Induction Of Skin Allograft Tolerance In Mice By Recombinant Adenovirus AdCD40Ig With Donor Resting B Lymphocytes Pretreatment

[Objective] To investigate the efficacy of recombinant adenovirus AdCD40Ig with donor resting B lymphocytes pretreatment in prolonging the survival time of murine skin graft and in inducing the establishment of donor-specific transplant tolerance.[Methods] Total RNA was isolated from spleens of BALB/c mice, extracellular domain of CD40 and Fc domain of IgG2a were cloned with respective primers by reverse transcriptase—polymerase chain reaction. The product of these two reactions had a 17bp overlapping domain, and CD40Ig fusion gene could be polymerized with overlapping-elongation polymerase chain reaction. Then this fusion gene was inserted into pMD18-T and sequenced. The CD40Ig gene was inserted into the shuttle plasmid pAdTrack-CMV of adenoviral vector AdEasy system, followed by homologous recombination with bone plasmid pAdEasy-1 in E.coli BJ5183. Recombinant adenoviral vector AdCD40Ig with murine CD40Ig came into being after packaged with 293 cells. Recombinant virus was verified with polymerase chain reaction, and the titre of recombinant virus was measured with fluorescent counter; protein level of CD40Ig in the supernatant of the examined cells was measured with SDS-PAGE electrophoresis andenzyme-linked immunosorbent assay. The murine skin transplantation model was established. The animals were allocated into the following groups: control (group 1), third-partner control (group 2), AdCD40Ig (group 3), C57BL/6 resting B lymphocytes (group 4), AdCD40Ig with resting B lymphocytes (group 5) and third-partner experimental (group 6); mice was treated 1 week prior to skin transplantation; the survival of graft was monitored closely.[Results] Overlapping-elongation polymerase chain reaction produced an 1257bp segment, and this segment was proved to be murine CD40Ig fusion gene. The titre of recombinant adenovirus AdCD40Ig reached 5><109 efu/ml, segments of CD40Ig fusion gene could be cloned from the DNA of the adenovirus, and the supernatant of virus-infected 293 cell culture contained CD40Ig fusion protein. Seven days after AdCD40Ig was injected via tail veins of mice, serum CD40Ig peaked at 130±11.2 ug/ml and remained elevated in 8 weeks. Mean survival times (days) of all six groups were 10.33±0.46, 9.83±0.53, 10.83±0.25, 10.00±3.24, 63.50±10.45 and 11,50±3.39 respectively. Survival time of skin graft in group 5 is significantly prolonged compared with that in the other five groups (P<0.01); the differences between other pretreatments and their controls were not significant, indicating recombinant adenovirus AdCD40Ig with donor resting B lymphocytes pretreatment could induce donor-specific tolerance and improve the survival of skin graft. Recombinant adenovirus AdCD40Ig or donor resting B lymphocytes pretreatment alone had no detectable effect. [Conclusion] 1. Recombinant adenovirus AdCD40Ig with murine CD40Ig gene could be successfully constructed, the titre of virus could reach 5 * 109efu/ml. In vitro and in vivo experiments all showed that this vector could express CD40Ig stably and effectively. 2. The pretreatment strategy adopted in this experiment—recombinant adenovirus AdCD40Ig with donor resting B lymphocytes—could successfully induce the establishment of specific transplant tolerance in murine, and significantly improve the survival of donor-specific skin grafts, suggesting that this strategy possibly be applied in clinical setting and worth further study.