Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and de...Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.展开更多
在证实分子改进能增强人绒毛膜促性腺激素 β链 C-末端肽 (β h CG-CTP)的免疫原性后 ,进一步观察其抗血清对 h CG生物活性的影响。利用蛋白质固相合成技术 ,将β h CG-CTP的两个 B细胞表位 (β8,β9)以及一个源自麻疹病毒融合蛋白的“...在证实分子改进能增强人绒毛膜促性腺激素 β链 C-末端肽 (β h CG-CTP)的免疫原性后 ,进一步观察其抗血清对 h CG生物活性的影响。利用蛋白质固相合成技术 ,将β h CG-CTP的两个 B细胞表位 (β8,β9)以及一个源自麻疹病毒融合蛋白的“通用”T细胞表位合成于一体 ,以此多肽免疫家兔 ,以 WHO的 β h CG-CTP疫苗标准品为对照 ,制备抗血清 ,利用小鼠子宫增重试验检测抗血清对 h CG生物活性的影响。结果 :该嵌合多肽免疫家兔后产生的抗血清原液、1∶ 1 0及 1∶ 5 0倍稀释时 ,能显著抑制 h CG诱导的小鼠子宫增重。结果表明 ,β h CG-CTP经分子改进后不仅免疫原性增强 ,且能在体内中和 h CG的生物学效应 。展开更多
Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed b...Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic ex-pression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vac-cinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vacci-nated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive DNA vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future.展开更多
基金This study was supported by the National Key Technologies R and D Program （No. 2012BAI31B07 and No. 2006BAI03B12）, the National Science Foundationof China （No. 81172694）. This project was also funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
文摘Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.
文摘在证实分子改进能增强人绒毛膜促性腺激素 β链 C-末端肽 (β h CG-CTP)的免疫原性后 ,进一步观察其抗血清对 h CG生物活性的影响。利用蛋白质固相合成技术 ,将β h CG-CTP的两个 B细胞表位 (β8,β9)以及一个源自麻疹病毒融合蛋白的“通用”T细胞表位合成于一体 ,以此多肽免疫家兔 ,以 WHO的 β h CG-CTP疫苗标准品为对照 ,制备抗血清 ,利用小鼠子宫增重试验检测抗血清对 h CG生物活性的影响。结果 :该嵌合多肽免疫家兔后产生的抗血清原液、1∶ 1 0及 1∶ 5 0倍稀释时 ,能显著抑制 h CG诱导的小鼠子宫增重。结果表明 ,β h CG-CTP经分子改进后不仅免疫原性增强 ,且能在体内中和 h CG的生物学效应 。
基金Grants from the Key Research Projects of Science and Technology of Fujian Province,China (Grant No. 2002Y032)Chinese PLA Foundation of Medical Sciences during the Tenth Five–Year Period (Grant No. 01MA032)
文摘Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic ex-pression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vac-cinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vacci-nated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive DNA vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future.
