小鼠精子蛋白Sp17的克隆,表达及其免疫原性研究.docVIP

小鼠精子蛋白Sp17的克隆、表达及其免疫原性研究 张巧玉1 ,2 梁志清1 李玉艳1 李晋涛3 史常旭1 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 第三军医大学免疫研究所，重庆，400038 摘　要目的 获得鼠精子蛋白Sp17基因，构建重组表达载体并在大肠杆菌中表达。方法 提取Balb/c小鼠睾丸总RNA，通过PCR扩增Sp17全长序列，克隆至pMD-T质粒载体中，Hind Ⅲ/ XhoⅠ双酶切克隆原核表达载体pET-28a（+）中转化大肠杆菌BL21(DE3)，异丙基-β-硫代半乳糖苷（IPTG）诱导表达SDS）、Western blotSp17免疫小鼠后血清特异抗体滴度。结果 获得了Sp17的全长cDNA，成功构建了重组表达载体pET/Sp17，在的诱导下高效表达。结论 小鼠精子蛋白Sp17序列已被克隆至原核表达载体pET-28a（+）中，并在大肠杆菌BL21(DE3)中获得高效表达。关键词 小鼠；精子蛋白；克隆；原核表达and expression of mouse sperm protein Sp17 and its immunological effects ZHANG Qiao-yu1 ,2，LIANG Zhi-qing1，LI Yu-yan1，LI Jin-tao1，SHI Chang-xu1 （1 Department of Obstetrics and Gynecology，Southwest Hospital，Third Military Medical University，Chongqing，400038，China 2.Department of Obstetrics and Gynecology，the second clinic Department of PLA,Beijing,100091；;3 Institute of Immunology,Third Military Medical University，Chongqing，400038，China） Abstract：Objectives To clone mouse sperm Sp17 gene and fulfill its expression in Escherichia 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 Escherichia coli BL21（DE3）with induction of IPTG and analyzed by SDSand Western blot, the mice were immunized intranasally with the recombinant proteins and the titers of specific antibodies IgG in serum. Result Sequencing and restriction digestion of the recombinant plasmid demonstrated that the coding sequence of mouse protein Sp17 was successfully cloned. SDSand Western blot analysis showed that a recombinant protein with molecular weight of 24 KD, which is corresponding to the theoretical molecular weight of Sp17 protein, existed in the extract of Escherichia coli BL21 (DE3) cells, and after immunization, Sp17-specific antibody IgG response was present in the mice. Conclusion The coding sequence of