Design, expression, and characterization of a novel dendritic cell-targeted proteins.

In vivo approaches to inducing an effective immune response focus on targeted antigen (Ag) delivery to dendritic cells (DCs). In this study, we developed a new method of targeting plasmid DNA and/or the antigen (Ag)-antibody (Ab) complex to DCs via the DC receptor DEC-205, also known as cluster of differentiation CD205. We cloned and expressed a recombinant protein composed of mouse DEC-205-specific single-chain fragment variable region (mDEC-205-scFv), the streptococcal protein G (SPG) IgG-binding domain and cationic peptide (CP), which named mDEC205-scFv-SPG-CP (msSC). In vitro, the recombinant protein msSC can specifically bind to DCs through the section of mDEC-205-scFv, and bound the Ag-Ab complex via SPG as well as plasmid DNA through electrostatic bonding with CP in vitro. In addition, msSC functioned in a manner similar to anti-DEC-205 monoclonal Ab and bound to mouse bone marrow-derived DCs. It was demonstrated in vivo that msSC can target plasmid DNA to DCs, resulting in efficient uptake and expression. Moreover, msSC can form a complex with pGL3-CMV and transport it to draining lymph nodes when injected in vivo. These results indicate that msSC can be used as a carrier protein for vaccine delivery to DCs via formation of plasmid DNA-Ag-Ab ternary complexes.

Increasing plasmid-based DNA vaccine construct (16 kb pSVK-HBVA) production in Escherichia coli XL10-Gold through optimization of media component.

At present, there are production processes to produce protein by Escherichia coli (E. coli) fermentation. Research on the design and optimization of the plasmid fermentation medium, however, is less advanced. The fermentation medium that is optimized for plasmid DNA production is different from the medium that is optimized for protein production. So, establishing a scientific and rational method to optimize the fermentation medium used for plasmid production is very important. Previously, our laboratory developed a novel therapeutic DNA vaccine (named pSVK-HBVA) for hepatitis B based on the alphavirus replicon, and found that E. coli XL10-Gold was the optimal host strain for the production of plasmid pSVK-HBVA. The aim of this study was to establish a scientific and rational method to optimize the fermentation medium used for plasmid production, and investigate the effect of growth medium composition on the production of plasmid pSVK-HBVA harboured in E. coli XL10-Gold, as well as to optimize the medium composition. The one-factor-at-a-time experiments demonstrated that Luria-Bertani (LB) was the optimal basic medium. The optimal carbon source and nitrogen source were glycerol and home-made proteose peptone, respectively. Based on the Plackett-Burman (PB) design, proteose peptone, glycerol and NH4Cl were identified as the significant variables, which were further optimized by the steepest ascent (descent) method and central composite design. Growth medium optimization in 500-mL shake flasks by response surface methodology resulted in a maximum volumetric yield of 13.61 mg/L, which was approximately 2.5 times higher than that obtained from the basic medium (LB).

Association of H3K9me3 with breast cancer prognosis by estrogen receptor status.

BACKGROUND: Cellular experiments revealed that a decreased histone H3 lysine 9 trimethylation (H3K9me3) level was associated with the upregulation of oncogenes in breast cancer cells. Moreover, the role of H3K9me3 in breast cancer was closely associated with estrogen receptor (ER) status. Therefore, we aimed to examine the prognostic value of H3K9me3 on breast cancer by ER status. The level of H3K9me3 in tumors were evaluated with tissue microarrays by immunohistochemistry for 917 women diagnosed with primary invasive breast cancer. Hazard ratios (HRs) and their 95% confidence intervals (CIs) for overall survival (OS) and progression-free survival (PFS) were estimated using Cox regression models. Interaction between H3K9me3 and ER on the prognosis was assessed on multiplicative scale. RESULTS: The level of H3K9me3 in tumor tissues was lower than that in adjacent tissues. The high level of H3K9me3 was associated with a better OS (HR = 0.43, 95% CI: 0.21-0.86) and PFS (HR = 0.49, 95% CI: 0.29-0.81) among only ER-positive but not ER-negative tumors. Moreover, the interaction between the level of H3K9me3 and ER status (negative and positive) on the prognosis was significant (Pinteraction = 0.011 for OS; Pinteraction = 0.022 for PFS). Furthermore, the ER-positive tumors were stratified by ER-low and ER-high positive tumors, and the prognostic role of H3K9me3 was significant among only ER-high positive patients (HR = 0.34, 95% CI: 0.13-0.85 for OS; HR = 0.47, 95% CI: 0.26-0.86 for PFS). CONCLUSIONS: Our study showed that the prognostic value of H3K9me3 on breast cancer was related to ER status and expression level, and the high level of H3K9me3 was associated with a better prognosis among ER-positive tumors, particularly ER-high positive tumors.

[Construction and expression of the efficient recombinant plasmid pEE14.1-IFN-α expressing human IFN-α].

AIM: To construct the eukaryotic expression plasmid pEE14.1-IFN-α expressing human IFN-α gene, and to detect the expression of the plasmid in eukaryotic cells. METHODS: The human IFN-α gene amplified by PCR and was linked into pCI-GPI, then inserted into the eukaryotic expression vector pEE14.1. The recombinant plasmid was transfected into the 293T cells, the IFN-α expression was detected by ELISA and Western blotting. RESULTS: Enzyme digestion and sequence analysis showed that the bicistronic eukaryotic expression vector pEE14.1-IFN-α was constructed successfully. The expression of plasmid was detected by ELISA, and the production of IFN-α in supernatant of transfected cells was about 3.15 ng/mL. Also, Western blotting could reveal the characteristic band of IFN-α gene. CONCLUSION: The vector is constructed successfully, which provide a new selection for HBV immunotherapy.

[Construction of a replicative anti-tumor DNA vaccine PSCK-2PFcGB and its expression in vivo and in vitro].

OBJECTIVE: To construct a replicative anti-tumor DNA vaccine PSCK-2PFcGB based on Semliki Forest Virus (SFV) replicon vector and observe its expression in vivo and in vitro. METHODS: The plasmid pVAX1-2PFcGB was digested with Nhe I, and the digestion product was blunted prior to further digestion with BssH II to obtain the fragment 2PFcGB, a fusion gene containing the multitarget complex antigen 2PAG encoding both the most cytotoxic T lymphocyte epitopes of human survivin and chorionic gonadotropin ß chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I. The products with the expected size were extracted and ligated, and the positive clones were screened by kanamycin and amplified. The recombinant PSCK-2PFcGB, following identification by colony PCR and restriction endonuclease Nde I, was transfected into 293T cells via lipofectamine 2000 and its expression was detected. The recombinant plasmid was also transfected into mouse quadriceps femoris muscle to observe its expression in vivo by immunohistochemistry. RESULTS: Nde I digestion resulted in a fragment of the expected size. Transfection with the recombinant plasmid PSCK-2PFcGB resulted in successful expression of the antigen and adjuvant molecular protein in 293T cells, with the positivity rates of 5.70% and 19.75%, respectively. The fusion tumor antigen survivin and hCGß-CTP37 were also detected in the muscular tissues of the mice. CONCLUSION: A novel replicative anti-tumor DNA vaccine PSCK-2PFcGB has been successfully constructed and can be expressed in 293T cells and in the muscular tissues of immunized mice, which provide a basis for further studies of the antitumor activity and immunological mechanism of the DNA vaccine.

[Construction of eukaryotic expression vector of human hCGbeta and establishment of stably transfected B16 cell line].

AIM: To construct the eukaryotic expression vector of human hCGbeta and stably transfect B16 cell line with it. METHODS: The full length of hCGbeta cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pIRES-neo, added the restriction enzyme position and 6xHis tag. After identification of restriction digestion and PCR, The recombinant plasmid pIRES-neo-hCGbeta-(His)6; was obtained. Then transfected it into B16 cells by lipofectamine 2000. After screening culture by G418, a stably transfected cell line was established, the transcription and expression of the hCGbeta gene was identified by RT-PCR, Western blot and immunofluorescence assay. RESULTS: The eukaryotic expression vector pIRES-neo-hCGbeta-(His)6; was successfully constructed. A stably transfected cell line was established and the expression rate of hCGbeta gene was higher than 90%. CONCLUSION: The established cell line can highly express hCGbeta stably, the expression of the target gene provide a solid experimental foundation for further studies on the function of the hCGbeta, and which will contribute to the research of hCGbeta gene in the tumor immunotherapy.