聚-γ-谷氨酸合成酶复合体A的原核表达及其多克隆抗体的制备

Prokaryotic Expression and Polyclonal Antibody Preparation of the Poly-γ-Glutamic Acid Synthetase A Protein

以pgsBCA质粒为模板对已知的pgsA进行PCR扩增,得到长度为1 116 bp的片段。将获得的PgsA基因连接到pQE-30表达载体,转化大肠埃希菌后用IPTG诱导其表达,经SDS-PAGE和Westernblot分析,均可见约42 ku目的蛋白带。通过Ni-NTA agarose纯化,获得较高纯度的重组蛋白,经基因定量仪测定其浓度可达1.5 mg/mL;以其免疫新西兰兔获得免疫血清,Western blot分析证实,该重组蛋白具有良好的反应原性,用间接ELISA检测抗体效价可高达1∶12 800。结果表明,成功获得了pgsA的多克隆抗体。

The DNA fragment of PgsA gene was amplified by PCR from the DNA of pgsBCA . After DNA cloning and sequencing, the fragment was inserted into expression vector pQE-30. The recombinant expression plasmid pQE30-pgsA was transferred into E. colis strain XL1-Blue, and the high level expressed recombinant protein was detected in the inclusion body protein of the expression strain induced by IPTG. The expressed protein could be recognized by antibodies of Anti-His-Tag using Western blot method. The recombinant protein was highly purified by Ni-NTA agarose,and its concentration was 1.5 mg/mL determined by GeneQuant. Then the purified recombinant protein was injected into rabbits to obtain polyclonal sera. The result of Western blot analysis showed that the recombinant protein had good antigenicity, pgsA-specific antibody titer was up to 1 ： 12 800 by indirect ELISA. The results showed that we successfully obtained pgsA polyclonal antibodies.