【作者】 黄丹；

【导师】 潘善培；

【作者基本信息】 暨南大学 ， 发育生物学， 2010， 硕士

【摘要】 目的：构建重组hCatsperl特异抗原(含胞外区、钙选择孔区和破伤风类毒素通用T细胞表位TT580-599)原核表达载体并表达重组抗原。用重组抗原免疫新西兰雄兔,评价其免疫及抗生育效能,为进一步以Catsperl的免疫避孕研究积累资料。方法：设计引物,以RT-PCR法扩增人CatSperlS1-S6的整个跨膜区DNA片段,利用重叠PCR法合成重组人CatSperl特异抗原DNA片段,并分别插入到载体pET-21b和pET-21b-Trx构建重组表达质粒。经PCR、酶切和测序鉴定后转化工程菌株E.coli BL21 (DE3)进行诱导表达。表达产物用Tricine-SDS-PAGE和Western blotting分析。含重组hCatsperl特异抗原的包涵体经Tricine-SDS-PAGE、染色和脱色处理后,将目的蛋白条带切下,磨碎,与弗式佐剂乳化后免疫新西兰雄兔,制备抗重组hCatsperl特异抗原的抗血清。含重组Trx(硫氧还蛋白)-hCatsperl特异抗原的包涵体用2mol/L尿素变性,经Ni螯合层析柱纯化重组Trx-hCatsperl特异抗原。用ELISA法检测抗血清的抗体反应。用Western blotting检测抗血清与重组Trx-hCatsperl特异抗原及人睾丸组织蛋白的反应。抗血清与人精子的反应用间接免疫荧光法检测。抗血清处理人精子1、2和4小时后,用连续曝光法记录精子运动轨迹结合统计学分析抗血清对精子运动的影响。免疫后雄兔的睾丸及附睾做石蜡组织切片,H.E染色后作组织病理学观察。结果：构建了pET-21b-hCatsper1特异抗原和pET-21b-Trx-hCatsper1特异抗原原核表达质粒。PCR、酶切和测序的结果显示插入DNA片段的大小和序列与预期的重组hCatsper1特异抗原DNA一致。重组表达质粒转化E.coli BL21 (DE3)获得阳性克隆,经IPTG诱导表达重组蛋白。Tricine-SDS-PAGE和Western blotting结果显示,重组hCatSperl特异抗原和重组Trx-hCatsperl特异抗原主要以包涵体形式存在。其大小分别为11kDa、24kDa,与预期目的蛋白大小一致。对重组Trx-hCatsperl特异抗原包涵体进行变性、Ni螯合层析柱纯化及复性处理,可获得较纯的可溶性重组蛋白。用重组hCatSperl特异抗原免疫雄兔可诱发抗体反应。兔抗重组hCatSperl特异抗原的抗血清能与重组Trx-hCatsperl特异抗原结合,并能与人睾丸组织蛋白反应,显示大小相应于hCatSperl (80kDa)的特异反应带。间接免疫荧光法测定表明抗血清能与人精子反应,在精子头部末端、中部及尾部呈现绿色荧光。精子体外运动分析结果显示,抗血清能显示一定程度的降低精子运动速度,3例精液标本的运动降低率分别为14.2%、15.1%、17.6%。免疫雄兔睾丸曲细精管见形态正常的各级生精细胞,附睾管组织结构完整,管内充满精子。结论：成功构建重组hCatsperl特异抗原和Trx-hCatsperl特异抗原的原核表达系统并表达重组hCatsperl特异抗原和Trx-hCatsperl特异抗原。用重组hCatsperl特异抗原免疫新西兰雄兔能诱发特异的抗体反应；抗重组hCatSperl特异抗原抗血清能与人睾丸组织蛋白及人精子反应；抗血清在体外能一定程度的减弱精子运动；免疫雄兔的睾丸及附睾未见病理变化。重组hCatsperl特异抗原免疫的抗生育效能有待进一步的研究。更多还原

【Abstract】 Aim:To construct the prokaryotic expression vectors of recombinant human Catsperl-specific antigen (including the extracellular region, the pore region and the promiscuous T cell epitope TT580-599) and express the recombinant antigens. To evaluate antibody response, antifertility effect of immunization with the recombinant hCatsperl-specific antigen and accumulate data for further study of immunocontraception targetting hCatsperl.Methods:The DNA fragment of human Catsperl coding the transmembrane region (S1-S6) was amplified by RT-PCR with total mRNA extracted from human testis.The DNA fragment of human Catsperl-specific antigen was synthesed by overlapping PCR and inserted into prokaryotic expression vector pET-21b and pET-21b-Trx, constructing the recombinant expression plasmids pET-21 b-hCatsperl and pET-21 b-Trx-hCatsperl. After identified by restriction endonuclease digestion, PCR and sequence analysis, the recombinant plasmids were transformed into E. Coli BL21 (DE3) and the recombinant proteins were expressed with IPTG induction. The expressed proteins were analysed by Tricine-SDS-PAGE and Western blotting. The inclusion bodies of hCatSperl specific antigen were separated by Tricine-SDS-PAGE. After staining, destaining and equilibrating with phosphate buffer, the gel containing hCatsperl-specific antigen were cut, rubbed, emulsified with freund’s adjuvant and used to immune male rabbits by multi-point subdermal injection and muscle injection. The recombinant Trx-hCatsperl specific antigen was separated and purified from inclusion body by denatured with 2 mol/L urea and Ni-chelating chromatography. Anti-hCatSperl specific antigen antibody response of the immunized rabbits were monitored by ELISA with the purified Trx-Catsperl specific antigen as coating antigen. Reactions of antiserum specific to recombinant Trx-hCatsperl and human testis tissue protein were detected by Western blotting. Antiserum’s binding to human sperm were detected by indirect immunofluorescence (ⅡF). The effect of antiserum on human sperm motility was determined by co-culturing sperm with antiserum in vitro, and recording the sperm moving trajectory using continuous exposure photography. Paraffin sections of the rabbits testis and epididymis were prepared and stained by HE to observe pathological changes in testis and epididymis of immunized rabbits.Results:The prokaryotic plasmid pET-21b-hCatsperl specific antigen and pET-21b-Trx-hCatsperlspecific antigen were successfully constructed. Results of PCR, restriction endonuclease digestion and sequence analysis showed that the length and sequence of inserted DNA fragment coincided with those of the designed recombinant hCatsperl specific antigen. The recombinant proteins were expressed after the recombinant plasmid transformed to E. coli BL21(DE3). Analysis by Tricine-SDS-PAGE and Western blotting showed that recombinant human CatSperl specific antigen and the recombinant Trx-hCatsperl specific antigen were mainly in inclusion bodies, with 11kDa、24kDa size respectively and consistent with the expected protein size. The pure soluble recombinant Trx-hCatsperl specific antigen was separated and purified by denaturation, Ni-chelating chromatography and renaturation from inclusion body. Antibody response could be induced in male rabbit by immunization with recombinant hCatSperl-specific antigen. By Western blotting, antiserum was able to bind to Trx-hCatsperl specific antigen and also bind to human testis protein showing a band with size-80kDa consistent with the size of CatSperl protein. Antiserum could bind to the human sperm, showing green fluorescence in the middle, tail and the region behind the head. Antiserum could depress human sperm motility in a certain extent in vitro.No pathological changes were found in the testis and epididymis of immunized rabbits. Spermatogenic cells in defferent developing stages and with normal appearance could be observed in testicular seminiferous tubules. The epididymis tubules exhibited integrated histological structureand were full of sperm.Conclusion:The prokaryotic plasmid pET-21b-hCatsperlspecific antigen and pET-21b-Trx-hCatsperlspecific antigen were constructed and recombinant proteins were successfully expressed in prokaryotic system. Anti-hCatSperl specific antigens antibody response was induced after immunization of New Zealand male rabbits with the recombinant hCatsperl-specific antigen. The antiserum was able to react with the 80kDa protein in human testis and could bind to human sperm. The antiserum treatment in vitro could depress sperm motility in a certain extent. No significant testis and epididymis pathological injury can be seen in the immunizesd rabbits during this study. The antifertility effect of immunization with the recombinant hCatSperl-specific antigen needs to be further studied.更多还原