Objective To clone amastin coding genes from different strains of Leishmania parasites. Methods Using amastin cDNA sequence as the reference, dbEST data base established by National Center Biotechnology Information (NCBI), USA, was searched by BLAST tool. A 309 bp DNA fragment of Leishmania major was found and used as the probe for the screening of a DNA library. The amastin gene of Leishmania major Abdou was cloned and sequenced. Specific primers were designed and amastin genes for Leishmania mexicana WR972, Leishmania brizeliensis and Leishmania amazonensis joseph were amplified by polymerase chain reaction. Results The amastin genes from 4 strains of Leishmania parasites were cloned and sequenced. It was found that all 4 amastin genes contained unique open reading frame of 552 bp and encoded amastin protein of 183 amino acid residues. Conclusion The amastin genes of 4 strains of Leishmania parasites were successfully cloned.

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.

Articular cartilage repair is limited. Current treatments for cartilage defect are less satisfactory, and rarely restore full function or return the tissue to its native normal state. The rise of tissue engineering holds great promise for the generation of functional cartilage tissue substitutes. The history of cartilage tissue engineering and highlights the applications and advantages of various kinds of scaffolds in cartilage tissue engineering, such as native scaffolds, synthesis scaffolds, composite scaffolds and nanometer scaffolds had been introduced. But native scaffolds have weak strength and immunogenicity insufficiency, synthesis scaffolds degrade quickly, whose degrading products have cytotoxicity,which need further improvement. The application of superficial decoration overcomes the disadvantage of some scaffolds to an extend. Composite scaffolds possess the advantages of several scaffolds, it points out the direction of future scaffolds research. The development of Nanometer technique endows newly-synthesis scaffolds with nano-grade, thus it has some advantages and give a new way for the development of tissue engineering. At the end, the problems of these scaffolds, their trend of development and perspective studies were discussed.

Objective To construct a cDNA subtractive library of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus(MHBs t) with suppression subtractive hybridization technique for cloning genes associated with transactivation. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-Mt167 and pcDNA3.1(-) empty vectors, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small-size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by MHBs t was constructed successfully. The amplified library contained 94 positive clones. Colony PCR showed that these clones contained 200-800bp inserts. Sequence analysis was performed in 50 clones,and the full length sequences were obtained with bioinformatics method. 23 coding sequences were obtained in total, which consisted of 19 known and 4 unknown ones.Conclusions The obtained sequences may be target genes transactivated by MHBs t, among which some genes coding proteins may involve in cell cycle regulation, immune response and tumour genesis.

In order to screen genes transregulated by core protein of hepatitis C virus, cDNA microarray technology was employed to detect the gene expression change between HepG2 cells transfected with pcDNA3 1(-) core and the empty vector, respectively. Among 1152 genes, there were 95 genes with different expressions, of which 45 genes were upregulated and 50 genes were downregulated in HepG2 cells transfected with core protein expression plasmid. These genes transregulated by HCV core protein included human genes encoding proteins involved in cell proliferation, differentiation, apoptosis, signal transduction and immune regulation. Therefore, the results provided some new clues for further clarifying the molecular biology mechanism of pathogenesis and tumorigenesis of HCV core protein

Hepatitis C virus (HCV) non structure 3 (NS3) protein and hepatitis B virus (HBV) X protein expressing plasmids were constructed with the vector pcDNA3 1(-). The plasmids were transfected into HepG2 cells and the viral proteins expressed in HepG2 cells were identified using Western blotting methods. Then the two recombined plasmids were transfected into HepG2 cells and were cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter. The activity of CAT enzyme was detected by a CAT ELISA assay kit, which reflected the transactivating function of two proteins on SV40 early promoter. The findings indicated that the expression of two viral proteins were successfully detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV NS3 protein transactivated the CAT enzyme expressed at a value 3 5 fold higher than the control, while HBX protein transactivated at a value 4 4 times. It arrived at 8 5 times when transfected with two plamids simultaneously. The activating effect was increased in relation to the amount of plasmids. It was suggested that the two kinds of viral proteins had a transactivating effect on SV40 early promoter, and they acted synergistically. These results might contribute to explaining the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection

Hepatitis C virus (HCV) non structure 3 (NS3) gene was amplified from plasmid pBRTM3011 by employing polymerase chain reaction (PCR), and the amplified product was cloned into pcDNA3 1(-) vector. Then the recombinant plasmid pcDNA3 1(-) NS3 was transfected into HepG2 cells and was cotransfected into HepG2 cells with reporter plasmid pCAT3 promoter, respectively. HCV NS3 protein expressed in HepG2 cells was detected by reverse transcription PCR (RT PCR) and Western blotting method. The activity of CAT was detected by a ELISA kit, which reflected the transactivating function of HCV NS3 protein. The results showed that HepG2 cells transfected with pcDNA3 1(-) NS3 could express HCV NS3 protein. The expression of CAT in HepG2 cells transfected with the pcDNA3 1(-) NS3 was 4 6 fold higher than that of control plasmid. It was suggested that the recombinant plasmid pcDNA3 1(-) NS3 could be expressed in mammalian cell line, and had transactivating effect on SV40 early promoter

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.