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 explore the effects of dendritic cells(DCs) pulsed by hepatitis B surface antigen(HbsAg) on the proliferation and function of cytokine-induced killer(CIK) cells.Methods The peripheral blood mononuclear cells(PBMCs) were isolated by the conventional method,pulsed by HbsAg,and added into the CIK cells.The ~3H-TdR incorporation was used to determine the proliferation of CIK cells and lactate dehydrogenase(LDH) release was used to measure the specific killer activity of CIK cells induced by HBsAg-pulsed DCs.Results The HBsAg-pulsed DCs could induce the proliferation of CIK cells and strengthen the killer activity of CIK cells induced by HBsAg-pulsed DCs(P

AIM: To purify and refold the inclusion body of a human anti-HBsAg scFv with a 6?His tag, and to determine the affinity constant of the purified recombinant product.METHODS: Solubilizing in buffers containing urea or guanidine hydrochloride (GuHCl), the inclusion body was purified by IMAC, and then refolded by dialysis against urea or GuHCl, at the same time, Ni 2+ charged chelate column was utilized for in situ refolding. The affinity constant of the refolded scFv, polished by immune-affinity chromatography, was determined by non-competitive ELISA. RESULTS: The refolded scFv with highest specific bioactivity was produced by dialysis against GuHCl. Under this condition, the recovery of target protein reached (61.08?1 45)%. The affinity constant of the polished scFv was confirmed to be(2.30?0.32) ?10 7 L/mol. CONCLUSION: The inclusion body studied in this paper can be refolded efficiently under optimal dialysis condition in vitro . The antigen-binding property of this recombinant scFv is not affected by the purification tag fused to the N terminal of the protein.

BACKGROUND: Intrapulmonary vascular abnormalities result in the right-to-left shunting and severe hypoxemia in liver transplantation candidates. Currently, a convenient, sensitive and effective method is absent to screen the intrapulmonary vascular dilatations.OBJECTIVE: To evaluate the role of contrast-enhanced echocardiography on clinical diagnosis of intrapulmonary shunting in liver transplantation candidates.DESIGN, TIME AND SETTING: The experiment, prospective controlled observation based on cases, was performed at the Hepatology Unit of the 458 Hospital of PLA (Guangzhou, Guangdong, China) from February 2004 to February 2006.PARTICIPANTS: Twenty-four consecutive liver transplantation candidates were recruited from the Hepatology Unit of the 458Hospital of PLA.METHODS: Routine examination was conducted under the condition without any regimen of vascular dilatation drugs.Contrast-enhanced echocardiography was applied to detect the prevalence of right-to-left shunting in the patients with end-stage liver disease. The microvesicle of the left ventricle in patients was qualitatively assessed by a score from 1+ to 3+. Accordingly, all patients were divided into two groups: intrapulmonary shunting and non-intrapulmonary shunting.MAIN OUTCOME MEASURES: The prevalence of right-to-left shunting and clinical characteristics of liver transplantation candidates were determined.RESULTS: Ten (41.7%) of 24 patients with positive contrast-enhanced echocardiography were proved to develop the intrapulmonary right-to-left shunting, including 6 for l+ and 4 for 2+ by left ventricle abnormality, which emerged after 6-10 cardiac cycles of right ventricle abnormality. There were no significant differences in age, gender, arterial blood gas analysis and liver function tests between the two groups (P > 0.05). Echocardiography results demonstrated that, the upper digestive tract hemorrhage,spleen thickness that indicated portal hypertension, pulmonary artery systolic pressure and Tei index were significandy higher in the patients of intrapulmonary shunting than in those of non-intrapulmonary shunting (P<0.05-0.01 ).CONCLUSION: Intrapulmonary vascular dilatation occurs frequently in liver transplantation candidates associated with intrapulmonary shunting but without hypoxemia. Contrast-enhanced echocardiography is a sensitive and non-invasive method for the early diagnosis of intrapulmonary vascular dilatation. The pathogenic cause is portal hypertension. Tel index can be used as an important parameter for evaluating right ventricular function in patients of intrapulmonary vascular dilatation.