Objective To explore the distinction between measles vaccine strain Shanghai-191 (S- 191) and wild-type strains in China at molecular level. Methods After having been amplified by RT-PCR, the nucleotides of S-191 was sequenced and analyzed with reference to the sequences obtained from Gen- Bank, which includes 24 whole genome sequences, 211 sequences of wild-type strains isolated from China and WHO reference strains. Results The sequence of S-191 strain had been submitted to GenBank. Phylo- genetic analysis of the COOH terminal 456 nucleotides coding for the nucleoprotein (N) indicated that most of the measles virus strains from China were members of clade H1. The genetic distances between the virus strains detected in a number of provinces and S-191 strain were 6.7% -8.2%. It is noteworthy that the muta- tion rate of non-coding regions was considerably higher than that of coding regions. The mutation rate of N gene was the highest among all of the coding regions. Conclusion This report shows that genotype H1 is widely distributed throughout the country. The circulation has no apparent geographic and temporal restric- tion. The evolution of measles virus should be closely followed.

Objective To investigate the influence of murine cytomegalovirus on the expansion of CD+CD25+ Foxp3+ regulatory T cell (Treg) and the activation of CD4+ CD25+Foxp3 - effector T cell (TE) in vivo. Methods Forty-two BALB/c mice were intraperitoneally inoculated with appropriate amount ofMCMV Smith strain for establishing the model of infection, another 42 mice served as mocked-infected con-trois. Day 28 post MCMV infection was determined to be the demarcation point of the acute and chronic in- fection based on the viral load of major visceral organs. On day 1,3, 7, 14, 28, 45 and 60 post infection, splenocytes were prepared by means of routine method. The proportions of CD4+CD25+ Foxp3+Treg and CD4+CD25+Foxp3- activated TE in T lymphocyte were measured by flow cytometry. Results The propor- tion of CD+CD25+ Foxp3+ T cells in T lymphocytes was persistently suppressed since day 7 post infection, and fell to the lowest level on day 28 post infection (P <0.01), then zoomed and reached the peak value on day 60 post infection (P < 0.05). CD4+CD25+ Foxp3 - TE proportion was up to the highest on day 3 post infection(P < 0. 01), then suppressed and in significantly lower level since day 45 post infection (P < 0.05). Treg/CD+TE ratio was in lower level on day 3 to 14 post infection(P <0.05) ; but on day 45 and day 60 post infection Treg/CD+ TE ratio was markedly increased (P < 0.05). Conclusion MCMV infec- tion can increase the CD+CD25+Foxp3+ Treg proportion, and inhibit CD4+T cells activation in chronic in- fection phase, which is likely to suppress the function of antiviral immunity in the infected host to cause a persistent latent infection.

Objective To identify the protein interacting with hepatitis E virus(HEV) recombi-nant capsomeric particles(P239). Methods Protein interacting with HEV was analyzed by the pull-down, MALDI-TOF-MS, co-immunoprecipitation (Co-IP) and CONFOCAL. Results A protein interacting with HEV recombinant particle (P239) was identified as HSP90 by MALDI-TOF-MS. The interaction between HSP90 and P239 was further confirmed by Co-IP. The protein level and localization of HSP90 and P239 in HepG2 were detected. The total quantity of HSP90 didn't change, and the movement of HSP90 from plasma membrane to perinuclei region with P239 was observed. Conclusion HSP90 may play an important role in the trafficking of P239. It suggests that HSP90 participate in the transportation of HEV after infection, which may contribute to the prevention and control of the disease.

Objective To establish a stable cell line secreting human IgE Cε2-4 protein, and in-vestigate the binding capacity of receptor FcεR Ⅰ Methods The E24 gene was derived from SKO-O07 cell line, and was then cloned into pcDNA3.1 (+) (signal peptides were synthesized and fused at the 5'-end of E24 gene) or pCMV-L vector. After transient transfection into 293T cell, the secreted F24 protein was ana-lyzed by sandwich ELISA. The best vector was chosen to be transfected into CHO cells with LipofectAMI-NETM 2000 reagent. After being selected by G418 and subcloned three times by limited-dilution method, two stable cell lines were established. E24 gene was amplified by RT-PCR, and the E24 protein in the superna-tant was identified by ELISA. Besides, the binding capacity of FceR ⅠⅡ was analyzed by flow cytometry method. Results Three mammalian expression vector SP-E24-F3. 1, SP lI-E24-P3.1 and E24-PL were constructed and transient transfected to 293T cells. The output of E24 protein in the supernatant were 19.1, 19.4 and 8.7 μg/ml, respectively. Then the vector SP IX-E24-P3.1 was transfected into CHO cells. Final-ly, two single clones secreting E24 protein were stably obtained. The output of E24 were all at least 25 μg/ml. RT-PCR could detect the E24 gene from one of the two clones. Furthermore, flow cytometry results showed that E24 could bind the receptor in a dose-dependent manner. Conclusion Two stable cell line se- creting E24 protein were obtained, while E24 could specifically bind FcεR Ⅰ.

Objective To investigate the association of HLA-DRB1 alleles in Han population of Shanxi childrcn with nephrotic syndrome of non-IgA mesangial proliferative glomerulonephritis (MsPGN). Methods HLA-DRB1 was performed by polymerase chain reaction-sequence specific primers technique, and twenty patients with nephrotic syndrome of non-IgA MsPGN were detected. Results Analysis of the fre- quencies of specific at the HLA-DRB1 loci revealed significantly higher frequencies of HLA-DRB1 * 11 al- leles among the nephrotic syndrome patients of non-IgA MsPGN comparing with controls (22. 50% vs 8.33%, x2= 9. 544, P = 0.002, CI = 1. 674-9.995, RR = 4.09). Nine patients with HLA-DRB1 * 11 all accompanied hematuria, hypertension or short renal insufficiency. Conclusion The results suggested that HLA-DRB1 * 11 alleles contribute to genetic susceptibility to nephritic syndrome of non-IgA MsPGN. The pa- tients with HLA-DRB1 *11 easy accompanied hematuria, hypertension or short renal insufficiency.

Objective To explore a new peptide-based approach independent of HLA to generate antigen-specific CD+ CD8+T cells. Methods Peripheral blood mononuclear cells(PBMC) were stimula- ted for 6 h with IE-1 peptide pool. Then the activated IFN-γsecreting ceils were tested by immunomagnetic selection. And the selected cells were cultured with radio-inactivated PBMC in medium with 100 IU/ml IL-2 for 4 weeks. Results The generated T cell lines consisted of IE-1 specific CD4+ T (6.88%) and CD8+ T cells 92.99%, which demonstrated antigen-specific killing and cytokine secretion. Conclusion T ceils can be proliferated with this new procedure, and maintain its phenotype and antigen-specific function.

Objective To investigate the role of NF-KB binding element in regulation of NOD2. Methods Promoter region of NOD2 containing the NF-κB binding site was amplified by PCR from human genome DNA and correctly connected to the vector pEGFP-N3 which had been cut out promoter by restriction enzyme to obtain the GFP expression vector driven by human NOD2 gene promoter. The constructed plasmids were transiently transferred into cell line HeLa by LipofectAMINETM2000 and the GFP expression was ob- served by the inversion fluorescence microscope. The NF-κB binding site in the constructed vector pEGFP- N3-NOD2wt was deleted by the QuikChange site-directed mutagenesis kit. The recombinant plasmid mpEG- FP-N3-NOD2 was transiently transferred into cell line HeLa by LipofectAMINETM2000, and the GFP expres- sion was observed by the inversion fluorescence microscope. Results The constructed pEGFP-N3-NOD2wt plasmids and mpEGFP-N3-NOD2 were the same as the design confirmed by restriction digestion and se- quence analysis. The results of the cell transient transfection indicated that different strength of GEP ex- pressed by recombinant plasmids in HeLa cells could be observed. The GFP expression of constructed mpEGFP-N3-NOD2 was lower than that of pEGFP-N3-NOD2wt. Conclusion The GFP expression vector driven by human NOD2 gene promoter which contains the NF-κB binding site, and the site deleted plasmid were successfully constructed. The GFP expression of recombinant plasmid mpEGFP-N3-NOD2, deletion of the NF-KB binding site, was obviously weaken in HeLa. The results indicate that NF-KB binding element may play a positive role in regulation of NOD2 gene, which establishes favourable bases for further study on the mechanism of NOD2 gene expression and regulation.

Objective To compare the infectivity between laboratory adapted human inununodefi- ciency virus(HIV-1) and primary HIV-1 isolates for different mucosal epithelial cell lines. Methods Mu-cosal epithelial cells Caco-2, T-84, HeLa and lymphocyte MT-4 were infected with laboratory adapted HIV-1 SF33 and 2 primary HIV-1 isolates (02010561, 02010141). Culture supernatant and cells were collected respectively on 3-4 days interval after virus inoculation. The former was tested for HIV-1 antigen P24 level and viral load, and the latter was tested for total viral DNA and integrated viral DNA. Results All 3 virus strains could infect MT-4 cells and integrate into their genome. Only HIV-1 SF33 could infect Caco-2 cells but could not integrate into their genomic DNA. Both HIV-1 SF33 and 02010561 infected HeLa cells but only integration of HIV-1 SF33 was detected. All the 3 HIV-1 strains infected T-84 cells but only the integra-tion of HIV-1 SF33 and 02010141 was observed. Conclusion Although laboratory adapted and primary HIV-1 strains are able to infect human mucosal epithelial cell lines, transient or productive infection estab-lished in different mucosal epithelial cells is dependent on the character of cells and virus strains.

Objective To obtain better insights into transmission dynamics of macrolide resistance genes between human and animal Enterococcus strains.Methods The antimicrobial susceptibility to 8 anti-bioties of 52 Enterococci isolated from animal and 55 Enterococci isolated from human was determined.PCR was used to detect the macrolide resistance genes ermB and mefA,tetracycline resistance genes tetM,and the integrase gene int of Tnl545 of the total 107 strains.Forty-nine ermB positive strains were chosen to be se-quenced.Filter mating experiments were taken.Results The resistance rate to erythromycin were 89.09% and 80.77%for isolates from human and animal:and resistance rate to tetracycline were 80.00%and 67.3l%for isolates from human and animal.respectively.All isolated Enterococci strains were found sensi-tive to vancomycin ermB was detected in 61.82% human Enterococci and 53.85% porcine ones.Identical er-mB gene sequences were found in animal and human Enterococci.Transfer of the ermB gene from porcine E.faecalis to human E.feacalis was successful.and the transfer frequency is 1.2×10-5.Conclusion En-terococci have a high resistance rate to erythromycin and some other antibio tics,especially in pediatric iso-lates:but still very sensitive to glycopeptide.ermB was the predominant genes for macrolide and tetracy-cline.Identical ermB gene sequences were present in animal and human Enterococci and that transfer of the ermB gene from porcine E.faecalis to human E.faecalis and vice versa is possible.but probably occurs at a low frequency.

Objective To establish antigen capture ELISA methed to detect Kaposi's sarcoma-as-sociated herpesvirus(KSHV)antigen,and to evaluate its feasibility for clinical application.Methods The BALB/c mice and New Zealand white rabbits were injected with purified recombinant KSHV gpK8.1 proteins to prepare the monoclonal antibody(McAb)and polyclonal antibody(PcAb)anti-gpK8.1,respectively.A new antigen capture ELISA method was established for KSHV antigen detection.The detection reproducibili-ty as well as the sensitivity and specificity of this new assay were determined by the optimization test,which antibody pairs were analyzed to choose the best coating antibody and detecting antibody.The 3 KSHV posi-tive patients sera and 257 patients sera from sexually transmitted disease,cancers or gynecological diseases were detected with this assay to evaluate its value for clinical application.Results When the McAb as coat-ing antibody at concentration of 5 μg/ml and PcAb as detecting antibody at concentration of 1.6μg/ml were selected,the highest P/N value could be obtained.The sensitive analysis of this test could detect recombi-nant KSHV gpK8.1 antigen of 31.28 ng/ml.Meanwhile,it is highly specific to detect KSHV antigen with-out cross reaction to Epstein-Barr vims(EBV),herpes simplex virus(HSV)-1 or HSV-2.All of three KSHV-positive sera and 4 sera from 257 clinical samples were positive with this new assay.which indicated that it could be used for capturing KSHV antigen.Conclusion A sensitive and specific McAb-based anti-gen capture ELISA method to detect KSHV antigen were established successfully.It is of great potential val-ue to develop reagent for KSHV clinical serologic dingnosis.