SD雄鼠3种不同新蛋白构型GnRH去势抗原免疫学及生物学效果对比研究
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摘要
旨在对比研究SD雄鼠3种不同新蛋白构型GnRH抗原的免疫学及生物学效果。本研究新蛋白构型GnRH抗原设计采用增加GnRH拷贝数和增大抗原分子量方法。以化学合成法合成G6K-GnRH并列体(G6K-GnRH-tandem,G6KT)作为新蛋白GnRH抗原制备的基本单元。将G6KT二聚化,形成抗原Ⅰ(G6KTD),将G6KT和G6KTD分别与卵清蛋白(OVA)偶联,分别形成抗原П(G6KT-OVA)及抗原Ⅲ(G6KTD-OVA)。试验分5组:完整对照组(不做任何处理,intact control)、外科阉割去势组(surgical castrates)及3种新蛋白构型GnRH免疫组(G6KTD、G6KT-OVA及G6KTD-OVA免疫组)。将(G6KTD、G6KT-OVA、G6KTD-OVA)3种不同新蛋白构型GnRH抗原配于Specol佐剂,分别免疫SD雄鼠,6周龄时初免,腿部肌肉注射1 mL乳化抗原(含相应新构型GnRH蛋白100μg),8周后加免,注射剂量与方法同初次免疫,每试验组12只雄鼠。放免法及酶联免疫法测定血清抗体及生殖激素浓度变化,qPCR检测垂体-睾丸生殖相关基因mRNA表达变化。结果显示:1)SD雄鼠主动免疫3种不同新蛋白构型GnRH抗原后均能产生良好的抗体反应。2)G6KT-OVA及G6KTD-OVA免疫组仅初免就能诱发较高水平的抗体产生,并显著降低雄鼠血清LH、FSH、睾酮(T)及抑制素B (INHB)浓度(P<0.05);试验结束时,该两种抗原免疫组雄鼠睾丸重量及体积均降低,精子发生完全被终止,垂体GnRHR、LHβ、FSHβ及睾丸LHR、FSHR、INHα、INβA及INHβB mRNA表达显著下调(P<0.05)。3)G6KTD免疫组12只雄鼠中,9只(75%)在加免后血清LH、FSH、T及INHB浓度显著降低(P<0.05),试验结束时睾丸重量及体积均显著降低(P<0.05),精子发生受到明显抑制(P<0.05)。综上表明:新蛋白构型G6KT-OVA和G6KTD-OVA具有极强的免疫原性,初次免疫就能达到抑制生殖激素分泌的目的,在作为单剂量GnRH疫苗方面具良好的开发前景;同时G6KTD也具有较强的免疫原性,可不与载体蛋白偶联单独作为GnRH去势抗原,继而避开GnRH分子与载体蛋白偶联所面临的GnRH抗原损失严重及与载体蛋白偶联的后续纯化等问题。本研究结果为研发高效GnRH去势疫苗及其大规模应用提供了理论参考。
The study was aimed to study the immunological and biological efficacy of 3-different types of GnRH-like constructs in male Sprague Dawley(SD) rats. The designing strategies for the new GnRH-like peptide constructs are based on: 1) increasing the number of GnRH copy; 2) increasing the molecular weight of GnRH antigen. The new GnRH-like peptide constructs were designed based on a GnRH-tandem peptide, which was modified by relacing Gly at position 6 of the decapeptide with D-Lys(G6 KT) to allow the conjugation of the GnRH peptide to ovalbumin(OVA). The first new GnRH-like construct was prepared by the dimerization of G6 KT(G6 KTD); the second and third were prepared by conjugating G6 KT and G6 KTD to OVA, respectively(G6 KT-OVA and G6 KTD-OVA). Sixty male SD rats at the age of 6 weeks, were randomly allocated to 5 groups(n=12):intact control(no treatment), surgically castrated or vaccinated against 100 μg peptide equivalent of G6 KTD, G6 KT-OVA and G6 KTD-OVA in Specol adjuvant, respectively, at 6 weeks of age(with a booster 8 weeks later). Serum anti-GnRH antibody titers and reproductive hormone concentrations were assayed by RIA and ELISA, respectively. mRNA expressions of reproduction-related genes in pituitary-testicular axis were quantified using qPCR.The results shown that: 1) active immunization against all of the 3 different new GnRH-like constructs could intrigue a good antibody responses in male SD rats; 2) Only with the first injection, active immunization against both G6 KT-OVA and G6 KTD-OVA intrigued a high antibody generation(P<0.05) and reduced serum concentrations of LH, FSH, testosterone and inhibin B(P<0.05), and further caused antrophy of testes with completely disrupted spermatogenesis and downregulated mRNA expressions of pituitary GnRHR, LHβ, FSHβ and testicular LHR, FSHR, INHα, INβA and INHβB(P<0.05) with the booster injection; 3) in contrast, the significant decrease of serum concentrations of LH, FSH, testosterone and inhibin B in rats actively immunized against G6 KTD only occurred after the booster injection(P<0.05), but which still caused antrophy of testes with obviously disrupted spermatogenesis at decapitation(P<0.05) in 9 of 12(75%) rats. In conclusion,based on those results, we concluded that both G6 KT-OVA and G6 KTD-OVA were highly efficacious GnRH antigen, which could be used as one-shot immunocastration vaccine in the furthure, whereas G6 KTD itself was also effective, which might be used as GnRH antigen so as to avoid the drawbacks derived from the conjugation and purification processes of GnRH-carrier conjugates. Our findings promoted the further development and large scale commercial applications of GnRH vaccine.
The study was aimed to study the immunological and biological efficacy of 3-different types of GnRH-like constructs in male Sprague Dawley(SD) rats. The designing strategies for the new GnRH-like peptide constructs are based on: 1) increasing the number of GnRH copy; 2) increasing the molecular weight of GnRH antigen. The new GnRH-like peptide constructs were designed based on a GnRH-tandem peptide, which was modified by relacing Gly at position 6 of the decapeptide with D-Lys(G6 KT) to allow the conjugation of the GnRH peptide to ovalbumin(OVA). The first new GnRH-like construct was prepared by the dimerization of G6 KT(G6 KTD); the second and third were prepared by conjugating G6 KT and G6 KTD to OVA, respectively(G6 KT-OVA and G6 KTD-OVA). Sixty male SD rats at the age of 6 weeks, were randomly allocated to 5 groups(n=12):intact control(no treatment), surgically castrated or vaccinated against 100 μg peptide equivalent of G6 KTD, G6 KT-OVA and G6 KTD-OVA in Specol adjuvant, respectively, at 6 weeks of age(with a booster 8 weeks later). Serum anti-GnRH antibody titers and reproductive hormone concentrations were assayed by RIA and ELISA, respectively. mRNA expressions of reproduction-related genes in pituitary-testicular axis were quantified using qPCR.The results shown that: 1) active immunization against all of the 3 different new GnRH-like constructs could intrigue a good antibody responses in male SD rats; 2) Only with the first injection, active immunization against both G6 KT-OVA and G6 KTD-OVA intrigued a high antibody generation(P<0.05) and reduced serum concentrations of LH, FSH, testosterone and inhibin B(P<0.05), and further caused antrophy of testes with completely disrupted spermatogenesis and downregulated mRNA expressions of pituitary GnRHR, LHβ, FSHβ and testicular LHR, FSHR, INHα, INβA and INHβB(P<0.05) with the booster injection; 3) in contrast, the significant decrease of serum concentrations of LH, FSH, testosterone and inhibin B in rats actively immunized against G6 KTD only occurred after the booster injection(P<0.05), but which still caused antrophy of testes with obviously disrupted spermatogenesis at decapitation(P<0.05) in 9 of 12(75%) rats. In conclusion,based on those results, we concluded that both G6 KT-OVA and G6 KTD-OVA were highly efficacious GnRH antigen, which could be used as one-shot immunocastration vaccine in the furthure, whereas G6 KTD itself was also effective, which might be used as GnRH antigen so as to avoid the drawbacks derived from the conjugation and purification processes of GnRH-carrier conjugates. Our findings promoted the further development and large scale commercial applications of GnRH vaccine.
引文
[1] CLARKE H,DHILLO W S,JAYASENA C N.Comprehensive review on kisspeptin and its role in reproductive disorders[J].Endocrinol Metab,2015,30(2):124-141.
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[4] ALUWé M,TUYTTENS F A M,MILLET S.Field experience with surgical castration with anaesthesia,analgesia,immunocastration and production of entire male pigs:performance,carcass traits and boar taint prevalence[J].Animal,2015,9(3):500-508.
[5] HAN X F,ZHOU Y Q,ZENG Y,et al.Effects of active immunization against GnRH versus surgical castration on hypothalamic-pituitary function in boars[J].Theriogenology,2017,97:89-97.
[6] APONTE P M,GUTIERREZ-REINOSO M A,SANCHEZ-CEPEDA E G,et al.Active immunization against GnRH in pre-pubertal domestic mammals:testicular morphometry,histopathology and endocrine responses in rabbits,guinea pigs and ram lambs[J].Animal,2018,12(4):784-793.
[7] ZENG X Y,TURKSTRA J A,TSIGOS A,et al.Effects of active immunization against GnRH on serum LH,inhibin A,sexual development and growth rate in Chinese female pigs[J].Theriogenology,2002,58(7):1315-1326.
[8] ZENG X Y,TURKSTRA J A,MELOEN R H,et al.Active immunization against gonadotrophin-releasing hormone in Chinese male pigs:effects of dose on antibody titer,hormone levels and sexual development[J].Anim Reprod Sci,2002,70(3-4):223-233.
[9] HAN X F,CAO X H,TANG J,et al.Active immunization against GnRH reduces the synthesis of GnRH in male rats[J].Theriogenology,2013,80(9):1109-1116.
[10] 韩兴发,曹晓涵,杜小刚,等.GnRH主动免疫对SD雄鼠下丘脑GnRH合成及性腺反馈系统的影响[J].畜牧兽医学报,2013,44(4):562-569.HAN X F,CAO X H,DU X G,et al.Effects of active immunization against GnRH on the hypothalamus GnRH biosynthesis and gonadal feedback system in sprague-dawley male rats[J].Acta Veterinaria et Zootechnica Sinica,2013,44(4):562-569.(in Chinese)
[11] BOKHOUT B A,VAN GAALEN C,VAN DER HEIJDEN P J.A selected water-in-oil emulsion:composition and usefulness as an immunological adjuvant[J].Vet Immunol Immunopathol,1981,2(5):491-500.
[12] FAGERSTONE K A,MILLER L A,KILLIAN G,et al.Review of issues concerning the use of reproductive inhibitors,with particular emphasis on resolving human-wildlife conflicts in North America[J].Integr Zool,2010,5(1):15-30.
[13] LEVY J K.Contraceptive vaccines for the humane control of community cat populations[J].Am J Reprod Immunol,2011,66(1):63-70.
[14] OONK H B,TURKSTRA J A,SCHAAPER W M M,et al.New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH[J].Vaccine,1998,16(11-12):1074-1082.
[15] DEMYASHKIN G A.Inhibin B in seminiferous tubules of human testes in normal spermatogenesis and in idiopathic infertility[J].Syst Biol Reprod Med,2018,doi:10.1080/19396368.2018.1478470.
[16] HAN X F,LI J L,ZHOU Y Q,et al.Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations,testicular development and spermatogenesis[J].Asian J Androl,2016,18(3):485-491.
[17] CLAUS R,LACORN M,DANOWSKI K,et al.Short-term endocrine and metabolic reactions before and after second immunization against GnRH in boars[J].Vaccine,2007,25(4):4689-4696.
[2] D’OCCHIO M J.Immunological suppression of reproductive functions in male and female mammals[J].Anim Reprod Sci,1993,33(1-4):345-372.
[3] THOMPSON D L Jr.Immunization against GnRH in male species (comparative aspects)[J].Anim Reprod Sci,2000,60-61:459-469.
[4] ALUWé M,TUYTTENS F A M,MILLET S.Field experience with surgical castration with anaesthesia,analgesia,immunocastration and production of entire male pigs:performance,carcass traits and boar taint prevalence[J].Animal,2015,9(3):500-508.
[5] HAN X F,ZHOU Y Q,ZENG Y,et al.Effects of active immunization against GnRH versus surgical castration on hypothalamic-pituitary function in boars[J].Theriogenology,2017,97:89-97.
[6] APONTE P M,GUTIERREZ-REINOSO M A,SANCHEZ-CEPEDA E G,et al.Active immunization against GnRH in pre-pubertal domestic mammals:testicular morphometry,histopathology and endocrine responses in rabbits,guinea pigs and ram lambs[J].Animal,2018,12(4):784-793.
[7] ZENG X Y,TURKSTRA J A,TSIGOS A,et al.Effects of active immunization against GnRH on serum LH,inhibin A,sexual development and growth rate in Chinese female pigs[J].Theriogenology,2002,58(7):1315-1326.
[8] ZENG X Y,TURKSTRA J A,MELOEN R H,et al.Active immunization against gonadotrophin-releasing hormone in Chinese male pigs:effects of dose on antibody titer,hormone levels and sexual development[J].Anim Reprod Sci,2002,70(3-4):223-233.
[9] HAN X F,CAO X H,TANG J,et al.Active immunization against GnRH reduces the synthesis of GnRH in male rats[J].Theriogenology,2013,80(9):1109-1116.
[10] 韩兴发,曹晓涵,杜小刚,等.GnRH主动免疫对SD雄鼠下丘脑GnRH合成及性腺反馈系统的影响[J].畜牧兽医学报,2013,44(4):562-569.HAN X F,CAO X H,DU X G,et al.Effects of active immunization against GnRH on the hypothalamus GnRH biosynthesis and gonadal feedback system in sprague-dawley male rats[J].Acta Veterinaria et Zootechnica Sinica,2013,44(4):562-569.(in Chinese)
[11] BOKHOUT B A,VAN GAALEN C,VAN DER HEIJDEN P J.A selected water-in-oil emulsion:composition and usefulness as an immunological adjuvant[J].Vet Immunol Immunopathol,1981,2(5):491-500.
[12] FAGERSTONE K A,MILLER L A,KILLIAN G,et al.Review of issues concerning the use of reproductive inhibitors,with particular emphasis on resolving human-wildlife conflicts in North America[J].Integr Zool,2010,5(1):15-30.
[13] LEVY J K.Contraceptive vaccines for the humane control of community cat populations[J].Am J Reprod Immunol,2011,66(1):63-70.
[14] OONK H B,TURKSTRA J A,SCHAAPER W M M,et al.New GnRH-like peptide construct to optimize efficient immunocastration of male pigs by immunoneutralization of GnRH[J].Vaccine,1998,16(11-12):1074-1082.
[15] DEMYASHKIN G A.Inhibin B in seminiferous tubules of human testes in normal spermatogenesis and in idiopathic infertility[J].Syst Biol Reprod Med,2018,doi:10.1080/19396368.2018.1478470.
[16] HAN X F,LI J L,ZHOU Y Q,et al.Active immunization with GnRH-tandem-dimer peptide in young male rats reduces serum reproductive hormone concentrations,testicular development and spermatogenesis[J].Asian J Androl,2016,18(3):485-491.
[17] CLAUS R,LACORN M,DANOWSKI K,et al.Short-term endocrine and metabolic reactions before and after second immunization against GnRH in boars[J].Vaccine,2007,25(4):4689-4696.