The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

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.

A Novel Contraceptive Vaccine: Design and Synthesis of the Chimeric Peptide Containing Multivalent Sperm-Specific Epitopes

ve To develop a novel multivalent chimeric peptide vaccine for bisexual fertility regulation

Immunocontraceptive effects of ACRBP epitope-based vaccines in mice

Acrosin binding protein(ACRBP)is restrictedly expressed in sperm acrosomes and involved in many important reproductive processes.Here we investigated the immunocontraceptive effects of ACR BP peptide-based vaccines.B-cell epitope vaccines of mouse ACRBP were predicted by bioinformatics and prepared by vector coupling and adjuvant combination.Subsequently,female mice were actively immunized with the epitope vaccines subcutaneously,and the titers and specificity of anti-ACRBP antibodies were detected.Then the efficancy,safety and reversibility of epitope vaccines for contraception were evaluated in vivo and in vitro.

Research on immune effect of two gene vaccines containing brZPC′and brLDHC_4′when in combined inoculation

ZPC and LDHC_4 play a key role in the process of recognition between sperm and egg or sperm movement,respectively.In this study,partial cDNA sequences of ZPC and LDH-C_4 of Microtus branditi(brZPC and brLDHC_4,respectively) were cloned by RT-PCR,and directly inserted into pCR3.1 vector to construct two gene vaccines(pCR3.1-brZPC' and pCR3.1-brLDHC'_4).pCR3.1-brZPC' and pCR3.1-brLDHC,' could express corresponding proteins in transiently transfected CHO cells.The adult female BALB/c mice were inoculated with the recombinant vaccine alone on in combination.The immunized mouse can produce specific antibody that recognizes the corresponding recombinant protein expressed by BL21 in vitro.Moreover,antibodies produced by combinedly immunized mouse were specific and direct,with no inhibitory effect between two vaccines observed when in combined inoculation.The test of cytokines indicated that the expression of IFNγin pCR3.1-brZPC'- and combinedly inoculated mouse increased obviously and the expression of IL2 in pCR3.l-brLDHC'_4- and combinedly inoculated mouse increased obviously,while the expression of IL4 in pCR3.1-brZPC,pCR3.l-brLDHC'_4 and combinedly inoculated group increased obviously.It was suggested that the combined inoculation with pCR3.1-brZPC' and pCR3.l-brLDHC'_4 could induce both humoral immune response and CTL response.In some sense,the combined inoculation may achieve a better contraceptive effect.The results also showed that,when used alone or in combination,these two recombinant vaccines did not do much harm to the follicular development of immunized mouse.So the combined inoculation with these two recombinant vaccines could be a better way to immunocontraception.This study may provide a theoretical basis for the following tests on antifertility in vivo.

国产第一代β-hCG避孕疫苗免疫恒河猴的抗体反应及免疫安全性研究

用国产第一代ｈＣＧ避孕疫苗（以β－ｈＣＧ－ＴＴ为免疫原制备的氢氧化铝吸附制剂）免疫成年雌性恒河猴，评价疫苗在实验猴体内诱发抗ｈＣＧ抗体的能力和免疫安全性．结果显示疫苗能在实验猴诱发抗ｈＣＧ抗体，但抗体滴度较低（峰滴度为１４０００），持续时间较短，加入Ｆｒｅｕｎｄ’ｓ佐剂免疫能显著增强动物的抗体反应（峰滴度可达１６４０００），免疫猴对强化免疫有较好的记忆反应．免疫前后各免疫猴血液红细胞数、血红蛋白含量、白细胞数、白细胞分类计数均未见异常，血清谷丙转氨酶、总蛋白、白蛋白等肝脏功能指标也未见异常．免疫猴心、肝、肾、脾、胰腺、腺垂体、卵巢、子宫等器官未发现组织病理学改变．．

hCGβ-C3d3融合蛋白免疫BALB/c小鼠及其抗血清对hCG诱导鼠子宫增重的抑制效应

目的:通过分子佐剂C3d3增强hCGb避孕疫苗的免疫原性。方法:采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达质粒phCMV1-6His-hCGb-C3d3和phCMV1-6His-hCGb,在CHO细胞中获得稳定、高效表达的重组蛋白,并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGb-C3d3融合蛋白和单用hCGb间隔4周两次免疫生育期雌性BALB/c小鼠,ELISA测定血清中抗hCGb抗体滴度,并对各组小鼠产生的抗血清拮抗hCG诱导的小鼠子宫增重效应进行比较。结果:C3d3使hCGb蛋白疫苗的免疫原性增强1 995倍,hCGb-C3d3融合蛋白免疫小鼠产生的抗血清具有很强的抑制小鼠子宫增重作用。结论:通过分子佐剂C3d3可以大幅提高机体对hCGb的体液免疫应答能力,hCGb-C3d3融合蛋白免疫小鼠产生的抗血清对hCG的生物学作用具有更强的抑制效应。

分子佐剂C3d增强hCGβ基因免疫体液免疫效应

目的 :该实验室前期工作已成功构建真核表达质粒pcDNA3 hCGβ、pcDNA3 hCGβ C3d3且证明其能在真核表达系统中有效表达。通过动物DNA免疫 ,以期证实分子佐剂C3d增强免疫避孕疫苗免疫原性。方法 :抽提与纯化pcDNA3、pcD NA3 hCGβ、pcDNA3 hCGβ C3d3质粒 ,进行动物DNA免疫。免疫剂量分别为 5、10、2 0pmol,共免疫 2次 ,每次间隔 3周。末次免疫后 6周采血 ,用间接ELISA分析实验动物外周血抗hCGβ抗体效价。 结果 :C3d分子佐剂能明显提高抗hCGβ抗体滴度 ;且其作用呈剂量依赖性。结论 :分子佐剂确实能增强hCGβ免疫原性 ;此结果将有助于免疫避孕疫苗的技术进步及实际应用。

分子佐剂C3d3增强hCGβ蛋白疫苗的体液免疫效应

目的:通过分子佐剂C3d3增强hCGβ避孕疫苗的免疫原性。方法:采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达载体phCMV16HishCGβC3d3和phCMV16HishCGβ,在CHO细胞中获得重组蛋白的稳定、高效表达并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGβC3d3融合蛋白、hCGβ联合C3d3和单用hCGβ对BALBc与C57BL6小鼠进行免疫,共免疫两次,间隔4周,ELISA测定血清中的抗体滴度,比较各组的免疫效果。结果:无论是BALBc小鼠还是C57BL6小鼠,hCGβC3d3融合蛋白诱导产生的抗hCGβ抗体的出现时间都明显早于hCGβ与C3d3联合免疫和hCGβ单独免疫组。在BALBc小鼠,初次和再次免疫结果显示,C3d3使hCGβ的免疫原性增强了1995倍。C57BL6小鼠所显示出的体液免疫反应强度虽不如BALBc小鼠,但C3d3也使hCGβ的免疫原性增强了1259倍。结论:通过分子佐剂C3d3可以大幅地提高hCGβ避孕疫苗的免疫原性,在个性差异很大的不同品系小鼠,C3d3均能增强机体对hCGβ的体液免疫应答能力。

分子佐剂C3d3增强hCGβ蛋白疫苗免疫原性及其抗血清中和hCG生物学活性的作用

本文观察分子佐剂C3d3增强hCGβ蛋白避孕疫苗体液免疫效力的能力。采用分子生物学技术以phCMV1为载体分别构建分泌型、带有6个组氨酸纯化标签的真核表达质粒phCMV1-6His-hCGβ-C3d3和phCMV1-6His-hCGβ,在CHO 细胞中获得重组蛋白的稳定、高效表达,并用镍柱和凝胶过滤层析对其进行分离、纯化。分别用hCGβ-C3d3融合蛋白、hCGβ+弗氏佐剂和单用hCGβ免疫生育期雌性BALB/c 小鼠,共免疫两次,间隔4周。ELISA 测定血清中抗hCGβ抗体滴度,并对各组小鼠产生的抗血清中和hCG 生物学活性的能力进行比较。结果表明hCGβ单独免疫组在加强免疫后才见抗体生成,其抗体滴度比hCGβ-C3d3融合蛋白免疫组低1995倍,C3d3的佐剂能力是弗氏佐剂的10倍(初次免疫)-32倍(再次免疫),并且hCGβ-C3d3融合蛋白免疫小鼠产生的抗血清具有很强的中和hCG 生物学活性的作用。实验证明通过分子佐剂C3d3可以大幅提高机体对hCGβ的体液免疫应答能力。

hCG抗原／缓释微球对C_（57）小鼠免疫应答的研究

本文描述了常规免疫ｈＣＧ和一次性接种ｈＣＧ／缓释微球（ｈＣＧ／ＰＧＬＡ）对Ｃ５７ＢＬ小鼠产生免疫应答的比较研究，对它们所产生的抗ｈＣＧ血清作了三种参数的比较：１．抗ｈＣＧ血清效价的动态变化；２．抗体在体内体外中和ｈＣＧ生物活性的能力；３．抗原抗体亲和常数的测定。结果表明：一次性接种ｈＣＧ／缓释微球能在Ｃ５７小鼠体内产生高效价的ｈＣＧ抗体，对三种参数的分析表明：一次性接种ｈＣＧ／缓释微球系统所产生的抗血清和常规免疫ｈＣＧ所产生的抗血清基本上是相似的，抗体浓度与中和ｈＣＧ生物活性的能力成正比。本研究提示：一次性接种疫苗的缓释微球完全可以代替普通疫苗的常规免疫方法，为今后的β－ｈＣＧ避孕疫苗和其它疫苗的研究和临床推广应用提供了新的便利方法。

hCGβ避孕疫苗研究进展

h CGβ避孕疫苗的研究已从结合疫苗进入到了基因工程及 DNA疫苗阶段 ,载体、佐剂和免疫途径是进一步增强 h

Construction of sperm-specific lactate dehydrogenase DNA vaccine and experimental study of its immuno-contraceptive effect on mice

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.

HCGβ抗肿瘤核酸疫苗的纯化工艺

对HCGβ抗肿瘤核酸疫苗的预处理和层析纯化工艺进行了研究。用碱裂解法对湿菌体进行预处理,当菌体湿重（g）∶悬浮液体积（mL）∶裂解液体积（mL）∶中和液体积（mL）为1∶2∶2∶2,碱裂解时间为8-12 min时,质粒的产量最高,可达32.94 mg/g。用Sepharose 6 FF凝胶过滤层析去除RNA和蛋白质,再用Plasmidselect亲和层析捕获超螺旋质粒DNA,质粒DNA的得率为48.3%。用L-Histidine-agarose亲和层析可从澄清的碱裂解液中一步分离纯化超螺旋质粒DNA,质粒DNA得率为51.62%。经琼脂糖核酸电泳检测,HCGβ抗肿瘤核酸疫苗超螺旋质粒DNA的纯度为100%电泳纯。

The Study on Immunological Response of Long－term Release System hCG Microsphere in C_(57） Black Mouse

This paper describes the study of the immunological response of C57 Black mouse to hCG by multiple inoculations with hCG and single injection with a long-term release system of hCG/poly(lactide-co-glycolide) microsphere(hCG/PGLA).Three parameters including1)the dynamic variation of hCG antibody titres,2)the capacity of biological neutralization by hCG antibodies in vitro and in vivo,and3)assay of the binding affinity constants of hCG antibodies,are compared.The results showed that the single injection with hCG/PGLA was capable of producing high titres of anti-hCG antibodies.The analyses of three parameters of antisera from C57 Black mouse showed that the properties of the antisera elicited using a single injection with hCG/PGLA were generally similar to those of the antisera raised by the multiple inoculations with hCG,and concentration of the antibodies was directly proportional to hCG bioneutralization capacity. Meanwhile,all of these results suggest us that a further long-term release system of β-hCG/PGLA as a vaccine may substitute for conventional vaccine and it also provides a new convenient inoculative method for widespread clinical application and research of hCG vaccine and other vaccines.

卵透明带DNA避孕疫苗pVAX1-pZP3α的构建及其在体外的表达

目的:构建pVAX1-pZP3α,探讨其在体外的表达,研制卵透明带DNA避孕疫苗.方法:利用基因工程的方法构建卵透明带DNA避孕疫苗pVAX1-pZP3α,用脂质体法转染Hela细胞并采用RT-PCR和Westernblot检测卵透明带抗原pZP3α的体外表达.结果:构建了卵透明带DNA避孕疫苗pVAX1-pZP3α,并在体外表达系统检测到卵透明带抗原pZP3αmRNA和pZP3α的表达.结论:正确构建了卵透明带DNA避孕疫苗pVAX1-pZP3α.

表达鼠精子Cyritestin蛋白的减毒沙门氏菌疫苗株的构建及其鉴定

目的 构建表达鼠精子cyritestin蛋白的可口服减毒沙门氏菌活疫苗株。方法 应用质粒TAZ83/ 13,将之进行双酶切获得含有成熟cyritestin蛋白cDNA序列的酶切片段。将其插入表达载体 pYA314 9中 ,构建重组质粒pCFL。将该重组质粒转入鼠沙门氏伤寒杆菌疫苗株X4 6 32和X4 5 5 0中 ,以获得重组菌株X4 6 32 (pCFL)和X4 5 5 0 (pCFL)。结果 该两种无抗药性的重组菌株在体外营养选择压力下 ,可较稳定地携带重组质粒传代繁殖。X4 5 5 0 (pCFL)在体内可较稳定地定居于Peyer’s结、肠系膜淋巴结和脾脏 ;X4 6 32(pCFL)可较稳定地定居于Peyer’s结。二者均可表达被抗cyritestin单抗 (mAb)识别的重组cyritestin蛋白。口服该疫苗菌株无明显的毒性作用。结论 X4 6 32 (pCFL)、X4 5 5 0(pCFL)疫苗株的构建 。

男性避孕纳米粒的制备与体外实验研究

目的构建聚乳酸-羟乙酸共聚物(polylactic acid-glycolic acid,PLGA)包载促卵泡生成素受体表位肽(FSHR32-44)的纳米粒,探讨PLGA材料作为避孕疫苗载体的可行性。方法使用PLGA为载体,制备出包载促卵泡生成素受体的B细胞表位(FSHR32-44)的纳米粒,透射电镜(TEM)观察表观形态,粒径仪检测粒径分布范围和Zeta电位,体外释放试验检测其理化特性。激光共聚焦显微镜观察小鼠骨髓来源树突状细胞(DCs)吞噬FSHR可溶性多肽和FSHR/PLGA NP的能力,流式细胞仪检测FSHR、PLGA NP和FSHR/PLGA NP促骨髓来源DCs成熟能力。结果单乳-溶剂挥发法制备的FSHR/PLGA NP大小粒径分布均匀、表面规整,粒径(296.3±3.5)nm,Zeta电位(-16.1±0.4)m V,FSHR包封率(62.71±2.83)%,并具有缓慢释放抗原的能力;体外细胞水平实验结果显示FSHR表位肽纳米粒易于被DCs摄取,有剂量和时间依赖性,能显著上调DCs表面CD40、CD86、CD83和MHC-Ⅱ功能分子的表达。结论 PLGA包载FSHR多肽有良好的包封率,易被DCs吞噬,并有强的促进DCs成熟作用,可以作为FSHR32-44避孕疫苗的载体。

不同人群血清中抗透明带抗体的检测

为了探讨抗透明带抗体的临床意义 ,本研究用纯化的猪卵透明带ZP3抗原制备抗卵透明带抗体酶联免疫检测试剂盒 ,采用ELISA方法检测不同人群血中抗透明带抗体 ,并用阳性血清与人卵巢组织切片进行免疫组化分析。结果显示不同年龄组妇女抗透明带抗体阳性率差异不显著 (P >0 0 5 ) ,不孕组阳性率 15 % ,与其他组相比有显著性差异 (P <0 0 5 )。ELISA检测到的透明带抗体阳性血清不与人卵巢组织切片中的透明带产生肉眼可见的沉淀反应。除了早孕组和老龄妇女 ,任何年龄阶段的女性均可出现抗透明带自身抗体 ,鉴于这些抗体不引起周期紊乱现象 ,却可能阻止受精 。

精子膜抗原的研究新进展

精子表面的膜抗原是精子发生成熟过程中的重要标志分子 ,对其深入研究有助于揭示精子发生成熟及受精过程的生理机制和不育的病理改变。本文回顾了近年来随着分子生物学技术的广泛应用 ,传统的一些重要的精子膜抗原相继得到了克隆和测序 ,一些新的表达精子膜抗原的基因也陆续被发现 ,为从蛋白水平进一步深入研究其功能以及免疫避孕疫苗的筛选奠定了基础。

基因枪介导人精子顶体膜相关蛋白32基因疫苗在小鼠骨骼肌中的表达

目的:观察pcDNA3.1(+)-人精子顶体膜相关蛋白32(hSAMP32)基因疫苗经基因枪介导肌肉接种后在小鼠体内的表达。方法:BALB/c雌性小鼠6只,实验组鼠(4只)经后腿股内侧肌以基因枪多点注射重组质粒pcDNA3.1(+)-hSAMP325μg,空载体对照组(1只)注射pcDNA3.1(+)载体5μg,空白对照组(1只)注射无菌生理盐水200μl。接种后第7d取小鼠股内侧肌注射部位肌肉,RT-PCR和原位杂交法检测目的基因mRNA的表达,以自制抗血清进行免疫组织化学染色检测目的基因蛋白的表达。取小鼠心、肝、脾、脑、肾、胚胎组织,PCR扩增,行基因疫苗的安全性检测。结果:RT-PCR和原位杂交检测结果显示实验组小鼠注射部位肌肉组织有目的基因及其蛋白的表达,对照组未见表达。实验组小鼠心、肝、脾、脑、肾、胚胎的基因组DNA均未扩增出目的基因条带,说明外源性基因没有整合到宿主染色体。结论:该基因疫苗可在小鼠骨骼肌内有效、安全地表达。