中华绒螯蟹组蛋白H3基因保守序列的克隆、表达及抗体制备

Cloning,Expression and Polyclonal Antibody Preparation of Histone H3 Gene Conservative Sequence From Eriocheir sinensis

为了研究组蛋白H3在中华绒螯蟹(Eriocheir sinensis)精子发生中的动态特征,采用PCR技术扩增中华绒螯蟹H3基因编码区的DNA片段,将其重组至含有6个His标签序列的原核表达质粒pET-30a(+)上,转化入大肠杆菌(Escherichia coli)Rosetta(DE3)感受态细胞中,以0.2 mmol/L的异丙基β-D硫代半乳糖苷(IPTG)诱导产生pET-30a-H3重组蛋白。SDS-PAGE表明,重组菌成功表达出了分子量约为21 ku的目标蛋白质,融合蛋白以上清和包涵体的形式存在。采用镍柱亲和层析的方法纯化目的蛋白,免疫新西兰大白兔(Oryctolagus cuniculus),制备兔抗血清。Western blot结果表明,制备的多克隆抗体具有较好的特异性。至此,成功地克隆了中华绒螯蟹H3编码区基因并制备了多克隆抗体,为进一步研究其在精子发生中的动态特征提供了基础。

During spermatogenesis of most animals, the basic proteins associated with DNA are continuously changing： somatic-typed histones are partly replaced by spermatoza-specific histones, which are then replaced by transition proteins, which in turn are finally replaced by protamines. With replacement of sperm basic nuclear proteins （SBNP） , nuclei progressively undergo condensation. The nuclei of spermatozoa in Chinese crab Eriocheir sinensis （Decapoda, Crustacea） are found to be uncondensed. However, the mechanism of nuclear uncondensation is not clear. To study histone H3 dynamics during E. sinensis spermatogenesis, H3 coding sequence of E. sinensis was amplified by PCR and cloned into prokaryotic expression vector pET-30a （ ＋ ） , which was then transferred into Escherichia coli Rosetta （DE3） and induced to express the recombinant His/H3 protein by 0. 2 mmol/L isopropyl [^-D-thiogalactoside （IPTG）. Meanwhile, the recombinant protein was purified through Ni-NTA Agarose affinity chromatography and purified protein was used as antigen to inoculate Oryctolagus cuniculus to produce antiserum. The polyclonal antibody was tested using western blotting method. Finally, H3 gene coding region from E. sinensis was cloned, prokaryotic expression vector pET-30a-H3 was constructed. SDS-PAGE showed that a 21 ku protein was expressed successfully in Rosetta （ DE3 ） , and the recombinant protein existed in the form of supernatant and inclusion body, and recombinant protein was purified well through Ni-NTA Agarose affinity chromatography. Western blot showed that the polyclonal antibody had high specificity. In summary, coding sequence of histone H3 in E. sinensis was successfully cloned, prokaryotic expression vector pET-30a-H3 was constructed and recombinant protein was purified. The polyclonal antibody with high specificity was produced, which might provide fundamental basis for further study of histone H3 dynamic changes during E. sinensis spermatogenesis.