Effect Of Ag85A DNA Vaccine On FasL-encoding MRNA Expression In Intestinal Intraepithelial Lymphocytes Of Mice

PrefaceDNA vaccines are made of a modified form of an infectious organism's DNA altered by special techniques to provide what are known as eukaryotic promoters in firont of the gene or genes on this DNA. This material, when introduced into the body, usually leads to the expression of the genes with the help of processing within host cells. This gene expression ultimately leads to synthesis of infectious organisms' proteins inside the injected cells and then activates host immune responses. As a result, the host immune system responds in a protective manner to produce antigen molecule frequently almost the same way as would occur if the host were actually infected by the true organism, itself. In addition, target gene fragments can be easily selected and combined according to the research objective. Most importantly, DNA vaccines can stimulate not only humoral immunity but also cellular immunity which ordinary vaccines fail to activate. As to oral immunization, attenuated Salmonella typhimurium as a good carrier can stimulate both mucosal and systemic immune response. Up to now few reports can be found with regard to effect of oral DNA vaccines on intestinal intraepithelial lymphocytes (iIEL).iIEL, which exist in the basolateral surfaces of intestinal epithelial cells, are a group of heterogeneous lymphocytes and constitute the first lymphoid compartment to encounter intestinal pathogens. iIEL are great different from the conventional T lymphocytes in maturing place, subsets constitution, cellular pheno-type and antigen recognition, ect. There is a predominance of CD8+ T lymphocytes ( CD8+ CTL) in mice with a relatively large proportion expressing the TCR and TCR CD8 . With their limited TCR repertoire, iIEL recognize antigen molecules in non - classical MHC restriction mode. Recently, we have done some research on the biological activities of iIEL and found that activated iIEL mainly secreted Th1 type cytokines and exhibited highly cytolytic and pro-liferative function. It has been reported that FasL is the effective molecule medi-ating cytotoxicity of ilEL. In this research, we used semiquantitive RT - PCR technique to test the expression of FasL - encoding mRNA in ilEL following oral Ag85 A DNA vaccine immunization and to analyze the effect of oral DNA vaccine on ilEL. At the same time we also compared the ilEL and conventional spleen lymphocytes in order to have a full insight into ilEL biological activities.Materials and methodsAccording to protocol of EZ Spin Column Plasmid Mini - Preps Kit, Ag85A - Vljns. tPA plasmid were extracted and then digested by Bglll restriction en-donuclease. The digested plasmid was analyzed by agarose gel electrophoresis. 30 C57BL/6 mice, 6 to 8 weeks, were randomly divided into 3 groups. Group I (Ag85A DNA plasmid group) ;12 mice for attenuated Salmonella ry-phimurium containing Ag85A - Vljns. tPA plasmid;Group II (empty plasmid control group) : 12 mice for attenuated Salmonella typhimurium containing Vljns. tPA empty plasmid; Group .01 (normal control group) :6 normal mice. For oral immunization, 1 x 108 cfu attenuated Salmonella typhimurium harboring the empty expression plasmid or the Ag85A - encoding plasmid were once administered intragastrically in a lOOjjJ volume of 0.2M NaHC03 into each mouse from Ag85A DNA plasmid group and empty plasmid control group. Immunizations were 3 times at 2 - week intervals. ilEL and spleen lymphocytes from 3 mice in each group were collected on day 1st, 3rd , 5th, and 8th after last immunization. The total RNA were isolated from both ilEL and spleen lymphocytes in the direction of RNeasy Mini Kit and analyzed by UV300 ultraviolet spectro-photometer and then reverse transcribed in a 20jxl reaction mix using M - MuLV reverse transcriptase according to the manufacturer * s protocol. From this reaction mixture, 9(xl was used for PCR to amplify FasL gene with housekeeping gene p - actin as internal control. PCR products were visualized in 1 % agarose gel following staining with ethidium bromide. FasL - encoding mRNA expression were...