Fusion Expression Of Luteinizing Hormone Releasing Hormone In Escherichia Coli And Antigenicity Of The Fusion Protein

The mammalian luteinizing hormone releasing hormone (LHRH) gene had been synthesized by chemical method according to the amino acids sequence of mammalian LHRH and favourit genetic codes of E. coil. The gene was cloned into thioredoxin(TRX) fusion expression vector 梡ET?2c. After selection, the recombinant plasmid was transformed into F. co/i BL21(DE3). The effects of induction time, concentration of IPTG and Amp in the culture medium on the yield of TRX桳HRH fusion protein was studied. The expression of TRX桳HRH fusion protein reached to the highest yield(33. 2%) at 4 hours after induction with 1 mM IPTG, but the yield of TRX桳HRH fusion protein was not affected by the concentration of Amp and JPTG. The expression of fusion protein could also be induced by lactose. 1% lactose had the same induction effect as TPTG. It was demonstrated that the TRX桳HRH fusion protein was soluble and could be purified by osmotic shock method. The physiologic mammalian LHRH and avian LHRH was conjugated to bovine serum albumin(BSA),ovalbumin(OVA) or Staphylococcus aurous Cowan I(SAC) by glutaraldehyde method. The higher titer antibody to LHRH had been prepared and Ehe immunogenieity of BSA桳HRH and SAC桳HRH had been compared. It was shown that the BSA桳HRH was better than SAC桳HRH in induction of antibody response to LHRH. The reactinogenicity of TRX桳HRFI fusion protein with antibody to LHRH had been identified with indirect ELISA and indirect inhibitory ELISA. Four two梞onth old male rabbits were immunized with the TRX桳HRH fusion protein. The sera of all the rabbits had detected out antibody to LHRH after the booster injection. Compared with the control group, the rabbits immunized against TRX桳HRH fusion protein showed no sexual behavior, reduction of testis weight and volume. Histopathological changes of the testis showed atrophy of seminiferous tubules, 91 ?~ {LYdJ2 completely or incompletely arrested spermatogenesis and polykaryocyte were found in the spermatogenesis tubule. It was demonstrated that the TRX桳HRH fusion protein could be an candidate of immunocastration vaccine. The tandem gene, mLHRH梐LHRH梞LHRH, had been cloned into pET?2c vector. The tenth amino acid of?mammalian LHRH had been changed into Asn. The TRX梤riLHRH---aLHRH梞LHRH fusion protein was expressed in E. coil BL21(DE3). By indirect ELISA, indirect inhibitory ELISA and western 0 blot, it was shown that the fusion protein had good reactinogenicity with antibody to avian LHRH, but leak with antibody to mammalian LHRH.