Objective The aim of the study was to develop a purification procedure on Pichia pastoris GS115/Fab expressing human anti-HBsAg Fab fragment. Methods Purity and yield ratio and conjugated activity of purified Fab fragment were analyzed with three purified ways of goat anti human Fab affinity chromatography and 14F7 monoclonal antibody affinity column, as well as Ion exchange Size exclusion column. Results 98% purity was reached through 14F7 monoclonal antibody column, and 95% purity was gained after goat anti human Fab fragment column. But yield ratio of the two affinity columns was low, being 35% and 55%, respectively. ForIon exchange Size exclusion column, purity and yield ratio of Fab fragment were very good, being 93.8% and 80% or more, respectively. Results of ELISA analysis showed that purified Fab fragment through three columns could bind to HBsAg specifically. Conclusion The purification process of recombinant anti-HBsAg Fab fragment was established. It lays a foundation for industrialization and clinical research of human anti-HBsAg Fab fragment.

Objective To study the expression, purification and bioactivity of a recombinant fusion protein consisting of anti-HBs single chain Fv and interleukin-2. Methods The engineering bacterium M15[pQE-ScFv-IL-2] which can express the fusion protein consisting of anti-HBsAg single chain Fv and interleukin-2, was induced by IPTG, then a 43kD recombinant protein was identified by SDS-PAGE and Western-blot analysis. Results The ratio of target protein to total protein of host reached 18%. Further analysis confirmed that the recombinant protein formed inclusion body in the cytoplasm of bacteria. 95% purity could be achieved after two-step purification of ScFv-IL-2, including Ni metal chelating chromatography (the first) and ion-exchange chromatogram (the second). The bioactivity assay of the purified product showed that the antibody-cytokine fusion protein could bind to HBsAg specifically and stimulate the proliferation of CTLL-2. Conclusion These results suggest that the fusion protein retains the bioactivity of its parental molecules, and may be a potential gene-engineering targeting drug for the treatment of chronic hepatitis B and other relevant diseases.

Objective To study the expression in high density fermentation of anti HBsAg Fab fragment in Pichia pastoris , and the purification and activity detection of expressed target protein. Method The high density fermentation of genetically engineered Pichia pastoris was proceeded in a 5L bioreactor using fed batch fermentation. The fermentation temperature was set at 28-30℃,the pH was 5 0-5 5, and the DO was kept over 20%. When the absorbance (OD 600 ) of the broth reached 400-450 (the first time of fermentation), 200-250 (the second time), and 300-350 (the third time), the induced phase was initiated, and the methanol concentration was 0 5%-1%. The fermentation ended after 96h's induction, the target protein was purified by affinity chromatography, and its activity was assessed by ELISA. Results It showed that the optimum initial cell density during methanol induced phase should be 300-350, which was good for control of the fermentation process and the expression of recombinant Fab. At the end of the fed batch phase, a yield of about 245mg/l of Fab was reached, and 98% purity could be reached as demonstrated by affinity chromatography. The results of ELISA showed that the supernatant of fermentation and the purified recombinant Fab could bind to HBsAg specifically. Conclusion The success of high density fermentation lays a sound foundation of mass production and clinical applications of recombinant humanized anti HBsAg Fab fragment

Objective:To develop an effective therapeutic double HBV DNA vaccine with two eukaryotic expressing motif,namely pS2.S and pIIF,a fusion ORF of hIL-2/hIFN-?.Methods:Two genes were amplified,which separately encoded HBV preS2.S middle envelope protein as a vaccine and human IL-2/IFN-? fusion protein as an adjuvant by PCR technique from plasmids pcDNA3.1/preS2.S and pcDNA3.1/hIL-2/IFN-?.Then the genes subcloned into eukaryotic vector pVAX1.The new plasmid was analysed by restriction endonuclease and DNA sequencing.The constructed plasmids were transfected into COS-7 cells in vitro with LipofectamineTM 2000.The expressing products in supernatants were quantified by ELISA.Space structure of fusion hIL-2/IFN-? expressed by the adjuvant plasmid was simulated by CE software.The double plasmid association were injected into BALB/c mice by in situ electroporation method,and specific humoral and cellular immune responses were examined.Results:The gene segments and inserting direction of pVAX1-S2.S(pS2.S)and pVAX1-IL-2IFN-?(pIIF)were both correct by genetic analysises.At 48 h after transfection,HBsAg and the cytokines of IL-2 and IFN-? were respectively 45.1 ng/ml,10.03 ng/ml and 11.5 ng/ml by ELISA.The activity domains of the two cycokines in fusion protein were entirely exposed on surface by CE software.Experiments were pefrormed in mice by injection of plasmid pS2.S.Protective antibodies of anti-HBs were produced in terms of dose-dependment pattern for the efficacy.Co-injection of plasmid pS2.S together with adjuvant pIIF resulted in more evident immune responses and dose of the plasmid pS2.S needed was diminished.There was strongly specific humoral and cell immunity by pS2.S and pIIF co-injection with EP technique.Conclusion:The double plasmids pS2.S and pIIF of anti-HBV are constructed succeedly and induce strongly specific immune activity in vitro and vivo.