The study on dynamic analysis of human urinary proteome is the foundation that we discriminate certain various urinary proteins as potential biomarker derived from the disease itself or normal physiological change. In our results, based on RPLC-MS/MS and spectral count to study pooled and individual urine samples and other researchers' studies, it can be known that the content of many urinary proteins maintain relatively stable. We have reason to believe that the relatively stable urinary protein is a very valuable resource as biomarkers. Many similar proteins such as prostaglandin-H2 D-isomerase and apolipoprotein D proteins have been proved our hypothesis. The following field, the number, preservation and treatment methods of urine sample, the standardization of analysis method and data processing, and suitable quantitative method, is ought to the focus of future study.

Objective:To explore the strategies of high-level secretory expression in order to enhance the production of Pfs25 protein in Pichia pastoris.Methods:Four main strategies were established in this study.First-ly,two kinds of recombinant Pfs25-expressing strains were constructed using the basic technology of molecular biol-ogy,and the difference between two kinds of strain was compared.The expression conditions were optimized and the best conditions being suitable for Pfs25 protein were selected.The expression medium,carbon source,pH val-ue,expression time and so on were determined.The multiple copy recombinant strains were constructed in pAO815 to enhance the expression level of Pfs25 in Pichia pastoris.The chaperone protein PDI was recombined by gene en-gineering methods into the recombinant Pfs25-expressing strains to enhance Pfs25 expression.Results:The 2 kinds of recombinant Pfs25-expressing strains were successfully constructed,and the constitutive expression was somewhat higher than inductive expression.Through comparisons of various expression conditions,the multi-copy recombi-nant strains were successfully constructed,and the Pfs25 production was higher in the 8 copy recombinant strains than in other strains.The PDI recombination enhanced the Pfs25 secretory expression in the Pfs25-expressing strains.Conclusion:The Pfs25 secretory expression in Pichia pastoris has been obviously enhanced by five folds in the optimal conditions of high-level expression strategies.

Malaria is the mosquito-borne lethal infectious disease.The pfs25 protein was proved to be the antigen for transmission-blocking vaccine.The aim of our study is to construct eukaryotic expressive vector for expression of Plasmodium falciparum transmission-blocking vaccine antigen pfs25 in CHO cell.In this study,the novel vector,in which the expression cassette was integrated with weaken SV40 promoter and dihydrofolate reductase(dhfr),was constructed and pfs25 gene was cloned into the new vector.The novel vector was designed and accomplished based on the pBudCE4.1 vector and named as pCMV-WD.It included the artificially synthetica weaken SV40 promoter,dhfr expression cassette and newly designed multiclone sites(MCS).The vector was applied especially on the CHO cell.The pfs25 gene was inserted into the pCMV-WD vector and recombinant plasmid pfs25/pCMV-WD was constructed,and then transformed into the host CHO-DHFR-cell by electroporation.Positive CHO cells expressed pfs25 gene were obtained by selection of deleting hypoxomthine,thymioline and adding zeocin antibiotics in medium.The recombinant pfs25 protein was identified by ELISA and Western Blot.The positive reaction was found in the Western Blot assay.Seven positive CHO cell lines expressed pfs25 gene were obtained,and the 0.1 mg/mL pfs25 was detected in the culture medium of 5A4 CHO cell line.The pfs25 gene was expressed successfully in the CHO cell by the eukaryotic expressive vector construction.

Based on the natural human CNTF gene, Ying et al. acquired CNTF-T mutant and constructed Escherichia coli expressing CNTF. In order to increase the yield and enhance the protein expression in the constructed E. coli, the strains were irradiated with high-LET heavy ions. Some mutants were obtained in this work and the mechanisms underlying the enhanced expression of CNTF in E. coli after exposure to heavy ions are discussed in this paper.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important causes of economic losses of the swine industry. PRRS virus (PRRSV) infection poses a challenge to current vaccination strategies. In this study, three replication-defective adenovirus recombinants expressing fusion protein GP3–GP5, GP4–GP5, or GP3–GP4–GP5 were developed as potential vaccine against PRRSV in a mouse model. Six groups of BALB/c mice (24 mice per group) were inoculated subcutaneously twice at 2-week intervals with above mentioned recombinants and other adenoviruses expressing single GP3, GP4, or GP5 protein. The results showed that the mice inoculated with recombinant adenoviruses developed PRRSV-specific antibodies, cellular immune response by 2 weeks post-boost-immunization. However, mice immunized with recombinant adenoviruses rAd-GP3–GP5, rAd-GP4–GP5, and rAd-GP3–GP4–GP5 developed significantly higher titers of neutralizing antibodies to PRRSV and produced stronger lymphocyte proliferation responses compared to mice immunized with rAd-GP3, rAd-GP4 or rAd-GP5 alone. It was also found that mice immunized with rAd-GP3–GP5 and rAd-GP3–GP4–GP5 were primed for significant higher levels of anti-PRRSV CTL responses than mice immunized with rAd-GP3 and rAd-GP5. These findings suggested that the recombinant adenoviruses expressing fusion proteins GP3–GP5 or GP3–GP4–GP5 might be an attractive candidate vaccine for preventing PRRSV infection.