Objective To study the influence of the modification of the special neutralizing epitopes of the HIV-1 envelope (Env) on its assembly of functional pseudovirus and neutralizing activity. Methods Site-directed mutations were performed using cycling mutagenesis and selection of mutants with Dpn I . With this method, the 2G12 and 2F5 neutralizing epitopes were integrated into Env of subtype BC which was without the two epitopes, then the capability of forming pseudovirus and the neutralizing activity against 2G12 and 2F5 were compared with pre-modified Env. Results The special Env neutralizing epitopes of five HIV pseudovirus (BC02, BC03, BC04, BC05 and BC12) were modified. Among the five pseudovirus, BC04 and BC12 pseudovirus can't be formed after the 2G12 epitope was modified, whilst the BC02, BC03 and BC05 pseudovirus can be formed after the 2G12 and 2F5 epitopes were added, and there was no variation of the pseudovirus titer; On the aspect of neutralizing activity, BG03 pseudovirus against 2G12 and 2F5 was enhanced, BC02 and BC05 pseudovirus against 2F5 was enhanced while which against 2G12 was not changed. Conclusion The modification of 2G12 epitope influences the forming of pseudovirus and the addition of neutralizing epitopes can enhance the neutralizing activity of pseudovirus, which offers new approach for the optimization of HIV immunogen.

The serum samples and corresponding cervical swabs were collected fi'om 50 women with genital warts from Tianjin city,China.The neutralizing antibodies against HPV-16,-18,-58,-45,-6 and-11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV.The results revealed that 36%(18/50)of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560,of which 22%(11/50),12%(6/50),10%(5/50),4%(2/50),4%(2/50)and 2%(1/50)were against HPVs-6,-16,-18,-58,.45 and-11,respectively.Additionally,60%(30/50)of samples were HPV DNA-positive,in which the most common types detected were HPV-68(18%),HPV-16(14%),HPV-58(12%),HPV-33(8%)and HPV-6,HPV-11,HPV-18 and HPV-52(6% each).The concordance between HPV DNA and corresponding neutralizing antibodies was 56%(28/50)with a significant difference(P＜0.05).The full-length sequences of five HPV types(HPV-42,-52,-53,-58 and-68)were determined and exhibited 98%-100% identities with their reported genomes.The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.

Wild Cordyceps sinensis strain was isolated from Mt. Batang in Yush District of Qinghai Province. Its optimal culture conditions were 1.5% Glucose, 1% malt extract, 1 peptone, 0.5% yeasts extract, pH6.0, temperature 23℃, 5—10% inoculum, vibration velocity 80—90r/min.

Objective To study the influence of site-directed deglycosylation of the HIV-1 envelope (Env) on its immunogenicity and assembly of functional pseudovirus. Methods Site-directed deglycosylation were performed using cycling mutagenesis and selection of mutants with DpnⅠ. Single-cycle infection assay was employed to analyze the effect of the mutations on the ability of functional pseudovirus assembly. The influence of deglycosylations on the immunodeficiency of Env was evaluated using pseudovirusbased neutralization assay and ELISPOT assay. Results Mutant N197Q induced higher neutralization activities for both pseudoviruses, but lower Env-specific T-cell response. And N197Q rendered the Env to lose the ability of functional pseudovirus assembly. Mutant G2 induced higher neutralization activities for pseudovirus 74-2 but lower for pseudovirus Wt, and had almost no influence on Env-specific T-cell response and functional pseudovirus forming. Conclusion The site-directed deglycosylation of the HIV-1 Env affects the pseudovirus forming and its immunogenicity.

Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.

Objective Construct the reasonably effective performance assessment mode of clinical departments,which is suitable for the continuous development of modern hospital.Method Use of balanced scorecard (BSC) principle,using the analytic hierarchy process (AHP) and Rank sum ratio method (RSR)to establish a performance assessment system. Results Constructed the performance assessment mode of clinical departments,which can be dynamically adjusted based on management objectives strategy.The mode takes the BSC as the baseline,the strategy of hospital development as the guide,performance assessment index system as the key element. Conclusion The performance assessment mode of clinical departments,base on BSC,achieve the strategic performance management,and is conducive to the hospital for continuous improvement of medical care quality,in line with the long-term strategic needs of the hospital.

Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.

Objective To establish the mouse model for human papillomavirus types 16, 45 and 58 by the corresponding pseudovirions. Methods The 293FT cells were co-transfected with eodon-modified HPV eapsids genes together with a reporter plasmid containing the luciferase gene. The cells were collected and lysed, then the pseudovirus was collected and the titration was performed. The mouse was subcutaneous-ly injected with Depo-Provera. After 4 d and intravaginally injected with nonoxynol-9, and 6 h later pseud-oviruses were inoculated in intravaginal. After 7 d, the mouse was instilled luciferin substrate intravaginally, and the expression level of the lueiferase gene was detected by the in vivo Imaging System (IVIS). Results Three types (HPV16, HPV45 and HPV58) of pseudoviruses had been produced and the titer was 3.7×108 TU/ml, 1.5×108 TU/ml and 1.2×108 TU/ml, respectively. The luminescent regions could be detected in the mice which were infected with the pseudovirions, the luminescent signal intensity for types 16,45 and 58 was 1.779×106p/s, 5.738×105×p/s and 1.829×106p/s, respectively. Conclusion The mouse models for HPV16, 45 and 58 have been successfully established based on pseudovirions, which will be very useful for the research of HPV infection intervention, the evaluation of HPV vaccines and the screening of the prophylactic agents.

The ELISPOT assay is increasingly used for assessing cellular immune responses in clinical trials of HIV-1 or cancer vaccines. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints. This is due in part to the lack of assay standardization and validation across laboratories and therefore, a quality control panel is required to establish competency and comparability amongst different laboratories. In this study peripheral blood mononuclear cells (PBMCs) from healthy individuals were screened and frozen in liquid nitrogen. The recovery and viability of the PBMCs and the frequencies of interferon (IFN)-γ-secreting cells after CEF peptide pool stimulation were detected after various intervals in seven different laboratories. The recovery and viability did not differ significantly after different intervals. Although the frequencies of IFN (interferon)-γ-secreting cells among thawed PBMCs (peripheral blood mononuclear cells) fluctuated after CEF peptide pool stimulation at different intervals, they were not significantly decreased compared with those among fresh PBMCs. However, the viabilities, recoveries and frequencies of IFN-γ-secreting cells differed significantly among the seven laboratories. Our results indicate that cryopreserved PBMCs could be used as a quality control panel for ELISPOT. However, the procedures for ELISPOT need to be standardized amongst different laboratories.

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around Nab epitopes and deletions of variable regions in env.The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γELISPOT.Overall,five mutants(dWt,M2,M5-2,M5-1 and dM7)induced highed neutralization activities for both pseudoviruses than plasmid Wt,while only two of the mutants(dWt and M5-2)showed significant differences(P<0.05).Two mutants(M2 and dM2)induced more Env-specific T cells than plasmid Wt.Statistically however,significance was only reached for mutant M2.Thus,properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.