The preparation of the mouse monoclonal antibodies specific for the DNAH2 protein / 天津医药

Objective To prepare specific mouse monoclonal antibodies against Homo sapiens dynein,axonemal, heavy chain 2 (DNAH2). Methods Firstly, recombinant plasmid encoding His tagged immunogen, targeting N-terminal sequence of DNAH2 protein (1-300 aa), in E. coli was constructed. IPTG was used to induce the expression of His-immunogen, which was then purified and immunized in BALB/c mice. Hybridoma cells were obtained through the fusion between myeloma cells and splenocytes isolated from BALB/c mice. Finally, ELISA and Western blot assays were performed to screen the positive hybridoma. Results IPTG was used efficiently to induce the expression of DNAH2 immunogen in E. coli. DNAH2 protein bands were detected in screened positive hybridoma. Conclusion Mouse monoclonal anti-DNAH2 antibody is prepared successfully.

Construction and identification of recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosoma japonicum / 中华地方病学杂志

Objective To construct and identify recombinant vaccine Bifidobacterium bifidum (pGEX-Sj32) of Schistosomajaponicum (Sj).Methods The Sj32 gene amplified by PCR from template of plasmid pET28α-Sj32 that extracted from recombinant Escherichia coli(E.coli) BL21 (pET28α-Sj32) stored in our laboratory was cloned into E.coli-Bifidobacterium bifidum shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj32.The recombinant plasmid was transformed into E.coli BL21 (DE3).The plasmid was extracted and identified by restriction enzyme digestion.The recombinant Bifibacterium bifidum (pGEX-Sj32) vaccine was constructed by electroporating pGEX-Sj32 into Bb.The extracted plasmid was amplified and identified by PCR.Results The gene Sj32 of 1 270 bp in length was amplified by PCR.The restriction endonuclease digestion showed that the length of plasmid vector was 4 947 bp,and Sj32 gene was 1 270 bp.The Sj32 gene amplified by PCR from the template of pGEX-Sj32 extracted from recombinant Bb (pGEX-Sj32) vaccine was 1 270 bp in length which consistent with expected result.Conclusion The recombinant Bb (pGEX-Sj32) vaccine of Sj is successfully constructed.

Progress in role of breast cancer susceptibility gene 1 in DNA damage repair / 中国药理学与毒理学杂志

Breast cancer susceptibiIity gene 1( BRCA1)is a tumor suppressor,but mutated get BRCA1 is cIoseIy reIated to the deveIopment of breast cancer. Current study has reveaIed that BRCA1 can get invoIved in the DNA damage repair process as a key mediator. DNA doubIe-stranded break (DSB)is the most serious form in DNA Iesions,and BRCA1 pIays an important roIe in repairing DSB through reguIation of homoIogous recombination( HR). In this articIe,the moIecuIar mechanism of BRCA1 in reguIating HR is reviewed with reference to the activity of functionaI domains in BRCA1 struc-ture,the mutations occurring in main domains of BRCA1,the reIationship of BRCA1 with BRCA2, Rad51 or CtIP,and phosphoryIation of BRCA1. In addition,the association of the defective BRCA1-mediated HR repair mechanism with the sensitivity to different DNA damaging agents and synthetic IethaIity in tumor ceIIs is aIso addressed.

Curative effect of transplantation of mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene on intracerebral hemorrhage in rats / 中华神经科杂志

Objective To observe the curative effect of transplantation of mesenchymal stem cells (MSCs) transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor (BDNF) gene on intracerebral hemorrhage in rats.Methods MSCs were isolated from the rat bone marrow,cultured and transfected by recombinant lentiviral vectors carrying BDNF gene.Intracerebral hemorrhagic models were constructed and randomly divided into 4 groups:phosphate buffered saline transplanted (PBS) group,MSCs group,mesenchymal stem cells transfected with empty lentiviral vectors transplanted (MSCs-EGFP) group and mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene transplanted (MSCs-EGFP-BDNF) group.PBS and MSCs were transplanted according to the groups 72 hours after the establishment of models.The improvements of the neurological function were recorded of each group 7 d,14 d,and 21 d after the transplantation.Double labeling immunofluorescent staining were used to detect the migration and the differentiation of transplanted MSCs.Results MSCs-EGFP-BDNF group had significant higher levels of BDNF gene and protein expression than MSCs group and MSCs-EGFP group.All MSCs transplanted groups (MSCs groups:7 d:1.6 ±0.2,14 d:1.2 ±0.3,21 d:0.8 ±0.2; MSCs-EGFP groups:7 d:1.6 ±0.3,14 d:1.1 ±0.2,21 d:0.8 ±0.3; MSCs-EGFP-BDNFgroup:7 d:1.2 ±0.3,14 d:0.6 ±0.1,21 d:0.2±0.2) had more improvements in the neural function (F=6.667,18.417,20.882,all P ＜0.05) than PBS group(7 d:2.0 ±0.4,14 d:1.7 ±0.2,21 d:1.3 ±0.2),and MSCs-EGFP-BDNF group had the most significant improvement.With double labeling immunofluorescent staining,the MSCs-EGFP-BDNF group had significantly higher positive rates of glial fibrilary acidicprotein,neuron specific nuclear protein,2',3 '-cyclic nucleotide 3'-phosphodiesterase than MSCs group and MSCs-EGFP group,while there was no significant differences between the latter two.Conclusions The expression levels of gene and protein are higher for the MSCs modified with recombinant lentiviral vectors carrying BDNF gene.The modified MSCs can migrate to the perihematomal brain issue of intracerebral hemorrhage,express the characteristic molecules of neurons and improve the neural function after intracerebral hemorrhage.

Construction of human surfactant protein B promoter luciferase reporter gene vectors and detection of their activities / 中国医师杂志

Objective To construct human surfactant protein B (SP-B) gene promoter luciferase reporter plasmids and detect their transcriptional activities in H441 cells.Methods (1)The fragment of SP-B promoter (-218/+ 435 bp) was acquired from human genome DNA by polymerase chain reaction (PCR) amplification and then was inserted into pGM-T vector by the T4 DNA ligase.The vector was transfected into TOP10 E.coli.The positive clone was identified by DNA sequencing.The identified target SP-B promoter sequence was cloned into pGL3-basic vector to construct the recombinant vector pGL3-basic-SP-B-promoter and was identified by enzyme digestion and sequencing; (2)The pGL3-basic-SP-B-promoter vector was converted into pGL4.17-SP-B-promoter vector through enzyme digestion.The identified recombinant vectors and control plasmid pRL-TK were transfected into H441 cells by lipofectamine 2000,and luciferase assays was performed using the dual-luciferase reporter assay system.Results The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were consistent with the one published on Genebank.The firefly/renilla luciferase activity ratio of pGL3-basic/pGL4.17-SP-B-promoter vector (2.8 ± 1.1,66.5±3.8) was significantly higher than pGL3-Basic,pGL4.17 control vector (0.2 ±0.1,4.3 ±0.4) with statistical significance (t =4.182,27.419,P =0.000),respectively.The SP-B promoter activity of pGL4.17-SP-B-promoter vector was significantly higher than pGL3-basic-SP-B-promoter vector (t =27.712,P =0.000).Conclusions The pGL3-basic/pGL4.17-SP-B-promoter vectors are successfully constructed with SP-B promoter activity in H441 cells and pGL4.17-SP-B-promoter vector is the better choice for further study with higher luciferase activity.

Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells / 中国地方病学杂志

Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and cysteine protease inhibitor ( CPI ) gene from periodic Brugia malayi (Bm) and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 (+). pcDNA3.1 (+)/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE). Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass (Mr) of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell.

Preparation and evaluation of recombinant human ferritin light chain for quality control materials / 中华检验医学杂志

Objective To construct a prokaryotic expression system of human ferritin L which could be overexpressed in E.coli, and then prepare quality control materials of rFL and evaluate the homogeneity, stability, precision and application value of the rFL.Methods RT-PCR was used to clone ferritin L gene with total RNA from peripheral blood.Ferritin L gene was inserted into plasmid pGM-T to construct subclone plasmid pGM-T/ferritin L, which was identified by sequencing.The recombinant plasmid pET-30a/ferritin L was transformed into E.coli BL21 (DE3) and efficiently expressed under IPTG induction.rFL was identified by SDS-PAGE, and its antigenicity was examined by WB.The concentration of rFL was measured by ACCESS immunoassay system.Fifteen control samples were randomly selected using random number table.The homogeneity and stability of rFL were evaluated by one-way analysis of variance (ANOVA) and a linear regression model, respectively.The uncertainties of homogeneity, stability and the precision were evaluated.Results A prokaryotic expression system of ferritin L was successfully constructed.Sequence analysis showed that the ferritin L gene inserted was identical with the one from GenBank(NM_000146.3).The PCR products were 527 bp long, in complete agreement with length of ferritin L gene.The molecular weight of rFL highly expressed in E.coli induced by IPTG was 19 000 Da.The WB analysis indicated that this rFL protein had good antigenicity.The concentration of rFL(1 000 times dilution) measured by ACCESS immunoassay system was (1115.84±38.38) ng/ml.Homogeneity evaluation of low-concentration QC sample and high-concentration QC sample of rFL showed that there were no statistical significances in the within-groups and between-groups (for high-concentration F=2.336, P>0.05 and for low-concentration F=0.730, P>0.05).A linear regression based on the stability test indicated that there was no statistically significant trend of instability in five and a half months (F=1.755, P>0.05). Precision analysis showed the within-run CV, the between-run CV, the between-day CV and the total CV of high-concentration QC sample were 2.06%, 2.12%, 0% and 2.96% respectively; the within-run CV, the between-run CV, the between-day CV and the total CV of low-concentration QC sample 2.03%, 2.08%, 0% and 2.90%, respectively.Conclusion This rFL for quality control materials with independent intellectual property rights could meet the clinical requirement of homogeneity, stability and precision, and could provide raw materials for preparation of mixed ferritin for quality control.

The genetic study on a rare inter-allelic recombination of HLA-C locus in a Chinese Han family / 中华检验医学杂志

Objective To study the inter-allelic recombination event occurring in the HLA-C locus in a family of Chinese Han nationality, and to evaluate the molecular genetic background of the new HLA allele.Methods Peripheral blood samples were collected from a Chinese leukemia woman patient, as well as her healthy parents and two brothers.HLA-A, C, B, DRB1 and DQB1 alleles were typed by high-resolution PCR-sequence-based typing (SBT) method using Atria Genetic AlleleSEQR HLA SBT kits.The Protrans S4 HLA-C single allele-specific sequencing strategy was used to separate the two HLA-C alleles and to determine novelty of the allele.The full length sequences of HLA-C alleles of the patient and her parents were further analyzed using cloning and haplotype sequencing method. The HLA five loci linked haplotypes and the recombination site were analyzed by family study, meanwhile the full length sequences of the five HLA-C alleles were compared with the IMGT/HLA database by the program BLAST.Results The two haplotypes of the father and mother were a:A*0207-C*010201-B*550201-DRB1*090102-DRQ1*030302 and b:A*240201-C*120202-B*5201-DRB1*1502-DRQ1*0601, c:A*300101-C*060201-B*130201-DRB1*0405-DRQ1*0401 and d:A*110101-C*070201-B*4001-DRB1*080302-DRQ1*0601,respectively.The two brothers inherited their parent′s haplotypes a, d and b, c respectively.The two haplotypes of the patient were the maternal c and paternal recombinant a/b haplotype.The recombinant a/b haplotype A*240201-C*new-B*550201-DRB1*090102-DRQ1*030302, A*240201 came from the paternal haplotype b,while B*550201-DRB1*090102-DRQ1*030302 came from the other paternal haplotype a.When comparing the full length sequences of the HLA-C new allele with the father′s allele C*010201 and C*120202, it could deduce that the recombinant a/b haplotype derived from a recombination event occurring between the paternal chromosome 6 during meiosis.The crossover site was between genomic nt273 and nt330 of HLA-C alleles, which created a HLA-C new allele and the fifth haplotype of the family, and inherited it to the patient.The full length sequences of the new allele had been submitted to Genbank, and officially named C*0121 by WHO nomenclature committee.Conclusion This study demonstrates a rare inter-allelic recombination event occurring in the HLA-C locus within a Chinese Han family and illustrates the process of novel allele and haplotype, and provides direct theory for further studying the mechanisms of gene recombination and HLA polymorphism.

Construction and identification of recombinant adenovirus containing heat shock protein70 gene driven by carcinoembryonic antigen promoter / 中华胰腺病杂志

Objective To construct the recombinant adenoviros containing heat shock protein70 (Hsp70) gene driven by carcinoembryonic antigen (CEA) promoter. Methods Hsp70 gene and CEA promoter were amplified by RT-PCR and PCR, and then subcloned into the shuttle vector pDC316 to construct the recombinant vector PDC316-pCEA-Hsp70. The recombinant vector was co-transfected with adenoviral backbone plasmid into HEK293 cells to generate the recombinant adenovirus Ad5-pCEA-Hsp70. The recombinant adenovirus was purified by CsCl banding and titrated by 50％ tissue culture infective dose (TCID50) assay. After transfection of the recombinant adenovirus into human pancreatic cell lines SW1990 and BxPC3, the expression of mRNA and protein level of Hsp70 were determined by RT-PCR and ELISA,respectively. Results Digestion and DNA sequencing certified that the Hsp70 gene and CEA promoter was successfully inserted into pDC316 plasmid. Virus acquired through co-transfection with backbone plasmid was confirmed to be constructed successfully by PCR amplification. The particles finally expressed was 2.2 ×1011vp/ml, and the titer was 1.5 x 1010 PFU/ml. BxPC3 cancer cells with positive CEA expression showed increased expression of Hsp70 mRNA and protein after infected by recombinant adenovirus; while SW1990 cancer cells with negative CEA expression showed no change of expression of Hsp70 mRNA and protein after infected by recombinant adenovirus. Conclusions The recombinant adenovirus Ad5-pCEA-Hsp70 which can express Hsp70 gene in CEA positive cancer cells is constructed successfully.

Therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor and augmenter of liver regeneration on rats with hepatic fibrosis / 中华传染病杂志

Objective To evaluate the therapeutic effects of recombinant expression plasmid containing hepatocyte growth factor (HGF) and augmenter of liver regeneration (ALR) on rats with hepatic fibrosis. Methods Ninety Sprague-Dawley rats, which had been established into hepatic fibrosis models, were equally divided into 6 groups: blank group, pcDNA3.1 therapy group,pcDNA3.1-HGF therapy group, pcDNA3. 1-ALR therapy group, pcDNA3.1-HGF and pcDNA3. 1-ALR combined therapy group, and pcDNA3. 1-HGF-ALR therapy group. Zero point one μmol of blank or plasmid was injected into model rats in each group by tail vein once a day for 3 days. Model rats in blank group didn't receive any treatment. Additional 10 rats were chosen as control group, which were not given any interference during the experiment. All rats were sacrificed 4 days after end of treatment. Liver tissues were reserved for observing pathologic changes after HE staining and detecting proliferating cell nuclear antigen (PCNA) and c-jun by immunohistochemistry. Measurement data were compared by single-factor analysis of variance. Comparison between groups was done by SNK test. Enumeration data were analyzed by Fisher's exact test. Results In blank group and pcDNA3.1 therapy group, hyperplasia of fibrous connective tissue was very obvious, false lobules were formed. There was no significant difference between these two groups (x2 =0. 317,P= 1. 000).In the 4 remaining groups, hepatic fibrosis all achieved different degree of amelioration, and the therapeutic effect of pcDNA3.1-HGF-ALR was optimal. In control group, the expressions of PCNA and c-jun in liver tissues were low, with absorbance value of 8.6±1.9 and 3.2 ± 1.2, respectively. In blank group and pcDNA3. 1 therapy group, the expressions of PCNA and c-jun were obviously increased, with absorbance value of 24. 1±3.0, 24.5±4.3 and 23.8±3.1, 24.9±4.2, respectively,which were significant different from control group (all P＜0.01). In the 4 remaining groups, the expressions of PCNA were all obviously increased, and expressions of c-jun were all obviously decreased. The maximum change scope was observed in pcDNA3. 1-HGF-ALR therapy group.Conclusions The recombinant expression plasmid pcDNA3. 1-HGF-ALR can effectively ameliorate experimental hepatic fibrosis of rats. The anti-fibrosis effects are achieved probably by up-regulating PCNA expression and down-regulating c-jun expression.

Cloning, expression and identification of phage's capsid Vp3 protein of guinea pig inclusion conjunctivitis chlamydia / 中华传染病杂志

Objective To get phagc's capsid Vp3 gene and protein of guinea pig inclusion conjunctivitis (GPIC) chlamydia. Methods The genome DNA was extracted from the φCPG1 phage.The full sequence of Vp3 gene was amplified by polymerase chain reaction (PCR) from the above genome DNA. The Vp3 gene was digested by restriction endonuclease and then inserted into prokaryotic plasmid vector pET30a (+). The recombinant plasmid was transformed into E. coil BL21, and was identified by restriction endonuclease, PCR and sequencing. The E. coil BL21 with expected recombinant plasmid was induced and the expressed recombinant Vp3 protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, then purified by agarose gel. Results The recombinant gene was sequenced and proved to be 447 bp which was consistent with the φCPG1 Vp3 gene sequence in GenBank. A 25 000 capsid protein was expressed and confirmed by SDS-PAGE and Western blot. The purified protein was obtained. Conclusion The capsid Vp3 protein of φCPG1 is successfully expressed and purified, which is helpful for the further study on its mechanism and clinical applications.

Expression of Recombinant Human Thyroid Stimulating Hormone Subunit α in Escherichia Coli / 天津医药

Objective:To construct pGEX-3X/hTSHa Escherichia coli(E.coli)expression system and prepare purified recombinant GST-recombinant human thyroid stimulating hormone(rhTSH)α protein.Methods:The complete coding sequence of hTSHα was obtained by RT-PCR with total RNA extracted from fresh chorial tissue as the template,and thereafter cloned into expression vector pGEX-3X by EcoRl and BamHI digestion.The recombinant plasmid was transformed into E.coli Mach1-T1 and then induced expression by WrG.The GST-rhTSHα fusion protein was identified by SDS-PAGE and its antigenicity was verified by a modified competitive ELISA.Results:A specific protein band of 36 ku,in accordance with predicted molecular weight,could be visualized in SDS-PAGE.As the result of ELISA,the recombinant GST-hTSHα protein can inhibit the intact TSH molecular binding with anti-TSHα antibody in a dose dependent manner.Conclusion:The cDNA of hTSHα was cloned and the recombinant expression vector pGEX-3X/hTSHα was constructed successfully.The recombinant GST-rhTSHα protein could be highly expressed in E.coli Machl-T1 and was approved of possessing antigenicity.

Establishment of enzyme-linked immunosorbent assay one-step assay based on recombinant proteins derived from different genotypes of hepatitis E virus / 中华传染病杂志

Objective To establish an anti-hepatitis E virus (HEV) enzyme-linked immunosorbent assay (ELISA) one-step assay based on seven glutathione S-transferase (GST)-fusion recombinant proteins derived from different HEV genotypes and subtypes. Methods Concentration of the coating antigen was optimized by block titration. The cut-off values were determined for anti-HEV IgG and IgM, respectively. Assay sensitivity, specificity and reproducibility were investigated using samples with confirmed anti-HEV positive. Results An optimal concentration of mixture of recombinant proteins (Mix166) was 1.5 mg/L for antigen coating. Coefficient of variations (CV) of anti-HEV within-run and between-run were 8.67% and 10.85%, respectively. CV of anti-HEV IgM within-run and between-run were 4.56% and 5.99%, respectively. Positive rates of anti-HEV IgG and IgM were both 94% for 50 HEV-polymerase chain reaction (PCR) positive sera tested with the one step assay. Using one-step assay to detect 674 serum samples from healthy people, 52 samples were found anti-HEV IgG positive and 3 samples were anti-HEV lgM positive. A series of serum specimens collected at different time points until 76 weeks from a chimpanzee challenged with HEV Mexican strain were anti-HEV IgM positive during week 1--6 and anti-HEV IgG positive during week 2--76 determined by the one step ELISA. However, import ELISA kits were lack of both the IgM and lgG reactivity to all of the serial chimpanzee sera. Conclusions The sensitivity and specificity of anti-HEV ELISA one-step assay based on the Mix166 antigen are high and could be used for the diagnosis of HEV infection.

Construction and identification of recombinant human cytomegalovirus with ganciclovir-resistance associated mutations in UL97 gene / 中华传染病杂志

Objective To construct drug-resistant variant recombinant human cytomegalovirus (rHCMV)and identify drug susceptibility by phenotypic and genotypic assays.Methods The UL97 fragments containing Pine I recongnition site and resistant mutation were introduced by site-directed mutagenesis using gene splicing by overlap extension polymerase chain reaction(PCR),and blended with human cytomegalo-virus(HCMV)standard strain ADl 69 genomic DNA proportionally,then the DNA mixture were transfected into MRC-5 fibroblasts by the vector of liposomes.HCMV-PP65 antigen was detected by indirect-immunofluorescent assay to verify rHCMV infection of MRC-5 fibroblasts.When the eytopathic effect(CPE)of homologous recombinant virus reached 100%,the virus was harvested.The purified target virus was screened by plague with different concentrations of ganciclovir(GCV)and the recombinant virus was identified by plague reduction test and DNA sequencing of drug-resistant genes(UL97 and UL54).Results The UL97 fragments containing intended mutations for transfection were constructed successfully.After cotransfected with AD169DNA mixture for 7 days,rHCMV formed cytopathology was obviously visible,which was verified as rHCMV infected focus by HCMV-PP65 antigen test.The UL97 genotypic analysis of recombinant virus obtained by cloning was as expected.No mutation was found by UL54 gene sequencing.The GCV susceptibility of rHCMV positive clone was 15 μmol/L (50% inhibiting concentration),which was 12-fold of standard AD169 strain and was drug-resistant phenotype.Conclusions The rHCMV containing intended mutations is constructed successfully by cotransfeetion into MRC-5 cells and the recombinant virus strain is obtained by GCV screening and plaque purifying.The establishment of this method provides technique platform for identifications of new drug-resistant mutations of HCMV during anti-viral therapy.

Construction and identification of expressing human c-Jun N-terminal kinase(JNK)recombinant adenovirus / 中国医师杂志

Objective To construct replication deficient recombinant adenovirus expressing human c-Jun N-terminal kinase by homologous recombination.Methods The linearized recombinant shuttle vector pAdTrack-CMV-WT-JNK was co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for recombinant adenoviral vector.The recombinant adenoviral vector was transfected into HEK293 packing cells to construct replication deficient recombinant adenovirus,and then the recombinant adenovirus was detected by PCR and DNA sequencing.Results JNK recombinant adenoviral vector was effectively transfected into HEK 293 cells and was successfully packed by intracellular enzyme.The expression of green fluorescent protein(GFP)was observed on the 5th day after transfection.The fragment of JNK gene was amplified by PCR and identified by sequencing.The animal experiment confirmed that Ad-WT-JNK was effectivety expressed in liver tissue. Conclusion The research successfully constructed recombinant adenoviral vector and recombinant adenoviral particle.And the achievement laid a foundation for further investigation of the function and application of JNK.

Identification of two newly discovered subgenotypes of hepatitis B virus genotype C / 中华传染病杂志

Objective To analyze the genotypic characteristics of hepatitis B virus (HBV)C/D recombinant of two types of newly discovered HBV genotypea found in Western China.Methods The whole genomes of 17 HBV strains isolated from Western China were amplified by polymerase chain reaction (PCR). Bioinformatic softwares were used for the analysis of full genome structure,genetic distances and recombination points.Results The heterogenicity of the HBV C/D recombinant was more than 8% compared with genotype A,B,D,E or F,but 3.8% 0A-8.O% compared with genotype C Based on phylogenetie analysis, a11 C/D recombinant strains clustered within genotype C.but were rouped into two other clusters within the genotype C independently from C1-C5 subgenotypes,which were two kinds of new HBV/C genotypea.Condusion The HBV C/D recombinant could he considered as tWO kinds of new subgenotypea of HBV genotype C which are different from subgenotype C1-C5 based on the genetic distances analysis.

In vivo expression of exogenous hepatocyte growth factor inhibits hepatocyte apoptosis in mice / 中华传染病杂志

Objective To establish high level expression system of exogenous hepatocyte growth factor(HGF) protein in mouse livers by in vivo gene transfection and to observe the inhibition effect of exogenous HGF on hepatocyte apoptosis in mice. Methods Mice were divided into four groups, with 10 mice in each arm, which were injected with control solution, empty pcDNA3 plasmids, pCMV-HGF plasmid or 0.9% sodium chloride solution by tail vein. Enzyme-linked immunosorbent assay(ELISA) was used to determine the peak level and the expression duration of HGF protein in the peripheral blood and liver tissue. Western blotting was performed to measure the Caspase-3, tBid, Bax and Cytochrom C in the hepatocyte homogenatea and mitochondrion. Results HGF protein was detected in the mice blood as early as 4 hours after single injection of pCMV-HGF plasmid. The peak level of HGF protein in liver and plasma was respectively achieved by 8 hours and 12 hours after first injection while HGF protein was still detectable in the blood 6 days after the initial injection. D-Galactosamine/lipopolysaeeharide (LPS) led to obvious hepatocyte apoptnsis and induced an increased concentration of tBid, Bax, Caspase-3 and Cytochrom C in the hepatocyte homogenates and mitochondrion. Compared to sodium chloride control group and empty pcDNA3 protected group, the expression of tBid, Bax, Caspase-3 and Cytochrom C decreased in pCMV-HGF plasmid protecting group. Conclusions Hepatocyte apoptosis can be inhibited by exogenous HGF protein expression in mouse livers, which is induced by in vivo gene transfection. Moreover, it may inhibit the activation of downstream apoptotic proteins by blocking the expression of tBid.

Analysis and state of HCV genotype 6a infection / 北京大学学报(医学版)

Objective: To investigate the infection state of hepatitis C virus genotype 6a in China.Methods: Three(95,126,150)HCV genotype 6a serum samples were identified by digesting 5′NCR with compound enzyme method.Then,HCV 5′NCR and NS5B fragments were amplified from these samples by RT-PCR assay and sequenced.The phylogenetic trees of the samples were analyzed and compared with 24 HCV complete gene sequences from GenBank.Results: The sequencing reports on 5′NCR showed "CA" bases in 3 serum samples(95,126,150) were inserted into-145 site,and the sequences of 3 serum samples had the highest homology with sequence Y12083(0.934,0.930,and 0.926,respectively).The results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 6a.The sequencing reports on NS5B showed the 3 serum samples also had the highest homology with HC-J4(0.934,0.930,and 0.926,respectively),and the results of the phylogenetic trees suggested these 3 serum samples belonged to HCV genotype 1b.To exclude the influence of amplification efficiency of primers,NS5B fragments were amplified by HCV genotype 6a specific primers and no amplification products appeared.Conclusion: There are different results of HCV genotype by analyzing 5′NCR and NS5B in 3 samples infected with HCV genotype 6a.It may be related with gene recombination.It suggests HCV genotype should be analyzed on more than two regions.

Effect of recombinant human trefoil factor on protection of gastric mucosa and healing of chronic gastric ulcer in rats / 北京大学学报(医学版)

Objective: To study the effect of recombinant human intestinal trefoil factor ( rhITF) on the healing of rat chronic gastric ulcer , protect gastric mucosal and mechanisms are involved. Methods: (1) Acute gastric mucosal injury was induced by ethanol, stress, aspirin and pylorusl ligation. The injury index ,MDA, GMBL,hexosamine (Hex) and acid output were measure. (2) Chronic gastric ulcer was induced in rats by application of 50% glacial acetic acid to the serosa of the glandular stomach. After injury, rats received by rhITF or vehicle orally twice daily for 11 days. On day 12, gastric mucosal blood flow(GMBF)was measured under ether anesthesia. Then the pylorus was ligated for 3 hours and each stomach removed. The gastric acid output, ulcer index, Hex and nitric oxide(NO) content in gastric mucosa, as well as iNOSmRNA in the ulcer bed were determined. Results: (1) rhITF protected gastric mucosa from the acute lesion, and increased Hex content in gastric mucosa. (2) rhITF treatment significantly decreased the ulcer index and gastric acid output, but increased the GMBF, Hex and NO content in comparison with the control groups. In addition, rhITF also stimulated iNOSmRNA expression in the ulcer bed by situ hybridization analysis. Conclusion: rhITF can protect gastric mucosa against acute lesion, and enhance the healing of chronic gastric ulcer in the rats.This action may result from the inhibition of gastric acid output, increase of GMBF.Hex and NO content and rhITF stimulated iNOSmRNA expression.

Construction, identification and sequence analysis of eukaryotic expression reco mbinant plasmid contai-ning dense granules protein 8 gene from Toxoplasma gondii in BALB/c mice / 中华传染病杂志

Objective To clone the coding dense granules protein 8(GRA8) from Toxoplasma gondii RH isolate for the potential use in the development of DNA vaccination. Methods Amplifing gene fragment coding GRA8 from the genomic DNA of Toxoplasma gondii RH isolate by means of ploymerase chain reaction (PCR), the gene is inserted into cloning vector pUC-19 digesting with restrictive enzymes and linking react ions. The positive colon is screed on LB plates containing ampicilline and IPTG, Xgal identified by blue-white and restrictive enzyme digestion. The inserted GR A8 gene was recombined with pcDNA3.1(+) eukaryotic expression vector by digestion with restrictive enzyme and linking reactions. The positive coloe is screene d o n LB plates containing ampicillin and identified by restrictive enzymes and link ing reactions. Results The GRA8 gene with about 804 base is specifically amplified from genomic DNA of Toxoplasma GRA8/RH and pcDNA3.1(+)-GRA8 recombinant is successfull y constructed. The sequencing results showed that GRA8 gene of isolate RH and R H from genebank shares quite high homology. Conclusion The gene encoding GRA8 is amplified from genomic DNA of Toxoplasma gene isolate GRA8/RH and pcDNA3.1 (+)-GRA8 recombinant is successfully constructed.