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

At present, there are production processes to produce protein by () 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.

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).

Enhancement of antitumor immunity using a DNA-based replicon vaccine derived from Semliki Forest virus.

A DNA-based replicon vaccine derived from Semliki Forest virus, PSVK-shFcG-GM/B7.1 (Fig. 1a) was designed for tumor immunotherapy as previously constructed.

[Preliminary observation on antitumor effect of HPV58 composite DNA vaccine].

Objective: To initially observe the antitumor immune of PVAX1-HPV58mE6E7FcGB composite DNA vaccine.

Methods: Before detecting immune effect of the vaccine, the B16-HPV58E6E7 tumor cell line was built which could steadily express HPV58E6E7 fusion gene. Then, HPV58E6E7-GST fusion protein as an antigen was expressed and purified.

[Transformation activity and antigenicity of the human papillomavirus type 58 E6E7 fusion gene mutant].

Objective: To develop a prophylactic and therapeutic vaccine against human papillomavirus (HPV) type 58-associated cervical carcinoma, and explore its transformation activity and antigenicity.

Methods: The E6 and E7 three amino acid codons in the HPV 58 virus were modified respectively and fused. The modified and fused gene was named HPV58 mE6E7.

[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.

Intraarticular gene delivery of CTLA4-FasL suppresses experimental arthritis.

T lymphocytes are key inflammatory cells contributing significantly to the pathogenesis of Rheumatoid arthritis (RA). Biological treatments targeting T lymphocytes may provide an efficient approach for treatment of RA. CTLA4-FasL, a fusion product of extracellular domains of CTLA4 and FasL, integrating two inhibitory elements against T cells into one molecule, might be a desirable derivative of engineered soluble FasL or CTLA4 and have therapeutic potential in RA.

[A study on the construction, expression and immunosterility of Lagurus laguru zona pellucida 3 DNA vaccine pVAX1-sig-LTB-lZP3-C3d3].

Aim: To enhance the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 DNA vaccine, and to achieve the prospect of application through the pVAX1-sig-LTB-lZP3-C3d3 different immunity pathway.

Methods: Two adjuvant molecules were constructed into the recombinant plasmid pVAX1-sig-LTB-lZP3-C3d3 as DNA vaccine which contains Escherichia coli heat-labile enterotoxin B subunit and the molecular adjuvant 3 copies of C3d. The results of RT-PCR and western blot showed that the DNA vaccine was expressed in mRNA and protein level.

[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 β chain-CTP37 of human and monkey. The 2PFcGB fragment was inserted into the PSCK vector digested by Sma I.

Puerarin: a novel antagonist to inward rectifier potassium channel (IK1).

Puerarin, isolated from the root of pueraria, had been widely used to prevent and treat arrhythmia. We show that puerarin effectively prevents and reverses aconitine-induced arrhythmias in perfused heart in vitro and in rats in vivo. To study the mechanisms of antiarrhythmic action of puerarin, we investigated the electrophysiological actions of puerarin using whole-cell clamp in isolated rodent ventricular myocytes and two electrode voltage-clamp (TEV) in I(K1)-expressing Xenopus oocytes.

[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.

Association between the frequency of class II HLA antigens and the susceptibility to intrauterine infection of hepatitis B virus.

Multiple factors determine the susceptibility to intrauterine hepatitis B virus (HBV) infection. These factors include the HBV structure, HBV mutation, HBV DNA level, placental barrier, the immune status of the mother, and the genetic make-ups of the newborn infants. Since HLA system is an integral component of the immune response, we hypothesized that the highly polymorphic HLA genes are the key determinants of intrauterine HBV infection.

[Development of an anti-rTNF-alpha chimeric antibody].

Aim: To develop an human-mouse chimeric antibody against rTNF-alpha.

Methods: Expression vector of human-mouse chimeric antibody CZ12 gene was constructed by using VL and VH genes of mAb Z12 with neutralizing activity, and then COS7 cells were transfected. Supernatant containing CZ12 was detected by RT-PCR, ELISA, Western blot and in-vitro neutralization assay, respectively.

[Construction of GPI-CTLA4Ig chimeric molecule and its expression on CHO-dhfr(-) cells].

Aim: To develop a novel immunosuppressant GPI-CTLAIg modified by glycosyl-phosphatidylinositol(GPI).

Methods: GPI-modified CTLAIg was produced by linking-up of CTLA4Ig with GPI-modification signal sequences from decay-accelerating factor (DAF). Chimeric molecule GPI-CTLA4Ig gene was cloned into eukaryotic expression vector pCI-dhfr.