人g2型溶菌酶的多克隆抗体制备及其在睾丸中的表达分析  

Preparation of the polyclonal antibody against human goose-type lysozyme 2 and its expression in human testis

作　　者：黄鹏[1] 杨智昉[1] 谢君[2] 徐一新[3] 鲍建瑛 曹晓娥 张宁[1] 余龙[2] 

机构地区：[1]上海健康医学院基础医学院,200237 [2]复旦大学遗传工程国家重点实验室,上海200433 [3]上海健康医学院药学院,200237

出　　处：《免疫学杂志》2016年第10期842-847,共6页Immunological Journal

基　　金：上海市自然科学基金(15ZR1421800)

摘　　要：目的以真核表达的人g2型溶菌酶(human goose-type lysozyme 2,LysG2)为免疫原制备抗LysG2的兔多克隆抗体,检测LysG2在睾丸组织中的定位,为阐明其生理功能提供线索。方法对LysG2毕赤酵母GS115转化菌株进行高密度培养,甲醇诱导目的蛋白表达并通过甲壳素亲和色谱纯化,以重组蛋白为免疫原制备兔抗LysG2多克隆抗体。通过ELISA检测抗体效价后,对人体各组织、精子及精液标本进行Western印迹分析,并对人睾丸组织进行免疫组化检测。结果以毕赤酵母表达LysG2为免疫原制备的抗体具有高效价,表达谱分析表明LysG2在睾丸中有表达,在精子蛋白提取物中没有检测到其存在,在精浆中检测到了LysG2。免疫组化检测结果显示LysG2在睾丸中主要定位于间质组织的Sertoli细胞。结论本研究成功以真核表达的LysG2制备了该蛋白多克隆抗体,LysG2在睾丸Sertoli细胞有表达,并分泌到精浆中,提示LysG2可能在男性生殖系统先天性免疫中发挥作用。This study designed to prepare rabbit antibody against human goose-type lysozyme 2 （LysG2） by using the recombinant protein expressed in Pichia pastoris as immunogen and to identify its distribution in the testis. The recombinant P. pastoris strain was cultured and induced with methanol to express LysG2, followed by purification using chitin affinity chromatography, and then the recombinant protein was used as an immunogen to generate polyclonal antibody （pAb）, whose specificity was examined by enzyme-linked immunosorbent assay （ELISA）. The distribution of LysG2 in human tissue and semen was identified by Western blotting and the subcellular localization in testis was investigated by immunohistochemistry （IHC）. ELISA analysis showed that anti- LysG2 polyclonal antibody could bind recombinant protein with high specificity. Western blotting indicated that LysG2 was existed in the testis, but not in other tissues. And in semen, LysG2 was detected in seminal plasma, but not in the spermatozoa. Immunodetection with a specific antiserum showed that the LysG2 protein localized in the Sertoli cells of testis. In conclusion, the rabbit pAb of LysG2 has prepared successfully by the recombinant LysG2 from P. pastoris. LysG2 is mainly expressed in the Sertoli cells of testis and secreted into the seminal plasma, suggesting that LysG2 might play a role in the innate immunity of the male reproductive tract.

关 键 词：人g2型溶菌酶 毕赤酵母 多克隆抗体 睾丸 先天性免疫 

分 类 号：R392[医药卫生—免疫学]