Preliminary DNA Vaccine. Sry Pathway

Nucleic acid vaccine is a recombination plasmid containing the code region of antigen gene. As the third generation of vaccine,it seems to be advantageous over classic immunization with purified proteins,which can induce completely immunal responses against a number of infectious agents and can be used safely,economically and conveniently.Consequencently, nucleic acid vaccine has been used widely in the study on treating infective disease.Now,our group introduced this method to changing mouse sex in order to benefit animal production relatied to the sexual feture.In mammal,SRY/Sry is a putative sex determining gene.All relative researches have shown that the presence of SRY/Sry gene leads to male phenotype.Or else,female phenotype forms. The sex determination is started by gene SRY/Sry,which happens between 10.5dpc and 12.5 dpc, and SRY/Sry synchronously presents during this period.So,the strategy causing sex reverse can be focused on the Sry gene.In the previous experiment,male pheynotype of XY genotype embryoes was successfully developed into female phenotype mice(sex reversal mice) through injecting the Sry nucleic acid vaccine into mouse's muscle.The aims of present investigations are to find the mechanism of the vaccine,which is foundational and essential to the further research.In the present experiments,firstly,expression of the nucleic acid vaccine,recombinant plasmid pCR3.1-Sry,was determined in vitro and in vivo,respectively.In vitro,the recombinant plasmid was transiently transfected into HeLa cells.The mRNA and protein in HeLa cells were detected successfully by RT-PCR and immunofluorescence assay,respectively.In vivo,the plasmid was injected into mouse's muscle.1 week later,RNA of the mouse's liver,spleen, muscle and uterus were extracted and detected by RT-PCR assay.Secondly,sandwich ELISA assay was used to detect the production of the anti-Sry antibody in antiserum.Sry antigen was obtained by resorting to prokaryotic expression of the recombinant plasmid pET21b-Sry.The antiserum was prepared from the mouse that was immunized with pCR3.1-Sry twice.The results indicated that nucleic acid vaccine pCR3.1-Sry can be expressed both in vitro and in vivo,and the specific antibody,anti-Sry,was produced in mice after vaccination.We can draw a conclusion that Sry nucleic acid vaccine does induce mouse's humoral immunity response,and its mechanism may be involved in the binding of Sry protein with anti-Sry,which might reversed male to female by blocking function of Sry protein in fetal development.