小鼠精子蛋白Sp17的克隆、表达及其免疫原性研究  

Cloning and expression of mouse sperm protein Sp17 and its immunological effects

作　　者：张巧玉[1,2] 梁志清[1] 李玉艳[1] 李晋涛[3] 史常旭[1] 

机构地区：[1]第三军医大学西南医院妇产科,重庆400038 [2]解放军总医院第二临床部妇产科,北京100091 [3]第三军医大学基础医学部全军免疫学研究所,重庆400038

出　　处：《第三军医大学学报》2008年第24期2281-2284,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30000183)~~

摘　　要：目的获得鼠精子蛋白Sp17基因,构建重组表达载体并实现其在大肠杆菌中的高效表达,为制备免疫性避孕疫苗奠定基础。方法提取BALB/c小鼠睾丸组织总RNA,通过RT-PCR扩增Sp17基因全长cDNA序列,并将其克隆至pMD 18-T质粒载体中,再经HindⅢ/XhoⅠ双酶切将Sp17基因片段亚克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),异丙基-β-硫代半乳糖苷(IPTG)进行诱导表达,采用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot对表达产物进行初步鉴定,并检测重组蛋白Sp17免疫小鼠后血清特异抗体滴度。结果获得了小鼠Sp17的全长cDNA,成功构建了重组原核表达载体pET/Sp17,在IPTG的诱导下实现了目的蛋白在大肠杆菌中的高效表达,免疫后小鼠血清中检测到特异性抗体。结论小鼠精子蛋白Sp17基因序列已被克隆至原核表达载体pET-28a(+)中,并在大肠杆菌BL21(DE3)中获得高效、正确的表达,重组蛋白Sp17能够诱导产生特异性免疫反应,具有一定程度的免疫原性。Objective To clone mouse sperm Spl7 gene and fulfill its expression in E. coli BL21 （DE3）. Methods The coding sequence of mouse protein Sp17 was amplified from mouse testis RNA through reverse transcription-polymerase chain reaction （RT-PCR）, and subsequently cloned into pET-28a （ ＋ ）. Recombinant Sp17 was then expressed in E. coli BL21 （DE3） with induction of IPTG and analyzed by SDS-PAGE and Western blotting. The mice were immunized intranasally with the recombinant proteins and the titers of specific antibodies IgG in serum were detected. Results Sequencing and restriction digestion of the recombinant plasmid demonstrated that the coding sequence of mouse protein Sp17 was successfully cloned. SDS-PAGE and Western blot analysis showed that a recombinant protein with molecular weight of 24 000, which is corresponding to the theoretical molecular weight of Sp17 protein, existed in the extract of E. coli BL21 （ DE3 ） cells. After immunization, Sp17-specific antibody IgG response was present in the mice. Coclclusion The coding sequence of mouse Sp17 was cloned into pET-28a （ ＋ ） plasmid vector, and high level of recombinant Sp17 protein expressed in E. coli BL21 （DE3） was obtained. The recombinant Sp17 protein can induce Sp17-speeifie antibody IgG response.

关 键 词：小鼠 精子蛋白 克隆 原核表达 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] R341[医药卫生—基础医学]