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polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

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Journal of Geriatric Cardiology 2005年第2期2卷 118-122页

作者： Yaling HAN Haiwei LIU Jian KANG Xiaozeng WANG Ye HU Lianyou ZHAO Shaohua LI Department of Cardiology General Hospital of PLA Shenyang Command Shenyang 110016 China Department of Cardiology General Hospital of PLA Shenyang Command Shenyang 110016 China Department of Cardiology General Hospital of PLA Shenyang Command Shenyang 110016 China Department of Cardiology General Hospital of PLA Shenyang Command Shenyang 110016 China Department of Cardiology General Hospital of PLA Shenyang Command Shenyang 110016 China Department of Cardiology Tangdu Hospital Fourth Military Medical University Xi''an 710038 China Department of Pathology and Laboratory Medicine Robert Wood Johnson Medical School New Jersey 08854 USA￡(C)

Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

关键词： E1A cellular repressor polyclonal antibody vascular smooth muscle cells differentiation

Prokaryotic expression, purification of a novel candidate tumor suppressor gene FUS1 and characterization of its polyclonal antibodies

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Journal of Biomedical Science and Engineering 2010年第4期3卷 397-404页

作者： Dong-Mei Zhang Han-Shuo Yang Xin-Yu Zhao Wen Zhu Zhi-Hua Feng Yang Wan Zhi-Wei Zhao Ming-Hai Tang Nong-Yu Huang Yu-Quan Wei 不详

FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.

关键词： FUS1 polyclonal Antibody Prokaryotic Expression Recombinant Protein Tumor Suppressor Gene

Production of human polyclonal antibodies by transgenic animals

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Advances in Bioscience and Biotechnology 2011年第3期2卷 138-141页

作者： Louis-Marie Houdebine 不详

polyclonal antibodies collected from the blood of animals and humans experimentally immunised or spontaneously immunised respectively can be injected into patients to protect them against pathogens, toxins, tumours etc. This approach is severely limited by the availability of human polyclonal antibodies of interest. Moreover, polyclonal antibodies from animals are recognised as antigens by patients and are thus rapidly rejected and inactivated. To circumvent this problem, animals (essentially rabbits, chicken, pigs and cows) are being genetically engineered. Their immunoglobulin genes are being inactivated and the corresponding human immunoglobulin genes are being transferred to them. These animals will be immunized and it is expected that large amounts of pure human polyclonal antibodies will be extracted from their blood to be administered to patients. The possible acceptability problem of this approach is under a case study of the European Union Pegasus project.

关键词： Human polyclonal Antibodies Transgenic Animals Acceptability

Evaluation of rabbit antibody response against 8 and 16 kDa recombinant subunits of antigen B from Echinococcus granulosus

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Asian Pacific Journal of Tropical Medicine 2012年第5期5卷 355-357页

作者： Jahangir Abdi Bahram Kazemi Mohammad Hasan Karimfar Mohammad Bagher Rokni Department of Medical Parasitology and Mycology School of ParamedicineIlam University of Medical SciencesHornIran Cellular and Molecular Biology Research Centre Shahid Reheshti University(Medical Campus)Tehran.Iran Department of Medical Parasitology and Mycology School of ParamedicineIlam University of Medical SciencesIlam.Iran Department of Medical Parasitology and Mycology School of Public HealthTehran University of Medical SciencesTehranIran

Objective:To immunize rabbits with 12 and 16 kDa recombinant subunits of antigen B from Echinococcus granulosus(***) and measuring polyclonal antibody and humoral immune response using ELISA and gel ***:Two mentioned antigens were cloned and expressed in expression vector and purified by affinity *** young rabbits were selected and challenged intradermallt with yielded recombinant ***’ sera were collected post infection and were tested using ELISA and gel diffusion for polyclonal antibody detection 10 days after last ***:The specific antibody against the recombinant peptides was efficiently produced within 4 weeks post ***: Produced recombinants proteins could induce the immune response of the rabbits successfully. This process might improve the clarification of diagnosis and vaccination as regards hydatidosis.

关键词： Cystic echinococcosis Hydatidosis polyclonal antibody

Intravenous immunoglobulin suppresses IL-10 production by activated B cells in vitro

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Open Journal of Immunology 2012年第4期2卷 149-160页

作者： Jun Tanaka Kumiko Hirano Yuzuru Sakamoto Akiko Sugahara-Tobinai Shota Endo Yumi Ito-Matsuoka Atsushi Nakano Masanori Inui Lars Nitschke Toshiyuki Takai Department of Experimental Immunology Institute of Development Aging and Cancer Tohoku University Sendai Japan Department of Genetics University of Erlangen Erlangen Germany Osaka Research Laboratory Benesis Corporation Osaka Japan

A therapeutic preparation of polyclonal human IgG, i.e., intravenous immunoglobulin (IVIg), has been employed to treat several inflammatory and autoimmune disorders. B cells are supposed to be a target of IVIg, but the molecular mechanism is elusive because of the lack of a suitable experimental system. To gain an insight into the beneficial effect of IVIg on B cells, we first established an experimental setting in which IVIg modulates a murine B cell function in vitro, and then aimed at identifying the mechanistic features at the molecular level. Here we show that IVIg down-regulates IL-10 production by CpG-activated B cells in vitro. The responsible component of IVIg was identified as the F(ab’)2 portion, whose polyclonality is mandatory for the suppressive effect. IVIg, bound to the surface of activated B cells, was found to be co-localized with intracellular SHP-1 on confocal laser microscopy, suggesting that B cell-surface immunoreceptor tyrosine-based inhibitory motif-harboring receptors that recruit SHP-1 are target molecules for IVIg in our experimental setting. Overall, we postulate a scenario in which IVIg attenuates B cells by suppressing IL-10 production, a B cell growth factor, and thus down-regulates the production of pathogenic antibodies.

关键词： F(ab’)2 Fragment Immune Inhibition ITIM Receptors polyclonal IgG SHP-1

Protein expression and preparation of polydonal antibody of AD-004 and study on its expression in the adrenal and testis

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China Medical Abstracts(Internal Medicine) 2006年第4期23卷 202-203页

作者：乔洁 Shanghai Instit Endocrine & Metab Dis Shanghai Endocrine & Metab Dis Center Ruijin HospShanghai Jiaotong Univ Sch MedShanghai 200025

Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni2+ -NTA column. The purified protein was used as an immunogene to prepare

关键词： adrenal prokaryotic polyclonal purified testis rabbit prepare cloned adding plasmid

Liver and Biliary System Hepatitis

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China Medical Abstracts(Internal Medicine) 2009年第3期26卷 166-169页

Cloning, Expression, and Immunogenicity Analysis of the CpsE Protein from Group B Streptococcus Isolated from Tilapia (Oreochromis Niloticus)

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Engineering（科研） 2012年第10期4卷 167-171页

作者： Xi Fu Kaiyu Wang Jinlu Huang Jun Wang Hai Lian Yang He Lanmin Li Kaiyu Wang Fish Disease Research Center College of Veterinary Medicine of Sichuan Agricultural University Ya’anChina Key Laboratory of Animal Disease and Human Health of Sichuan Province College of Veterinary Medicine of Sichuan Agricultural University Ya’an China

Group B Streptococcus (GBS) is a major cause of serious bacterial infection in numerous animal species. The production of capsular polysaccharide(CPs) is vital to GBS to evade host immunity. One of the genes that required for production of CPs, cpsE, has been determined to be well conserved in capsule gene cluster (cps).This study cloned the cpsE gene from Tilapia of GBS clinical isolate (serotype Ia) and expressed this gene with aid of pET-32a(+) in Escherichia coli BL21(DE3) competent cells to obtain high levels of the recombinant protein for further study about CpsE in fish and examination of its immunogenicity. The optimization of induction conditions (IPTG concentration, temperature and time) in *** was accomplished and let us to perform the recombinant protein induction at 37℃ for 3h,with 0.2mM IPTG in Luria Bertani (LB) medium. At the optimal conditions, recombinant protein was expressed in an insoluble form (inclusion bodies) and accounted for approximately 23% of the total protein. Purification by affinity chromatography yielded about 480mg fusion protein per liter culture.

关键词： Recombinant CpsE Expression Purification polyclonal Antibody

Expression of Fas-estrogen receptor fusion protein in COS-7 cells

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中国实验血液学杂志 1997年第3期5卷 304-305页

作者： Lei Sun, Huixin Wang, Tingchong ZhouBeijing Institute of Basic Medical Sciences., Beijing 100850

Anti-Fas or Fas ligand can induce apoptosis whenthey bind Fas antigen on cell surfaces, and have potentialtherapeutic uses. But the disadvantage is obvious asnormal cells can also be killed. In this paper, weconstructed a fusion protein between the transmembraneand cytoplasmic domains of hfas and the HBD (hormonebinding domain) of hER (referred to as MfasER).Glioma cells transfeced with MfasER could be indued

关键词： estrogen killed cytoplasmic disadvantage cytotoxic polyclonal referred transmembrane purified glioma