1. College of Life Sciences, Nanjing Normal University, Nanjing 210046, China; 2. Institute of Military Medical Science, Nanjing Command, Nanjing 210002, China
摘要目的 利用原核表达系统诱导表达2型猪链球菌(Streptococcus suis 2,S.suis 2)分裂相关因子GpsB重组蛋白,为后续研究奠定基础。方法 以S. suis 2菌株05ZYH33全基因组DNA为模板,经PCR扩增得到目的基因片段。目的基因经双酶切后连接至表达载体pET32a,转化大肠杆菌(Escherichia coli, E. coli)DH5α感受态细胞。重组质粒经测序鉴定正确后转化E. coli BL21感受态细胞。获得的重组表达菌经IPTG诱导表达目的蛋白。利用Ni离子亲和层析柱纯化重组蛋白,并进行SDS-PAGE和Western blot 鉴定。利用重组蛋白免疫小鼠制备多克隆抗体。结果 成功构建出重组表达载体pET32agpsB,并经IPTG诱导表达出目的蛋白。重组蛋白主要存在于表达菌裂解液上清中,分子质量约30 kD,与预期大小一致。Western blot 检测发现,该蛋白能被His-Tag 单克隆抗体特异性识别。制备的多克隆抗体能特异性识别重组GpsB蛋白(rGpsB)。结论 成功表达和纯化了rGpsB并获得了该蛋白的多克隆抗体,为进一步研究该蛋白在S.suis 2 分裂过程中的作用鉴定了基础。
Abstract：GpsB is a protein that has been found to involve in cell division and growth of several bacteria. In the present study, we expressed the gpsB gene from Streptococcus suis 2 (S. suis 2) in the Escherichia coli (E. coli) BL21 system and prepared polyclonal antibodies against the recombinant GpsB protein. The gpsB gene was amplified from the genomic DNA of S. suis 2 strain 05ZYH33 with specific primers. The obtained PCR product was then digested and ligated into the expression vector pET32a. After sequencing, the recombinant plasmid, defined as pET32agpsB, was transformed into E. coli BL21 competent cells. The bacteria containing pET32agpsB were induced to express the target protein by IPTG. The recombinant protein was purified by Ni2+ NTA affinity chromatography and identified by SDS-PAGE and Western blot. Our data revealed that the S. suis 2 gpsB was successfully expressed in the E. coli BL21 system and presented mainly in the supernatant of the bacterial lysate. SDS-PAGE demonstrated that the molecular mass of the recombinant protein was about 30 kD, which is consistent with the expected size. Western blot analysis showed that this protein could be recognized by a monoclonal antibody against His-Tag. The polyclonal antibodies against recombinant GpsB(rGpsB) was prepared with purified rGpsB. In conclusion, this study provides fundamental resources for further exploring the role of GpsB in the process of S. suis 2 cell division.
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2019, Vol. 35 
