Study On The Dynamic Distribution Of GPV-VP3 Gene Vaccine In Mice And Gosling

In this study,According to VP3 gene sequence of GPV in GenBank,we designed a set of primers with the oligo software.The amplification product is a 441bp fragment as expected with PCR.We also relied on oligo software to select a 34bp oligonucleodide probe which was labelled with digaoxin at 5' end.With the primers and the probe,we developed in situ hybridization and indirect in situ PCR successfully to specifically detect pcDNA-GPV-SC-VP3 nucleic acids in paraffin-embedded tissues of Mouse and gosling.4-week-old BALB/c Mouse and Goslings were immunized with 200μg,100μg,50μg and 6μg,3μg,1μg,respectively via intramuscular injection and gene-gun,and with PBS,pcDNA3.1(+)as control,then heart,liver,spleen,lung,kidney,brain,intestine,and immue site(cutis or muscle) of mouse and heart,liver,spleen,lung,kidney,brain,intestine,immue site(cutis or muscle),bursa of fabricius,thymus,harderian gland,duodenum,jejunum,caecum,recta and pancreas of goslings were collected at different time after administration and used the in situ hybridization and indirect in situ PCR detect the distribution and location of pcDNA-GPV-SC-VP3 in these tissues.The results showed that:1.the established in situ hybridization and in situ PCR was specific,highly sensitive and exactly locative,and can detect 13pg homology DNA.2.The pcDNA-GPV-SC-VP3 could distribute into all the tissues of mouse and goslings;All tissues except brain were positive at 1h post administration,but brain was positive at 3h of mouse and gosling;The duration time in each organ had difference,and the muscle of immune site was longest,liver secondly in intramuscular group.Whereas liver was longest in gene-gun group,and spleen,heart took second place.Brain was most short in all tissues.3.The signals and duration time of different dose group were:6μg group＞3μggroup＞1μg group and 200μg group＞100μg group＞50μg group.This showed that gene vaccine immunizing dose and distribution had definite positive correlations.4.Both intramuscular injection and gene gun bombard were good immunity approach for pcDNA-GPV-SC-VP3,but gene gun bombard was better in contrast.5.The tissues were detected by ISH for the pcDNA-GPV-SC-VP3 location.Results showed that:positive signal mostly dispersed over hepatocyte,hepatic sinusoid cell, Kupffer's cell,spleen dispersivity positive,intestinal villi epithelia,goblet cell and intestinal gland cell,alveolar epithelial cells,kidney renal tubule department cell,medulla thymi and cortices,bursa of fabricius medulla.6.We compared the two molecular methods mentioned above.The results reveal that in situ PCR is more sensitive than ISH,and the duration time was longer and the signals were more with in situ PCR.