Humans ; Medicine, Chinese Traditional ; Respiratory Tract Diseases ; prevention & control ; therapy ; virology ; Virus Diseases ; prevention & control ; therapy

Asthma ; psychology ; therapy ; Humans ; Integrative Medicine ; Stress, Psychological

Objective To evaluate the influence of expression of pre S1 protein on the genomic expression of hepatitis B virus (HBV) infected hepatocyte with microarray. Methods The differentially expressed genes between the hepatoblastoma cell line HepG2 transfected by pcDNA3.1(-) and pcDNA3.1(-)-preS1, were respectively compared by cDNA microarray technique. The HBV pre-S1 coding DNA fragment was amplified with polymerase chain reaction (PCR) technique by using G376-7 plasmid DNA containing the full length of HBV genome as the template. The expressive vector of pcDNA3.1-preS1 was constructed by routine molecular biological methods. HepG2 cells were transfected by pcDNA3.1(-) and pcDNA3.1-preS1, respectively, using FuGENE6 Transfection Reagent. The total RNA was isolated and reversely transcribed. Results The cDNAs were subjected for microarray screening with 1152 cDNA probes. From the scanning results, it was found that 30 genes were up-regulated and 38 genes were down-regulated by pre-S1 protein of HBV. Conclusion The expression of pre-S1 protein affected the genomic expression spectrum of HBV infected hepatocyte(HepG2 cell line).

Asthma ; drug therapy ; metabolism ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Inflammation Mediators ; metabolism ; Phytotherapy ; Signal Transduction

It was found that pre-X region was present in five clones of hepatitis B virus (HBV) genome from 2 patients with chronic HBV infection. The open reading frame (ORF) consisted of 168 base pair (bp), and could be transslated with the X gene in frame. To investigate the activity of pre-X gene promoter, promoter DNA sequence was amplified from HBV-DNA by polymerase chain reaction (PCR), The amplified product was cloned into pCAT3 vector, The HepG2 cells were transfected by pCAT3-Pre-X-p. The CAT activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit. It was found that pCAT3-Pre-X-p had higher activity of CAT, which was 3.5 fold over that of pCAT3-promoter. This result implicated that pCAT3-Pre-X-p possessed promoter activity.

To investigate the promoter activity of pre-pre-S gene, which was found in five clones of hepatitis B virus (HBV) genome from 2 patients with chronic HBV infection, promoter DNA sequence was amplified from HBV-DNA by polymerase chain reaction (PCR).The amplified product was cloned into pCAT3 reporter vector. The HepG2 cells were transfected by pCAT3-pre-pre-S-p. The CAT activity was detected by an enzyme-linked immunosorbent assay (ELISA) kit. It was found that pCAT3-pre-pre-S-p had higher activity of CAT which was 10 fold over that of pCAT3-basic. This result implicated that pCAT3-pre-pre-S-p had promoter activity.

Objective T7 cDNA phage display system and bioinformatics methods were employed to find the binding protein to the PreS1 protein of hepatitis B virus (HBV). Methods PreS1 protein was coated in ELISA plate as the target protein, and then T7 cDNA library phage display system was used to scan the binding protein or peptide. A piece of cDNA was found to have the function to bind the PreS1 protein, and the product was named as PreS1 binding protein (PreS1BP). Using BLAST in GenBank, the amino acid sequence of PreS1BP was compared in the protein sequence database. Results The amino acid sequence of PreS1BP was identified as a piece of glioma tumor suppressor candidate region gene 2 (GLTSCR2), and the length of cDNA of PreS1BP was proved to be 1436 nt. The gene was located at chromosome 19q arm (19q13.3) with a length of 11445 base pair between 10403483 and 10414989, containing 13 exons and 12 introns. Conclusion HBV PreS1BP gene could be obtained by T7 cDNA phage display system in combination with bioinformatics methods.

Polymerase chain reaction was employed to amplify the HBV X gene from plasmid pCP10, and the product was cloned into pVR1012, then transfected HepG2 cells and cotransfected HepG2 cells with reporter plasmid pSV lacZ HBx protein produced by HepG2 cells was measured by ELISA method The activity of ? galactosidase was measured by a kit, which reflected the transactivating function of HBx protein The results showed that HepG2 cells transfected by pVR1012 X could express HBx protein The expression of ? galactosidase in HepG2 cells transfected by the pVR1012 X was 3 2 fold higher as that of control plasmid It is suggested that the recombinant plasmid pVR1012 X can be expressed in mammalian cell line, and has transactivating effect on SV40 early promoter

A set of specific primers was synthesized according to DNA sequence of HBV found in China, the reverse transcriptase (RT) region in polymerase gene was amplified by PCR method from the serum of 3 patients with chronic HBV infection, and then the PCR products were subcloned into pGEM Teasy vectors. 13 clones were sequenced. Sequence comparison of the selected clones was made to look for the difference. After being compared, 13 sequences of RT were found different. Besides 2 clones with long sequence deletion, the different rate of RT coding nucleic acid sequences, RT and HBsAg amino acid sequences of the 11 clones is 5 1%, 4 9% and 7 5%, respectively. Many mutation types, including point substitution and deletion mutation, were found in this region. There is quasispecies population and defective HBV genome in patients with chronic HBV infection.

Objective To construct plasmid pVR1012 M as nuclei acid vaccine for hepatitis B,was constructed to immunize mice with or without plasmid pcDNA 3.1 - IL 18 to identify the effect of Interleukin 18(IL 18). Methods Polymerase chain reaction method was used to amplify the PreS2 and S region of HBV and reconstruct plasmid pVR1012 M as nuclei acid vaccine. Plasmid pcDNA 3.1 - IL 18 was used as a co stimulator. Twenty five Balb/c mice were divided into 3 groups, group 1 immunized with 100 ?g plasmid pVR1012 group 2 pVR1012 M,Group 3,pVR1012 M with pcDNA 3.1 - IL 18, respectively, every 2 weeks for 3 times. Anti HBs were detected in serum 2 weeks after each injection. Lactated ehydrogenase (LDH) cytotoxicity assay was done to analyze the cytotoxic T lymphocytes funciton. Results The positive rate and the antibody titer of serum from mice injected pVR1012 M increasing gradually with the increasing frequency of inoculation, while those from mice injected pVR1012 M and pcDNA 3.1 - IL 18(joint group) were lower than those injected with pVR1012 M alone (Difference after 3rd inoculation was significant, P