- SHI Yan-chun,LIANG Hao,BOU Shorgan (The Key Laboratory of Ministry of Education of China for Mammal Reproduction Biology and Biotechnology,The Biological Postdoctoral Programme of Inner Mongolia University,Huhhot 010021,China)
Objective To construct a retrovirus vector for the fusion expression of IL-2 and EGFP.Methods Design the adaptor of plasmids pRevTet-On and pEGFP-C1,digest with restriction endonuclease and ligate to recombine a transition retrovirus vector pRevEGFP-C1.Meanwhile,digest plasmids pBV220-IL-2 and pEGFP-C1 with restriction endonuclease and ligate to recombine another transition retrovirus vector pEGFP-C1-IL-2.Digest plasmids pRevEGFP-C1 and pEGFP-C1-IL-2 with restriction endonuclease respectively.Recover target gene fragment IL-2 by agarose gel electrophoresis,insert into vector pRevEGFP-C1 and transform to E.coli DH5α for proliferation.Extract plasmid DNA to obtain a recombinant pRevEGFP-C1-IL-2.Results Analaysis proved that vector pRevEGFP-C1-IL-2 was correctly constructed.Conclusion A retrovirus vector pRevEGFP-C1-IL-2 for the fusion expression of IL-2 and EGFP was successfully constructed.
2006 01 [Abstract][OnlineView][Download 517K] - WANG Xin-ying~△,FAN Hong-xue,YAN Jie ( ~△School of Public Health,Jilin University,Changchun 130021,China)
Objective To clone the flagellar biosynthesis genes flhA,flhB_2 and fliR of Leptospira interrogans and construct their prokaryotic expression system.Methods Extract genomic DNA from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain 56601 by phenol-chloroform method and amplify flhA,flhB_2 and fliR gene fragments by high fidelity PCR.After T-A cloning,the amplified genes were identified by sequencing and used for the construction of prokaryotic expression vector.Identify the expressed fusion protein by SDS-PAGE and Western blot.Results The homologies of nucleotide sequences of amplified flhA,flhB_2 and fliR genes to those reported were 100%,99.9% and 99.9%,and those of the deduced amino acid sequences were 100%,100% and 99.8%,respectively.Fusion proteins Trx-FlhA,Trx-FlhB_2 and Trx-FliR were effectively expressed in the constructed prokaryotic expression system under induction of IPTG.The expressed product contained about 10% of total somatic protein.Conclusion The prokaryotic expression system of flagellar biosynthesis genes flhA,flhB_2 and fliR of Leptospira interrogans was successfully constructed.
2006 01 [Abstract][OnlineView][Download 380K] - LI Hao, YU Xiang-hui,JIANG Chun-lai,et al (College of Life Science,Jilin University, Changchun 130012,China)
Objective To explore the difference between the expressions of potassium ion channels in the myocardial tissues of healthy dog and the dog with heart failure,and lay a foundation of further study on the pathogenic mechanism of heart failure.Methods Establish the dog model of heart failure,and analyze the expressions of potassium ion channels Kv4.3 and Kv1.4 as well as accessory subunit Mink in the myocardial tissues of healthy dog and the dog with heart failure by Western blot.Results The expression level of Kv4.3 in myocardial tissue of dog with heart failure decreased significantly,but that of Kv1.4 increased at a certain extent.The expression levels of Kv1.4 in the external and internal layers of myocardial tissue were different significantly,and that of Mink in the external layer decreased significantly.Conclusion There might be relationship between the alterations of expressions of I_(to)-related potassium ion channels Kv4.3,Kv1.4 and accessory subunit Mink in the myocardial tissues of patients with heart failure.
2006 01 [Abstract][OnlineView][Download 463K] - LV Feng,HU Ning-zhu,SHI Hai-jing,et al (Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming 650118,China)
Objective To study the inhibitory effect of DNAzyme on expression of H-ras gene.Methods Seven DNAzyme gene fragments containing motif 10-23(10-23 DNAzyme,No.1-7),specific to H-ras mRNA sequence,were designed and synthesized,then used for extracellular cleavage test separately.The transfection efficiency of DNAzyme in the mediation of lipofectamine~ TM was observed by fluorescent microscopy.The inhibitory effect of DNAzyme on expression of H-ras gene was determined by real-time quantitative PCR and MTT methods at cellular level.Results All the seven 10-23 DNAzyme fragments effectively cleaved H-ras mRNA. Hep-2 cells were transfected with fluorescently-labeled DNAzyme No.1 in the mediation of lipofectamine~ TM .After transfection,DNAzyme was observed in both nucleus and cytoplasm.However,the DNAzyme content in perinuclear space was relatively low.Real-time quantitative PCR proved that the H-ras RNA content in Hep-2 cells 24 and 48 hours after transfection with DNAzyme No.1 decreased significantly.The determination result by MTT method showed that the proliferation and differentiation of Hep-2 cells 24 and 48 hours after transfection with DNAzyme No.1 was inhibited significantly.Conclusion DNAzyme showed both intracellular and extracellular inhibitory effects on the expression of H-ras gene.
2006 01 [Abstract][OnlineView][Download 362K] - YANG Ze-hua,XIE Jun,YANG Qi,et al (Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)
Objective To construct a high prokaryotic expression system of globular domain of human adiponection (gAd) and purify the expressed product.Methods Extract human genomic DNA from lymphocytes and amplify the gene fragment containing adiponectin-encoding sequence.Clone the amplified gene fragment into pMD18-T vector by T-A cloning,then introduce start codon,His 6-encoding sequence at C-terminus,end codon and the corresponding restriction site by designing appropriate primers,and subclone gAd gene into expression vector pBV220.Identify the recombinant by gene sequencing and transform to E.coli DH5α for expression of fusion protein at 42℃.Results The gAd fusion protein containing about 19% of total somatic protein,in form of inclusion body,was successfully and stably expressed in E.coli.After ultrasonication and purification by immobilized metal chelation affinity chromatography,homogenous protein was obtained and reached a high purity as proved by SDS-PAGE.Conclusion The globular domain of human adiponectin was successfully expressed.It laid a foundation of further study on its biological function and action mechanism.
2006 01 [Abstract][OnlineView][Download 402K] - SUN Ya-li, LIU You-sheng, WANG Chang-song(Institute of Pathology Research,Southwest Hospital,Third Military Medical University,Chongqing 400038,China)
Objective To analyze the primary structure of parental human endotoxin binding peptide(EBP),induce mutation at base sites corresponding to the amino acids which might influence the biological activity of EBP,clone the mutant of EBP(mEBP) and study its expression.Methods mEBP gene was predicatively analyzed by DNASIS software,and the mutations(Gln→Lys) at sites 5 and 18 were induced by PCR site-directed mutagenesis.The mEBP gene was cloned into prokaryotic vector pinpoint Xa-3 and transformed to E.coli BL21(DE3) pLysS for fusion expression.The expressed product was identified by Western blot.Results The nucleotides at sites 13 and 52 of mEBP gene were mutated from C to A.The constructed recombinant showed identical sequence to that designed,as proved by digestion with restriction endonuclease and sequencing.The biotinylated fusion protein was expressed in E.coli BL21(DE3) pLysS under induction of IPTG.Western blot showed the binding capacity of expressed product to McAb against biotin.Conclusion The mutant of EBP gene was obtained and successfully expressed in E.coli in a form of fusion protein.
2006 01 [Abstract][OnlineView][Download 315K] - WEI San-hua,YIN Wen,LEI Ying-feng,et al (Department of Microbiology,Fourth Military Medical University,Xi'an 710032,China)
Objective To construct an eukaryotic expression vector of compound multi-epitope gene of HCV and express the gene in COS7 cells.Methods Amplify the cDNA sequence encoding truncated HCV gene,with a part of carboxyl-terminus deleted,by PCR.Synthesize the mimic epitope at E2 region of HCV and seven T or Th cell epitope genes of NS3-NS5 respectively.Bind HCV core gene with a part of carboxyl-terminus deleted to the synthetic epitope gene by PCR,then clone into eukaryotic expression vector pcDNA3.1(-) and transiently transfect COS7 cells.Results Eukaryotic expression vector pcDNA3.1(-)-CtEm was constructed and successfully transfected into COS7 cells.Both indirect IFA and RT-PCR proved that the compound multi-epitope gene of HCV was expressed in COS7 cells.Conclusion The eukaryotic expression vector of compound multi-epitope gene of HCV was constructed and transiently expressed in COS7 cells.It laid a foundation of further study on compound multi-epitope genetic immunization.
2006 01 [Abstract][OnlineView][Download 275K] - LIU Chang,WANG Zhi-wu,TAN Fang,et al (Changchun Institute of Biological Products,Changchun 130062,China)
Objective To interfere the expression of HBsAg gene in recombinant CHO cells by vector-mediated RNAi.Methods Design and synthesize two pairs of 64 nt oligo nucleotide fragments according to the sequence of HBV gene and insert downstream to H1 promoter to construct plasmids pHs-9 and pHs-170 for expression of short hairpin RNA(shRNA).Transfect CHO cells integrated with HBsAg gene by the two plasmids and determine the expression level of HBsAg by ELISA.Results Decreased expression levels of HBsAg were determined in the culture supernatant of CHO cells 24-120 h after transfection with plasmids pHs-9 and pHs-170 respectively.Both the inhibitory effect of HBsAg expression reached about 70%.Conclusion The constructed plasmids pHs-9 and pHs-170 successfully expressed shRNA and inhibited the HBsAg expression in recombinant CHO cells.
2006 01 [Abstract][OnlineView][Download 91K] - YIN Juan,XIAO Jian,ZHOU Zhi-jun,et al (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To compare the structures and antigen epitopes of glycoprotein of aG(PG) and CTN strains used for production of rabies vaccine in China.Methods Search the amino acid sequences of glycoprotein of aG and CTN strains in GenBank and compare the homology of the two strains by Vector NTI Suite 8 software.Analyze the secondary structures and potential antigen epitopes of the two strains by DNAStar 5 software.Results The homology of amino acid sequences of glycoprotein of aG and CTN strains were 88.2%,and the sites and numbers of their characteristic secondary structures were basically consistent.Both the glycoprotein contained thirteen epitopes.Conclusion The amino acid sequences of glycoprotein of aG and CTN strains showed a certain difference.However,both the vaccines prepared with the two strains showed good protective effect.It indicated that some antigen epitopes played key roles in the induction of neutralizing antibody.
2006 01 [Abstract][OnlineView][Download 294K] - WEI Bao-jun,HAN Yue-wu (Department of Biochemistry and Molecular Biology,Basic Medical College of Lanzhou University,Lanzhou 730000,China)
Objective To induce the site-directed mutation of human β-defensin-3(hBD-3) gene and express the mutant in E.coli.Methods Extract total RNA from normal human skin tissue,then amplify the cDNA encoding the mature peptide of β-defensin-3 by RT-PCR and identify by sequencing.Design a pair of primers containing mutation site and induce the site-directed mutation of the amplified cDNA by over-lap extension PCR.Clone the mutant into vector pUC18 and express in E.coli.Results The gene sequence of the amplified mature peptide of β-defensin-3 was completely consistent with that reported in GenBank.After site-directed mutation,the codon at site 29 of β-defensin-3 mature peptide changed from CAG encoding glutamine to CGA encoding arginine,however,no change of the rest nucleotide sequence was observed.The mutant of human β-defensin-3 was expressed in E.coli.Conclusion The site-directed mutant of human β-defensin-3 gene was successfully cloned and expressed.
2006 01 [Abstract][OnlineView][Download 294K] - HU Xing-bin,XU Zhi-kai,YIN Wen,et al (Department of Microbiology,The Fourth Military Medical University,Xi'an 710032,China)
Objective To construct the shuttle plasmid for the secretory expression of pre-S1 gene and truncated core gene of hepatitis B virus(HBV).Methods Amplify the pre-S1 and truncated core gene fragments by PCR using the sequence of HBV genomic plasmid pCP10.Clone the amplified gene fragments into shuttle expression vector pDE22.Transform the recombinant plasmid to M.smegmatics by electroporation.Screen positive clones with hygromycin and identify by PCR.Induce the expression of target protein by heat shock.Identify the expressed product by SDS-PAGE.Results The two target gene fragments at the expected lengths were amplified,and the shuttle vector for secretory expression was constructed.SDS-PAGE profile showed a protein band with a relative molecular weight of 23 000.Conclusion The shuttle vector for secretory expression of pre-S1 and truncated core gene of HBV in M.smegmatics was successfully constructed.It laid a foundation of development of recombinant BCG vaccine containing pre-S and truncated core gene.
2006 01 [Abstract][OnlineView][Download 202K] - LI Shi-min,LIU Dong,ZHANG Li-jun,et al (Shenzhen Polytechnic,Shenzhen 518055,China)
Objective To obtain the recombinant angiotensin-converting enzyme inhibitory peptide(ACEIP) with high biological activity.Methods Synthesize the gene encoding angiotensin-converting enzyme inhibitory peptide,insert into expression vector pGEX-4T-2 and transform to E.coli BL21 for expression.Purify the expressed product by Glutathione Sepharose 4B affinity chromatography and HiTrap Benzmidine FF column chromatography.Determine the expression level of GST-ACEIP fusion protein by thin layer chromatogram scanning,then analyze the protein and polypeptide contents of expressed product by BCA method,and its activity by an improved HPLC method.Results Restriction analysis,PCR and sequencing proved that the target gene was successfully cloned to expression vector pGEX-4T-2.The expression level of GST-ACEIP fusion protein reached 24.6%.The protein and polypeptide contents of the expressed product were 1.1 g/L and 0.14 g/L respectively.The inhibiting activity of expressed product to angiotension-converting enzyme was 85%.Conclusion The recombinant angiotensin-converting enzyme inhibitory peptide was successfully prepared.It laid a foundation of further development of drugs for hypertension.
2006 01 [Abstract][OnlineView][Download 183K] - LIU Fang-lei,WU Bing,DU Lin,et al (Lanzhou Institute of Biological Products,lanzhou 730046,China)
Objective To compare the immunities of meningococcal serogroup A polysaccharide(GAMP)-protein conjugate vaccine prepared with different carrier proteins(TT,DT and rEPA).Methods Activate GAMP with cyanogens bromide(CNBr) and prepare GAMP-protein conjugates using ADH as a linker and EDAC as a coupling agent.Immunize mice with the conjugates and determine the antibodies against GAMP and carrier proteins in their sera as well as the bactericidal activity of antibody against GAMP.Results Both GAMP derivatives and GAMP-protein conjugates showed GAMP antigen specificity.Compared with GAMP alone,the GAMP-protein conjugates induced high serum anti-GAMP IgG antibody level and strong bactericidal activity in vitro.The conjugate also induced immunological memory.Conclusion All the GAMP-protein conjugates prepared with carriers proteins TT,DT and rEPA enhanced the immunogenicity of GAMP.
2006 01 [Abstract][OnlineView][Download 349K] - MA Shao-hui~△,LIU Long-ding,LI Jian-feng,et al ( ~△Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming 650118,China)
Objective To explore the effect of hepatitis C virus(HCV)/hepatitis B virus(HBV) bivalent vaccine by a prime-boost strategy.Methods Immunize BALB/c mice with the constructed eukaryotic expression vectors pcDNA-PCXS,pcDNA-PCSS+S/PCX protein and pcDNA3.0 respectively.Determine anti-HBs and anti-HCV antibodies by ELISA,T lymphocyte proliferation by ~3H-TdR incorporation and specific killing activity of CTLs by ~ 51 Cr release assay.Results Compared with those immunized with pcDNA-PCXS,high anti-HBs and anti-HCV titers were induced early in mice immunized with pcDNA-PCXS+S/PCX protein.The specific CTL response level induced by pcDNA-PCXS+S/PCX protein was significantly higher than that induced by pcDNA-PCXS.Conclusion DNA prime-protein boost immunization enhanced the humoral and cellular immunological functions of immunized mice.It provided a novel strategy for the practical application of DNA vaccine.
2006 01 [Abstract][OnlineView][Download 99K] - SONG Zong-ming,LIU Shuang-jun,WANG Li-na,et al (Changchun Institute of Biological Products,Changchun 130062,China)
Objective To screen an appropriate virus seed for the preparation of Vero cell-derived tick-borne encephalitis vaccine.Methods The current virus strain Sen-Zhang for preparation of tick-borne encephalitis vaccine was subcultured in Vero cells,and the titers,immunogenicities and stabilities of virus of various passages were studied.Results The virus titer of Sen-Zhang strain adapted in Vero cells reached 8.5 LgLD_ 50 /ml.The serum neutralizing antibody titer of mice immunized with the strain reached more than 1∶20 .All the quality indexes of the adapted strain met the requirements for virus seeds for vaccine production.Conclusion The Vero cell-adapted Sen-Zhang stain might be used as a virus seed for preparation of Vero cell-derived tick-borne encephalitis vaccine.
2006 01 [Abstract][OnlineView][Download 57K] - RAO Gui-rong,CHEN Wen-yin,SU Kuan-yuan,et al (Research Center of Liver Disease of PLA,The 458th Hospital of PLA,Guangzhou 510600,China)
Objective To explore the purification procedure of Fab fragment of recombinant human anti-HBs and analyze the structure of the fragment.Methods Purify the Fab fragment of recombinant human anti-HBs expressed in yeast cells by ion exchange-molecular sieve chromatography.Determine the biological activity of the Fab fragment by ELISA,its pI by isoelectric focusing electrophoresis and its relative molecular weight and peptide map by matrix assisted laser desorption ionization time of flight mass spectrometry(MADLI-TOF-MS).Results The purity of Fab fragment purified by ion exchange column chromatography reached more than 90%.However,after further purification by Sephacryl-100 chromatography,the purity reached more than 99%.The total recovery rate of Fab fragment reached more than 80%.The Fab fragment was a basic protein with a pI value of 7.6 and a relative molecular weight of 50 494 ,and showed good antigen binding activity.After digestion with endoglycosidase H,its relative molecular weight was 49 609.It proved the glycosylation in both H and L chains of the fragment.Peptide map proved that the Fab fragment was digested into 40 segments,of which 21 showed the same amino acid sequences as those of theoretical segments.In addition,one disulfide bond joint correctly was observed.Conclusion A stable purification procedure of Fab fragment of recombinant human anti-HBs was developed,and the Fab fragment showed correct primary structure.
2006 01 [Abstract][OnlineView][Download 293K] - GUO Qiao,XIAO Yan-ling,WANG Xiao-bo,et al (Changchun Institute of Biological Products,Changchun 130062,China)
Objective To control the quality and study the clinical effect of recombinant human interferon(rhIFN) α2b suppository.Methods Prepare three serial batches of rhIFN α2b suppository.Perform control tests on the suppository and observe its curative effect on cervical erosion in clinical trial,using the commercial rhIFN α2b suppository as control.Results All the quality indexes of rhIFN α2b suppository met the relevant requirements.The effective rate of the suppository in cervical erosion was 74.8%,which was significantly higher than that of control(60.0%).The incidence rates of adverse reactions in test and control groups were 9.1% and 8.3% respectively.Conclusion The prepared rhIFN α2b suppository was of good stability and safety and definite curative effect.
2006 01 [Abstract][OnlineView][Download 90K] - XU Yan-ping,ZHAN Xue-jun,YANG Shu-hua,et al (Jiangxi Institute of Medical Science,Nanchang 330006,China)
Objective To prepare egg yolk-derived hepatitis B virus specific transfer factor(EY_(HBV)-TF) and determine its immunological activity.Methods Immunize hens with hepatitis B vaccine and collect the eggs.Extract egg yolk and dialyze to prepare EY_(HBV)-TF,then study its physical and chemical properties by spectral analysis,HPLC and Lowry method et al.according to the requirements in Chinese Pharmacopoeia.Determine the immunological activity of EY_(HBV)-TF by leukocyte adherence inhibition(LAI) and delayed type hypersensitivity tests.Results EY_(HBV) -TF was a dialyzable substance(pH 6.9±0.5) with a relative molecular weight of less than 12 000,containing 18 kinds of amino acids.The polypeptide and ribose contents of EY_(HBV-TF were 1.2 mg/ml and 152.94 μg/ml respectively .The EY_(HBV)-TF was negative in protein reaction test and showed the maximum ultraviolet absorption at 270 nm.It inhibited the leukocyte adherence in mice significantly(P<0.01) and induced the DTH in metatarsophalangeal skin of mice (P<0.01) .The LAI and DTH effects of EY_(HBV)-TF showed no significant difference from those of pig splenic lymphocyte-derived hepatitis B virus specific transfer factor(PS_(HBV)-TF,P>0.05).However,the extract of normal egg yolk,non-specific transfer factor and hepatitis A antigen showed no LAI or DTH effects(P>0.05).Conclusion The major physical and chemical property and effect of EY_(HBV)-TF were similar to those of PS_(HBV)-TF.Both of them showed hepatitis B specific cell immunological activity.
2006 01 [Abstract][OnlineView][Download 134K] - HAO Yan-ling~△,ZHANG Meng-hua,HUANG Yun-jian,et al ( ~△National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 100050,China)
Objective To prepare the major antigen fragment of GP120 of HIV-1 virus strain RL42 epidemic in China and develop a diagnostic kit for HIV antibody.Methods The gene encoding the 250 amino acids at C-terminus of GP120 of strain RL42 was amplified and subcloned into E.coli expression vector pBV220.After themo-induction,the recombinant protein was purified by washing of inclusion body,precipitation with ammonium sulfate and ion exchange chromatography(IEC).The purified protein was prepared into antigen strips for immunoblot assay with HIV reference sera at various dilutions.Eight HIV-positive and seven HIV-negative serum specimens isolated in clinic were tested with the antigen strips.Results The expressed product contained about 10% of total somatic protein and reached a purity of more than 95% after purification.Strip immunoblot assay showed a clear reaction band of the recombinant GP120 antigen with 1∶20 000 diluted HIV-positive serum.The antigen strip prepared with the purified protein reacted specifically with eight HIV-positive serum specimens but showed no cross reaction with seven HIV-negative specimens.Conclusion The major antigen fragment of HIV-1 GP120 with good specificity and sensitivity was prepared.It laid a foundation of development of diagnostic kit for HIV antibody.
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2006 01 [Abstract][OnlineView][Download 52K] - WEI Jian~△,LIU Da-tao,TAN Xiao-ding,et al ( ~△State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)
Objective To develop a purification procedure of rProEMAPⅡ/P43.Methods The rProEMAPⅡ/P43 protein expressed in E.coli was purified by procedure 1,consisting of hydrophobic interaction chromatography,ion exchange chromatography and heparin affinity chromatography,and procedure 2,consisting of ion exchange chromatography and heparin affinity chromatography,respectively.The purified rProEMAPⅡ/P43 was determined for total protein content and analyzed for the content and purity of target protein by SDS-PAGE.Results Compared with that purified by procedure 2,the recovery rate of rProEMAPⅡ/P43 protein purified by procedure 1 at pH 7.4~7.5 increased by 28.4%,and its purity reached 92%.Conclusion The introduction of hydrophobic interaction chromatography into purification procedure increased the recovery rate of rProEMAPⅡ/P43 protein expressed in E.coli significantly.
2006 01 [Abstract][OnlineView][Download 244K] - LI Qun-liang,CAI Hai-bo,LIU Qi-wei,et al (The State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)
Objective To study the influence of culture environment in vitro on the gene expression of CD34~+ hematopoietic stem/progenitor cells from umbilical cord blood(UCB).Methods Cultivate the mononuclear cells(MNCs) from UCB in static and stirred culture systems for 7 d,then collect CD34~+ hematopoietic stem/progenitor cells(HSPCs).Extract total RNA from the collected HSPCs using Trizol reagent and amplify sufficient cDNA by SMART-PCR for the labeling of probe.Screen the differentially expressed genes with Atlas cDNA arrays covering 588 genes.Results The differential expression of a total of 45 genes were found in CD34~+HSPCs before and after cultivation,of which 20 genes were up-regulated and 25 genes were down-regulated.In addition,the expression levels of 12 genes were significantly different in the CD34~+HSPCs cultivated in static and stirred systems.With the exception of CCL2,the rest genes were highly expressed in the CD34~+HSPCs in static culture.Conclusion Quantitative molecular understanding could provide new insights into the physiological responses of CD34~+HSPCs to culture environments.
2006 01 [Abstract][OnlineView][Download 133K] - ZHANG Wen-chao,CHEN Chang-hua,LI Su-xia,et al (The State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)
Objective To study the influence of magnesium ion and amino acid on the growth of recombinant E.coli BL21(DE3) and the expression of procarboxypeptidase B.Methods Culture recombinant E.coli BL21(DE3) in spinner and fermentor respectively,add magnesium ion and amino acids to the medium and observe the growth of recombinant E.coli BL12(DE3) and the expression of procarboxypeptidase B.Results The stability of plasmid was improved by adding magnesium ion to a final concentration of 1 g/L.After amino acids were added,the expression levels of procarboxypeptidase B increased from 18.7 g/L to 24 g/L.Conclusion The addition of a suitable amount of magnesium ion and amino acid improved the growth of recombinant E.coli BL21(DE3) and expression of procarboxypeptidase B.
2006 01 [Abstract][OnlineView][Download 254K] - YANG Li-jun,YANG Tao,XIE Jun,et al (Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)
Objective To optimize the fermentation procedure of recombinant E.coli strain expressing Echistatin fusion gene.Methods Study the influence of medium and time for incubation and induction on the growth of recombinant E.coli and expression of target protein in a 15 L fermentor,and explore the genetic stability of recombinant plasmid.Results After the recombinant E.coli strain cultured in 2×YT medium(pH 7.4) was induced for 4 h,the wet weight of bacteria reached 75 g/L.The expressed product contained about 35% of total somatic protein.The constructed recombinant plasmid was inherited steadily in host bacterial strain E.coli BL21.Conclusion The condition for fermentation of recombinant E.coli strain and expression of Echistatin fusion gene was optimized.It laid a foundation of large-scale production of Echistatin protein.
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2006 01 [Abstract][OnlineView][Download 54K] - LIAO Qun,WU Ying (Department of Clinical Laboratory,Chongqing Emergency Medical Center,Chongqing 400014,China)
Objective To analyze the characters of serum protein patterns of three kinds of patients with hepatitis B.Methods Determine the marker of hepatitis B by ELISA,total protein by biuret method,albumin by bromocresol green method,and the component of serum protein by agarose gel electrophoresis.Results HBsAg carrier showed no significant difference(P>0.05) with healthy person in the component of serum protein.However,significant difference(P<0.01) was observed between the serum protein components of the patients with hepatitis B and healthy person.Compared with that of patients positive in HBsAg,HBeAg and HBcAb,the Alb levels of patients positive in HBsAg,HBeAb and HBcAb decreased significantly,and their globulin-γ levels increased significantly.However,the globulin α_1,α_2 and β levels of the patients showed no significant diffeence(P>0.05).Conclusion The combination of serum protein electrophoresis and tests for total protein and albumin was benefit to the overall study on character of serum protein pattern of patients with hepatitis B and was of important reference value in the clinical diagnosis and treatment of hepatitis B.
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