附睾蛋白酶抑制蛋白的B细胞表位筛选及其抗生育效应的实验研究
摘要
免疫性避孕一直是男性避孕节育研究的重要领域,具有巨大的临床应用前景和良好的社会经济价值。然而迄今为止没有理想的避孕疫苗问世,究其原因最大的可能在于没有理想的靶抗原。作为避孕疫苗应用的的靶抗原应当至少满足两个条件,即抗原特异表达于生殖系统且参与生殖过程中的重要环节。
近年来,附睾蛋白酶抑制蛋白(Epidydimal protease inhibitor,Eppin)因在非人灵长类动物实验中显示出可靠的避孕效应而备受关注。它特异表达于睾丸和附睾,在精子成熟和生殖过程中具有重要作用。利用重组Eppin蛋白免疫雄猴获得了78%抗生育效果,停止免疫后,71%的受试雄猴恢复了生育力。体外实验发现来自不育雄猴血清中的抗Eppin抗体可直接破坏人Eppin-精囊凝固蛋白(Semenogelin,Sg)复合体,干扰Eppin调控前列腺特异性抗原PSA对Sg的水解作用,影响人精子活动力,进而抑制生育力。因此,Eppin可能成为理想的避孕疫苗靶抗原。
虽然对Eppin的研究有了一定的进展,但仍存在如下问题:(1)蛋白抗原分子量较大,难以实现质量控制;(2)未对抗原中可能导致远期毒副反应的不良表位做剔除或加工。(3)抗体产生不稳定;(4)隐匿的保护性表位功能未能表达,所引发的保护性免疫不够强大,免疫避孕效果不理想。因此,为了提高Eppin抗原的保护性,就不得不从天然抗原整个分子水平向表位水平过渡:摒除不良表位,保留、改善保护性表位以得到更理想的疫苗分子。表位是蛋白质抗原性的基础,正确而详细地绘制蛋白质表位图谱对设计疫苗分子结构及免疫干预治疗等具有重要意义。
本研究首先通过RT-PCR法从人新鲜睾丸组织扩增获得Eppin cDNA,构建入原核表达载体,诱导表达并经纯化获得重组人Eppin蛋白(第一部分)。同时,利用生物信息学方法分析Eppin蛋白的二级结构及抗原性、亲水性、柔韧性、表面可及性等理化特性,预测Eppin蛋白的主要功能区域,根据该区域抗原趋势指数分析结果,采用错位重叠套式方法设计该区域的肽段。利用人工合成肽技术固相合成多肽,并与载体蛋白钥孔血蓝蛋白(KLH)、牛血清蛋白(BSA)偶联,用KLH偶联肽与弗氏完全佐剂、不完全佐剂等体积混合,充分乳化后足垫皮下注射免疫小鼠,通过间接ELISA方法观察各个表位肽刺激抗体的产生,通过体外、体内抗生育实验观察表位肽的抗生育效应,以此筛选出其优势中和B细胞表位,并以rhEppin免疫为阳性对照,分析其抗生育潜能及安全性(第二部分)。最后,设计并制备基于Eppin优势B细胞表位的2分支MAP抗原肽,以弗氏佐剂为佐剂,腹部皮下免疫雄性恒河猴,ELISA方法监测免疫后被免疫雄猴血清抗体滴度,CBA方法检测Th1/Th2细胞因子的改变,睾丸活检术取睾丸标本电镜观察免疫后睾丸超微结构的变化,并与发情期雌猴合笼2个周期,观察Eppin的MAP抗原肽对恒河猴的抗生育效果(第三部分)。
结果显示:①成功构建了pET-32a-Eppin重组质粒,并获得了具有抗原特异性的可溶性Eppin蛋白。②通过分析Eppin蛋白二级结构及表面特性,初步预测出Eppin蛋白的89～133位氨基酸为主要功能区域,设计合成8条代表该氨基酸区域的肽段,并与载体蛋白KLH偶联,命名为F1-F8。③采用偶联肽足垫皮下注射免疫小鼠,发现8个候选表位肽都能激发高滴度的抗体,但仅F5肽所获得的抗血清能与人精子天然Eppin蛋白结合,并能显著抑制精卵结合,使雄鼠的生育力受到很大限制,从而获得Eppin优势中和B细胞表位为F5:FVYGGCQGNN(105～114区段)。④Eppin的MAP抗原肽免疫的10只雄猴中有2只血清IgG抗体滴度始终无明显提高,淘汰,剩余8只抗体滴度可达1:400~1600,但需2~3周加强免疫一次。合笼后发现对照组5只有4只受孕,实验组8只仅3只受孕,显示了较肯定的避孕效果。电镜结果未发现免疫后睾丸组织超微结构的损害,CBA细胞因子检测未见Th1/Th2极化改变,各细胞因子免疫前后无明显差异。提示合成肽对T细胞功能的影响甚微,今后的剂型改造可以考虑增强T细胞功能,在佐剂、递呈方式上做优化。
本研究结果表明生物信息学方法表位预测并合成偶联肽免疫策略是筛选蛋白优势中和B细胞表位的有效方法;Eppin蛋白的第105～114区段是Eppin优势中和B细胞表位区域;Eppin表位肽用于免疫避孕具有较好的安全性和有效性;基于Eppin优势表位的避孕疫苗尚需要在表位水平进一步修饰改造才能满足人用避孕疫苗的要求。
The world’s population is growing at a tremendous rate and despite of the availability of various contraceptive modalities there is an urgent requirement for a better contraception method, which is reversible, safe and highly effective, easily obtained, inexpensive and suitable for all stages of reproductive life. Contraceptive vaccines can fulfill most of the properties of an ideal contraceptive. Up to now, there has been no indication of an ideal contraceptive vaccine being developed probably due to the lack of an ideal target antigen. The target antigen applied in contraceptive vaccine must meet at least two conditions. First, it should be expressed specifically in the reproductive system; second, it should act as an important component in the reproductive process.
Recently, epididymal protease inhibitor (Eppin), a sperm specific antigen, was identified as an ideal and promising target for the development of a series of safer and more effective contraceptive vaccines. Human-derived Eppin is specifically expressed in epididymis and testis. Eppin was detected in Sertoli cells in 12 day-old mice and afterwards expresses during the maturation process of spermatids. When used for active immunization of adult male Macaca monkeys, seven out of nine males (78%) developed high titers to recombinant Eppin, and all these high titer monkeys were infertile. Five out of seven (71%) high anti-Eppin titer males recovered fertility when immunization was stopped. This study revealed that Eppin is a promising target for male contraception.
However, previous studies showed that the contraceptive effect of the full-length protein was not sufficient to satisfy clinical demand. This may ascribe to: Eppin protein not only has B-cell and T cell epitopes providing immune recognition, but also has toxic or inhibitory epitopes, non-neutralized dominant epitopes, pathological epitopes and cross-reactive epitopes with auto-antigen. Therefore, in order to induce an immune response with stronger specificity and an increased immune effect, we must effectively avoide the former and modify the latter, allowing more ideal vaccine molecules to be developed. Epitope vaccine, synthesized with antigen epitope, is a newly developed vaccine technique with the advantages of a simple manufacturing processes, relatively low cost, stable chemical property and strong immune specificity with minimal toxicity or side-effects. Screening as well as identification of epitopes is the primary and most important task during preparation of an epitope vaccine.
Eppin cDNA was obtained by RT-PCR from fresh human testis, and then cloned into expression vector pET-32a, the recombinant vector was transformed into BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni2+-NTA-column.The antigenic activity of purified recombinant protein was assessed with Western blot (Part I).The epitope prediction was performed according to the correlation of B-cell epitope with physico- chemical property or secondary structures. After reviewing references, analyzing the homology and rejecting the crossing part of Eppin,8 sequential overlapping decapeptides (six amino acids overlap) targeting the Kunitz domain were designed and synthesized by solid-phase synthesis using a synthesis peptide technique.The dominant cellular epitopes were screened and identified by further experiments in vitro and in vivo. It was theoretically confirmed that the antigens had strong antigenicity and prevented potentially adverse immune reactions caused by pathological antigen and cross-reactive epitopes (Part II).Finally, the two branches of multiple antigen peptide (MAP) was designed and synthesized by solid-phase synthesis using a synthesis peptide technique. The sexually mature male rhesus monkeys were immunized with MAP by abdominal subcutaneous injection in Freund’s adjuvant. The presence and reactivity of antibody (IgG) against peptides in the serum were analyzed using ELISA. The levels of Th1/Th2 cytokine in serum were detected by non-human primate Th1/Th2 cytokine kit (BDTM cytometric bead array) (Part III).
The results showed that:①Enzyme digestion analysis and sequencing showed that the target fragment was successfully inserted into the vector pET-32a (+).Soluble Eppin protein possessing specific antigenicity was expressed and purified successfully.②B-cell epitope prediction of the human testis and epididymis specific sequence of Eppin was made based on the characteristics of hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface and turns, using DNASTAR’S Lasegene software (DNAStar Inc.). Eight B-cell epitopes corresponding to 89-133 sites amino acids domain were selected, synthesized and coupled with keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA) by Xi’an Huachen Bio-technology Co., Ltd, China. Homology analysis using the BLAST function of NCBI didn’t show any peptide sequences which share significant homology with other proteins with the exception of Eppin protein itself. The purity of each peptide was above 90% as analyzed by reverse phase high-performance liquid chromatography (HPLC).③All eight peptides can elicit similar specific antibody titers, however,only the antisera from F5 showed the capacity to combined with natural Eppin in semen,and significantly decreased the rate of pregnancy in mice. Therefore the immunodominant neutralizing B-cell epitope of Eppin is F5: FVYGGCQGNN (Eppin 105-114).④High–anti-Eppin titers of 1:400 ~ 1600 were detected in eight of the ten monkeys immunized with Eppin MAP; two monkeys were low responders with titers <1:400 on postimmunization day 120. Because the purpose of this study was to test the efficacy of antibodies to Eppin MAP on fertility, the two low-titer monkeys were dropped from the study group and their fertility was not tested. Each male monkey in the immune and control groups were subjected to fertility testing by cohabiting with a proven fertile female during her two ovulatory cycles. Three out of eight males in the immune group were infertile and one out of five males in the control group was infertile. This revealed the MAP of Eppin had an affirmative contraceptive effect. The electron microscope examination of testis biopsy also showed no obvious abnomality. But in the detecting of Th1/Th2 cytokines, there was no significant difference in the males of immune and control groups.
The results of this research indicates that epitope prediction by bioinformatics method combinated with synthetic peptide coupling carrier protein immunization strategy is an an effective way and a beneficial alternative to analysis B-cell epitope.The immunodominant neutralizing B-cell epitope of Eppin located at the 105-114 amnio acids.Epitope peptide of Eppin is an effective and safe modality for male contraception.However contraceptive vaccine based on Eppin dominant epitope needs further modification on the level of epitope to meet the requirements of contraceptive vaccine for human use.
近年来,附睾蛋白酶抑制蛋白(Epidydimal protease inhibitor,Eppin)因在非人灵长类动物实验中显示出可靠的避孕效应而备受关注。它特异表达于睾丸和附睾,在精子成熟和生殖过程中具有重要作用。利用重组Eppin蛋白免疫雄猴获得了78%抗生育效果,停止免疫后,71%的受试雄猴恢复了生育力。体外实验发现来自不育雄猴血清中的抗Eppin抗体可直接破坏人Eppin-精囊凝固蛋白(Semenogelin,Sg)复合体,干扰Eppin调控前列腺特异性抗原PSA对Sg的水解作用,影响人精子活动力,进而抑制生育力。因此,Eppin可能成为理想的避孕疫苗靶抗原。
虽然对Eppin的研究有了一定的进展,但仍存在如下问题:(1)蛋白抗原分子量较大,难以实现质量控制;(2)未对抗原中可能导致远期毒副反应的不良表位做剔除或加工。(3)抗体产生不稳定;(4)隐匿的保护性表位功能未能表达,所引发的保护性免疫不够强大,免疫避孕效果不理想。因此,为了提高Eppin抗原的保护性,就不得不从天然抗原整个分子水平向表位水平过渡:摒除不良表位,保留、改善保护性表位以得到更理想的疫苗分子。表位是蛋白质抗原性的基础,正确而详细地绘制蛋白质表位图谱对设计疫苗分子结构及免疫干预治疗等具有重要意义。
本研究首先通过RT-PCR法从人新鲜睾丸组织扩增获得Eppin cDNA,构建入原核表达载体,诱导表达并经纯化获得重组人Eppin蛋白(第一部分)。同时,利用生物信息学方法分析Eppin蛋白的二级结构及抗原性、亲水性、柔韧性、表面可及性等理化特性,预测Eppin蛋白的主要功能区域,根据该区域抗原趋势指数分析结果,采用错位重叠套式方法设计该区域的肽段。利用人工合成肽技术固相合成多肽,并与载体蛋白钥孔血蓝蛋白(KLH)、牛血清蛋白(BSA)偶联,用KLH偶联肽与弗氏完全佐剂、不完全佐剂等体积混合,充分乳化后足垫皮下注射免疫小鼠,通过间接ELISA方法观察各个表位肽刺激抗体的产生,通过体外、体内抗生育实验观察表位肽的抗生育效应,以此筛选出其优势中和B细胞表位,并以rhEppin免疫为阳性对照,分析其抗生育潜能及安全性(第二部分)。最后,设计并制备基于Eppin优势B细胞表位的2分支MAP抗原肽,以弗氏佐剂为佐剂,腹部皮下免疫雄性恒河猴,ELISA方法监测免疫后被免疫雄猴血清抗体滴度,CBA方法检测Th1/Th2细胞因子的改变,睾丸活检术取睾丸标本电镜观察免疫后睾丸超微结构的变化,并与发情期雌猴合笼2个周期,观察Eppin的MAP抗原肽对恒河猴的抗生育效果(第三部分)。
结果显示:①成功构建了pET-32a-Eppin重组质粒,并获得了具有抗原特异性的可溶性Eppin蛋白。②通过分析Eppin蛋白二级结构及表面特性,初步预测出Eppin蛋白的89～133位氨基酸为主要功能区域,设计合成8条代表该氨基酸区域的肽段,并与载体蛋白KLH偶联,命名为F1-F8。③采用偶联肽足垫皮下注射免疫小鼠,发现8个候选表位肽都能激发高滴度的抗体,但仅F5肽所获得的抗血清能与人精子天然Eppin蛋白结合,并能显著抑制精卵结合,使雄鼠的生育力受到很大限制,从而获得Eppin优势中和B细胞表位为F5:FVYGGCQGNN(105～114区段)。④Eppin的MAP抗原肽免疫的10只雄猴中有2只血清IgG抗体滴度始终无明显提高,淘汰,剩余8只抗体滴度可达1:400~1600,但需2~3周加强免疫一次。合笼后发现对照组5只有4只受孕,实验组8只仅3只受孕,显示了较肯定的避孕效果。电镜结果未发现免疫后睾丸组织超微结构的损害,CBA细胞因子检测未见Th1/Th2极化改变,各细胞因子免疫前后无明显差异。提示合成肽对T细胞功能的影响甚微,今后的剂型改造可以考虑增强T细胞功能,在佐剂、递呈方式上做优化。
本研究结果表明生物信息学方法表位预测并合成偶联肽免疫策略是筛选蛋白优势中和B细胞表位的有效方法;Eppin蛋白的第105～114区段是Eppin优势中和B细胞表位区域;Eppin表位肽用于免疫避孕具有较好的安全性和有效性;基于Eppin优势表位的避孕疫苗尚需要在表位水平进一步修饰改造才能满足人用避孕疫苗的要求。
The world’s population is growing at a tremendous rate and despite of the availability of various contraceptive modalities there is an urgent requirement for a better contraception method, which is reversible, safe and highly effective, easily obtained, inexpensive and suitable for all stages of reproductive life. Contraceptive vaccines can fulfill most of the properties of an ideal contraceptive. Up to now, there has been no indication of an ideal contraceptive vaccine being developed probably due to the lack of an ideal target antigen. The target antigen applied in contraceptive vaccine must meet at least two conditions. First, it should be expressed specifically in the reproductive system; second, it should act as an important component in the reproductive process.
Recently, epididymal protease inhibitor (Eppin), a sperm specific antigen, was identified as an ideal and promising target for the development of a series of safer and more effective contraceptive vaccines. Human-derived Eppin is specifically expressed in epididymis and testis. Eppin was detected in Sertoli cells in 12 day-old mice and afterwards expresses during the maturation process of spermatids. When used for active immunization of adult male Macaca monkeys, seven out of nine males (78%) developed high titers to recombinant Eppin, and all these high titer monkeys were infertile. Five out of seven (71%) high anti-Eppin titer males recovered fertility when immunization was stopped. This study revealed that Eppin is a promising target for male contraception.
However, previous studies showed that the contraceptive effect of the full-length protein was not sufficient to satisfy clinical demand. This may ascribe to: Eppin protein not only has B-cell and T cell epitopes providing immune recognition, but also has toxic or inhibitory epitopes, non-neutralized dominant epitopes, pathological epitopes and cross-reactive epitopes with auto-antigen. Therefore, in order to induce an immune response with stronger specificity and an increased immune effect, we must effectively avoide the former and modify the latter, allowing more ideal vaccine molecules to be developed. Epitope vaccine, synthesized with antigen epitope, is a newly developed vaccine technique with the advantages of a simple manufacturing processes, relatively low cost, stable chemical property and strong immune specificity with minimal toxicity or side-effects. Screening as well as identification of epitopes is the primary and most important task during preparation of an epitope vaccine.
Eppin cDNA was obtained by RT-PCR from fresh human testis, and then cloned into expression vector pET-32a, the recombinant vector was transformed into BL21 (DE3) and induced by IPTG. The recombinant protein was purified by Ni2+-NTA-column.The antigenic activity of purified recombinant protein was assessed with Western blot (Part I).The epitope prediction was performed according to the correlation of B-cell epitope with physico- chemical property or secondary structures. After reviewing references, analyzing the homology and rejecting the crossing part of Eppin,8 sequential overlapping decapeptides (six amino acids overlap) targeting the Kunitz domain were designed and synthesized by solid-phase synthesis using a synthesis peptide technique.The dominant cellular epitopes were screened and identified by further experiments in vitro and in vivo. It was theoretically confirmed that the antigens had strong antigenicity and prevented potentially adverse immune reactions caused by pathological antigen and cross-reactive epitopes (Part II).Finally, the two branches of multiple antigen peptide (MAP) was designed and synthesized by solid-phase synthesis using a synthesis peptide technique. The sexually mature male rhesus monkeys were immunized with MAP by abdominal subcutaneous injection in Freund’s adjuvant. The presence and reactivity of antibody (IgG) against peptides in the serum were analyzed using ELISA. The levels of Th1/Th2 cytokine in serum were detected by non-human primate Th1/Th2 cytokine kit (BDTM cytometric bead array) (Part III).
The results showed that:①Enzyme digestion analysis and sequencing showed that the target fragment was successfully inserted into the vector pET-32a (+).Soluble Eppin protein possessing specific antigenicity was expressed and purified successfully.②B-cell epitope prediction of the human testis and epididymis specific sequence of Eppin was made based on the characteristics of hydrophilicity, flexibility/mobility, accessibility, polarity, exposed surface and turns, using DNASTAR’S Lasegene software (DNAStar Inc.). Eight B-cell epitopes corresponding to 89-133 sites amino acids domain were selected, synthesized and coupled with keyhole limpet hemocyanin (KLH), or bovine serum albumin (BSA) by Xi’an Huachen Bio-technology Co., Ltd, China. Homology analysis using the BLAST function of NCBI didn’t show any peptide sequences which share significant homology with other proteins with the exception of Eppin protein itself. The purity of each peptide was above 90% as analyzed by reverse phase high-performance liquid chromatography (HPLC).③All eight peptides can elicit similar specific antibody titers, however,only the antisera from F5 showed the capacity to combined with natural Eppin in semen,and significantly decreased the rate of pregnancy in mice. Therefore the immunodominant neutralizing B-cell epitope of Eppin is F5: FVYGGCQGNN (Eppin 105-114).④High–anti-Eppin titers of 1:400 ~ 1600 were detected in eight of the ten monkeys immunized with Eppin MAP; two monkeys were low responders with titers <1:400 on postimmunization day 120. Because the purpose of this study was to test the efficacy of antibodies to Eppin MAP on fertility, the two low-titer monkeys were dropped from the study group and their fertility was not tested. Each male monkey in the immune and control groups were subjected to fertility testing by cohabiting with a proven fertile female during her two ovulatory cycles. Three out of eight males in the immune group were infertile and one out of five males in the control group was infertile. This revealed the MAP of Eppin had an affirmative contraceptive effect. The electron microscope examination of testis biopsy also showed no obvious abnomality. But in the detecting of Th1/Th2 cytokines, there was no significant difference in the males of immune and control groups.
The results of this research indicates that epitope prediction by bioinformatics method combinated with synthetic peptide coupling carrier protein immunization strategy is an an effective way and a beneficial alternative to analysis B-cell epitope.The immunodominant neutralizing B-cell epitope of Eppin located at the 105-114 amnio acids.Epitope peptide of Eppin is an effective and safe modality for male contraception.However contraceptive vaccine based on Eppin dominant epitope needs further modification on the level of epitope to meet the requirements of contraceptive vaccine for human use.
引文
[1] Hoesl CE,Saad F,Poppel M, et al. Reversible,non-barrier male contraception: status and prospects.Eur Urol 2005; 48(5): 712-22.
[2] Page ST, Amory JK, Bremner WJ.Advances in male contraception. Endocr Rev. 2008; 29 (4): 465-93.
[3] Naz RK. Status of contraceptive vaccines.Am J Reprod Immunol.2009; 61(1):11-8.
[4] Suri A. Sperm-based contraceptive vaccines: current status,merits and development. Expert Rev Mol Med 2005; 7(18): 1-16.
[5] Delves PJ, Lund T, Roitt IM.Antifertility vaccines.Trends-Immunol.2002; 23(4): 213-9.
[6] Naz RK,Gupta SK,Gupta JC,et al. Recent advances in contraceptive vaccine development: a mini-review [J]. Hum Reprod,2005,20(12): 3271-83.
[7] Naz RK,Chauhan SC. Human sperm-specific peptide vaccine that causes long-term reversible contraception[J]. Biol Reprod,2002,67(2):674-80.
[8] Chen H, Griffiths GS, Galileo DS & Martin-DeLeon PA.Epididymal SPAM1 is a marker of sperm maturation in mouse. Biology of Reproduction.2006; 74: 923-30.
[9] Hardy CM, Clydesdale G, Mobbs KJ, Pekin J, Lloyd ML, Sweet C, Shellam GR, Lawson MA. Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20.Reproduction. 2004; 127(3):325-34.
[10] Griffiths GS, Galileo DS, Aravindan RG, Martin-DeLeon PA.Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.Biol Reprod. 2009; 81(3):562-70.
[11] Kurth BE, Digilio L, Snow P, Bush LA, Wolkowicz M, Shetty J, Mandal A, Hao Z, Reddi PP, Flickinger CJ, Herr JC.Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.J Reprod Immunol. 2008; 77(2):126-41.
[12] Jagadish N, Rana R, Selvi R.Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.Biochem.J. 2005; 389:73–82.
[13] Williams J, Samuel A, Naz RK.Presence of antisperm antibodies reactive with peptide epitopes of FA-1 and YLP12 in sera of immunoinfertile women.Am J Reprod Immunol. 2008; 59(6):518-24.
[14] Naz RK.Effect of fertilization antigen (FA-1) DNA vaccine on fertility of female mice.Mol Reprod Dev. 2006; 73(11): 1473-9.
[15] Suri A.Contraceptive vaccines targeting sperm. Expert Opin Biol Ther 2005; 5(3): 381-92.
[16] O'Rand MG,Widgren EE,Wang Z,et al. Eppin: an effective target for male contraception. Mol Cell Endocrinol 2006; 250(1-2): 157-62.
[17] Richardson RT,Sivashanmugam P,Hall SH,et al. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 2001; 270: 93-102.
[18] Wang Z,Widgren EE,Richardson RT; et al. Eppin: a molecular strategy for male contraception. Soc Reprod Fertil Suppl 2007; 65: 535-42.
[19] Sivashanmugam P,Hall SH,Hamil KG,et al. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene 2003; 312: 125-34.
[20] Kirchhoff C,Osterhoff C,Pera I,et al. Function of human epididymal proteins in sperm maturation. Andrologia,1998,30(4-5): 225-32.
[21] Zinkernagel RM & Hengartner H. Regulation of the immune response by antigen. Science,2001,293(5528): 251-3.
[22] O'rand MG,Widgren EE,Sivashanmugam P,Richardson RT,Hall SH,French FS,VandeVoort CA,Ramachandra SG,Ramesh V,Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin.Science. 2004 ; 306:1189-90.
[23] Wang Z,Widgren EE,Sivashanmugam P,et al. Association of eppin with semenogelin on human spermatozoa[J]. Biol Reprod,2005,72 (5): 1064-70.
[24] O'Rand MG,Widgren EE,Beyler S,Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod.2009; 80(2):279-85.
[25] Romagnani S. Coming back to a missing immune deviation as the main explanatorymechanism for the hygiene hypothesis [J]. J Allergy Clin Immunol, 2007,119 (6): 1511-13.
[26] Romagnani S. Coming back to a missing immune deviation as the main explanatorymechanism for the hygiene hypothesis [J]. J Allergy Clin Immunol,2007,119 (6): 1511- 3.
[27] Barlow DJ,EdwardsMS,Thornton JM. Continous and discontinuous protein antigenic determinants[J]. Nature 1986,322(6081): 747.
[28] Bijker MS,Melief CJ,Offringa R,van der Burg SH. Design and development of synthetic peptide vaccines: past,present and future. Expert Rev Vaccines 2007; 6: 591-603. 11.
[29] Nardin E. The past decade in malaria synthetic peptide vaccine clinical trials. Hum Vaccin.2010 Jan 23; 6(1). [Epub ahead of print]
[30] Scott JK,Smith GP. Searching for peptide ligands with an epitope library. Science, 1990, 249(4967): 386-90.
[31]黄艳新,鲍永利,李玉新.抗原表位预测的免疫信息学方法研究进展.中国免疫学杂志,2008,24(9): 851-67.
[32]孙建宏,曹殿军.细胞的抗原表位研究方法.动物医学进展,2004,25(5):18-21.
[33]黄健,郭建巍,蔡美英. B细胞表位预测.微生物学免疫学进展,2004,32(1): 40-42.
[34]周光炎.免疫学原理[M].上海:上海科技出版杜,2000,36-7.
[35]吴玉章.免疫识别、分子设计与抗原工程.第三军医大学学报.2000.22:917-8.
[36] De Groot AS,Sbai H,Aubin CS,McMurry J,Martin W. Immuno-informatics: mining genomes for vaccine components [J]. Immunol Cell Biol.2002; 80(3): 255-69.
[37] Garnier J ,Osguthorpe DJ ,Robson B. Analysis of the accuracy and implications of simple method for predicting the secondary structure of globular proteins[J].J Mol Biol,1978,120:97-120.
[38] Chou PY,Fasman GD.Prediction of the secondary structure of proteins from their amino acid sequence[J].Adv Enzymol,1978,47: 45-8.
[39] Chou PY. Prediction of Protein Structure and the Principles of Protein Conformatio[M]. New York: Plenum Press,1990:549-86.
[40] Karplus PA,Schultz GE.Prediction of chain flexibility in proteins[J]. Naturwissenschaften, 1985,72 :212-213.
[41] Kyte J ,Doolittle RF. A simple method for displaying the hydropathic character of a protein[J]. J Mol Biol,1982,157:105-132.
[42] Emini EA,Hughes J,Perlow D,et al. Induction of hepatitis A virus neutralizing antibody by a virus-specific synthetic peptide [J]. J Virology,1985,55: 836-839.
[43] Jameson BA,Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants [J]. CABIOS,1988,4: 181-186.
[44] Kolaskar AS, Tongaonkar PC.A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett.1990; 276(1-2):172-4.
[45] Hoop J P,Wood K R.Prediction of protein antigenic determinants from amino acid sequences[J].Immunology,1981,78(6):3824.
[46]来鲁华.蛋白质的结构预测与分子设计.第1版[M].北京:北京大学出版社,1993.49.
[47] Tsche H.Modern methods in protein and nucleic acid research[M].1th ed.New York:Walter de Gruger Berlin,1990.231.
[48] Welling G W,Weijer W J,Vander Z R,et al.Prediction of sequential antigenic regions in proteins[J].FEBS Lett,1985,188(2):215.
[49] Gershoni J M,Stem B,Venisova G.Combinatorial libraries,epitope structure and the prediction of conformations[J].Immunol Today, 1997, 18(5):108.
[50] Wu YZ,Zhu XH. A new approach for B-cell epitope prediction in viral proteins [J].Clin Sci Bull.1995; 40(9):761-7.
[51]万涛,孙涛,吴家金.蛋白顺序性抗原决定簇的多参数综合预测[J].中国免疫学杂志,1997,13 (6): 329-33.
[52] Saha S,Raghava GPS. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network [J] . Proteins,2006,65 (1):40-8.
[53] McCrudden MT, Dafforn TR, Houston DF, Turkington PT, Timson DJ.Functional domains of the human epididymal protease inhibitor, eppin.FEBS J.2008,275(8): 1742-50.
[54] Reynolds SR, Dahl CE, Harn DA. T and B epitope determination and analysis of multiple antigenic peptides for the Schistosoma mansoni experimental vaccine triose-phosphate isomerase [J]. J Immunol, 1994, 152 (1): 193-200.
[55] HuebenerN, Lange B, Lemmel C, et al. Vaccination with minigenes encoding for novel’self’antigens are effective in DNA-vaccination against neuroblastoma [J].Cancer Lett, 2003, 197(1-2): 211-7.
[56] Williams KM, Bigley EC 3rd, Raybourne RB. Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides [J]. Infect Immun, 2000, 68 (5): 2535-45.
[57] Rammensee H, Bachmann J, Emmerich NP, et al. SYFPEITH I: database for MHC ligands and peptide motifs [J]. Immunogenetics, 1999, 50 (3-4): 213-9.
[58] Hagmann M. Computers aid vaccine design [J]. Science, 2000,290 (5489): 80-2.
[59]李全喜,王琰,李竟等,识别9E10单抗的噬菌体短肽的免疫学特性分析[J].中国免疫学杂志1997,13(6):365-368.
[60] Bronson RA.Antisperm antibodies: a critical evaluation and clinical guidelines.J Reprod Immunol. 1999 Dec;45(2):159-83.
[61] Hardy CM, Clarke HG, Nixon B, Grigg JA, Hinds LA, Holland MK. Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit. Biol Reprod. 1997; 57(4):879-86.
[62] Naz RK. Antisperm immunity for contraception. J Androl 2006; 27(2):153-159.
[63] Karle S, Nishiyama Y, Taguchi H, et al. Carrier-dependent specificity of antibodies to a conserved pep tide determinant of gp120 [ J ]. Vaccine, 2003, 21 (11-12): 1213-18.
[64] May RJ, Beenhouwer DO, Scharff MD. Antibodies to keyhole limpet hemocyanin cross-reactwith an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development [J].J Immunol, 2003, 171(9): 4905-12.
[65] Joshi MB, Gam AA, Boykins RA, et al. Immunogenicity of well-characterized synthetic Plasm odium falciparum multip le antigen peptide conjugates[ J ]. Infect Immun, 2001, 69: 4884-90.
[66] ZengW, Ghosh S, Lau YF, et al. Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines[ J ]. J Immunol, 2002, 169 (9): 4905-12.
[67] Li WM, Dragowska WH, Bally MB, et al. Effective induction of CD8+ T-cell response using CpG oligodeoxynucleotides and HER-2 /neu-derived peptide co-encapsulated in liposomes [ J ]. Vaccine, 2003, 21 (23): 3319-29.
[68] BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines--yesterday, today,and tomorrow [ J ]. Lancet Infect Dis, 2002, 2 (7): 425-31.
[69] Alice Banz, Magali Cremel, Audrey Rembert, Yann Godfrin.In situ targeting of dendritic cells by antigen-loaded red blood cells: A novel approach to cancer immunotherapy.Vaccine. 2010; 28(17):2965-72.
[70] McGuire MJ, Sykes KF, Samli KN, Timares L, Barry MA, Stemke-Hale K, Tagliaferri F, Logan M, Jansa K, Takashima A, Brown KC, Johnston SA. A library-selected, Langerhans cell-targeting peptide enhances an immune response. DNA Cell Biol. 2004; 23(11):742-52.
[71] Angelos JA, Lane VM, Ball LM, Hess JF.Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.Vet Res Commun. 2010; 34(3):229-39.
[72] Pandey RS, Dixit VK.Evaluation of ISCOM vaccines for mucosal immunization against hepatitis B.J Drug Target. 2010; 18(4):282-91.
[73] Madsen HB, Ifversen P, Madsen F, Brodin B, Hausser I, Nielsen HM.In vitro cutaneous application of ISCOMs on human skin enhances delivery of hydrophobic model compounds through the stratum corneum. AAPS J. 2009; 11(4):728-39.
[74] Rosenthal KS. Immune peptide enhancement of peptide based vaccines. Front Biosci. 2005; 10: 478-82.
[75] Marussig M, Renia L,Motard A,et al . Linear and multiple antigen peptides containing defined T and B epitopes of the plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection. Int Immunol, 1997, 9 (12):1817-24.
[76] De Groot N, Doxiadis GG, de Vos-Rouweler AJ, de Groot NG, Verschoor EJ, Bontrop RE.Comparative genetics of a highly divergent DRB microsatellite in different macaque species. Immunogenetics. 2008; 60(12):737-48.
[77] Doxiadis GG, de Groot N, Claas FH, Doxiadis II, van Rood JJ, Bontrop RE.A highly divergent microsatellite facilitating fast and accurate DRB haplotyping in humans and rhesus macaques. Proc Natl Acad Sci USA. 2007; 104(21):8907-12.
[78] Melnick DJ, Hoelzer GA, Absher R, Ashley MV.mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species.Mol Biol Evol. 1993; 10(2):282-95.
[79] Tosi AJ, Morales JC, Melnick DJ.Comparison of Y chromosome and mtDNA phylogenies leads to unique inferences of macaque evolutionary history.Mol Phylogenet Evol. 2000; 17(2):133-44.
[80] Zhang ZQ, Fu TM, Casimiro DR, et al. Mamu-A*01allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection.J Virol.2002; 76(24):12845-54.
[81] Ting J P, Trowsdale J. Genetic control of MHC class II expression.Cell, 2002, 109: S21.
[82] Otten L A, Tacchini-Cottier F M, Lohoff F, et al. Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes. J Immunol, 2003, 170:1150.
[1] Naz RK, Rajesh C. Novel testis/sperm specific contraceptive targets identified using gene knockout studies. Front. Biosci.2005; 10:2430–46.
[2] Naz RK, Gupta SK, Gupta JC, Vyas HK, Talwar AG.Recent advances in contraceptive vaccine development: a mini-review.Hum Reprod. 2005; 20(12):3271-83.
[3] Naz RK, Rajesh P.Gene knockouts that cause female infertility:search for novel contraceptive targets. Front. Biosci.2005; 10: 2447–59.
[4] Talwar G P, SinghO,Pal R,Chatterjee N,Sahai P,Dhall K,Kaur K,Das SK,Suri S,Bukshee K, Saraya L,Saxena BN.A vaccine that prevents pregnancy in women.Proc.Natl.Acad.Sci.1994; 91:8532–36.
[5] Baskin MJ. Temporary sterilization by injection of human spermatozoa:a preliminary report. Am.J.Obstet.Gynecol.1932; 24:892–7.
[6] Mancini RE,Andrana JA,Saracine D,Bachmann AE,Lavieri JC,Nemirovsky M. Immunological and testicular response in men sensitized with human testicular homogenate. J. Clin. Endocrinol.Metab.1965; 25:859–75.
[7] Ohl D,Naz RK. Infertility due to antisperm antibodies. J. Urol. 1995; 46:591–602.
[8] Inoue N, Ikawa M, Isotani A, Okabe M.The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.Nature 2005; 434: 234-8.
[9] O’Hearn PA,Liang ZG,Bambra CS,Goldberg E. Colinear synthesis of an antigen-specific B-cell epitope with a promiscuous tetanustoxin T-cell epitope: a synthetic peptide immunocontraceptive. Vaccine.1997; 15: 1761–6.
[10] Tollner TL,Overstreet JW,Branciforte D,Primakoff PD. Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titers but does not reduce fertility. Mol. Reprod. Dev. 2002; 62:257–64.
[11] O’Rand MG, Widgren EE,Sivashanmugam P,Richardson RT,Hall SH,French FS,Vande Voort CA,Ramachandra SG,Ramesh V,Jagannadha Ro A. Reversible immunocontraception in male monkeys immunized with eppin. Science.2004; 306: 1189.
[12] Inoue N,Ikawa M,Okabe M. Putative sperm fusion protein IZUMO and the role of N-glycosylation.Biochem Biophys Res Commun.2008; 377(3): 910-4.
[13] Naz RK.Immunocontraceptive effect of Izumo and enhancement by combination vaccination.Mol Reprod Dev. 2008; 75(2): 336-44.
[14] Cho C,Bunch DO,Faure JE,Goulding EH,Eddy EM,Primakoff P,Myles DG. Fertilization defects in sperm from mice lacking fertilin beta. Science.1998; 281: 1857–9.
[15] Dunman PM,Nessin M. Passive immunization as prophylaxis: when and where will this work? Curr.Opin.Pharmacol.2003; 20: 351–60.
[16] Koren E,Zuckerman LA,Mire-Sluis AR. Immune response to therapeutic proteins in humans-clinical significance, assessment and prediction. Curr.Pharm. Biotechnol. 2002;3: 349–60.
[17] Mirick GR,Bradt BM,Denardo SJ,Denardo GL.A review of human anti-globulin antibody (HAGA, HAMA, HACA,HAHA) responses to monoclonal antibodies.Not four letter words. Q. J. Nucl.Med. Mol. Imaging 2004; 48:251–7.
[18] Sidhu SS,Fellouse FA. Synthetic therapeutic antibodies. Nat.Chem.Biol. 2006; 2: 682–8.
[1] Richardson RT, Sivashanmugam P, Hall SH, et al. Cloning and sequencing of human Eppin: A novel family of p rotease inhibitors expressed in the ep ididymis and testis [ J ]. Gene, 2001, 270 (122) : 93-102.
[2] Sivashanmugam P, Hall SH, Hamil KG, et al. Characterization ofmouse Epp in and a gene cluster of similar p rotease inhibitors on mouse chromosome 2 [ J ]. Gene, 2003, 312: 125-34.
[3] Bian ZH, Zhang J, Ding XL, et al. Localization of epididymal protease inhibitor in adult rat and its transcription profile in testis during postnatal development [J]. Asian Journal of Andrology (2009): 731–9.
[4] Deloukas P, Matthews LH, Ashurst J, et al. The DNA sequence and comparative analysis of human chromosome 20 [J]. Nature, 2001, 414 (6866): 865-71.
[5] ClaussA, Lilja H, Lundwall A. A locus on human chromosome 20 contains several genes exp ressing p rotease inhibitor domains with homology to whey acidic p rotein[ J ]. Biochem J, 2002, 368(Pt 1): 233-42.
[6] Hagiwara K, Kikuchi T, Endo Y, et al. Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single whey acidic protein motif[ J ]. J Immunol, 2003, 170 (4): 19732-79.
[7] Yenugu S, Richardson RT, Sivashanmugam P, et al. Antimicrobial activity of human EPPIN, an androgen2regulated, sperm-bound p rotein with a when acidic protein motif[ J ]. Biol Rep rod, 2004, 71 (5) : 1484-90.
[8]库卓华.人精子在附睾中的成熟[J].中华男科学, 2001, 7(1) : 129.
[9]周宗瑶,王一飞.附睾精子成熟功能研究进展[J].生殖与避孕, 2003, 23 (4) : 238-43.
[10] Zhou CX, Zhang YL, Xiao L, et al. An epididymis-specific beta-defensin is important for the initiation of sperm maturation [ J ].Nat Cell Biol, 2004, 6 (5) : 458-64.
[11] Cornwall GA, Hsia N.胱蛋白酶抑制剂相关的附睾精子发生基因的调节和功能[J].中华男科学, 2002, 8 (5) : 313-8.
[12] Ellerman DA, Da Ros VG, Cohen DJ, et al. Exp ression and structure-function analysis of a sperm cysteine2rich secretory protein that mediates gamete fusion [ J ].Biol Rep rod, 2002, 67(4) : 1225-31.
[13] O’Rand MG, Widgren EE, Sivashanmugam P, et al. Reversible Immunocontracep tion in Male Monkeys Immunized with Eppin[ J ]. Science, 2004, 306 (5699): 1189-90.
[14] Wang ZJ, Esther E.W, Richard T, et al. Characterization of an Eppin Protein Complex from Human Semen and Spermatozoa[J]. BIOLOGY OF REPRODUCTION 77, 476–84 (2007).
[15] McLaughlin EA, HollandMK, Aitken RJ. Contraceptive vaccines[ J ]. Expert Op in Biol Ther, 2003, 3 (5): 829-41.
[16] Suri A. Sperm specific-proteins potential candidate molecules for fertility control[ J ]. Rep rod Biol Endocrinol, 2004, 2 (1): 10.
[17] Naz RK, Chauhan SC. Human sperm-specific pep tide vaccine that causes long2term reversible contracep tion [J]. Biol Rep rod, 2002, 67 (2): 674-80.
[18] Naz RK. Human synthetic pep tide vaccine for contraception targeting sperm[ J ]. Arch Androl, 2004, 50 (2): 113-119.
[19]崔毓桂,童建孙,王兴海.附睾相关的抗生育研究及进展[ J ].中华男科学,2002,8 (2) : 139-41.
[20] Zhang J, Ding XL, Bian ZH.The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa[J]. Human Reproduction, Vol.25, No.1 pp. 29–36, 2010.
[21] Mitra A, Richardson RT. Analysis of recombinant human semenogelin as an inhibitor of human sperm motility[J]. O'Rand MGBiol Reprod. 2010 Mar;82(3):489-96.
[22] Magnus J, Birgitta F and Johan M.Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance [J]. Published-Ahead-of-Print on April 8, 2010 by Journal of Andrology
[23] Couzin J. Medicine. Sperm-targeting vaccine blocksmale fertility in monkeys [ J ]. Science, 2004, 306 (5699): 1117.
[24] Karande A. Epp in: a candidatemale contracep tive vaccine[ J ] J Biosci, 2004, 29 (4) : 373-4.
[2] Page ST, Amory JK, Bremner WJ.Advances in male contraception. Endocr Rev. 2008; 29 (4): 465-93.
[3] Naz RK. Status of contraceptive vaccines.Am J Reprod Immunol.2009; 61(1):11-8.
[4] Suri A. Sperm-based contraceptive vaccines: current status,merits and development. Expert Rev Mol Med 2005; 7(18): 1-16.
[5] Delves PJ, Lund T, Roitt IM.Antifertility vaccines.Trends-Immunol.2002; 23(4): 213-9.
[6] Naz RK,Gupta SK,Gupta JC,et al. Recent advances in contraceptive vaccine development: a mini-review [J]. Hum Reprod,2005,20(12): 3271-83.
[7] Naz RK,Chauhan SC. Human sperm-specific peptide vaccine that causes long-term reversible contraception[J]. Biol Reprod,2002,67(2):674-80.
[8] Chen H, Griffiths GS, Galileo DS & Martin-DeLeon PA.Epididymal SPAM1 is a marker of sperm maturation in mouse. Biology of Reproduction.2006; 74: 923-30.
[9] Hardy CM, Clydesdale G, Mobbs KJ, Pekin J, Lloyd ML, Sweet C, Shellam GR, Lawson MA. Assessment of contraceptive vaccines based on recombinant mouse sperm protein PH20.Reproduction. 2004; 127(3):325-34.
[10] Griffiths GS, Galileo DS, Aravindan RG, Martin-DeLeon PA.Clusterin facilitates exchange of glycosyl phosphatidylinositol-linked SPAM1 between reproductive luminal fluids and mouse and human sperm membranes.Biol Reprod. 2009; 81(3):562-70.
[11] Kurth BE, Digilio L, Snow P, Bush LA, Wolkowicz M, Shetty J, Mandal A, Hao Z, Reddi PP, Flickinger CJ, Herr JC.Immunogenicity of a multi-component recombinant human acrosomal protein vaccine in female Macaca fascicularis.J Reprod Immunol. 2008; 77(2):126-41.
[12] Jagadish N, Rana R, Selvi R.Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein.Biochem.J. 2005; 389:73–82.
[13] Williams J, Samuel A, Naz RK.Presence of antisperm antibodies reactive with peptide epitopes of FA-1 and YLP12 in sera of immunoinfertile women.Am J Reprod Immunol. 2008; 59(6):518-24.
[14] Naz RK.Effect of fertilization antigen (FA-1) DNA vaccine on fertility of female mice.Mol Reprod Dev. 2006; 73(11): 1473-9.
[15] Suri A.Contraceptive vaccines targeting sperm. Expert Opin Biol Ther 2005; 5(3): 381-92.
[16] O'Rand MG,Widgren EE,Wang Z,et al. Eppin: an effective target for male contraception. Mol Cell Endocrinol 2006; 250(1-2): 157-62.
[17] Richardson RT,Sivashanmugam P,Hall SH,et al. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 2001; 270: 93-102.
[18] Wang Z,Widgren EE,Richardson RT; et al. Eppin: a molecular strategy for male contraception. Soc Reprod Fertil Suppl 2007; 65: 535-42.
[19] Sivashanmugam P,Hall SH,Hamil KG,et al. Characterization of mouse Eppin and a gene cluster of similar protease inhibitors on mouse chromosome 2. Gene 2003; 312: 125-34.
[20] Kirchhoff C,Osterhoff C,Pera I,et al. Function of human epididymal proteins in sperm maturation. Andrologia,1998,30(4-5): 225-32.
[21] Zinkernagel RM & Hengartner H. Regulation of the immune response by antigen. Science,2001,293(5528): 251-3.
[22] O'rand MG,Widgren EE,Sivashanmugam P,Richardson RT,Hall SH,French FS,VandeVoort CA,Ramachandra SG,Ramesh V,Jagannadha Rao A. Reversible immunocontraception in male monkeys immunized with eppin.Science. 2004 ; 306:1189-90.
[23] Wang Z,Widgren EE,Sivashanmugam P,et al. Association of eppin with semenogelin on human spermatozoa[J]. Biol Reprod,2005,72 (5): 1064-70.
[24] O'Rand MG,Widgren EE,Beyler S,Richardson RT. Inhibition of human sperm motility by contraceptive anti-eppin antibodies from infertile male monkeys: effect on cyclic adenosine monophosphate. Biol Reprod.2009; 80(2):279-85.
[25] Romagnani S. Coming back to a missing immune deviation as the main explanatorymechanism for the hygiene hypothesis [J]. J Allergy Clin Immunol, 2007,119 (6): 1511-13.
[26] Romagnani S. Coming back to a missing immune deviation as the main explanatorymechanism for the hygiene hypothesis [J]. J Allergy Clin Immunol,2007,119 (6): 1511- 3.
[27] Barlow DJ,EdwardsMS,Thornton JM. Continous and discontinuous protein antigenic determinants[J]. Nature 1986,322(6081): 747.
[28] Bijker MS,Melief CJ,Offringa R,van der Burg SH. Design and development of synthetic peptide vaccines: past,present and future. Expert Rev Vaccines 2007; 6: 591-603. 11.
[29] Nardin E. The past decade in malaria synthetic peptide vaccine clinical trials. Hum Vaccin.2010 Jan 23; 6(1). [Epub ahead of print]
[30] Scott JK,Smith GP. Searching for peptide ligands with an epitope library. Science, 1990, 249(4967): 386-90.
[31]黄艳新,鲍永利,李玉新.抗原表位预测的免疫信息学方法研究进展.中国免疫学杂志,2008,24(9): 851-67.
[32]孙建宏,曹殿军.细胞的抗原表位研究方法.动物医学进展,2004,25(5):18-21.
[33]黄健,郭建巍,蔡美英. B细胞表位预测.微生物学免疫学进展,2004,32(1): 40-42.
[34]周光炎.免疫学原理[M].上海:上海科技出版杜,2000,36-7.
[35]吴玉章.免疫识别、分子设计与抗原工程.第三军医大学学报.2000.22:917-8.
[36] De Groot AS,Sbai H,Aubin CS,McMurry J,Martin W. Immuno-informatics: mining genomes for vaccine components [J]. Immunol Cell Biol.2002; 80(3): 255-69.
[37] Garnier J ,Osguthorpe DJ ,Robson B. Analysis of the accuracy and implications of simple method for predicting the secondary structure of globular proteins[J].J Mol Biol,1978,120:97-120.
[38] Chou PY,Fasman GD.Prediction of the secondary structure of proteins from their amino acid sequence[J].Adv Enzymol,1978,47: 45-8.
[39] Chou PY. Prediction of Protein Structure and the Principles of Protein Conformatio[M]. New York: Plenum Press,1990:549-86.
[40] Karplus PA,Schultz GE.Prediction of chain flexibility in proteins[J]. Naturwissenschaften, 1985,72 :212-213.
[41] Kyte J ,Doolittle RF. A simple method for displaying the hydropathic character of a protein[J]. J Mol Biol,1982,157:105-132.
[42] Emini EA,Hughes J,Perlow D,et al. Induction of hepatitis A virus neutralizing antibody by a virus-specific synthetic peptide [J]. J Virology,1985,55: 836-839.
[43] Jameson BA,Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants [J]. CABIOS,1988,4: 181-186.
[44] Kolaskar AS, Tongaonkar PC.A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett.1990; 276(1-2):172-4.
[45] Hoop J P,Wood K R.Prediction of protein antigenic determinants from amino acid sequences[J].Immunology,1981,78(6):3824.
[46]来鲁华.蛋白质的结构预测与分子设计.第1版[M].北京:北京大学出版社,1993.49.
[47] Tsche H.Modern methods in protein and nucleic acid research[M].1th ed.New York:Walter de Gruger Berlin,1990.231.
[48] Welling G W,Weijer W J,Vander Z R,et al.Prediction of sequential antigenic regions in proteins[J].FEBS Lett,1985,188(2):215.
[49] Gershoni J M,Stem B,Venisova G.Combinatorial libraries,epitope structure and the prediction of conformations[J].Immunol Today, 1997, 18(5):108.
[50] Wu YZ,Zhu XH. A new approach for B-cell epitope prediction in viral proteins [J].Clin Sci Bull.1995; 40(9):761-7.
[51]万涛,孙涛,吴家金.蛋白顺序性抗原决定簇的多参数综合预测[J].中国免疫学杂志,1997,13 (6): 329-33.
[52] Saha S,Raghava GPS. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network [J] . Proteins,2006,65 (1):40-8.
[53] McCrudden MT, Dafforn TR, Houston DF, Turkington PT, Timson DJ.Functional domains of the human epididymal protease inhibitor, eppin.FEBS J.2008,275(8): 1742-50.
[54] Reynolds SR, Dahl CE, Harn DA. T and B epitope determination and analysis of multiple antigenic peptides for the Schistosoma mansoni experimental vaccine triose-phosphate isomerase [J]. J Immunol, 1994, 152 (1): 193-200.
[55] HuebenerN, Lange B, Lemmel C, et al. Vaccination with minigenes encoding for novel’self’antigens are effective in DNA-vaccination against neuroblastoma [J].Cancer Lett, 2003, 197(1-2): 211-7.
[56] Williams KM, Bigley EC 3rd, Raybourne RB. Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides [J]. Infect Immun, 2000, 68 (5): 2535-45.
[57] Rammensee H, Bachmann J, Emmerich NP, et al. SYFPEITH I: database for MHC ligands and peptide motifs [J]. Immunogenetics, 1999, 50 (3-4): 213-9.
[58] Hagmann M. Computers aid vaccine design [J]. Science, 2000,290 (5489): 80-2.
[59]李全喜,王琰,李竟等,识别9E10单抗的噬菌体短肽的免疫学特性分析[J].中国免疫学杂志1997,13(6):365-368.
[60] Bronson RA.Antisperm antibodies: a critical evaluation and clinical guidelines.J Reprod Immunol. 1999 Dec;45(2):159-83.
[61] Hardy CM, Clarke HG, Nixon B, Grigg JA, Hinds LA, Holland MK. Examination of the immunocontraceptive potential of recombinant rabbit fertilin subunits in rabbit. Biol Reprod. 1997; 57(4):879-86.
[62] Naz RK. Antisperm immunity for contraception. J Androl 2006; 27(2):153-159.
[63] Karle S, Nishiyama Y, Taguchi H, et al. Carrier-dependent specificity of antibodies to a conserved pep tide determinant of gp120 [ J ]. Vaccine, 2003, 21 (11-12): 1213-18.
[64] May RJ, Beenhouwer DO, Scharff MD. Antibodies to keyhole limpet hemocyanin cross-reactwith an epitope on the polysaccharide capsule of Cryptococcus neoformans and other carbohydrates: implications for vaccine development [J].J Immunol, 2003, 171(9): 4905-12.
[65] Joshi MB, Gam AA, Boykins RA, et al. Immunogenicity of well-characterized synthetic Plasm odium falciparum multip le antigen peptide conjugates[ J ]. Infect Immun, 2001, 69: 4884-90.
[66] ZengW, Ghosh S, Lau YF, et al. Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontraceptive vaccines[ J ]. J Immunol, 2002, 169 (9): 4905-12.
[67] Li WM, Dragowska WH, Bally MB, et al. Effective induction of CD8+ T-cell response using CpG oligodeoxynucleotides and HER-2 /neu-derived peptide co-encapsulated in liposomes [ J ]. Vaccine, 2003, 21 (23): 3319-29.
[68] BenMohamed L, Wechsler SL, Nesburn AB. Lipopeptide vaccines--yesterday, today,and tomorrow [ J ]. Lancet Infect Dis, 2002, 2 (7): 425-31.
[69] Alice Banz, Magali Cremel, Audrey Rembert, Yann Godfrin.In situ targeting of dendritic cells by antigen-loaded red blood cells: A novel approach to cancer immunotherapy.Vaccine. 2010; 28(17):2965-72.
[70] McGuire MJ, Sykes KF, Samli KN, Timares L, Barry MA, Stemke-Hale K, Tagliaferri F, Logan M, Jansa K, Takashima A, Brown KC, Johnston SA. A library-selected, Langerhans cell-targeting peptide enhances an immune response. DNA Cell Biol. 2004; 23(11):742-52.
[71] Angelos JA, Lane VM, Ball LM, Hess JF.Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.Vet Res Commun. 2010; 34(3):229-39.
[72] Pandey RS, Dixit VK.Evaluation of ISCOM vaccines for mucosal immunization against hepatitis B.J Drug Target. 2010; 18(4):282-91.
[73] Madsen HB, Ifversen P, Madsen F, Brodin B, Hausser I, Nielsen HM.In vitro cutaneous application of ISCOMs on human skin enhances delivery of hydrophobic model compounds through the stratum corneum. AAPS J. 2009; 11(4):728-39.
[74] Rosenthal KS. Immune peptide enhancement of peptide based vaccines. Front Biosci. 2005; 10: 478-82.
[75] Marussig M, Renia L,Motard A,et al . Linear and multiple antigen peptides containing defined T and B epitopes of the plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection. Int Immunol, 1997, 9 (12):1817-24.
[76] De Groot N, Doxiadis GG, de Vos-Rouweler AJ, de Groot NG, Verschoor EJ, Bontrop RE.Comparative genetics of a highly divergent DRB microsatellite in different macaque species. Immunogenetics. 2008; 60(12):737-48.
[77] Doxiadis GG, de Groot N, Claas FH, Doxiadis II, van Rood JJ, Bontrop RE.A highly divergent microsatellite facilitating fast and accurate DRB haplotyping in humans and rhesus macaques. Proc Natl Acad Sci USA. 2007; 104(21):8907-12.
[78] Melnick DJ, Hoelzer GA, Absher R, Ashley MV.mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species.Mol Biol Evol. 1993; 10(2):282-95.
[79] Tosi AJ, Morales JC, Melnick DJ.Comparison of Y chromosome and mtDNA phylogenies leads to unique inferences of macaque evolutionary history.Mol Phylogenet Evol. 2000; 17(2):133-44.
[80] Zhang ZQ, Fu TM, Casimiro DR, et al. Mamu-A*01allele-mediated attenuation of disease progression in simian-human immunodeficiency virus infection.J Virol.2002; 76(24):12845-54.
[81] Ting J P, Trowsdale J. Genetic control of MHC class II expression.Cell, 2002, 109: S21.
[82] Otten L A, Tacchini-Cottier F M, Lohoff F, et al. Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes. J Immunol, 2003, 170:1150.
[1] Naz RK, Rajesh C. Novel testis/sperm specific contraceptive targets identified using gene knockout studies. Front. Biosci.2005; 10:2430–46.
[2] Naz RK, Gupta SK, Gupta JC, Vyas HK, Talwar AG.Recent advances in contraceptive vaccine development: a mini-review.Hum Reprod. 2005; 20(12):3271-83.
[3] Naz RK, Rajesh P.Gene knockouts that cause female infertility:search for novel contraceptive targets. Front. Biosci.2005; 10: 2447–59.
[4] Talwar G P, SinghO,Pal R,Chatterjee N,Sahai P,Dhall K,Kaur K,Das SK,Suri S,Bukshee K, Saraya L,Saxena BN.A vaccine that prevents pregnancy in women.Proc.Natl.Acad.Sci.1994; 91:8532–36.
[5] Baskin MJ. Temporary sterilization by injection of human spermatozoa:a preliminary report. Am.J.Obstet.Gynecol.1932; 24:892–7.
[6] Mancini RE,Andrana JA,Saracine D,Bachmann AE,Lavieri JC,Nemirovsky M. Immunological and testicular response in men sensitized with human testicular homogenate. J. Clin. Endocrinol.Metab.1965; 25:859–75.
[7] Ohl D,Naz RK. Infertility due to antisperm antibodies. J. Urol. 1995; 46:591–602.
[8] Inoue N, Ikawa M, Isotani A, Okabe M.The immunoglobulin superfamily protein Izumo is required for sperm to fuse with eggs.Nature 2005; 434: 234-8.
[9] O’Hearn PA,Liang ZG,Bambra CS,Goldberg E. Colinear synthesis of an antigen-specific B-cell epitope with a promiscuous tetanustoxin T-cell epitope: a synthetic peptide immunocontraceptive. Vaccine.1997; 15: 1761–6.
[10] Tollner TL,Overstreet JW,Branciforte D,Primakoff PD. Immunization of female cynomolgus macaques with a synthetic epitope of sperm-specific lactate dehydrogenase results in high antibody titers but does not reduce fertility. Mol. Reprod. Dev. 2002; 62:257–64.
[11] O’Rand MG, Widgren EE,Sivashanmugam P,Richardson RT,Hall SH,French FS,Vande Voort CA,Ramachandra SG,Ramesh V,Jagannadha Ro A. Reversible immunocontraception in male monkeys immunized with eppin. Science.2004; 306: 1189.
[12] Inoue N,Ikawa M,Okabe M. Putative sperm fusion protein IZUMO and the role of N-glycosylation.Biochem Biophys Res Commun.2008; 377(3): 910-4.
[13] Naz RK.Immunocontraceptive effect of Izumo and enhancement by combination vaccination.Mol Reprod Dev. 2008; 75(2): 336-44.
[14] Cho C,Bunch DO,Faure JE,Goulding EH,Eddy EM,Primakoff P,Myles DG. Fertilization defects in sperm from mice lacking fertilin beta. Science.1998; 281: 1857–9.
[15] Dunman PM,Nessin M. Passive immunization as prophylaxis: when and where will this work? Curr.Opin.Pharmacol.2003; 20: 351–60.
[16] Koren E,Zuckerman LA,Mire-Sluis AR. Immune response to therapeutic proteins in humans-clinical significance, assessment and prediction. Curr.Pharm. Biotechnol. 2002;3: 349–60.
[17] Mirick GR,Bradt BM,Denardo SJ,Denardo GL.A review of human anti-globulin antibody (HAGA, HAMA, HACA,HAHA) responses to monoclonal antibodies.Not four letter words. Q. J. Nucl.Med. Mol. Imaging 2004; 48:251–7.
[18] Sidhu SS,Fellouse FA. Synthetic therapeutic antibodies. Nat.Chem.Biol. 2006; 2: 682–8.
[1] Richardson RT, Sivashanmugam P, Hall SH, et al. Cloning and sequencing of human Eppin: A novel family of p rotease inhibitors expressed in the ep ididymis and testis [ J ]. Gene, 2001, 270 (122) : 93-102.
[2] Sivashanmugam P, Hall SH, Hamil KG, et al. Characterization ofmouse Epp in and a gene cluster of similar p rotease inhibitors on mouse chromosome 2 [ J ]. Gene, 2003, 312: 125-34.
[3] Bian ZH, Zhang J, Ding XL, et al. Localization of epididymal protease inhibitor in adult rat and its transcription profile in testis during postnatal development [J]. Asian Journal of Andrology (2009): 731–9.
[4] Deloukas P, Matthews LH, Ashurst J, et al. The DNA sequence and comparative analysis of human chromosome 20 [J]. Nature, 2001, 414 (6866): 865-71.
[5] ClaussA, Lilja H, Lundwall A. A locus on human chromosome 20 contains several genes exp ressing p rotease inhibitor domains with homology to whey acidic p rotein[ J ]. Biochem J, 2002, 368(Pt 1): 233-42.
[6] Hagiwara K, Kikuchi T, Endo Y, et al. Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single whey acidic protein motif[ J ]. J Immunol, 2003, 170 (4): 19732-79.
[7] Yenugu S, Richardson RT, Sivashanmugam P, et al. Antimicrobial activity of human EPPIN, an androgen2regulated, sperm-bound p rotein with a when acidic protein motif[ J ]. Biol Rep rod, 2004, 71 (5) : 1484-90.
[8]库卓华.人精子在附睾中的成熟[J].中华男科学, 2001, 7(1) : 129.
[9]周宗瑶,王一飞.附睾精子成熟功能研究进展[J].生殖与避孕, 2003, 23 (4) : 238-43.
[10] Zhou CX, Zhang YL, Xiao L, et al. An epididymis-specific beta-defensin is important for the initiation of sperm maturation [ J ].Nat Cell Biol, 2004, 6 (5) : 458-64.
[11] Cornwall GA, Hsia N.胱蛋白酶抑制剂相关的附睾精子发生基因的调节和功能[J].中华男科学, 2002, 8 (5) : 313-8.
[12] Ellerman DA, Da Ros VG, Cohen DJ, et al. Exp ression and structure-function analysis of a sperm cysteine2rich secretory protein that mediates gamete fusion [ J ].Biol Rep rod, 2002, 67(4) : 1225-31.
[13] O’Rand MG, Widgren EE, Sivashanmugam P, et al. Reversible Immunocontracep tion in Male Monkeys Immunized with Eppin[ J ]. Science, 2004, 306 (5699): 1189-90.
[14] Wang ZJ, Esther E.W, Richard T, et al. Characterization of an Eppin Protein Complex from Human Semen and Spermatozoa[J]. BIOLOGY OF REPRODUCTION 77, 476–84 (2007).
[15] McLaughlin EA, HollandMK, Aitken RJ. Contraceptive vaccines[ J ]. Expert Op in Biol Ther, 2003, 3 (5): 829-41.
[16] Suri A. Sperm specific-proteins potential candidate molecules for fertility control[ J ]. Rep rod Biol Endocrinol, 2004, 2 (1): 10.
[17] Naz RK, Chauhan SC. Human sperm-specific pep tide vaccine that causes long2term reversible contracep tion [J]. Biol Rep rod, 2002, 67 (2): 674-80.
[18] Naz RK. Human synthetic pep tide vaccine for contraception targeting sperm[ J ]. Arch Androl, 2004, 50 (2): 113-119.
[19]崔毓桂,童建孙,王兴海.附睾相关的抗生育研究及进展[ J ].中华男科学,2002,8 (2) : 139-41.
[20] Zhang J, Ding XL, Bian ZH.The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa[J]. Human Reproduction, Vol.25, No.1 pp. 29–36, 2010.
[21] Mitra A, Richardson RT. Analysis of recombinant human semenogelin as an inhibitor of human sperm motility[J]. O'Rand MGBiol Reprod. 2010 Mar;82(3):489-96.
[22] Magnus J, Birgitta F and Johan M.Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance [J]. Published-Ahead-of-Print on April 8, 2010 by Journal of Andrology
[23] Couzin J. Medicine. Sperm-targeting vaccine blocksmale fertility in monkeys [ J ]. Science, 2004, 306 (5699): 1117.
[24] Karande A. Epp in: a candidatemale contracep tive vaccine[ J ] J Biosci, 2004, 29 (4) : 373-4.