MAC-1（marker for increased microgliosis） in the spinal cord remained *** blot analysis results also demonstrated that apocynin decreased NOX-p47 phox expression at both doses and catalase expression at 100 mg/kg per *** of diabetic sciatic nerve revealed marked degeneration.n NOS and i NOS immunoreactivities were increased,while S-100 immunoreactivity（Schwann cell marker） was decreased in sciatic *** treatment reversed these changes *** conclusion,decreased pain threshold of diabetic rats was accompanied by increased NOX and catalase expression in the spinal cord and increased degeneration in the sciatic nerve characterized by increased NOS expression and Schwann cell *** treatment attenua

关键词： apocynin diabetic complications experimental diabetes mellitus neuropathic pain NADPH oxidase sciatic nerve spinal cord western blotting peripheral nerve injury neural regeneration

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Expression of Interleukin-6 and Interleukin-6 Receptor in Ovine Oocytes During In vitro Maturation

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Journal of Integrative Agriculture 2012年第8期11卷 1333-1339页

作者： ZHAO Xi-an CANG Ming GAO Xiao-yu YANG Mei-ling YUAN Jian-long ZHU Bing WANG Zhi-gang LIU Dong-jun Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology Inner Mongolia UniversityHohhot 010021P.R.China

To study the effects of interleukin-6 （IL-6） and its receptor （IL-6R） during in vitro maturation of ovine oocytes, the mRNA and protein expression levels of IL-6 and IL-6R, along with their localization, were examined during ovine oocytes maturation in vitro through real-time PCR, western blotting, and immunohistochemistry. Specific patterns of expression of IL-6 and IL-6R were observed at both mRNA and protein levels at each stage of ovine oocytes maturation. IL-6 and IL-6R were distributed primarily on the surface of the cell membrane, with little expression in the cytoplasm or nucleus. IL-6 and IL-6R were expressed significantly at higher levels in the maturation around 4 h, and then decreased dramatically. However the level slightly elevated at 20-24 h. The role of IL-6 and IL-6R on oocytes maturation was studied through in vitro addition of recombinant human IL-6 in different concentrations. The addition of 10 ng mL-1 IL-6 significantly increased the rates of oocytes maturation （P〈0.05）, but did not affect the rates of development of the subsequence IVF ovine embryos. In summary, IL-6 is likely to play an important role in the early ovine oocytes maturation. The expression patterns of the IL-6 and IL-6R on the ovine oocytes maturation open up the possibility of regulatory role of the cytokine in ovine oocytes maturation.

关键词： IL-6 oocyte in vitro maturation real time PCR western blotting

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Thermal preconditioning protected cerebellar granule neurons of rats by modulating HSP70 expression

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Acta Pharmacologica Sinica 2004年第4期25卷 60-63页

作者： Li-jun CHEN, Xing-wen SU, Peng-xin QIU, Yi-junHUANG, Guang-mei YAN2 Department of Pharmacology, Zhongshan MedicalCollege, Sun Yat-Sen University, Guangzhou 510089, China Department of Pharmacology Zhongshan Medical College Sun Yat-Sen University

AIM: To explore the possibility that expressions of different heat shock proteins (HSPs) are specifically involved in the protection of thermal preconditioning against apoptosis of cerebellar granule neurons (CGNs) induced by repolarization. METHODS: western blotting was used to detect expressions of HSP27, HSP70, and HSP90 in- duced by thermal preconditioning (TP) in CGNs; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression level of HSP70 mRNA; apoptosis of CGNs was induced by switching culture medium containing KCl 25 mmol/L to one containing KCl 5 mmol/L. RESULTS: No expression of HSP27 in cerebellar granule neurons was observed with TP at 44 oC. Expression of HSP90 was obvious in CGNs both without and with TP at 44 oC for different periods. Expression of HSP70 protein in CGNs was lower with TP at 44 oC for 5 min, but it increased gradually after the period was prolonged to 30, 60, or 90 min. HSP70 mRNA was detected after TP 44 oC for 30, 60, and 90 min and increased gradually with time. HSP70 antisense oligodeoxynucleotides 10 μmol/Lfor 72 h inhibited the protective effects of TP at 44 oC on apoptosis of CGNs induced by repolarization. CONCLUSION: HSP70 is involved in protective effects of thermal preconditioning on apoptosis in cerebellar granule neurons induced by repolarization.

关键词： thermal preconditioning heat heat shock proteins cerebellum neurons apoptosis antisense oligodeoxynucleotides western blotting

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Design,Synthesis and Biological Evaluation of Pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione Derivatives as Novel Neuroprotective Agents

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Chemical Research in Chinese Universities 2021年第3期37卷 647-654页

作者： QUAN Jishun ZHANG Dongping ZHANG Zhuo WANG Jian MA Chao CHENG Maosheng Key Laboratory of Structure-based Drug Design&Discovery of Ministry of Education School of Pharmaceutical EngineeringShenyang Pharmaceutical UniversityShenyang110016P.R.China

A series of pyrrolo[2,1-c][1,4]benzodiazepine-3,11-dione derivatives was designed and synthesized,and their neuroprotective activity against SH-SY5Y cell injury induced by N-methyl-D-aspartic acid(NMDA)was *** the compounds showed significant neuroprotective effects,especially B16,which showed excellent performance and better activity than the positive control ifenprodil(B16:56.2%±0.6%;ifenprodil:41.0%±2.7%).Further investigation indicated that B16 could attenuate the Ca^(2+)influx induced by NMDA in SH-SY5Y cells and western blotting also showed that B16 could attenuate the NR2B upregulation in SH-SY5Y cells induced by *** molecular docking results showed that compound B16 fitted in the binding pocket of NR2B-NMDAR well and could interact with binding sites of compounds 1 and 2 *** ADME/Tox prediction results suggested that compound B16 had good blood-brain barrier(BBB)permeability and the zero alert of Pan Assay Interference Structures(PAINS)indicated that B16 could not elicit false-positive *** results strongly suggest that B16 is a promising and effective candidate neuroprotective compound,and that NR2B-NMDAR is a potential target of B16.

关键词： Neuroprotective activity Ca^(2+)influx western blotting NR2B-NMDA receptor Molecular docking

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