Protein protein binding is the basis of virus and host cell interactions. With the application of technology of studying of protein interactions, more knowledge of replication and pathogenesis of hepatitis C virus (HCV) could be acquired. Non structure protein NS5A is one of the important regulatory factors in virus replication , transcription and signal transduction, but there are controversy in effect on HCV pathogenesis and resistance to interferon ?(IFN?). In order to describe the relationship between NS5A and host proteins, we use yeast two hybrid system 3 to screen the gene encoding proteins that could interact with NS5A from human hepatocyte library. Thirty five clones were obtained including apo A1, apo A2, apo B100, haplotype mitochondrion complete genome, phosphatidic acid phosphatase type 2B, albumin similar to tumor endothelial marker 5 precursor, matrix metalloproteinase 14, and three of the Homo sapiens hypothetical proteins. The study paved a way for further studies on the pathogenesis of HCV NS5A

To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then 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. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

To clone the genes of hepatocyte protein interacting with hepatitis C virus NS3 protein, "bait" plasmids of hepatitis C virus NS3 were constructed. After verifying that hepatitis C virus NS3 protein could be steadily expressed in AH109 yeast strain, yeast two hybrid assay was berformed by mating AH109 with Y187 that pre transformed with liver cDNA library plasmids pACT2, and the diploidy yeast cells were plated on quadruple dropout (QDO) medium and assayed for X ? gal activity. Nineteen yeast colonies that could grow on QDO and had ? gal activity were obtained, then the library plasmids were extracted and sequenced. The gene sequences from the 19 positive colonies were aligned with the genes deposited in GenBank. It was found hepatitis C virus NS3 protein could interact with some proteins which have different functions.

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

To investigate the potential role of hepatitis B virus(HBV) preS1 protein in mediating HBV adhesion to liver cell, we prepared recombinant proteins of HBV preS1 in yeast. PCR was performed to amplify the gene of HBV preS1 from the plasmid pCP10/HBV ayw subtype containing the whole fragment of HBV and the PCR product was cloned into pGEM T vector. The gene of HBV preS1 was cut from pGEM T vector and cloned into yeast expression plasmid pGBKT7, the pGBKT7 plamids containing preSl were transformed into yeast cell AH109. The yeast protein was isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting. The results showed that the presence of HBV presl proteins in yeast cells was confirmed by Western slot analysis. the molecular weight of the expressed product was about 30000 Da. The findings indicated that HBV preS1 was successfully expressed in yeast system.

Objective To construct recombinant human Bcl-2 adenoviral vector. Methods Sense human Bcl-2 cDNA fragment from plasimid pcDNA3 1/bcl-2 was cloned into adenovirus shuttle vector pAdCMV. pAdCMV/bcl-2 and plasmid pJM17 were cotransfected into 293 cells with lipofectamine 2000. Replication defective adenovirus was reproduced and purified. Adenoviral virus DNA was detected with polymerase chain reaction(PCR), and plaque assay was performed to determine the titer of virus. Results Recombinant adenovirus containing bcl-2 target gene was obtained. The titer of virus was 2 0?10 10 pfu/ml. Conclusion High titer of adenovirus containing target gene was successfully obtained by recombinant adenovirus vector.

In view of the current conditions and reasons about the reform of medical and health system in China,the paper expounds the feasibility of authorized operation with state-owned assets of public hospital and the concrete operational pattern from the angle of hospital operational mechanism, which proposes a new idea about how to effectively solve the contradiction between the limited investment on state-owned health resources and the growing demands on medical health.

We previously showed that adenvirus-mediated lymphotactin (Ltn) gene transfer in vivo could improve the an-titumor efficacy of cytosine deaminase (CD) gene therapy significantly. In the precent study, we investigated the im-munological mechanisms involved in the enhanced antitumor efficacy. Upregulation of CD80 and CD54 on murine CT26 colon carcinoma cells was observed after combined transfection with adenovirus encoding CD (AdCD) and adenovirus encoding murine Ltn ( AdLtn) followed by administration of 5-PC in vitro. IL-2 and IFN-? level secreted by splenocytes increased significantly after the combination therapy. In vivo depletion analysis showed that both CD4~+ and CD8~+ T cells participated in the antitumor effect of the host with CD8~+ T cells being the main T cell subset responsible for the enhanced antitumor immune response. These data suggested that increased irnmunogenicity and efficient induction of antitumor immunity of the host might contribute to the enhanced antitumor effects of the combined Ltn and CD suicide therapy.

Interleukin 18 (IL 18) is emerging as a powerful, pleiotropic cytokine involved in determining the polarization of T cell responses. To identify the effect of IL 18 on hepatitis B virus, mouse IL 18 gene was transferred and expressed in 2.2.15 cells. Meanwhile, the inhibition of HBsAg, HBeAg and HBV DNA was investigated. Reverse transcription polymerase chain reaction (RT PCR) was used to amplify the mouse IL 18 from spleen cell of Balb/c mouse challenged with lipopolysaccharide (LPS) and phytahematoagglutinin (PHA), and reconstruct plasmid pLXSN IL18 with the retroviral vector pLXSN. Both pLXSN IL18 and pLXSN were transfected into PA317 cells and then 2.2.15 cells were infected by using the supernatant containing pseudovirus released by PA317. HBsAg and HBeAg were detected by ELISA and the HBV DNA was determined by quantitative PCR. The 580bp of mIL 18 was cloned into retroviral vector pLXSN. The pseudovirus contained in the supernatant of transfected PA317 cells was identified by RT PCR. When the pseudovirus was infected into 2.2.15 cells and incubated for 3, 5, 7, 14 days, the P/N value of HBsAg and HBeAg decreased gradually and became negative on day 14. The copies of HBV DNA reduced markedly. The results indicate that mIL 18 gene expressed in 2.2.15 cells can significantly inhibit the replication expression of HBV and may be used as a potential agent for gene therapy against HBV infection.

OBJECTIVE:To observe the effects of parecoxib preemptive analgesia combined with postoperative epidural analgesia on hip replacement and immune function. METHODS:Totally 86 patients undergoing unilateral hip replacement selected from Xining Third People's Hospital during Jan. 2014-Jun. 2016 were divided into observation group and control group according to random num-ber table,with 43 cases in each group. Two groups underwent unilateral hip replacement under spinal epidural anesthesia. Observation group was given Parecoxib sodium for injection 40 mg intravenously 30 min before the end of surgery. Control group was given con-stant volume of normal saline intravenously. Both groups were given epidural analgesia pump(Ropivacaine hydrochloride injection 150 mg+Ondansetron hydrochloride injection 16 mg+Dezocine injection 10 mg,added into normal saline to 100 mL)after surgery with dripping speed of 2 mL/h and patient-controlled time of 15 min. The hemodynamic indexes,analgesic effect,T lymphocyte subsets and NK cells levels were compared between 2 groups at different time points as well as the occurrence of ADR after surgery. RE-SULTS:Two patients of observation group and one patient of control group withdrew from the study. Before surgery,there was no sta-tistical significance in MAP and HR between 2 groups(P>0.05);12,24 h after surgery,MAP and HR of control group were signifi-cantly increased and higher than observation group,with statistical significance(P<0.05). There was no statistical significance in MAP and HR of observation group at different time points compared to before treatment;there was no statistical significance in MAP and HR between 2 groups immediately after surgery,48 h after surgery(P>0.05). There was no statistical significance in VAS score between 2 groups immediately after surgery(P>0.05);6,12,24,48 h after surgery,VAS scores of observation group were signifi-cantly lower than those of control group,with statistical significance(P<0.05). There was no statistical significance in CD3+,CD4+, CD4+/CD8+and NK cells levels between 2 groups before surgery(P>0.05). CD3+,CD4+and NK cells levels of 2 groups immediately after surgery,CD3+,CD4+,CD4+/CD8+ and NK cells levels of 2 groups at 12,24 h after surgery,CD3+and CD4+of control group at 48 h after surgery all changed significantly;CD3+,CD4+,CD4+/CD8+and NK cells levels of observation group at 12,24 h after sur-gery were significantly higher than those of control group,with statistical significantly(P<0.05). There was no statistical significance in the incidence of ADR between 2 groups(P>0.05). CONCLUSIONS:The parecoxib preemptive analgesia combined with postopera-tive epidural analgesia shows good analgesic effect for hip replacement patients,keeps their hemodynamics stable and protects immune function to certain extent with good safety.