Construction Of GnIH Gene Vaccine And It's Immunization Effect

GnIH is only known as hypothalamic inhibitory hormone in the HPG axis and antagonized with Gn RH to keep dynamic balance of HPG axis,which regulate the reproduction of animals.GnIH has 12-amino acid.With the deepening of the study,it was found that GnIH could affect Gn RH neurons directly or indirectly,which inhibit the secretion of gonadotropin in animals,and GnIH can be through the autocrine / paracrine act on the gonadal directly to regulate animal reproduction.In this study,GnIH as the target gene,we constructed double copies of the recombinant plasmid pt S2 GnIH-asd.And the expression of pt S2 GnIH-asd was detected at the cellular level.The GnIH gene vaccine was used to immunize ewe lambs of Hu sheep,and the level of antibody titer and related hormones in the serum were measured.And the behavioral changes of ewe lambs and microstructure change of ovarian tissue were also detected.The aim of this study was to investigate the immunization effect of GnIH gene vaccine on ewe lambs of Hu sheep.(1)Construction and identification of recombinant plasmid pt S2 GnIH-asdTwo pairs of complementary single-stranded nucleotide fragments of GnIH gene were chemically synthesized,the terminal and mid-terminal GnIH fragments were obtained by PCR polymerization.The Gn RH gene in p VAX-S-Gn RH-asd vector was replaced by the terminal GnIH fragment to construct single-copy plasmid p SGnIH-asd.Mid-terminal GnIH was subcloned into the codon of the 112 th and 113 th amino acids that encoded by the HBs Ag-S gene in p SGnIH-asd,and to get double copies plasmid p S2 GnIH-asd.The t PA gene was subcloned into the 5 'end of the HBs Ag-S gene in p S2 GnIH-asd plasmid.The recombinant plasmid pt S2 GnIH-asd was constructed and the target gene was identified as t S2 GnIH.The sequence,insertion site and direction of the fusion gene t S2 GnIH were identified correctly by restriction endonuclease digestion and sequencing.The recombinant plasmid pt S2 GnIH-asd was transfected into Hela cells by liposome-encapsulated method.Total RNA was extracted from Hela cells after 24 h,and the fusion gene t S2 GnIH was detected correctly by RT-PCR.The total protein of Hela cells was extracted after 48 h,the expression of the fusion gene t S2 GnIH was successfully detected by Western blot,and expressed fusion protein was about 30.91 k Da.The results showed that recombinant plasmid pt S2 GnIH-asd could be transcribed and expressed in eukaryotic cells.(2)GnIH gene vaccine immunized ewe lambs of Hu sheep and evaluation of immunization effectTen 8 week-old ewe lambs of Hu sheep were randomly divided into two equal groups.Vaccine group(V)were injected intramuscularly with pt S2 GnIH-asd while the control group(C)animals were treated with p VAX-asd,the same treatments were repeated three times and each time interval of 21 d.From primary immunization to weeks 14,and the blood and weight was collected every two weeks.Weight of the vaccine group did not change significantly compared with the control group(P > 0.05).The level of serum antibody titer was measured by Elisa,and the level of serum antibody was found in vaccine group at weeks 2-14 compared with control group(P < 0.05).The GnIH gene vaccine can immunize endogenous GnIH,the levels of LH and E2 in experimental group showed an increasing trend and significantly higher than in control group at weeks 10-14(P < 0.05),although there was no significant effect on the secretion of FSH and P in vaccine group(P > 0.05),the number of follicles in the ovary of the experimental group was significantly higher than that of the control group(P < 0.05),Although ewe lambs refused the rams to climb,the number of chase ewe lambs(V)was significantly higher than that in control group while ram into the group(P < 0.05).Result infers that ewe lambs(V)had more reproductive development and sexual signals,and stimulate the sexual excitement of ram significantly.