Antiviral Agents ; pharmacology ; Hepatitis B virus ; drug effects ; Interferons ; pharmacology

Objective Acquiring genetic information of Y-SNPs and Y-STRs genetic makers from samples with the surname of Kong, which is useful for exploring the correlation between surname and Y chromosome in forensic applications studies.Methods Two multiplex genotyping assays and SNaPshot assay were used to analyze 255 unrelated male blood samples who share the same surname Kong and 330 unrelated male blood samples obtained randomly. 17 Y-STRs were typed for the surname Kong population samples. The software Arlequin 3.5.1.2 and the program Network 4.6.1.1 were used for data statistical analysis.Results 13 haplogroups were observed. The highest haplogroup frequency in the two populations were O3a2c1a-M117 (21.57%, 14.85%). 196 haplotypes in Kong population deifned by 17 Y-STRs locus were obtained and the haplotype diversity was 0.9939. 14-12-25-28-19-15-12-19-12-11-12-22-12-11-14-10-19 is the typical haplotype. Median Joining algorithm and Mismatch Distribution were adopted to analyze the Y-STR haplotye under haplogroup O3-M122, and the result shows that there are two “central star” distribution. Conclusion Combined with Y-SNP and Y-STR analysis showed that the Kong population had experienced complicated exchanges and expansion or continued growth, which has more than one surname origin. Hence, its population genetic structure and historical differences have potential applications in forensic science.

We compared the RT-LAMP,LAMP and L-J for detecting MTB to provide the rapid diagnosis method for tu-berculosis.In this assay,NTM and other common respiratory bacteria were used to detect specificity,Mycobacterium tubercu-losis specific products were identified by restriction enzyme digestion.To detect sensitivity,we used RT-LAMP,LAMP and L-J to detect 100 cases sputum specimens from patients with tuberculosis and 22 cases control sputum specimens,the 10 times dilution of 1 ng/μL H37Rv standard strains was used to detect RT-LAMP limit.The results showed that the positive rate of RT-LAMP,LAMP and L-J were 100%,92%,88%.RT-LAMP and L-J,RT-LAMP and LAMP were statistically signifi-cant,RT-LAMP had 10 times higher sensitivity than LAMP,RT-LAMP not only to identify viable but also capable of detec-ting a single copy of MTB.So RT-LAMP is superior to LAMP and L-J and is practical for use in primary medical care institu-tion or peripheral laboratory.

OBJECTIVETo construct a stable cell line with permanent secretion of recombinant hepatitis B virus (HBV) vector, which express blasticidin resistant gene.

METHODSReplication-defective HBV vector, pCH-BsdR, which express blasticidin resistance gene was constructed by deleting the HBV genes and inserting the blasticidin resistance gene into the S region. The G418-resistant, the packaging signal deleted HBV helper plasmid, pcDNA3.1-CH3142, and the HBV vector pCH-BsdR were cotransfected into HepG2 cells. Cell clones were selected by the adding of both blasticidin and G418, then serial detection were done.

RESULTSAfter 36 cell clones were picked and expanded. Three cell clones were defined as the best. Quantity of their HBV DNA were 4.1 x 10(6), 3.6 x 10(6) and 1.2 x 10(6) copies/ml, respectively. Enveloped recombinant, but not wild type HBV were confirmed in the culture medium.

CONCLUSIONSThe stable cell lines can realize large preparation of recombinant HBV virions. This will contribute to the use of HBV vector for gene therapy and HBV susceptible cell lines screening.

Cell Line ; drug effects ; virology ; Clone Cells ; Drug Resistance ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Nucleosides ; pharmacology ; Transfection ; Virion ; genetics ; physiology ; Virus Assembly ; Virus Replication

Animals ; Carbon Tetrachloride ; adverse effects ; Chemical and Drug Induced Liver Injury, Chronic ; prevention & control ; Chronic Disease ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Mice ; Mice, Inbred BALB C

OBJECTIVETo explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein.

METHODSFull length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSThe mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium.

CONCLUSIONSIt has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Cell Line ; Genetic Engineering ; Genetic Vectors ; Hepatitis B virus ; genetics ; Mutation ; Plasmids ; RNA, Antisense ; physiology ; Transfection ; Viral Core Proteins ; physiology ; Virus Assembly ; Virus Replication

OBJECTIVETo cooperate with the study of HBV vector, hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles.

METHODSFree of packaging signal, HBV genome was inserted into plasmid pMEP4, which expresses the HBV structural proteins including core, pol and preS/S proteins. HepG2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 micrograms/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and hen selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSHygromycin-resistant HBV packaging cell line was generated, which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP, the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium.

CONCLUSIONAfter the packaging signal was deleted and transfected into HepG2 cell lines, the partial HBV genome lost its ability to form wild-type HBV, but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.

Cell Line ; Drug Resistance, Viral ; Genetic Vectors ; Genome, Viral ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Hygromycin B ; pharmacology ; Mutation ; Plasmids ; Retroviridae ; genetics ; Transfection ; Virus Assembly

BACKGROUNDTo explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein.

METHODSTwo kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR.

RESULTSThe mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP.

CONCLUSIONDominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

Cell Line, Tumor ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors ; Genome, Viral ; Hepatitis B Core Antigens ; biosynthesis ; physiology ; Hepatitis B virus ; genetics ; Humans ; Point Mutation ; Recombinant Fusion Proteins ; biosynthesis ; physiology ; Virus Replication

OBJECTIVETo evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.

METHODSA fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.

RESULTSThe HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.

CONCLUSIONIt is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

Cell Transformation, Viral ; Cells, Cultured ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; cytology ; virology ; Humans ; Liver ; cytology ; virology ; Recombinant Proteins ; genetics ; Transfection ; Virus Replication

Objective:Clinical study in the treatment of common bile duct stones combined with ERCP and EST.Methods:the October 2014-2017 year in February for hepatobiliary surgery inour hospital after ERCP combined with LC in the treatment of patients with cholecystolithiasis and choledocholithiasis in 158 cases.According to ERCP postoperative LC interval difference divided into ERCP definite operation LC group(104 cases) and ERCP Elective operation LC surgery group(54 cases).The definite operation for ERCP LC group is LC line 24h-72h,another is the patients were givenLC for 3 months after ERCP.The clinical efficacy,LC operation time,hemorrhage,the gallstone is discharged into the bile duct,recurrent cholecystitis,bile leakage,length of stay and medical fee were compared between the two groups (ERCP definite operation LC group of single hospitalization time;ERCP Elective operation LC surgery group of two hospital hours together),average ERCP times,medical expenses.Results:all of the two groups were cured after operation.length of sta is long and medical expensive which was selected a time to do (P＜0.05).Conclusion:ERCP definite operation of LC group in the treatment of common bile duct stones and gallbladder stones surgery is safe,less medical expenses,hospitalization time is short,patients recover quickly,the burden is small.