Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For pro...Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a （＋）-His6-tobacco etch virus （TEV）-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2＋ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 I of culture with a purity of 〉95%. The immunogenicity of the eppin was characterized using male Balb/c mice.展开更多
Objective To analyze the clinical features and outcome of patients who used different pretreatments before application of gonadotropin-releasing hormone antagonist protocol during in vitro fertilization - embryo trans...Objective To analyze the clinical features and outcome of patients who used different pretreatments before application of gonadotropin-releasing hormone antagonist protocol during in vitro fertilization - embryo transfer （IVF-ET） cycles, and to explore how effective to use the antagonist protocol. Methods A retrospective analysis was performed. All the ET cycles were divided into three groups, group A （n=125） used short acting GnRH agonist before GnRH antagonist treatment, group B （n=113） used short-acting oral contraceptives before GnRH antagonist treatment, group C （n=81） was untreated before GnRH antagonist treatment. All the patients had no tubal fluid, endometrial polyps and no anatomical abnormalities of the uterus, from April 2010 to December 2010. The patient＇s age, dose and duration of gonadotropin （Gn） treatment, the serum LH and E2 levels on the day of hCG injection, the number of oocytes retrieved, the rates of good-quality embryos, the clinical pregnancy rates were compared. At the same time, 261 GnRH agonist long protocol cycles （group D） were selected at the same period as further comparison. Results The patients in group C （32.9 ~ 4.8 years） were significantly older than those in groups A and B （31.6 ___＋3.7 years, 31.2 ___%4.1 years）（P 〈0.05）. The dose and the duration of Gn in group C were significantly lower than those in groups A and B. The serum LH level on the day of hCG injection in group A and group B was significantly lower than that in group C （P 〈0.05）, especially in group A. The endometrium was the thinnest in group B. There were no significant differences in the fertilization rates and the good-quality embryosrates among them. The clinical pregnancy rate of group B decreased significantly compared with groups A and C （P〈0. 05）. The clinical pregnancy rate of group C was the highest among them. There was no significant difference of clinical pregnancy rates between group C and group D （37% vs 40.2%,P〉0.05）. However, the dose （19.8 ±6.6 ampoule vs 26.4 ±8.1 ampoule） and the duration （9.0± 1.6 d vs 11.6±2.5 d） of Gn treatment in group C were decreased significantly than those in group D, P〈0.05. Conclusion The short acting GnRH agonist used before GnRH antagonist treatment during IVF-ET cycles failed to improve the pregnancy rates, the use of short-acting oral contraceptives before GnRH antagonist treatment makes the pregnancy rates decrease significantly, but untreated before GnRH antagonist protocol can get a better clinical outcome compared with agonist long protocol Untreated GnRH anagonist protocol is the best GnRH anagonist protocol.展开更多
基金ACKNOWLEDGEMENTS The authors are grateful to Dr Zi-Chun Hua （The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Science, Nanjing University, China） for kindly providing plasmid pET28a （＋） and TEV endoprotease and to Dr Michael G. O＇Rand （Laboratories for Reproductive Biology and Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, USA） for English-language editing. This study was supported by grants from the Key Project of the National Natural Science Foundation of China （No. 30930079） and the Chinese National Nature Science Foundation （No. 81001257）.
文摘Human epididymal protease inhibitor （eppin） may be effective as a male contraceptive vaccine. In a number of studies, eppin with an engineered His6-tag has been produced using prokaryotic expression systems. For production of pharmaceutical-grade proteins for human use, however, the His6-tag must be removed. This study describes a method for producing recombinant human eppin without a His6-tag. We constructed plasmid pET28a （＋）-His6-tobacco etch virus （TEV）-eppin for expression in Escherichia coli. After purification and refolding, the fusion protein His6-TEV-eppin was digested with TEV protease to remove the His6-tag and was further purified by NTA-Ni2＋ affinity chromatography. Using this procedure, 2 mg of eppin without a His6-tag was isolated from 1 I of culture with a purity of 〉95%. The immunogenicity of the eppin was characterized using male Balb/c mice.
文摘Objective To analyze the clinical features and outcome of patients who used different pretreatments before application of gonadotropin-releasing hormone antagonist protocol during in vitro fertilization - embryo transfer （IVF-ET） cycles, and to explore how effective to use the antagonist protocol. Methods A retrospective analysis was performed. All the ET cycles were divided into three groups, group A （n=125） used short acting GnRH agonist before GnRH antagonist treatment, group B （n=113） used short-acting oral contraceptives before GnRH antagonist treatment, group C （n=81） was untreated before GnRH antagonist treatment. All the patients had no tubal fluid, endometrial polyps and no anatomical abnormalities of the uterus, from April 2010 to December 2010. The patient＇s age, dose and duration of gonadotropin （Gn） treatment, the serum LH and E2 levels on the day of hCG injection, the number of oocytes retrieved, the rates of good-quality embryos, the clinical pregnancy rates were compared. At the same time, 261 GnRH agonist long protocol cycles （group D） were selected at the same period as further comparison. Results The patients in group C （32.9 ~ 4.8 years） were significantly older than those in groups A and B （31.6 ___＋3.7 years, 31.2 ___%4.1 years）（P 〈0.05）. The dose and the duration of Gn in group C were significantly lower than those in groups A and B. The serum LH level on the day of hCG injection in group A and group B was significantly lower than that in group C （P 〈0.05）, especially in group A. The endometrium was the thinnest in group B. There were no significant differences in the fertilization rates and the good-quality embryosrates among them. The clinical pregnancy rate of group B decreased significantly compared with groups A and C （P〈0. 05）. The clinical pregnancy rate of group C was the highest among them. There was no significant difference of clinical pregnancy rates between group C and group D （37% vs 40.2%,P〉0.05）. However, the dose （19.8 ±6.6 ampoule vs 26.4 ±8.1 ampoule） and the duration （9.0± 1.6 d vs 11.6±2.5 d） of Gn treatment in group C were decreased significantly than those in group D, P〈0.05. Conclusion The short acting GnRH agonist used before GnRH antagonist treatment during IVF-ET cycles failed to improve the pregnancy rates, the use of short-acting oral contraceptives before GnRH antagonist treatment makes the pregnancy rates decrease significantly, but untreated before GnRH antagonist protocol can get a better clinical outcome compared with agonist long protocol Untreated GnRH anagonist protocol is the best GnRH anagonist protocol.
