Construction And Immunization Effect Of KISS1 Gene Vaccine Without Antibiotic Resistance Gene

The KISS1 gene plays an important role in animal reproductive function by regulation of hypothalamic Gn RH secretion and the secretion of hormones at all levels of the reproductive axis.KISS1 gene has been shown to serve as a target for gene immunization.However,there are some problems in the previous experiment,such as the cumbersome and expensive preparation process of the plasmid.So it is necessary to construct a new KISS1 gene vaccine,this vaccine must be have the advantages of simple preparation process and Inoculation convenience.Recombinant plasmid(p VAX-KISS1-S-asd)was transformed into Salmonella enterica sv Choleraesuis C500 strain by electroporation,and then positive clones were chose by sequencing and enzyme digestion,the recombinant strain was named C500/p KS-asd.Strain were passaged 50 generation in sterile medium,the plasmid and bacterial genome DNA were extracted.Stability of plasmid p KS-asd and recombinant strain were detected by enzyme digestion and conserved Inv A gene.10 weeks old rams lamb were inoculated C500/p KS-asd and C500/p VAX-asd recombinant strain by nasal cavity,The dose was 2×1010CFU/sheep,three immunization in total,4 weeks interval and all animal were kill at the 14 weeks of primary immunization,anti-body and reproduction hormone were detected by ELISA,the slice of testis were made by HE.The difference to the levels of reproductive genes in hypothalamus pituitary testis axis were detected by q RT-PCR.The results showed plasmid p KS-asd and recombinant strain were stable in vitro culture.The titers of anti-body reached a peak at the 14 weeks after primary immunization;Testosterone concentration of treatment groups was significantly difference to control groups at 8 to 12 weeks of primary immunization(P < 0.05)and scrotal circumference was smaller than control groups at 2 to 14 weeks of primar y immunization(P>0.05);Sexual behavior(sniffing,mounting and bunting)and length,width,volume and weight of testis of treatment groups was significantly lower than control groups(P < 0.05);Histological examination of testis showed that spermatogenic cells and sperm production in seminiferous tubules were significantly less than the control groups and the stages of germ cells is vague,loosely arranged.The KISS1 m RNA and GPR54 m RNA levels of hypothalamus were significantly increased to control group(P < 0.05),and the GPR54 m RN A,Gn RHR m RNA and LH? m RNA levels of pituitary and A R m RNA level of testis were significantly decreased to control group(P < 0.05).In this study,we successfully constructed a non-resistant screening C500/p VAX-KISS1-S-asd recombinant strain,and the passage stability of t he strain and plasmid was good.The effect that lamb was inoculated C500/ p KS-asd by intranasal administration was obvious,and then development and spermatogenesis were inhibited,so the aim of castration was achieved.