ABSTRACT
To find out whether the integration of PERV in HEK293 cells influence the expression profile of HERV-W,based on the gene sequences of H ERV-W in GenBank,the primers of H ERV-W gag,pol,env,env from locus 7q21.2 genes as well as humanβ-actin were synthesized respectively and were used to develop the means of SYBR Green I real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the mRNA expression of these genes in HEK293 cells and PERV-infected HEK293 cells.Experiments showed that these RT-qPCR methods were of good specificity,sensitivity and stability:the standard curves could ensure the correlation coefficients to be all above 0.99 and the amplification efficiency were between 95％ and 110％,which verified that these methods could be used to detect the mRNA of HERV-W and humanβ-actin in culture cells.Through the detection and analysis of relative gene expression data using the 2-△△ct method,we found that the mRNA level of HERV-W gag,pol,env,env from locus 7q21.2 genes as well as humanβ-actin in PERV-infected HEK293 cells increased,after integration,by 37.08,42.56,2.49 and 13.17 times than in non-infected HEK293 cells,respectively.Results provide a reference to further evaluate the safety of PERV pathogen in xenotransplantation.
ABSTRACT
The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.