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Studies have shown that ABO blood group is related to the susceptibility and disease progression of SARS-CoV-2 infection, and most studies indicated that group O individuals were less likely to get infected while group A conferred a higher susceptibility to infection and propensity to severe disease. ABO blood group antigens are oligosaccharides expressed on red cells and other tissues. People with different ABO blood type have different susceptibility to a variety of pathogens, including SARS-CoV-2. There are several hypotheses to explain the differences in SARS-CoV-2 infection between ABO blood group individuals. Firstly, anti-A and/or anti-B antibodies could bind to corresponding antigens on the viral envelope and contribute to viral neutralization, thereby preventing target cell from being infected. The SARS-CoV-2 virus and SARS-CoV spike (S) proteins may be bound by anti-A isoagglutinin, which may block interactions between virus and angiotensin-converting-enzyme-2-receptor, thereby preventing entering into lung epithelial cells. Secondly, the receptor binding domain (RBD) of S protein domain can bind to antigen A expressed in respiratory epithelium and promote its infection to respiratory epithelial cells. In conclusion, most studies indicated that group O may be associated with a lower risk of SARS-CoV-2 infection while group A with a higher risk along with severe disease, and the related mechanism needs to be further studied.
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Objective To reduce immunogenicity of porcine skin by removingα-Gal epitopes expressed in cell surface and extracellular matrix using recombinant α-galactosidase produced by Bacteroides fragilis.Methods The porcine skin was harvested from healthy 2-month-old pigs without any skin disorders before being sterilized by iodine and 75%alcohol, respectively.Enzymatic removal of α-Gal antigen was followed by washing with PBS.The α-Gal antigen in the prepared porcine skin was measured with immunofluorostaining of cryosections and the residual enzyme was measured with a double-antibody sandwich ELISA method.Enzymatic removal procedures were optimized by detecting residual enzyme and the effi-cacy ofα-Gal removal under different enzymatic and washing conditions.Results Efficient enzymatic and washing methods were established to removeα-Gal antigen.Theα-Gal removal efficacy was above 90% and residual enzyme was undetect-able (αprescribed minimum ofα-galactosidase detection with indirect ELISA was 1 ng/ml) .Conclusion It is feasible to efficiently removeα-Gal antigen under these enzymatic and washing conditions, and a method of producing low-immunoge-nicity pig skin dressing for burn is established.
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Objective To establish a method of quantiying trace α-galactosidase from Bacteroides fragilis in enzymatic conversion of blood group B to O red blood cells ( B-ECO RBCs) .Methods BALB/c mice were immunized with purified recombinant B.fragilisα-galactosidase ( the purity>90%) to prepare monoclonal antibodies.The ascites were prepared using hybridoma cell lines stably secreting antibody and purified by HiTrap rProtein A column.The antibody titer and spe-cificity were detected by ELISA and Western blotting, respectively.Purified monoclonal antibody and rabbit polyclonal an-tibody were applied to detect residual enzyme in B-ECO RBCs and the washing solution was analyzed by indirect ELISA. Results A high titer and purity antibody was obtained.Western blotting showed that the antibody specifically reacted with B.fragilisα-galactosidase.Moreover, indirect ELISA was sensitive enough to detect the minimal amount of residualα-gal-actosidase at the concentration of 1 ng/ml.After four repeat washing cycles with 1∶4 ( v/v) phosphate-buffered saline, the amount of residual enzyme in B-ECO RBCs was less than 10 ng/ml.Conclusion An effective method of detecting the min-imal amount of residual α-galactosidase in blood conversion is established for safety evaluation of universal RBCs prepara-tion by enzymatic treatment.
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Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.
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AIM:To investigate the target killing effect of T lymphocytes with chimeric CD20scFv gene on Daudi cells and the activation of T lymphocytes.METHODS:Two kinds of plasmids were transfected into retrovirus-packed PA317 cell lines.The supernatant was collected from successfully transfected PA317 culture and was used to infect peripheral blood T lymphocytes.After one-week screening with G418,the cells were used to kill Daudi and K562 cells.The positive rates of AnnexinⅤ in Daudi cells were measured at different times points respectively by flow cytometry.Meanwhile,the level of IL-2 and IFN-? were determined by ELISA.RESULTS:The Annexin V positive rate was significant higher in Daudi cells compared to control K562 cell lines at 24 h.No difference of AnnexinV in Daudi cells was observed in CD20 modification T lymphocyte groups.The secretions of IL-2 and IFN-? in CD20scFv-CD80-IgGFc-CD28-? gene modified T cells co-cultured with Daudi cells were dramatically higher than that in CD20scFv-IgGFc group at 72 h.CONCLUSION:① The two kinds of genetic modified specific T cells have no significant difference in inducing early apoptosis of Daudi cells.CD28-? can't affect Daudi cell early apoptosis at the CD20scFv target killing.② The increase in the secretions of IL-2 and IFN-? is more obvious in CD20scFv-IgGFc-CD28-? group,indicating that the self-activation takes place in CD3? and CD28 modified T cells without MHC restriction and then increases the activation and killing function of T cells.
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AIM:To construct a recombinant eukaryotic expression vector pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/? and detect its expression in NIH 3T3 cells.METHODS:CD28-? cDNA was amplified from the plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/IgGFc/CD80 gene.The recombinant plasmids were transfected into NIH 3T3 cells,and resistant clones were obtained by G418 selection.The gene expression of the fusion protein was determined by RT-PCR and FACS.RESULTS:The recombinant eukaryotic vector was constructed successfully,determined by PCR and enzyme digestion analysis.The target gene was amplified from NIH 3T3 cells transfected with the vectors by RT-PCR.The FACS showed that recombinant protein was expressed in NIH 3T3 cells.CONCLUSION:Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/? expression vector and its expression in NIH 3T3 cells lay the foundation for further research of generation of modified T lymphocytes to CD20 positive lymphoma.