1.Preparation and application of rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB).
Lu YUAN ; Wenhua XU ; Tingting GE ; Huiping ZHOU ; Ling YANG ; Fan YANG ; Changmin NIU ; Ying ZHENG
Chinese Journal of Cellular and Molecular Immunology 2023;39(9):846-851
Objective To prepare rabbit polyclonal antibody against mouse IQ and ubiquitin-like domain-containing protein (IQUB) and detect its expression in the mouse testis. Methods Full-length coding sequence of IQUB was inserted into the pET-30a(+) vector to construct pET-30a-IQUB recombinant prokaryotic plasmid. Transformation of pET-30a-IQUB plasmid into E. coli BL21 was performed, and protein expression was induced with isopropyl-beta-D-thiogalactoside (IPTG). The protein was purified through histidine-tagged fusion protein purification column, then denatured by treatment of urea with gradient concentration. New Zealand rabbits were immunized with the denatured protein to produce IQUB polyclonal antibody. Antibody titer was detected by ELISA, and Western blot analysis and immunofluorescence assay were employed to validate the effectiveness and specificity of IQUB antibody. Results pET-30a-IQUB recombinant plasmid was constructed, and protein expression of IQUB was induced successfully with IPTG. The titer of IQUB polyclonal antibody reached 1:1 000 000. The antibody specifically recognized the endogenous IQUB protein of testis in the wild-type adult mouse. IQUB was expressed in spermatogenic cells of different stages. It was localized in the acrosome and flagellum of mature sperms. Conclusion The highly specific rabbit anti-mouse IQUB polyclonal antibody is successfully prepared, which can be used for Western blot and immunofluorescence histochemistry.
Male
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Rabbits
;
Animals
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Mice
;
Ubiquitins
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Escherichia coli/genetics*
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Isopropyl Thiogalactoside
;
Antibodies
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Enzyme-Linked Immunosorbent Assay
2.Clinical Significance and Outcomes of Initial No Growth Peritonitis from Peritoneal Dialysis Patients: Role of Mycobacterial or Fungal Peritonitis.
Seong Woo LEE ; Jae yoon PARK ; Jong Cheol JEONG ; Shin young AHN ; Jin Joo PARK ; Won woo SEO ; Chi hoon KIM ; Sahmin LEE ; Jin ho HWANG ; Kwon Wook JOO ; Dong Ki KIM ; Curie AHN ; Kook Hwan OH
Korean Journal of Nephrology 2010;29(6):742-751
PURPOSE: Peritoneal dialysis associated peritonitis (PD peritonitis) is an important complication in maintaining. There have been only a few reports on the clinical outcome of initial no-growth peritonitis (INGP). METHODS: We reviewed 332 episodes of PD peritonitis between January 2002 and August 2009. INGP was defined as PD peritonitis with no growth of etiologic microorganism within 3 days of peritonitis. INGP was compared with initial positive growth peritonitis (IPGP) in view of clinical manifestations and outcomes. RESULTS: We divided PD peritonitis episodes into two groups: INGP (n=90) and IPGP (n=242). Peritonitis-related mortality was 5.6% in INGP, while 0.8% in IPGP (p=0.017). Further relapse was noted in INGP (10.0%) than in IPGP (vs. 4.1%; p=0.041). Salvage antibiotics were used more frequently in INGP (21.1%) than in IPGP (vs. 11.6%; p=0.027). Odds ratio of INGP to IPGP for peritonitis-related mortality was 7.14 (95% CI 1.36-37.51; p=0.017). Growth of mycobacteria or fungi increased the risk of peritonitis-related mortality with an odds ratio of 18.11 (95% CI 2.99-109.89; p=0.013). In multivariate analysis, growth of mycobacteria or fungi was the only independent risk factor for peritonitis-related mortality with an odds ratio of 10.63 (95% CI 1.27-88.75; p=0.029). CONCLUSION: INGP revealed poorer outcome than IPGP. Higher growth rate of mycobacteria or fungi in INGP than in IPGP accounted for the poor outcome. Thus one should make vigorous efforts to detect surreptitious organism when there is no growth by 3 days, especially for the possibility of either mycobacteria or fungi.
Anti-Bacterial Agents
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Fatal Outcome
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Fungi
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Isopropyl Thiogalactoside
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Multivariate Analysis
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Mycobacterium
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Odds Ratio
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Peritoneal Dialysis
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Peritonitis
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Recurrence
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Risk Factors
3.Site-Specific Mutagenesis in Human Cells by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives.
Cancer Research and Treatment 2004;36(2):151-156
PURPOSE: 7-Bromomethylbenz[a]anthracene is a well- known mutagen and carcinogen. The aim of this study is to determine the mutagenic potency of its two major DNA adducts [N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[a]a2G) and N6-(benz[a]anthracen-7- ylmethyl)-2'-deoxyadenosine (b[a]a6A)] and the simpler benzylated analogs [N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A)] in Ad293 human cells and to compare to their mutagenicity in human cells and E. coli. MATERIALS AND METHODS: The shuttle vector pGP50 is capable of replicating in E. coli and human cells. Modified nucleotides were positioned in the plasmid pGP50 in a manner similar to pGP10 as described (8). Adenovirus transformed human embryonic kidney cells (line 293) were transfected with a shuttle vector containing an adduct. Two days later, the plasmids were recovered and treated with DpnI to remove unreplicated DNA. DH10B E. coli were transformed with the plasmids. Bacteria were cultured with the media containing X-gal, IPTG and ampicillin. Bacteria transformed by the plasmid with the adduct-induced mutation in the initiation codon of lacZ' form white colonies whereas bacteria transformed by the plasmid without mutation form blue colonies. RESULTS: In the human cell site-specific mutagenesis system, bn2G exhibited weak mutagenicity and bn6A was not mutagenic, although b[a]a2G or b[a]a6A produced 8% and 7% mutant colonies, respectively. At the site of the adduct, b[a]a2G induced the G--> T transversion mutation while b[a]a6A produced the A--> G transition mutation. CONCLUSION: These data indicate that bulkier b[a]a2G and b[a]a6A exhibit significantly greater mutagenicity in human cells than in E. coli.
Adenine*
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Adenoviridae
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Ampicillin
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Bacteria
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Codon, Initiator
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DNA
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DNA Adducts
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Genetic Vectors
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Guanine*
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Humans*
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Isopropyl Thiogalactoside
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Kidney
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Mutagenesis
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Mutagenesis, Site-Directed*
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Nucleotides
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Plasmids
4.Cloning and expression of spinach glycolate oxidase in Escherichia coli.
Jian-Feng JIN ; Tian-Wei TAN ; Guo-Fu SU
Chinese Journal of Biotechnology 2002;18(2):212-215
The cDNA coding spinach glycolate oxidase (GO) was amplified by RT-PCR using the total RNA of spinach leaves as the template, and was cloned into cloning vector pMD18-T. After the DNA sequence was determined, the go gene was subcloned into E. coli expression vector pBV220, pET-22b(+), pTIG-Trx and pThioHisC. SDS-PAGE analysis revealed that the recombinant E. coli BL21 (DE3) (pTIG-Trx-GO) and E. coli BL21 (DE3) (pET-22b(+)-GO) expressed the predicted 38 kD glycolate oxidase, and the enzyme activity was also detected.
Alcohol Oxidoreductases
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genetics
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isolation & purification
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metabolism
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Cloning, Molecular
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Escherichia coli
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Gene Expression
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drug effects
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Isopropyl Thiogalactoside
;
pharmacology
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Spinacia oleracea
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enzymology
;
genetics
5.Cloning and expression of Buthus martensii Karsch scorpion toxin gene (BmK IT3) in Escherichia coli.
Ji-Bin YU ; Ping JI ; Xin-Min ZHA ; Wei-De SHEN ; Xiang-Fu WU
Chinese Journal of Biotechnology 2002;18(1):106-108
According to the reported sequence of Buthus martensii Karsch scorpion toxin gene (BmK IT3), we synthesized two primers, which were complementary in a region. By the means of PCR, we got the gene. The gene was fused in expression vector pET-28a, which gave rise to a recombinant plasmid pET(IT3R). Then it was transformed into E. coli BL21 (DE3). With IPTG induction, the gene was efficiently expressed. And the fusion product was soluble.
Animals
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Cloning, Molecular
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Escherichia coli
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genetics
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Gene Expression
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drug effects
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Isopropyl Thiogalactoside
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pharmacology
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Recombinant Proteins
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biosynthesis
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genetics
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Scorpion Venoms
;
biosynthesis
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chemistry
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genetics
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Scorpions
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chemistry
;
genetics
6.Preparation and identification of recombinant maxadilan.
Tianhong YI ; An HONG ; Shanshan XIE ; Ling ZHANG ; Qiuling XIE ; Yun DAI ; Rongjie YU
Chinese Journal of Biotechnology 2008;24(12):2049-2055
To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.
Animals
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Base Sequence
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Escherichia coli
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genetics
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metabolism
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Insect Proteins
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biosynthesis
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genetics
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Inteins
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genetics
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Isopropyl Thiogalactoside
;
pharmacology
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Molecular Sequence Data
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Recombinant Proteins
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analysis
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biosynthesis
;
genetics
7.Construction of fusion expression vector of human-derived neurotrophin-6 gene encoding mature peptide and purification of its expressed product.
Chengwu ZHANG ; Qingsong CAI ; Zicheng LI ; Chaoyang ZHAI ; Yu ZHENG
Journal of Biomedical Engineering 2005;22(6):1241-1244
To get the mature peptide of human-derived neurotrophin-6 (NT-6), NT-6 gene encoding mature peptide was amplified by PCR, using the NT-6 cDNA that had been cloned as templet. The gene encoding mature peptide of NT-6 gene was cloned into pGEX1-lambdaT plasmid to construct the fusion expression vector. Expression of fusion protein in Escherichia coli was defected after induction by isopropyl beta-D-thiogalactoside(IPTG). The mature peptide of NT-6 was collected with GST fusion protein purifying kit. It was shown that a fragment of 460bp was gained by PCR. With the techniques of double-cleave and electrophoresis, the recombinant vector was identified as pGEX1-NT-6. The recombinant vector pGEX1-NT-6 transformed Escherichia coli expressed fusion protein of 41KD after induction by IPTG. Cleaved by thrombin, the mature peptide of NT-6 was obtained; its molecular weight was about 15KD. The cloning and expression of human-derived NT-6 gene encoding mature protein has provided a basis for further studies on the function and clinical application of NT-6.
Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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genetics
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Humans
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Isopropyl Thiogalactoside
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pharmacology
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Nerve Growth Factors
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biosynthesis
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genetics
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Peptides
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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isolation & purification
8.The influences of lactose as an inducer on expression of plant des-pGlu1-brazzein in Escherichia coli.
Chun-Li LI ; Qi-Xin CHEN ; Guo-Qing HE
Chinese Journal of Biotechnology 2006;22(6):1021-1025
Expression strain of des-pGlu1-brazzein was constructed and the conditions using lactose as inducer was also optimized. The Influences of three factors which were lactose concentration, induction time and inducing temperature on the growth of strain and on the yield of des-pGlul-Brazzein was analyzed in detail. The result indicated that high lactose concentration inhibit the growth of strains (P < 0.01) but made no difference on expression of target protein between 0.5%-5% (P > 0.05), Biomass would be improved as time passed (P < 0.01), but the yield of target protein didn't increase obviously at 30 degrees C compared with at 37 degrees C. Further result showed that the greater expressed level of des-pGlul-Brazzein, as high as about 20% of total cell protein, could be achieved after the strain had been induced with 0.5% lactose under 28 degrees C - 30 degrees C for 4 h.
Dose-Response Relationship, Drug
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Escherichia coli
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drug effects
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genetics
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Isopropyl Thiogalactoside
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pharmacology
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Lactose
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pharmacology
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Plant Proteins
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genetics
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Plasmids
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genetics
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Temperature
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Time Factors
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Up-Regulation
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drug effects
9.Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein.
Ruodong YUAN ; Yangchao DONG ; Fuxing WU ; Tian DUAN ; Pan XUE ; Jian ZHANG ; Mingcheng YUAN ; Zhifeng XUE ; Haijun ZHANG ; Qianqian ZHANG ; Xiaopeng GAO ; Yingfeng LEI
Chinese Journal of Cellular and Molecular Immunology 2023;39(6):544-551
Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.
Animals
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Mice
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Humans
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Adenoviruses, Human/genetics*
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Escherichia coli/genetics*
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HEK293 Cells
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Isopropyl Thiogalactoside
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Blotting, Western
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Immunoglobulin G
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Antibodies, Monoclonal
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Antibody Specificity
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Mice, Inbred BALB C
10.Production of IL-15 and lts Functional Study in Mouse Splenocyte Activation.
Korean Journal of Immunology 1999;21(4):297-302
After the synthesis of IL-15 cDNA from the total RNA of mouse spleen, it was inserted into the prokaryotic expression vector, pRseta, and eukaryotic expression vector, pcDNA3.0, respectively. Subsequently, the insertion of gene and open reading frame were confirmed by sequencing of each plasmid, respectively. Using pRseta- IL-15 plasmid, the recombinant IL-15 protein was induced by IPTG under BL21 (DE 3) host cells and recombinant IL-15 was expressed at 14.5 KDa with time. Then, IL- 15 was separated by His-tag affinity chromatography and analyzed by SDS-PAGE to yield soluble IL-15 at 14.5 KDa as monomer and 29.0 KDa as dimer. In order to inspect the function and contribution of IL-15, the in vitro experiment was established using mononuclear cells separated from the mouse spleen. After 48h exposure of PHA to mouse splenocyte and 24h treatment with recombinant IL-15, the effects of cytokine inductions inspected against IL-2, IL-6, IL-10, IL-12, IFN-r, and GM-CSF. The results showed that comparing with the control, IL-6 increased, IL-2, IL-12 and IFN-r increased and similar, and GM-CSF decreased. In addition, the direct injection of pcDNA3.0-IL-15 plasmid into mice gave the similar results to in vitro studies. Namely, IL-6 and IL-12 increased, and IL-2, IFN-r and GM-CSF were similar or decreased. IL-10 was not induced in in vitro and in vivo experiments. These results suggested that the IL-15 induce the splenocyte activation and can be an important factor in proliferation and fuction recovery of weakened T-cell.
Animals
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Chromatography, Affinity
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DNA, Complementary
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Electrophoresis, Polyacrylamide Gel
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Granulocyte-Macrophage Colony-Stimulating Factor
;
Interleukin-10
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Interleukin-12
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Interleukin-15*
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Interleukin-2
;
Interleukin-6
;
Isopropyl Thiogalactoside
;
Mice*
;
Open Reading Frames
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Plasmids
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RNA
;
Spleen
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T-Lymphocytes