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Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its polyclonal antibodies

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Chemical Research in Chinese Universities 2008年第3期24卷 344-347页

作者： CHEN Yang YANG Su-juan FU Yao WANG Jia-peng ZHAO Zhi-zhuang FU Xue-qi Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Jiltin University Changchun 130012 P R. China University of Oklahoma Health Science Centel Oklahoma 73104 USA

A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPα.

关键词： Receptor-like protein tyrosine phosphatase Clone polyclonal antibodies

Preparation and Characterization of polyclonal antibodies against VLDL Receptor

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2004年第3期24卷 205-207页

作者：屈伸陈涛吴凡尹燕华毕昊 Department of Biochemistry and Molecular Biology School of Basic Medical Sciences Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

Summary: The polyclonal antibodies against VLDL receptor were prepared and identified. Rabbits were immunized with polypeptide fragment of VLDL receptor as antigen. The collected blood serum of the immunized rabbits was analyzed and identified by using ELISA and Western Blot. The results showed that the rabbit against mouse and human VLDL receptor antibodies were obtained with high titer and could recognize the natural VLDL receptors through Western blot. The prepared polyclonal antibodies against VLDL receptor provide a new tool to study the protein of VLDL receptor.

关键词： VLDL receptor polyclonal antibodies

Effects of the Sheep polyclonal antibodies Against the Porcine Adipocyte Plasma Membrane Proteins on Porcine Carcass Composition and Meat Quality

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Agricultural Sciences in China 2007年第10期6卷 1256-1261页

作者： GAO Shi-zheng HU Hong-mei LIU Ling-yun ZHANG Xi LIU Yong-gang GE Chang-rong Key Laboratory of Animal Nutrition and Feed of Yunnan Province Yunnan Agricultural University Kunming 650201 P.R.China Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences Jinan 250100 P.R.China Kunming Normal School Kunming 650201 P.R.China

To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin （ASIg） or sheep nonimmune serum immunoglobulin （NSIg）. At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lurrg weight, dressing percentage, and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate, cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH, meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.

关键词： polyclonal antibodies pig carcass composition meat quality

Protective Efficacy and Mechanism of Passive Immunization with polyclonal antibodies in a Sepsis Model of Staphylococcus aureus Infection

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Protective Efficacy and Mechanism of Passive Immunization wi...

CDH1, a Novel Surface Marker of Spermatogonial Stem Cells in Sheep Testis

第十届全国免疫学学术大会

作者： Jinyong Zhang National Engineering Research Center of Immunological Products Department of Microbiology and Biochemical Pharmacy College of Pharmacy Third Military Medical University

Introduction:Staphylococcus aureus(***)is an opportunistic bacterial pathogen responsible for a diverse spectrum of human ***,it is a leading cause of nosocomial and community acquired infections,resulting in considerable yearly mortality *** to its’rapid acquisition of antibiotic resistance,such as methicillin-resistant ***(MRSA)and vancomycin-resistant ***(VRSA),it becomes increasingly difficult to cure *** infections with conventional *** represents a promising alternative strategy to prevent and/or treat the infection by using vaccines or therapeutic *** and Results:In the present study,polyclonal antibodies targeting three possible *** antigens,Hla,SEB and Mnt C(termed"ASvac-pc Ab"),were raised in rabbits and *** immunization with ASvac-pc Ab after challenge with lethal dose of *** resulting in reduced bacterial loads,inflammatory cell infiltration and decreased pathology,and was able to provide nearly complete protection in a murine sepsis *** vitro studies confirmed the direct binding of ASvac-pc Ab with *** *** studies validated that ASvac-pc Ab contained both opsonic and neutralizing antibodies that contributed to its protective *** former mediated opsonophagocytosis in a neutrophil-dependent manner,while the later inhibited the biological functions of Hla and SEB,two major virulence factors that are directly involved in the pathogenicity of ***,we demonstrate that ASvac-pc Ab is crossreactive with clinical strains of ***:These results confirmed the efficacy for treatment of *** infection by antibodies against multiple targets,which may become an important therapeutic option with the increasing antimicrobial resistance of ***.

关键词： Staphylococcus aureus Passive Immunization polyclonal antibodies sepsis

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Journal of Integrative Agriculture 2014年第8期13卷 1759-1765页

作者： ZHANG Yan WU Sachula LUO Fen-hua Baiyinbatu LIU Lin-hong HU Tian-yuan YU Bo-yang LI Guang-peng WU Ying-ji The Key Laboratory of National Education Ministry for Mammalian Reproductive Biology and Biotechnology Inner Mongolia University School of Basic Medical Sciences Inner Mongolia Medical University

Spermatogonial stem cells（SSCs） are unique stem cells in adult body that can transmit genetic information to the next generation. They have self-renewal potential and can continuously support spermatogenesis throughout life of a male animal. However, the SSC population is extremely small, isolation and purification of the SSCs is challenging, especially for livestock animals. It has been confirmed that CDH1（cadherin-1, also known as E-cadherin） can be expressed in undifferentiated SSCs of mouse and rats, but it has not been verified in sheep. Here, CDH1 was found as a novel surface marker for sheep SSCs. In this paper, sheep antiCDH1 polyclonal antibodies were prepared and its activity was checked. Using the obtained antibodies and immunohistochemistry analysis, we confirmed that CDH1 can be expressed by SSCs in sheep testis.

关键词： sheep spermatogonial stem cells CDH1 polyclonal antibodies immunofluorescent staining

Purification and Characterization of the Catalytic Domain of Protein Tyrosine Phosphatase SHP-1 and the Preparation of Anti-ΔSHP-1 antibodies

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Chemical Research in Chinese Universities 2008年第5期24卷 592-596页

作者： LI Wan-nan ZHUANG Yan LI He SUN Ying FU Yao WU Xiao-xia ZHAO Zhi-zhuang FU Xue-qi Edmond H. Fischer Signal Transduction Laboratory College of Life Sciences Key Laboratory for Molecular Enzymology ＆ Engineering Ministry of Education dilin UniversilT Changchun 130021 P. R. China Department of Pathology The University of Oklahoma Health Science Center OK 73104 USA

This study is focused on the expression of an SH2 domain-truncated form of protein tyrosine phosphatase SHP-1（designated ΔSHP-1） and the preparation of its polyclonal antibodies. A cDNA fragment encoding ΔSHP-1 was amplified by PCR and then cloned into the pT7 expression vector. The recombinant pT7-ΔSHP-1 plasmid was used to transform Rosetta（DE3） E. coli cells. ΔSHP-1 was distributed in the exclusion body of E. coli cell extracts and was purified through a two-column chromatographic procedure. The purified enzyme exhibited an expected molecular weight on SDS-gels and HPLC gel filtration columns. It possesses robust tyrosine phosphatase activity and shows typical enzymatic characteristics of classic tyrosine phosphatases. To generate polyclonal anti-ΔSHP-1 antibodies, purified recombinant ΔSHP-1 was used to immunize a rabbit. The resultant anti-serum was subjected to purification on ΔSHP-1 antigen affinity chromatography. The purified polyclonal antibody displayed a high sensitivity and specificity toward ΔSHP-1. This study thus provides the essential materials for further investigating the biological function and pathological implication of SHP-1 and screening the inhibitors and activators of the enzyme for therapeutic drug development.

关键词： SHP-1 Protein tyrosine phosphatase polyclonal antibodies

Prokaryotic Expression and Potential Application of the Truncated PCV-2 Capsid Protein

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Virologica Sinica 2010年第2期25卷 86-97页

作者： Zhong-zi LOU Zhi-yong LI Gang WANG Jian-qiang LI Xi LAN Xue-rui LI Xiang-ping YIN Ji-xing LIU Si-dang LIU Animal Infectious Diseases Research Laboratory Lanzhou Veterinary Research Institute Chinese Academy of Agricultural Sciences State Key Laboratory of Veterinary Etiological Biology Key Laboratory of Grazing Animal Diseases of Ministry of Agriculture Key Laboratory of Veterinary Public Health of Ministry of Agriculture Lanzhou 730046 China College of Veterinary Medicine Shandong Agricultural University Taian 271018 China

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 （PCV-2）. The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a（＋） vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta（DE3） E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % （73/264）. Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-20RF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.

关键词： Porcine circovirus type 2 Capsid protein Fusion expression polyclonal antibodies Virus detection

Analysis of Carbamates Pesticides： Immunogical Technique by Local Development of Enzyme-linked Immuno-Sorbent Assay

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Journal of Pharmacy and Pharmacology 2018年第4期6卷 395-402页

作者： N. Bellemjid A.Iddar A.Moussaif N.El Abbadi A.Mesfioui National Centre for Nuclear Energy Sciences and Technologies Rabat 10112 Morocco Faculty of Sciences University lbnTofail Kenitra 14000 Morocco

Carbamates insecticides are used increasingly in organochlorine insecticides for the control of insect pests. It agriculture as a replacement for environmentally more persistent was the most used pesticides in Loukkous-Morocco. Conventional methods employed to detect/analyze carbamates residues are time consuming and require sophisticated equipment only available in well-equipped laboratories. In addition, the conventional methods usually require a lot of complex pre-treatment of samples. Therefore, convenient and rapid pesticide detection system is urgently needed. In this order, we aimed to develop a rapid ELISA （enzyme-linked immuno-Sorbent Assay） for detection of some carbamates such as carbendazim [methyl 2-benzimidazole carbamate] and carbofuran [2,3-dihydro-2,2-dimethyl-7-benzofuranylmethyl carbamate]. To develop an immunoassay for carbendazim and carbofuran we have synthesized molecules with acid function （haptens with 4 and 5 carbons） that are coupled with BSA protein and injected to the rabbits, collected antibodies are used for the achievements of the immunoanalytical assay.

关键词： Carbamates polyclonal antibodies ELISA.

Aberrant expression of the IFN regulatory factor 4-binding protein (IBP) in human colorectal cancer

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Aberrant expression of the IFN regulatory factor 4-binding p...