ABSTRACT
Objective@#To investigate the hepatitis C prevention and control knowledge among clinicians in Jiaxing City, Zhejiang Province, so as to provide the evidence for intensified training and improved diagnosis and treatment of hepatitis C among clinicians.@*Methods@#In November, 2021, clinicians were sampled using a stratified random sampling method from a city-level and a county (district)-level hepatitis C designated hospital in Jiaxing City. A questionnaire survey was performed using the Questionnaire for Hepatitis C Prevention and Control Knowledge among Clinicians, and the awareness of basic knowledge, professional knowledge and related knowledge about hepatitis C prevention and control among clinicians were descriptively analyzed. @*Results@# A total of 186 questionnaires were allocated and 179 valid questionnaires were recovered, with an effective recovery rate was 96.24%. The respondents included 107 men (59.78%) and 72 women (40.22%) and had a mean age of (37.06±9.46) years. There were 107 respondents with a bachelor degree (59.78%), 56 with junior professional titles (31.28%), and 170 from non-infectious disease departments (94.97%). The awareness of basic hepatitis C prevention and control knowledge was 96.09%, and the awareness of “Transfusion of blood containing hepatitis C virus may acquire hepatitis C” was high (98.88%), and the awareness of “Hepatitis C can be cured” was low (77.09%). The awareness of professional hepatitis C prevention and control knowledge was 3.91% to 100.00%, and the awareness of “Pathogens of hepatitis C” (100.00%) and “Recommended screening populations for hepatitis C” (86.59%) was high, while the awareness of “There are two categories of hepatitis C cases: clinically diagnosed cases and confirmed cases” (3.91%) and “Clinical diagnosis of hepatitis C: positive anti-HCV antibody + any one of abnormal liver function or epidemiological history or clinical symptoms” (3.91%) was low. The awareness rates of “The state has included antiviral agents against hepatitis C into medical insurance” was and “Antiviral agents against hepatitis C are reimbursed in outpatient and inpatient departments of our hospital” were 81.56% and 59.78%, respectively. There were 69 clinicians participating hepatitis C-related training within one year (38.55%), and the awareness of clinicians that had participated in hepatitis C-related training had a higher awareness rate of basic hepatitis C prevention and control knowledge than those without participation (100.00% vs. 93.64%, P<0.05).@*Conclusion@#The awareness of basic hepatitis C prevention and control knowledge is high among clinicians in Jiaxing City; however, the training on diagnosis and classification criteria of hepatitis C and related medical insurance policy require to be improved.
ABSTRACT
@#Abstract: Objective ， To explore the effects of lead exposure on copper level copper transporter protein expression and Methods oxidative stress in mouse cerebral cortex. The specific pathogen free adult male C57BL/6 mice were randomly ， - - - divided into control group low lead exposure group and high lead exposure group with 10 mice in each group. The mice in low - ， and high lead exposure groups were respectively given 250 and 500 mg/L lead acetate in drinking water every day and the mice - ， in the control group were given double distilled water for 12 weeks. Twenty four hours after exposure Morris water maze and ， elevated cross maze were used to test the neurobehavioral function of mice. The cerebral cortex of mice was isolated and the levels of lead and copper were detected by inductively coupled plasma mass spectrometry. The activities of glutathione （ - ）， （ ） （ ） peroxidase GSH Px catalase CAT and malondialdehyde MDA were detected by histochemical method. The relative （ ） ， ， expression levels of copper transporter such as synthesis of cytochrome C oxidase SCO 1 SCO 2 and cytochrome C oxidase （ ） Results - - assembly protein 11 COX11 were detected by western blot. The escape latencies of mice in the low and high lead （ P ）， ， - exposure groups were prolonged all <0.05 while the number of crossing the platform the percentage of open arm entry - （ P ） times and the percentage of open arm retention time decreased all <0.05 compared with the control group. Mice in both the - - （ P ）， - low and high lead exposure groups increased levels of lead and copper in the cerebral cortex all <0.05 decreased GSH Px （ P ）， （ P ） and CAT activity all <0.05 and increased SCO1 relative expression all <0.05 compared with the control group. Mice in - （P ）， - the high lead exposure group showed prolonged escape latency <0.05 reduced GSH Px and CAT activities in the cerebral （ P ）， （ P ） - cortex all <0.05 increased MDA level and relative expression of SCO1 and SCO2 all <0.05 compared to mice in the low Conclusion - lead exposure group. Lead exposure increased the expression of copper and copper transport related proteins in mouse cerebral cortex and induced oxidative stress leading to central nervous system damage resulting in neurobehavioral abnormalities in mice.
ABSTRACT
Objective:To explore the correlation and mechanism between thalamic network abnormality and cognitive decline in patients with temporal lobe epilepsy (TLE).Methods:A total of 53 patients with unilateral TLE were consecutively enrolled through the epilepsy clinic of the First Affiliated Hospital of Guangxi Medical University from December 2018 to February 2020. During the same recruitment interval, 37 health controls(HC) with matching demographic characteristic were recruited. All subjects were received the Montreal cognitive assessment(MoCA) test and multimodal MRI scanning. Voxel-based morphometry method was used to study the changes of thalamic gray matter volume in patients with unilateral TLE. The structural covariance network and functional connectivity network based on seed points were used to analyze the changes of thalamic network in TLE patients. In addition, the correlations among abnormal thalamic structure, thalamic network and cognitive function score were analyzed. SPSS 22.0 software was used for statistical analysis. Independent sample t-test and Mann Whitney U test were used for inter group comparison. In order to explore the relationship between thalamus and thalamic network and cognitive performance in TLE patients, thalamic volume and gray matter volume and functional connection value of brain areas with abnormal synergistic changes were extracted and correlated with MoCA score. Results:The total score of MoCA in TLE patients (27.0(25.0, 29.0)) was significantly decreased compared with HC (29.0(28.0, 30.0))( Z=-4.601, P<0.001). Whole brain gray matter volume analysis showed that compared with HCTLE patients showed significant volume reduction in left cerebellum, right temporal pole, right fusiform gyrus, straight gyrus, bilateral middle temporal gyrus, thalamus, medial and paracingulate gyrus (GRF adjusted, voxel-level P<0.001 and cluster-level P<0.05). The thalamus-associated structural covariance network analysis revealed that compared with healthy controls, TLE patients exhibited decreased connectivity in right fusiform gyrus (MNI: x=28.5, y=-15.0, z=-34.5), left insula (MNI: x=-33.0, y=-18.0, z=-1.5), right middle temporal gyrus (MNI: x=55.5, y=-51.0, z=9.0), left complementary motor area (MNI: x=-10.5, y=1.5, z=57.0) and right posterior central gyrus (MNI: x=31.5, y=-33.0, z=51.0) ( P<0.001, cluster > 100). The thalamus-associated functional connectivity network analysis revealed that TLE patients exhibited decreased connectivity in left insula (MNI: x=-38, y=-7, z=-7), left lingual gyrus (MNI: x=-6, y=-81, z=-12), right lingual gyrus (MNI: x=15, y=-105, z=0) and left triangular inferior frontal gyrus (MNI: x=-39, y=36, z=-6) (GRF correction, voxel-level P<0.001 and cluster-level P<0.05). Volume of left insula which had decreased structural connectivity with thalamus were positively correlated with the MoCA score in TLE patients( r=0.279, P=0.043). Volume of left complementary motor area which had decreased structural connectivity with thalamus was positively correalated with the MoCA score and language score in TLE patients( r=0.323, P=0.018; r=0.334, P=0.015). Volume of left lingual gyrus which had decreased functional connectivity with thalamus was negatively correalated with the memory score in TLE patients ( r=-0.331, P=0.016). Conclusion:Thalamic volume, thalamic structural covariant network and functional connection network are changed in TLE patients. The abnormality of thalamic network is associated with cognitive performance in TLE patients, which may be the neural mechanism of thalamus participating in the cognitive impairment of TLE patients.
ABSTRACT
Objective@#To understand the epidemiological characteristics and differences of HIV-positive cases among 15-24 years old in Jiaxing city and provide evidence for the development of targeted prevention and control measures.@*Methods@#A descriptive epidemiological method was used to analyze the data of HIV cases aged 15-24 reported in Jiaxing from 1999 to 2018.@*Results@#A total of 375 cases of young HIV were reported in 1999-2018, with an average age of 21.29±1.90 years, of which 42 were students. The ratio of male to female was 2.47∶1. The proportion of foreign household registration was higher (76%, 285 cases). The proportion of off-campus youth cases in total cases showed a downward trend(χ2=8.26, P=0.00), but the proportion of student cases showed an upward trend(χ2=15.73, P<0.01). Off-campus youth cases were mainly heterosexual transmission(59.16%, 197 cases), and the students’ cases were mainly homosexual transmission(88.10%, 37 cases). There were significant differences in gender, age, household registration, education level, route of transmission, late detection, CD4 level and source of detection among students and off-campus adolescents(P<0.05).@*Conclusion@#The prevalence of AIDS in adolescents and students is worthy of attention. The characteristics of adolescents inside and outside the school are different. Targeted prevention measures should be taken to reduce the harm of AIDS to young people.
ABSTRACT
@#A biomarker is defined as a biological molecule found in the blood, other body fluids, or tissues that is a sign of normal or abnormal processes or a condition or disease. In cancer research, biomarkers are classified as diagnostic, prognostic, or predictive. The identification and application of biomarkers in clinical practice are important for evaluating their usefulness for clinical diagnosis, treatment and prognostic warning and for determining the biological effects of anti-cancer drugs, and they are currently one of the hottest topics in oncological translational research. Currently, translational research on biomarkers mostly focus on oncological diagnosis and molecular typing, targeted therapy, treatment protocol selection and optimization, prognostic prediction, etc. Here, we review the progress of translational research on treatments based on biomarkers in oral squamous cell carcinoma as well as the clinical application of inhibitors targeting EGFR, PD1, PI3K, WEE1, the Wnt/β-catenin pathway, the SHH pathway, and the ERK pathway. The prospect of research strategies for personalized treatments based on biomarkers in oral squamous cell carcinoma is also discussed.
ABSTRACT
Objective To investigate the status of syphilis and HCV infection and the influencing factors among community drug addicts in Jiaxing City.Methods HCV and syphilis were detected among community drug addicts in Jiaxing City from 2014-2015 and a questionnaire survey were conducted.Influencing factors of syphilis and HCV was analyzed in this study.Results A total of 449 drug users including 356 males (79.29％) and 93 females (20.71％) were investigated,with the age ranged from 16 to 47 and averaged 27.50 ± 12.28.A total of 370 of them take addictive drugs (82.41％),including 42 of them take more than 2 kinds of drugs.15 cases of Syphihs (double positive) were found,and the positive rate was 3.34％.There were significant difference among community drug addicts with different education level (P ＜0.05).The positive rate of HCV among males were lower than that of females (P ＜ 0.05),and the positive rate of HCV among heroin addicts were higher than other drug addicts (P ＜ 0.05).The positive rate of HCV of injection drug users were higher than oral drug addicts(OR =17.341,95％ CI:8.387-35.857,P ＜ 0.01).There was associations among condom use with double positive syphilis and HCV (OR =0.210,95％ CI:0.064-0.689,P ＜0.01;OR =0.131,95％ CI:0.063 -0.273,P ＜ 0.01),respectively.Conclusion Community drug addics are mainly young adults,drug injection and not using condom may increase the risks of TP and HCV infection.
ABSTRACT
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
Subject(s)
Humans , Antineoplastic Agents , Chemistry , Pharmacology , Carbolines , Chemistry , Pharmacology , Cell Line, Tumor , Cell Proliferation , Inhibitory Concentration 50 , Kinesins , Pharmacology , Molecular StructureABSTRACT
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
ABSTRACT
Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
ABSTRACT
Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.
ABSTRACT
The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.
Subject(s)
Animals , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Fermentation , Liver , Chemistry , Peptides , Genetics , Metabolism , Recombinant Proteins , Genetics , Sharks , MetabolismABSTRACT
With the development of the research on biotechnology and modern pharmacy, the application of enzyme drugs have grown rapidly and enzyme drugs have become an important branch of biopharmaceutics. In this article, some new varieties of therapeutic enzymes, enzyme targets, mechanisms and new technologies of application in therapeutic enzymes were reviewed, and the direction of development of therapeutic enzymes were discussed.
Subject(s)
Adenosine Deaminase , Genetics , Therapeutic Uses , Antineoplastic Agents , Therapeutic Uses , Enzyme Replacement Therapy , Methods , Fibrinolytic Agents , Therapeutic Uses , Protein C , Genetics , Therapeutic Uses , RNA, Catalytic , Genetics , Therapeutic Uses , Streptokinase , Genetics , Therapeutic Uses , Urokinase-Type Plasminogen Activator , Genetics , Therapeutic UsesABSTRACT
The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.
Subject(s)
Amino Acid Sequence , Chromatography, Liquid , Methods , Chymotrypsin , Chemistry , Fibrinolytic Agents , Chemistry , Hirudins , Chemistry , Molecular Sequence Data , Molecular Weight , Peptide Fragments , Recombinant Fusion Proteins , Chemistry , Recombinant Proteins , Chemistry , Spectrometry, Mass, Electrospray Ionization , Methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Methods , Tandem Mass Spectrometry , Methods , Tissue Plasminogen Activator , Chemistry , Trypsin , ChemistryABSTRACT
Kinesin spindle protein (KSP/Eg5) is essential for the formation and maintenance of bipolar spindles during mitosis. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, a series of tetrahydro-beta-carboline derivatives 5a - k were synthesized as kinesin spindle protein inhibitor. Their structures were confirmed with 1H NMR, ESI-MS and elemental analysis. The synthesized compounds were evaluated for their inhibition of KSP.
Subject(s)
Antineoplastic Agents , Chemistry , Pharmacology , Carbolines , Chemistry , Pharmacology , Kinesins , Metabolism , Molecular StructureABSTRACT
Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for insulin and insulin chain B was studied. The solution of intact insulin or insulin chain B was added to the solution of carboxypeptidase P (CPP) and carboxypeptidase Y (CPY). Fractions of appropriate volume were removed at some appointed time points, acidified with the same amount of 1% formic acid to stop the digestion, and then briefly vortexed for HPLC-ESI-ITMS analysis. Mobile phase A consisted of 0.02% TFA in 98% ultra-pure water and 2% acetonitrile. Mobile phase B consisted of 0.02% TFA in 98% acetonitrile and 2% ultra-pure water. The solution used for post-column fix consisted of propionic acid and isopropyl alcohol (20 : 80, v/v). Chromatographic separation was carried out on a reversed-phase column (Zorbax Prosphere C18, 300A, 5 microm, 2.1 mm ID x 150 mm length). The molecular weights of the multiply charged ions representing consecutive truncated losses of carboxyterminal amino acids were determined by the use of HPLC-ESI-ITMS. The differences between the consecutive truncated peptides are the experimental weights of the carboxyterminal amino acid residues. The carboxyterminal amino acid residue Ala, which released from chain B of intact insulin, was confirmed in the nanomolar concentration range by analyzing the molecular weight of the truncated peptides. Another one carboxyterminal amino acid Ala was confirmed in the nanomolar concentration range of insulin chain B. In the quality control for recombinant DNA product or natural protein, the confirmation of 1 - 3 carboxyterminal amino acid residues is regarded to be up to standard. One amino acid residue of insulin or insulin chain B could be confirmed accurately in the nanomolar concentration range. The results showed that intact insulin could be directly sequenced in the quality control without separating chain B from chain A. There would be no need to separate chain A from chain B to identify carboxyterminal of intact insulin. Furthermore, the method saved us a lot of trouble from the preparation and purification of insulin chain A and chain B.
Subject(s)
Amino Acid Sequence , Carboxypeptidases , Chemistry , Cathepsin A , Chemistry , Chromatography, High Pressure Liquid , Methods , Insulin , Chemistry , Reference Standards , Molecular Sequence Data , Molecular Weight , Peptide Fragments , Chemistry , Quality Control , Spectrometry, Mass, Electrospray Ionization , MethodsABSTRACT
AIM:To construct nine novel L-asparaginase mutants and study their enzyme-activity.METHODS:The mutants were constructed using overlap extension PCR according to the principle of alanine-scanning mutagenesis. The enzyme-activity was detected by Nessler's method. RESULTS:The DNA sequencing showed that the mutagenesis was consistent with the theoretical prediction. The enzyme-activity assay demonstrated that each mutant possessed enzyme activity equal to the original enzyme. CONCLUSION:Through gene modification,epitop of L-asparaginase was changed without activity loss.These results provide foundation for further study of the structure-function relationship of L-asparaginase.
ABSTRACT
<p><b>AIM</b>To investigate the protective effects of shark hepatic stimulator substance (sHSS) against acute hepatic injury induced by acetaminophen (AAP) in mice.</p><p><b>METHODS</b>Acute hepatic injury model of Balb/c mice was induced by a single intraperitoneal injection of AAP (200 mg.kg-1, i.p.). Serum ALT and AST activities were analyzed. The changes of microstructure and ultrastructure of hepatocyte were observed under optical and electronic microscope. The hepatocyte apoptosis was analyzed by flow cytometer and the expression level of Fas mRNA was determined by RT-PCR.</p><p><b>RESULTS</b>The activities of serum ALT and AST were significantly decreased and both necrosis and inflammatory infiltration were improved in the mice treated with sHSS 3.0 and 1.5 mg.kg-1. sHSS (3.0 mg.kg-1) prevented the ultrastructural changes of hepatocytes caused by AAP, decreased the percentage of apoptotic cells, and downregulated the expression level of Fas mRNA.</p><p><b>CONCLUSION</b>sHSS protected hepatocytes from AAP-induced injury, which might be associated with its protection of the mitochondria and inhibition of apoptosis and expression of Fas mRNA in hepatocytes.</p>
Subject(s)
Animals , Female , Mice , Acetaminophen , Apoptosis , Chemical and Drug Induced Liver Injury , Pathology , Growth Substances , Pharmacology , Mice, Inbred BALB C , Peptides , Pharmacology , Protective Agents , Pharmacology , RNA, Messenger , Genetics , Random Allocation , Sharks , fas Receptor , GeneticsABSTRACT
This study was intended to establish a method of preparation of recombinant human insulin, with (His)6-Arg-Arg-human proinsulin (RRhPI) expressed by Escherichia coli. After DEAE-Sepharose Fast Flow ion-exchange chromatography, Sephadex G-25 chromatography and refolding, enzyme cleavage and Superdex 75 size exclusion chromatography,the RRhPI expressed by Escherichia coli in inclusion body form was converted to human insulin. The obtained recombinant human insulin was analyzed by SDS-PAGE, HPLC, amino acid composition analysis and bioidentity test (mouse convulsion test). The results indicate that our obtained preparation is highly purified, active recombinant human insulin.
Subject(s)
Humans , Chromatography, High Pressure Liquid , Escherichia coli , Genetics , Metabolism , Insulin , Genetics , Proinsulin , Genetics , Recombinant Proteins , GeneticsABSTRACT
<p><b>AIM</b>To establish antibody sandwich enzyme-linked immunoadsorbent assay for determination of recombinant E. coli L-asparaginase in rat plasma and study its pharmacokinetics.</p><p><b>METHODS</b>A Japanese white rabbit was immunized with recombinant E. coli L-asparaginase. Immunoglobulin G was separated and purified by using DEAE-cellulose chromatography. Conjugation of horseradish peroxidase to immunoglobulin G was obtained using the two-step glutaraldehyde method. Recombinant E. coli L-asparaginase protein in plasma was measured by antibody sandwich enzyme-linked immunoadsorbent assay. Pharmacokinetic parameters were assessed with model-dependent method.</p><p><b>RESULTS</b>The linearities was 1-64 U.L-1. Concentration-time profile after i.v. of 1.25, 2.50, 5.00 kU.kg-1 of recombinant E. coli L-asparaginase fitted with a two-compartment model. The first and terminal elimination T1/2 were 0.50-0.57 h and 2.45-3.02 h, respectively. The AUC was linearly related to the doses.</p><p><b>CONCLUSION</b>Antibody sandwich enzyme-linked immunoadsorbent assay was constant, reliable, sensitive, and suitable for the determination of recombinant L-asparaginase. Pharmacokinetics of recombinant E. coli L-asparaginase in rats is warranted for the design of future clinical trails.</p>
Subject(s)
Animals , Rabbits , Rats , Area Under Curve , Asparaginase , Blood , Pharmacokinetics , Enzyme-Linked Immunosorbent Assay , Methods , Escherichia coli , Metabolic Clearance Rate , Random Allocation , Rats, Wistar , Recombinant Proteins , Blood , PharmacokineticsABSTRACT
The ocean is the treasure house by which human being live. The variety and particularity of marine creature provide people many physiological active substances, which are novel and unique in structure and effect. The recent research of marine active antitumor proteins and peptides was summarized in this article.