Development of recombinant ovalbumin-luteinizing hormone releasing hormone as a potential sterilization vaccine.
The objective was to develop an immunogenic chimeric ovalbumin-LHRH (ova-LHRH) molecule using genetic engineering. Hybrid ova-LHRH genes with either four or seven LHRH inserts were constructed by cassette mutagenesis and oligonucleotide mismatch mutagenesis. Recombinant ova-LHRH proteins were over-expressed in E. coli strain BL21 (DE3) using a pET expression system, which expresses a target protein with a C-terminal His-Tag. The C-terminal His-Tag allows purification by metal chelation chromatography. The antigenicity and biological effects of these recombinant proteins were tested in mice. In experiment 1, 17 female 7 wk old BALB/c mice were randomly divided into three groups. Six mice were injected with 50 microg of the recombinant ovalbumin (ova) protein. Five mice were injected with 50 microg of the recombinant protein with four LHRH inserts (ova-LHRH-7). Six mice were injected with 50 microg of the recombinant protein with seven LHRH inserts (ova-LHRH-7). One primary immunization using Freund's complete adjuvant was followed by one booster using incomplete adjuvant. Mice were killed 2 wk after the booster, blood collected, and the reproductive tract removed and weighed. Only ova-LHRH-7 decreased (P < 0.01) uterine-ovarian weight (89+/-11 mg) vs control (138+/-6 mg) and ova-LHRH-4 (126+/-16 mg). The genetically engineered molecule with seven LHRH inserts induced LHRH antibody titers which were significantly correlated (r = -0.79) with biological response. In experiment 2, the recombinant ova-LHRH-7 was evaluated at two doses with the adjuvants Zmax and Immumax. Seventy female 6-8 wk old BALB/c mice were randomly divided into seven groups of 10 mice each. Anti-LHRH titers were detected in all of the ova-LHRH-7 immunized mice. Significant decreases were shown in uterine-ovarian weight of the mice by the immunization with 30 microg of ova-LHRH-7 and Zmax (P < 0.005) or 10 microg of ova-LHRH-7 with Immumax (P < 0.025). These data show that the recombinant ova-LHRH-7 protein could have potential as an effective sterilization vaccine.
Zhang
Y
Y
Department of Animal Sciences, Washington State University, Pullman 99164-6353, USA.
Rozell
T G
TG
deAvila
D M
DM
Bertrand
K P
KP
Reeves
J J
JJ
D000818:Animals; D000941:Antigens / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005260:Female; D005620:Freund's Adjuvant / Q000276:immunology / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D016254:Mutagenesis, Insertional; D010047:Ovalbumin / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D012995:Solubility; D013245:Sterilization, Reproductive / Q000662:veterinary*; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000941:Antigens; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D007987:Gonadotropin-Releasing Hormone; D010047:Ovalbumin; D005620:Freund's Adjuvant