Construction And Identification Of CD112R-CD28 Lentivirus Expression Vector

Background In recent years,along with the research and development of modern medical technology,adoptive immunotherapy(AIT)showed the treatment effect is more significant,a chimeric antigen receptor(CAR)and the proliferation of life lasting is better for T lymphocytes,more targeted killing of cancer,and for the modification of T lymphocytes targeted immune therapy strategies,and from giving the AIT more significant therapeutic effect[1].In recent years,CD112 R has been recognized as a potential detection point of immunity,this study intends to design the CD112R-CD28 co stimulatory antigen conversion strategy,will inhibit intracellular CD112 R signal conversion for CD28 activation signal,construct the lentiviral expression vector,modified T cells,so as to further enhance the immune function and the effect of T cells.Objective Construction of chimeric antigen receptor CD112R-CD28 lentiviral expression vector and lentiviral packaging expression of chimeric antigen receptor gene,sub T1 infection and Jurkat cells in vitro,so as to make the CD112R-CD28 high expression in the two cell lines,successfully provide a theoretical basis for the anti-tumor efficacy enhancement of adoptive immunotherapy[2].Methods Targeting the CD112 R recombinant lentivirus by CD112R(579 bp,containing the extracellular and transmembrane region),CD28(123 bp)composition,method of chemical synthesis of poly nucleotides by PCR amplification and oligonucleotide,will chimeric antigen receptor CD112R-CD28 gene was synthesized successfully.Then the target gene was cloned into vector p LVX-IRES-Zs Green and Eco R I the Xba I site,after double enzyme digestion and sequencing,and lentiviral packaging plasmids Gag/Po1,Rev,VSV-G were transfected into 293 T cells to package lentivirus,lentivirus infected sub T1 and Jurkat cells,flow cytometry was used to detect EGFP gene expression.Results CD112 R and CD28 fragment by overlapping PCR spliced CD112R-CD28,CD112R-CD28 gene and the expression vector p LVX-IRES-Zs Green by Xba I and Eco R I digestion and then linked,after double digestion and sequencing correctly,the gene CD112R-CD28 was cloned into the lentiviral expression vector in vivo,and the sequence is correct,will slow virus packaging,infection sub T1 and Jurkat cells,the green fluorescence flow cytometry analysis of the expression level of EGFP.Conclusion Chimeric antigen receptor CD112R-CD28 lentiviral expression vector was successfully constructed,and successfully packaged lentivirus infected sub T1 and Jurkat cells,these two cells are able to target gene expression efficiently,is mediated by lentivirus chimeric antigen receptor T lymphocyte target to lay the foundation for cancer therapy[3].