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细胞转印技术（Cell－blotting）

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解剖学报 1996年第3期27卷 303-303页

作者：张世馥中国协和医科大学基础医学研究所细胞生物室

细胞转印技术（Ｃｅｌｌ－Ｂｌｏｔｔｉｎｇ）（中国协和医科大学基础医学研究所细胞生物室，北京１００００５张世馥）自１９７５年ＳｏｕｔｈｅｒｎＥＭ首先建立了Ｓｏｕｔｈｅｒｎ转印技术用于ＤＮＡ的研究之后，相继建立了用于ＲＮ... 详细信息

细胞转印技术（Ｃｅｌｌ－Ｂｌｏｔｔｉｎｇ）（中国协和医科大学基础医学研究所细胞生物室，北京１００００５张世馥）自１９７５年ＳｏｕｔｈｅｒｎＥＭ首先建立了Ｓｏｕｔｈｅｒｎ转印技术用于ＤＮＡ的研究之后，相继建立了用于ＲＮＡ研究的Ｎｏｒｔｈｅｒｎ转印以及用...

关键词：转印技术 blotting 蛋白分子培养的细胞聚丙烯酰胺基因克隆蛋白带单克隆抗体膜蛋白蛋白酶抑制剂

棉花咖啡酸-O-甲基转移酶基因的原核表达及蛋白纯化鉴定

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西北植物学报 2010年第9期30卷 1738-1743页

作者：李波倪志勇石庆华范玲新疆农业科学院核技术生物技术研究所乌鲁木齐830091 新疆农业大学农学院乌鲁木齐830052

为获得大量高纯度的GhCOMT2蛋白以便研究其功能和性质,以pMD18-GhCOMT2质粒为模板,PCR扩增GhCOMT2基因的cDNA编码区,构建原核表达载体pET-28a-GhCOMT2,经酶切鉴定并测序后转化到大肠杆菌BL21(DE3)中进行诱导表达,并采用Western blottin... 详细信息

为获得大量高纯度的GhCOMT2蛋白以便研究其功能和性质,以pMD18-GhCOMT2质粒为模板,PCR扩增GhCOMT2基因的cDNA编码区,构建原核表达载体pET-28a-GhCOMT2,经酶切鉴定并测序后转化到大肠杆菌BL21(DE3)中进行诱导表达,并采用Western blotting方法鉴定表达产物。结果表明:在大肠杆菌BL21(DE3)菌株中成功表达了与标签蛋白融合的GhCOMT2蛋白,大小约为40.062 kD,浓度为0.62 mg/mL。重组蛋白的最佳诱导条件为:0.2 mmol/LIPTG在16℃诱导12 h。重组蛋白以可溶形式高效表达,用蛋白标签亲和层析柱(HisTrapTMHP)获得纯化重组蛋白,Western blotting分析表明其能与His多克隆抗体起特异性反应。

关键词： GhCOMT2 原核表达蛋白纯化 Western blotting

Prevalence of HIV Indeterminate Western Blot Tests and Follow-up of HIV Antibody Sero-Conversion in Southeastern China

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Virologica Sinica 2019年第4期34卷 358-366页

作者： Shouli Wu Min Gao Jian Zheng Pingping Yan Wei Wang Xiaoli Lu Yuefeng Qiu Yansheng Yan Fujian Provincial Center for Disease Control and Prevention Fuzhou 350001China Fujian Provincial Key Laboratory of Zoonosis Research Fuzhou 350001China School of Public Health Fujian Medical UniversityFuzhou 350004China Mengchao Hepatobiliary Hospital of Fujian Medical University Fuzhou 350025China Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention Wuxi 214064China Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology Wuxi 214064China Jiangsu Institute of Parasitic Diseases Wuxi 214064China

HIV-indeterminate Western blotting(WB)results are typically obtained in WB confirmatory assays,and the number of indeterminate samples may increase with the detection of HIV infections,which will present considerable challenges for the management of HIV/*** acid detection has been used as a laboratory test for screening suspected or indeterminate ***,the effectiveness of these assays for the differential diagnosis of HIV-indeterminate WB samples remained *** this study,210 subjects with HIV-indeterminate WB results were detected from 6360 positive HIV screening samples between 2015 and 2016 in southeastern China,in which HIV-indeterminate WB results accounted for 3.30%.The highest proportion of indeterminate results was observed in pregnant and lying-in women receiving physical examinations(16.67%),followed by that in voluntary blood donors(8.82%).The most common WB band patterns were p24,gpl60 and p24,and *** follow-up study revealed that the highest negative and positive conversion rates of HIV antibodies were in samples with a single p24 band(80.28%),and with gpl60 and p24 bands(86.21%),*** the Env,Gag,and Pol antibodies,samples with a Gag band showed the highest negative conversion rate(81.25%),whereas the highest positive conversion rate was observed in samples with an Env band(56.76%).In addition,quantitative and qualitative HIV nucleic acid testing exhibited the highest sensitivity(96.3%)and specificity(97.85%),*** results indicate a lower proportion of HIV indeterminate WB results in southeastern China compared to previous reports,and the follow-up re-examination of patients with HIV indeterminate results should be *** acid testing facilitates the identification of HIV infections.

关键词： Human immunodeficiency virus(HIV) HIV indeterminate result Western blotting Nucleic acid test Differential diagnosis

Immunodetection of ephrin receptors in the regenerating tail of the lizard Podarcis muralis suggests stimulation of differentiation and muscle segmentation

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Zoological Research 2019年第5期40卷 416-426页

作者： Lorenzo Alibardi Comparative Histolab Padova and Department of Biology University of Bologna

Ephrin receptors are the most common tyrosine kinase effectors operating during development. Ephrin receptor genes are reported to be up-regulated in the regenerating tail of the Podarcis muralis lizard. Thus, in the current study, we investigated immunolocalization of ephrin receptors in the Podarcis muralis tail during regeneration. Weak immunolabelled bands for ephrin receptors were detected at 15–17 kDa, with a stronger band also detected at 60–65 kDa. Labelled cells and nuclei were seen in the basal layer of the apical wound epidermis and ependyma, two key tissues stimulating tail regeneration. Strong nuclear and cytoplasmic labelling were present in the segmental muscles of the regenerating tail, sparse blood vessels, and perichondrium of regenerating cartilage. The immunolocalization of ephrin receptors in muscle that gives rise to large portions of new tail tissue was correlated with their segmentation. This study suggests that the high localization of ephrin receptors in differentiating epidermis, ependyma, muscle, and cartilaginous cells is connected to the regulation of cell proliferation through the activation of programs for cell differentiation in the proximal regions of the regenerating tail. The lower immunolabelling of ephrin receptors in the apical blastema, where signaling proteins stimulating cell proliferation are instead present, helps maintain the continuous growth of this region.

关键词： Lizard Regenerating blastema Ephrin receptors Immunolabelling Western blotting

Treatment of chronic proliferative cholangitis with c-myc shRNA

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World Journal of Gastroenterology 2009年第1期15卷 95-101页

作者： Fu-Yu Li Nan-Sheng Cheng Jing-Qiu Cheng Hui Mao Li-Sheng Jiang Ning Li Sheng He Department of Hepatobiliary Surgery West China Hospital of Sichuan University Chengdu 610041 Sichuan Province China Key Laboratory of Transplantation Engineering and Immunology West China Hospital of Sichuan University Chengdu 610041 Sichuan Province China

AIM: To investigate the feasibility and effectiveness of c-myc shRNA in inhibiting the hyperplastic behavior and lithogenic potentiality of chronic proliferative cholangitis (CPC), in order to prevent stone recurrence and biliary ***: An animal model of CPC was established by giving intralumenally 0.5 mL of c-myc shRNA. Then, the effects of c-myc shRNA on hyperplastic behavior and lithogenic potentiality of CPC were evaluated by histological observation, immunohistochemistry, real-time PCR and Western blotting for c-myc, proliferating cell nuclear antigen (PCNA), procollagen TM mucin5AC, enzymatic histochemistry for β-glucuronidase, and biochemistry for hydroxyproline in the diseased bile ***: Treatment with c-myc shRNA efficiently suppressed the hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expression of c-myc. More importantly, it decreased the lithogenic potentiality of CPC by inhibiting the expression of mucin 5AC and the secretion of endogenous β-glucuronidase. Further investigation indicated that c-myc shRNA-3 had a better inhibitory effect on ***: Treatment with c-myc shRNA-3 can control CPC and reduce the lithogenic potentiality of CPC.

关键词： myc基因慢性增生性胆管炎治疗 blotting 葡萄糖醛酸酶增殖细胞核抗原结石复发

Expression and location of α-fetoprotein during rat colon development

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World Journal of Gastroenterology 2009年第14期15卷 1738-1743页

作者： Xiao-Yan Liu Dan Dong Peng Sun Jun Du Luo Gu Ying-Bin Ge Department of Physiology Nanjing Medical University Nanjing 210029 Jiangsu Province China

AIM: To investigate the expression of α-fetoprotein (AFP), a cancer-associated fetal glycoprotein, and its involvement during rat colon development. METHODS: Colons from Sprague-Dawley rat fetuses, young and adult (8 wk old) animals were used in this study. Expression levels of AFP in colons of different development stage were detected by reversetranscriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed using antibodies to specific cell markers and AFP, respectively. RESULTS: The highest levels of AFP mRNA were detected in colons of rats at embryonic day 18.5 (e18.5). Compared to e18.5 d, the AFP expression was significantly decreased during rat development [85% for e20.5, P blotting, the expression pattern was similar to AFP mRNA and conformed to the results of mRNA expression. The AFP positive staining was identical to different distribution patterns in fetuses, young and adult animals and positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage *** that AFP is localized in the mesenchyme of rat colon from the embryo to the weaning stage by immunofluorescence and presents 72-kDa isoform in the developing rat colons by Western blotting. The dynamic expression of AFP in the various developmental stages of the colon indicates that AFP might be involved in many aspects of colon development.

关键词：甲胎蛋白检测 SD大鼠 Western印迹甲胎蛋白mRNA 逆转录聚合酶链反应 blotting 免疫荧光染色发展中国家

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway

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World Journal of Gastroenterology 2009年第14期15卷 1751-1758页

作者： Yu Yao Chen Huang Zong-Fang Li Ai-Ying Wang Li-Ying Liu Xiao-Ge Zhao Yu Luo Lei Ni Wang-Gang Zhang Tu-Sheng Song Department of Oncology First Affiliated Hospital of Medical College of Xi'an Jiaotong University Xi'an 710061 Shaanxi Province China Department of Genetics and Molecular Biology Medical School Xi'an Jiaotong University/Key Laboratory of Environment and Genes Related to Diseases Ministry of Education Xi'an 710061 Shaanxi Province China Second Affiliated Hospital of Xi'an Jiaotong University/Key Laboratory of Environment and Genes Related to Diseases Ministry of Education Xi'an 710004 Shaanxi Province China

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

关键词：人肝癌HepG2细胞 bcl-2/bax 外源性磷脂线粒体跨膜电位诱导细胞凋亡 blotting 流式细胞仪分析

Immunochemical characterization of antigens of Brucella canis and their use in seroprevalence study of canine brucellosis

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Asian Pacific Journal of Tropical Medicine 2011年第11期4卷 857-861页

作者： Sharma Barkha Sinha Dharmendra Kumar Singh Dhirendra Kumar Department of Epidemiology and Preventive Medicine College of Veterinary SciencePandit Deen Dayal Upadhaya Veterinary UniversityMathuraUttar PradeshPin-281001India Division of Epidemiology Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122India Division of Veterinary Public Health Indian Veterinary Research InstituteIzatnagarBareillyUttar Pradesh243122 India

Objective:To explore immunochemical characterization of antigens of Brucella canis(B. canis),and the use in seroprevalence study of canine ***:External hot phosphate buffer saline extract(HPBSE) and internal sonicated(SA) antigens were prepared from *** strain MEX 51 and imniunochemically *** antigens were used to test 527 serum samples of dogs by 2-mercaptoethanol-tubc agglutination test(2 ME-TAT), agar gel immunodiffusion test(AGID).dot-ELISA and indirect enzyme-linked immunosorbent assay(I-ELISA) to assess the seroprevalence of canine ***:The protein content of HPBSE and SA antigens was 0.387 mg/*** 0.195 mg/mL,respectively,whereas carbohydrate content was 0.174 mg/mL and 0.150 mg/mL,*** sodium dodecyl sulfate-polyacrylamide gel electrophoresis(12.5%) of HPBSE and SA,revealed 6 and 8 visible peptide bands ranging from 18-80 kDa and 12-45 kDa,*** blot analysis showed immunodominant bands of MW 12.28.39 and 45 kl)a for HPBSE and 20-24 kl)a for SA. The AGII) revealed HPBSE as more specific antigen than SA but both I-ELISA and dot-ELISA indicated SA antigen to be more specific and reliable than *** seroprevalence of canine brucellosis was 2.27%by 2ME-TAT.1.5%by AGID.3.03%by dot-ELISA and 16.12%by I-ELISA. Conclusions:On the basis of the results of present study,we concluded that HPBSE is suitable antigen for AGID,which is more specific:whereas SA antigen is suitable for I-ELISA,which is highly ***,initial screening of serum samples should be carried out by I-ELISA followed by confirmation with AGID.

关键词： Brucella canis SDS-PAGE Western blotting Agar gel immunodiffusion test Dot-ELISA I-ELISA

Arsenic trioxide inhibits metastatic potential of mouse hepatoma H₂₂ cells in vitro and in vivo

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Hepatobiliary & Pancreatic Diseases International 2009年第5期8卷 510-517页

作者： XianShu Zhao PengLong Song Bei Sun HongChi Jiang TieFu Liu Department of Gastroenterology Fourth Affiliated Hospital Harbin Medical University Harbin 150010 China.

BACKGROUND:It has been pointed out that only low-dose arsenic trioxide(ATO)presents therapeutic benefits outweighing the toxic side ***-dose ATO can effectively alleviate acute promyelocytic leukemia(APL). However,it is quite challenging in treating solid tumors. The purpose of this study was to investigate the effect of ATO at low concentrations on the metastatic potential of mouse hepatoma H22 cells and the anti-metastatic mechanism of ATO. METHODS:The metastatic potential of H22 cells was evaluated by adhesion,migration and invasion assays after exposure to a low dose of ATO in *** mouse lung metastatic model induced by injection of H22 cells via the tail vein was adopted for the evaluation of metastatic potential. Different proteins in the lysate of H22 cells exposed to ATO at different concentrations were investigated by surface- enhanced laser desorption and ionization time-of-flight mass spectrometry(SELDI-TOF-MS).Finally,Western blotting analyses were made to detect the expression pattern of MMP-2 and nm23-M1 proteins. RESULTS:Significant cell death started at ATO concentrations above 2μmol/*** growth and adhesion potential of H22 cells was inhibited in a time-and dose- dependent manner,and the migration and invasion potential of H22 cells was inhibited in a dose-dependent manner while ATO concentration was below 2μmol/L. Mice injected with ATO at a dose of 0.5 mg/kg had fewer lung ***,mice injected with ATO at a dose of 2 mg/kg or 4 mg/kg had a high mortality rate and more liver injuries.A total of 15 different protein peaks were identified between the lysate of H22 cells treated with ATO and *** proteins that peaked atm/z 5302 and 17207 coincided with MMP-2(fragment) and nm23-M1,*** blotting analyses demonstrated that MMP-2 and MMP-2 fragments were down-regulated and nm23-M1 was up-regulated in H22 cells treated with 2μmol/L ATO for 48 hours. CONCLUSIONS:ATO at a low dose inhibits the metastatic pote

关键词： arsenic trioxide hepatocellular carcinoma neoplasm metastasis surface-enhanced laser desorption and ionization time-of-flight mass spectrometry Western blotting