| Objective: Hepatitis B-related liver disease is a leading cause of end-stage liver disease and a leading indication for liver transplantation in Asia. The result of liver transplantation for these patients was seriously affected by a high incidence of recurrent graft infection and subsequent graft failure. Passive immunoprophylaxis using hepatitis B immune globulin (HBIG) was the first prophylactic measure shown to be clinically effective in reducing HBV reinfection and improving graft survival after liver transplantation. But its clinical application was limitation for many reasons, for example, seldom patients in China were able to bear the heavy financial burden of lifelong high-dose HBIG. Anti-HBs-Fab, a kind of genetically engineered humanize micromolecule antibody, was composed of one heavy chain Fd segment and one light chain from HBsAb. It possesses the binding activity, and has more merit than serum original antibody. In this study, we constructed recombinant adeno-assciated virus(rAAV) type 1 vector containing anti-HBs-Fab genes derived from phage antibody library, rAAV1-HBs-Fab. And we investigated anti-HBs-Fab eukaryotic soluble expressing in vitro and in vivo and its protective role in inhibit HBV infection in vitro.Methods: A set of oligonucleotide primers were designed and used to amplify the VH and Vk gene from anti-HBsAg Fab antibodies screened from phage antibody library. A synthesized human Vk leader sequence was spliced to anti-HBs VH and Vk by overlap PCR. The pIRES-Fab was constructed by inserting coding sequences of Fd and k chain into pIRES. Fd-IRES-K fragments which was cut from pIRES-Fab and pXXUF1 were ligated together. And pXXUF1-HBs-Fab was constructed. It was identified by a series of restriction enzyme analyses, PCR and sequencing. Subconfluent packaging 293 cells were cotransfected using calcium phosphate method with the pXXUF1-HBs-Fab in combination with the helper plasmids PXX6 and PXX12. Then the recombinant virus rAAV1-HBs-Fab was propagated. Forty-eight hours after transfection, the anti-HBs-Fab expressed products were detected in the culture supernatant through ELISA. The virions were isolated on two sequential CsCl gradients. And viral titers were determined by dot blot analysis. The pXXUF1-HBs-Fab was transfected into the host cell HEK293 by liposome and into C57BL/6 mice by hydrodynamics-based administration via tail vein within 5 seconds. The ability of genetically engineered Fab antibody expression in vitro or in vivo was identified by RT-PCR, ELISA, Western blot and immunohistochemical. The expression of anti-HBs-Fab in vitro or in vivo was identified by RT-PCR, ELISA, Western blot and immunohistochemical. The soluble anti-HBs-Fab protein was purified from 293-anti-HBs-Fab cells' culture supernatant by affinity chromatography. And its function was analyzed in the following experiments: 1. The HBsAg primed by anti-HBs-Fab adhesive HepG2 cells were determined by indirect fluorescent staining. 2. The death rate of Hep3B cells primed by anti-HBs-Fab, anti-human Ig Fab goat antibody and complement were calculated. 3. The HBV positive human serum primed by purified Fab was mixed cultured human hepatocyte. PCR, ELISA and immunohistochemistry detected the expression of HBV-cccDNA and HBsAg. Result: The coding sequence of the Vk leader sequence, VH and Vk in pXXUF1-HBs-Fab were correct and Fd-IRES-K was successfully inserted into pXXUF1 by endonuclease digestion and PCR indicated. The rAAV1-HBs-Fab vector was obtained successfully and viral titers were up to 1 × 1011 plaque forming units (pfu)/ml after purification. The mRNA of Fd and k chain and anti-HBs-Fab protein in transfection cells was detected by RT-PCR, Western blot and ELISA. The lever of anti-HBsAg Fab expression was up to 1396 pg/ml. The plasma of mice administrated 20ug pXXUF1-HBs-Fab was confirmed to express anti-HBs-Fab stably by ELISA and level of Fab protein was 513.6±10.5 pg/ml. Brown positive pellets was found in the liver cells' endochylema and minority cohered on the inner walls of glomerular capillary lumen and collectors by immunohistochemistry. But the parenchyma cells of kidney had none expression. The concentration of purified anti-HBs-Fab was 5.65mg/L.Immunofluorescence was demonstrated that the anti-HBs-Fab conferred a significant protection against HBsAg adhesions HepG2 cells. 87.98% Hep3B cells were killed by complement after incubated with purified Fab antibodies and anti-human Ig Fab goat antibody. None HBV-cccDNA and HBsAg were detected in human hepatocytes which cultured with HBV positive serum primed by purified Fab. Conclusion: rAAV type 1 vector containing anti-HBs-Fab genes, rAAV1-HBs-Fab, was successfully constructed. pXXUF1-HBs-Fab was stably expressed soluble anti-HBs-Fab in eukaryotic cell. Study in vivo also showed the Fab protein could be filtrated through glomerular. The genetically engineered anti-HBs-Fab could prevent human hepatocytes free to HBV infection. This result lays the foundation for further study on rAAV1-HBs-Fab's function of prophylaxis HBV recurrence after liver transplantation.
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