兔抗大鼠LM23蛋白合成肽多克隆抗体的制备与初步应用  

Generation and preliminary application of rabbit anti-rat polyclonal antibodies against synthetic peptides of rat LM23 protein

作　　者：成毅明[1,2] 刘美玲[3] 石心泉[3] 王介东[3] 贾孟春[1] 

机构地区：[1]北京协和医学院研究生院,北京100730 [2]空军航空医学研究所,北京100142 [3]国家人口计生委科学技术研究所,北京100081

出　　处：《生殖医学杂志》2009年第3期279-283,共5页Journal of Reproductive Medicine

基　　金：国家自然科学基金资助项目(30670784)

摘　　要：目的制备大鼠精子发生相关蛋白LM23的合成肽多克隆抗体,对其特性进行初步鉴定,并对LM23多克隆抗体进行初步应用。方法利用生物信息学方法分析选择LM23蛋白的多肽序列。应用Fmoc法化学合成大鼠LM23蛋白N端第1～20和第274～291个氨基酸多肽(接近C端),经C18的RP-HPLC纯化后,通过高碘酸钠法,将纯化的LM23蛋白的多肽与KLH交联,免疫新西兰大耳白兔,获得LM23蛋白的多克隆抗体。用ELISA方法鉴定多克隆抗体效价。通过Western blot方法鉴定LM23多克隆抗体的特异性并研究LM23在大鼠睾丸中的表达;通过免疫组化方法研究LM23在大鼠睾丸组织和细胞中的定位。结果化学Fmoc法分别合成大鼠LM23蛋白N端第1～20,第274～291个氨基酸多肽,纯化后两条多肽纯度都达到90%以上,符合免疫用抗原标准。将多肽与KLH交联,用于免疫动物。经ELISA鉴定,两种多克隆抗体的效价分别达到1:64 000和1:128 000。应用大鼠睾丸组织切片进行LM23-18肽多克隆抗体的免疫组织化学研究,发现精母细胞有阳性颗粒反应,且LM23主要表达于细胞核。Western blot研究证实,LM23-18肽多克隆抗体可特异识别大鼠睾丸组织中相对分子量(Mr)约为36 kD的LM23蛋白。结论所制备的LM23合成肽多克隆抗体可用于ELISA、Western blot及免疫组化研究。LM23合成肽多克隆抗体的制备为进一步研究LM23的功能和作用机制提供了有力支持。Objective： To generate and characterize the COOH-terminal peptides of LM23 protein. rabbit polyclonal antibodies against NH2-terminal and Methods： According to the bioinformatics analysis and prediction of the possible high structure,hydrophilicity and antigenicity of LM23, two peptides （aa 1 to 20, aa 274 to 291） of rat LM23 were synthesized by using standard Fmoe solid phase synthesis. The synthesized peptides were purified by reversed phase high-performance liquid chromatography （RP-HPLC）, and cross-linked with keyhole limpet hemoeyanin （KLH） by EDC. Rabbits were immunized with conjugated peptides for 3 times （500 μg/rabbit）. The polyelonal antibodies were characterized by ELISA, Western blot and imrnunohistochemistry. Results： The two synthesized peptides with the purity of 90%were prepared respectively. The titers of purified polyclonal antibodies were 1 ： 64,000 and 1 ： 128,000 respectively. The immunopositive stain of LM23 was mainly located in spermatcoytes nucleus detected by immunohistochemistry analysis. Western blot results showed that the antibodies the total nucleoprotein of rat testis. recognized the protein with molecular weight of 36 kD in Conclusions： The polyelonal antibodies against LM23 generated and characterized in the present work are specific to LM23 protein and can be used in ELISA, Western blot and immunohistochemistry analysis. It would be useful tools for the functional study of rat LM23.

关 键 词：LM23 合成肽 精子发生 多克隆抗体 

分 类 号：R392-33[医药卫生—免疫学] R730.53[医药卫生—基础医学]