Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequences of the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. One of them was confirmed to be a new gene without homology with known genes in this database.Then, electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene, in conjunction with Kozak rule and the existence of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene with mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named TAHCCP1.The nucleotide sequence of the new gene and its corresponding protein-encoding amino acid, which was 2 001nt and composing 667aa, have been determined. The sequence of the TAHCCP1 gene has been registered in GenBank with its accession number AY038359. Conclusion TAHCCP1 gene transactivated by HCV core protein was cloned and identified successfully by a combination of molecular biological technology and bioinformatics technique. The results are expected to pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapies for chronic hepatitis C.

Objective To screen the genes differentially expressed in gene expression in human lymphoma cell line Jurkat cells treated with phosphonoformate (PFA). Methods cDNA microarray technique was employed to detect the mRNA expressed in Jurkat cells treated with PFA or 0.9 percent sodium chloride, respectively. Results Among 1 152 genes there were 94 genes with different expression, of which 38 genes were upregulated and 56 genes were downregulated in Jurkat cells treated with PFA compared with those treated with 0.9 percent sodium chloride. Conclusions cDNA microarray technique was successfully used to screen the genes with different expression in Jurkat cells treated with PFA, and the results brought some new clues for the study of the immune regulation mechanism of PFA.

Objective To screen and clone the target genes transactivated by nonstructural protein 5A (NS5A) of hepatitis C virus (HCV). Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-NS5A and pcDNA3.1(-) empty vector, respectively. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. The new gene with no homology with known genes in this database was confirmed, and electric polymerase chain reaction was conducted for cloning the full-length DNA of the new gene and in conjunction with Kozak rule and the terminus of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene from mRNA of HepG2 cell as the template. The coding sequence of the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named as NS5ATP3. The nucleotide sequence of the NS5ATP3 gene and its corresponding amino acid have been determined, which contained 1 572nt and 524aa. The sequence of the NS5ATP3 gene was deposited into GenBank, with the accession number AF529364. Conclusions NS5ATP3 gene transactivated by HCV NS5A protein was cloned and identified successfully by combining molecular biological technology and bioinformatics technique. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HCV NA5A protein and the development of new therapy for chronic hepatitis C.

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with phosphonoformate (PFA), and to clone genes associated with its immune regulation. Methods Using suppression subtractive hybridization (SSH) technique, the mRNA was isolated from Jurkat cells treated with phosphonoformate or 0 9 percent sodium chloride, respectively, and then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. The tester cDNA, which was hybridized with driver cDNA twice and amplified by nested polymerase chain reaction (PCR) twice, was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The clones, which were selected randomly, were amplified by PCR, and then they were sequenced and analyzed in GenBank with Blast search. Results The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA was constructed successfully. The amplified library contained 46 positive clones, which contained 200- 1 000 bp of inserts. Fourteen clones were analyzed by sequencing and bioinformatics, which were identified as eleven known genes and three genes with unknown function. Conclusions The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA using SSH technique was constructed successfully, which brought some new clues for the study of the immune regulation mechanism of PFA

Objective To explore the influence of XTP4 on genomic expression profile of hepatocyte through screening and cloning of genes trans-regulated by XTP4-expressing plasmid. Methods The expressive vector pcDNA3.1(-)-XTP4 was constructed by routine molecular biological methods, and suppression subtractive hybridization (SSH) method was employed to detect the mRNA differentially expressed by the HepG2 cells transfected with pcDNA3.1(-)-XTP4 and pcDNA3.1(-), respectively, using lipofectamine. The twice enriched PCR products were subcloned into T/A vectors to set up the subtractive library. Amplification of the library was carried out in E. coli strain JM109. The screened cDNA were sequenced and analyzed in GenBank with Blast search after PCR. Results The amplified subtractive library containd 21 positive clones. Colony PCR analysis showed that there were 16 clones containing 200-1000bp inserts. Sequence analysis was performed and 9 kinds of encoding sequences were achieved. These genes trans-regulated by XTP4 protein involved in hepatic fibrogenesis, tumorgenesis, mitochondrial function, and cell growth regulation. Conclusions The findings obtained by SSH provide significant data for a preliminary understanding of the biological function of a new identified gene-XTP4. These results will pave the way for the study of the molecular mechanism of the transactivating effects of HBxAg and the development of new therapy for chronic hepatitis B.

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with glycyrrhizin (GL), and to clone genes associated with its immunological regulation, and to further elucidate the molecular immune mechanism of GL. Methods The mRNA was isolated from Jurkat cells treated with either GL or 0.9 percent sodium chloride as a control, then cDNA was synthesized. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The cDNA was sequenced and analyzed in GenBank with Blast search after the amplification of the subtractive library by PCR. Results The amplified library contained 28 positive clones. Colony PCR analysis showed that there were 22 clones containing 200-1 000 bp inserts. Sequence analysis was performed, and the full length sequences were obtained with bioinformatics method. Altogether 11 kinds of encoding sequences were achieved, including interleukin-12, interleukin-18, and thymosin ?1, etc. Conclusions A subtractive cDNA library of genes differentially expressed in Jurkat cells treated with GL using SSH technique was constructed successfully, and it might give some new clues for the study of the immune regulation mechanism of GL.

A retrospective study and cause analysis on the emergency rescue and medical service during the 2015 Tianjin harbor explosion accident,probed into the defects of the contingency plan of the hospital.Problems found include impracticability or deficiency of the plan,lack of safety awareness,and absence of preventive measures.A cause analysis was thus made to propose the following improvements on the contingency plan:1.Development of specific standard operation procedures(SOPs)for medical emergency response,including SOP for emergency response organization and command,that for the procedure and responsibility of massive injuries examination,and that for emergency rescue;2. Development of the plan for data statistics,that for volunteer management,that for media communications and that for donation management.

Objective To investigate the transactivating effect of hepatitis C virus(HCV)core protein on inducible nitric oxide synthase(iNOS)gene promoter and the molecular biological mecha- nisms of HCV pathogenesis.Methods Polymerase chain reaction(PCR)technique was employed to amplify the sequence of iNOS promoter by using HepG2 genomic DNA as template,and the product was cloned into pGEM-T vector.The iNOSp gene was cut from T-iNOSp by KpnⅠand XhoⅠ,and then was cloned into pCAT3-Basic,the constructed vector was named as pCAT3-iNOSp,pCAT3-iN- OSp was transfected into the LO_2 cell line.LO_2 cell was also cotransfected with pcDNA3.1(-)-core and pCAT3-iNOSp by FuGENE 6 transfection reagents.The LO_2 cells transfected with pCAT3-Basic was used as negative control.The activity of CAT in LO_2 cells was detected by an ELISA kit after 48 hours,which reflected the transactivating function of HCV core protein to iNOS gene promoter.Re- sults The expressive vector pcDNA3.1(-)-core and report vector pCAT3-iNOSp had been construc- ted and confirmed by restriction enzyme digestion and sequencing.The expression of CAT in LO_2 cells transfected with pCAT3-iNOSp and peDNA3,1(-)-core was 11 times as higher as that of pCAT3-bas- ic,and 6 times as higher as that of pCAT3-iNOSp.Conclusion It is suggested that HCV core protein can transactivate iNOS gene promoter.