Objective: To study the function of the first extracellular membrane loop of the NH2-terminal of chemokine receptor 5(CCRS), and produce its specific antibody F(ab’ )2. Methods: Some amino acid sequences of β chemok...Objective: To study the function of the first extracellular membrane loop of the NH2-terminal of chemokine receptor 5(CCRS), and produce its specific antibody F(ab’ )2. Methods: Some amino acid sequences of β chemokine superfamily receptors were aligned by computer and the least homologous domain of the extracellular loops was located. In this paper, the first extracellular domain (114 nucleotides) was defined and amplified by PCR, and a expression vector of the recombinant GST fusion protein,pGEXIN/NR5, was constructed. After the inserted sequence was confirmed correct, the transformation and expression of fusion protein was performed in E. coli and the fusion protein was purified. Then 2 Newzealand rabbits were immunized. The anti-CCR5 NH2-terminal antibody F(ab’ )2 was made by protein A affinity chromatography, pepsin digestion and sepharose-12 column chromatography. Results: Reduced and unreduced SDS-PAGE and FAX analysis demonstrated that this F(ab’ )2 had a high specificity to combine with CCR5. Conclusion: A simple and quick method to prepare specific antibody F(ab’ )2 of certain functional domain is showed in this paper, by which we can get an improtant experiment material for studying gene expression, and it will provide a good idea and a technique to study other high similar superfamily members.展开更多
目的研究125I标记的抗炎抗凝双效融合蛋白TAP-SSL5单次静脉注射后在健康日本大耳兔体内的药代动力学过程。方法采用固相氧化法(Iodogen法)将Na125I直接标记于TAP-SSL5,由耳缘静脉给每只大耳兔注射18.5×103kBq的125I-TAP-SSL5,分别...目的研究125I标记的抗炎抗凝双效融合蛋白TAP-SSL5单次静脉注射后在健康日本大耳兔体内的药代动力学过程。方法采用固相氧化法(Iodogen法)将Na125I直接标记于TAP-SSL5,由耳缘静脉给每只大耳兔注射18.5×103kBq的125I-TAP-SSL5,分别于注射后1.5、3、5、10、30、60、120、240、480 min采血,称量并测定放射性计数(Counts per minute,cpm),换算为血液放射性浓度,经DAS软件分析得出最佳房室模型及相关药代动力学参数。结果纸层析法测得125I-TAP-SSL5的标记率为(67.32±9.91)%,放射化学纯度为(91.62±3.22)%,比活度为(30.2±4.4)TBq/μmol;TAP-SSL5在大耳兔体内的药代动力学过程符合权重为1的二室模型,分布相半衰期(t1/2α)及消除相半衰期(t1/2β)分别为(0.08±0.04)h和(4.97±0.75)h,清除率(Clearance,CL)为(0.015±0.011)ml/h,一室向二室转运常数(K12)为(6.651±3.642)/h,二室向一室转运常数(K21)为(4.072±1.737)/h。结论125I-TAP-SSL5在健康大耳兔体内的药代动力学过程符合权重系数为1的二室模型,自体清除率缓慢,可保证与组织有更多的结合几率。展开更多
OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were car...OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P展开更多
文摘Objective: To study the function of the first extracellular membrane loop of the NH2-terminal of chemokine receptor 5(CCRS), and produce its specific antibody F(ab’ )2. Methods: Some amino acid sequences of β chemokine superfamily receptors were aligned by computer and the least homologous domain of the extracellular loops was located. In this paper, the first extracellular domain (114 nucleotides) was defined and amplified by PCR, and a expression vector of the recombinant GST fusion protein,pGEXIN/NR5, was constructed. After the inserted sequence was confirmed correct, the transformation and expression of fusion protein was performed in E. coli and the fusion protein was purified. Then 2 Newzealand rabbits were immunized. The anti-CCR5 NH2-terminal antibody F(ab’ )2 was made by protein A affinity chromatography, pepsin digestion and sepharose-12 column chromatography. Results: Reduced and unreduced SDS-PAGE and FAX analysis demonstrated that this F(ab’ )2 had a high specificity to combine with CCR5. Conclusion: A simple and quick method to prepare specific antibody F(ab’ )2 of certain functional domain is showed in this paper, by which we can get an improtant experiment material for studying gene expression, and it will provide a good idea and a technique to study other high similar superfamily members.
文摘目的研究125I标记的抗炎抗凝双效融合蛋白TAP-SSL5单次静脉注射后在健康日本大耳兔体内的药代动力学过程。方法采用固相氧化法(Iodogen法)将Na125I直接标记于TAP-SSL5,由耳缘静脉给每只大耳兔注射18.5×103kBq的125I-TAP-SSL5,分别于注射后1.5、3、5、10、30、60、120、240、480 min采血,称量并测定放射性计数(Counts per minute,cpm),换算为血液放射性浓度,经DAS软件分析得出最佳房室模型及相关药代动力学参数。结果纸层析法测得125I-TAP-SSL5的标记率为(67.32±9.91)%,放射化学纯度为(91.62±3.22)%,比活度为(30.2±4.4)TBq/μmol;TAP-SSL5在大耳兔体内的药代动力学过程符合权重为1的二室模型,分布相半衰期(t1/2α)及消除相半衰期(t1/2β)分别为(0.08±0.04)h和(4.97±0.75)h,清除率(Clearance,CL)为(0.015±0.011)ml/h,一室向二室转运常数(K12)为(6.651±3.642)/h,二室向一室转运常数(K21)为(4.072±1.737)/h。结论125I-TAP-SSL5在健康大耳兔体内的药代动力学过程符合权重系数为1的二室模型,自体清除率缓慢,可保证与组织有更多的结合几率。
文摘OBJECTIVE: To screen the 5' regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. METHODS: The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells, and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. RESULTS: Two polymorphisms, C (-106) T and C (-12) G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C (-12) G and WT/C (-106) T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C (-106) T were 31.5% and 17.5% (P 0.05) respectively. The total frequency of WT/C (-12) G and WT/C (-106) T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P
