摘要
以pET22b为起始载体,构建了条斑紫菜6-磷酸海藻糖合成酶基因(PyTPS)0.6Kb片段的原核表达载体,并命名为pET22b-PyTPS0.6。重组菌BL21(pET22b-PyTPS0.6)经ITPG诱导后,SDS-PAGE分析显示菌体总蛋白的电泳图谱上有分子量约为23kDa的特异性蛋白条带,即PyTPS基因0.6Kb片段得到了原核表达。用溶菌酶裂解细菌提取包涵体,再利用镍离子亲和层析柱从包涵体中纯化出TPS重组蛋白。用获得的高纯度重组蛋白作抗原免疫家兔获得了抗血清,间接ELISA测定显示一份抗血清的效价为1∶16000,另一份为1∶32000。本研究制备的抗血清为进行转基因植物中PyTPS基因翻译产物的免疫学检测奠定了基础。
Using pET22b as initial vector,prokaryotic expression vector of 0.6Kb fragment of trehalose-6-phosphate synthase gene （PyTPS）from Porphyra yezoensis was constructed and named pET22b-PyTPS0.6.The recombinant E.coli strain BL21 （pET22b-PyTPS0.6） was induced with ITPG,and a specific protein band about 23kDa was observed in the SDS-PAGE pattern of total protein.The result showed that the PyTP gene fragment was successfully expressed in receptor E.coli strain.Bacterial cells were treated with lysozyme and inclusion bodies were extracted.The TPS recombinant protein was purified from inclusion body protein by nickel ion affinity chromatography,and high purity recombinant protein was obtained.The yielding TPS recombinant protein was used as antigen to immune two rabbits and prepare antisera,indirect ELISA analyses indicated that the titer of one antiserum was 1 ∶ 16000,and another was 1 ∶ 32000.Immunological detection of translation product of PyTPS gene in transgenic plant will be feasible by the use of the prepared antisera.
出处
《青岛农业大学学报（自然科学版）》
2013年第4期295-299,共5页
Journal of Qingdao Agricultural University(Natural Science)
基金
山东省良种工程马铃薯番茄资源创新利用研究
关键词
PyTPS基因
原核表达
镍离子亲和层析
重组蛋白
抗血清
PyTPS gene
prokaryotic expression
nickel ion affinity chromatography
recombinant protein
antiserum
