| 摘要: |
| 【目的】克隆大肠杆菌半胱氨酸合成酶(包括丝氨酸乙酰转移酶（cysE）和O-乙酰丝氨酸硫化氢解酶（cysM）)的基因，对其进行原核表达，并制备相应蛋白的抗体。【方法】用PCR方法从大肠杆菌BL21(DE3)中扩增cysE和cysM基因，构建其原核表达载体pET32a(+)-cysE和pET32a(+)-cysM，对其进行BamHⅠ和SacⅠ双酶切鉴定和测序验证后转入大肠杆菌中进行诱导表达，对表达产物进行SDS-PAGE和Western blot(以His抗体作为一抗)检测。用纯化的cysE和cysM蛋白作为抗原免疫新西兰大耳白兔，制备多克隆抗体，并通过酶联免疫吸附试验(ELISA)检测抗体效价。【结果】PCR获得了822 bp的cysE基因和912 bp的cysM基因。原核表达质粒pET32a(+)-cysE和pET32a(+)-cysM构建成功，并可在大肠杆菌BL21(DE3)中诱导表达，表达的cysE和cysM重组蛋白分子质量分别为56和58 ku。制备的多克隆抗体具有较强的免疫结合活性，抗体滴度均达到1∶102 400。【结论】成功克隆了大肠杆菌cysE和cysM基因，并实现了原核表达，同时制备出了相应的多克隆抗体。 |
| 关键词: 大肠杆菌 丝氨酸乙酰转移酶基因 O-乙酰丝氨酸硫化氢解酶基因 基因克隆 |
| DOI： |
| 分类号: |
| 基金项目:农业部转基因生物新品种培育重大专项（2008ZX08008-002） |
|
| Cloning, prokaryotic expression of Escherichia coli cysE,cysM and preparation of their antibodies |
|
|
| Abstract: |
| 【Objective】Cloning of cysE and cysM gene which encoded cysteine synthetase,prokaryotic expressing the enzymes and preparing their antibodies are preliminary work for the production of transgenic cashmere goat.【Method】cysE and cysM gene were amplified by PCR.Two recombinant prokaryotic expression vectors pET32a(+)-cysE and pET32a(+)-cysM were constructed by using molecular technique,and then they were transferred into Escherichia coli BL21 (DE3) to induce protein expression with IPTG.An anti-His antibody was used as the first antibody in Western blot.The purified recombinant proteins were used as antigens to immunize rabbits for preparation of polyclonal antibodies.The titers of cysE and cysM antibodies were detected by ELISA.【Result】The cysE and cysM genes are amplified by PCR.The cysE gene is 822 bp,and cysM gene is 912 bp.Prokaryotic expression vectors pET32a(+)-cysE and pET32a(+)-cysM are successfully constructed,and highly expressed in Escherichia coli BL21 (DE3) induced with IPTG,the molecular weights of recombinant cysE and cysM proteins are 56 and 58 ku.The expressed products are correctly identified by SDS-PAGE and Western blot.Two high titer antibodies (both 1∶102 400) are obtained by immunizing rabbit with the purified protein.【Conclusion】Cloning,prokaryotic expression and polyclonal antibody preparation methods and techniques of Escherichia coli cysE and cysM gene are successfully established.cysE and cysM protein from prokaryotic expression are identified and purified.The preparation of recombinant cysE,cysM and their polyclonal antibodies has provided reliable tools for the future study in the transgenic sheep of cysteine biosynthesis gene. |
| Key words: Escherichia coli cysE gene cysM gene gene cloning |
