| 陆春雪，吴移谋，彭波，刘良专，蔡恒玲，李忠玉.沙眼衣原体T细胞抗原CT143原核表达、纯化及免疫活性鉴定.[J].中南医学科学杂志.,2011,39(6):611-614. |
| 沙眼衣原体T细胞抗原CT143原核表达、纯化及免疫活性鉴定 |
| Cloning Expression,Purification and Antigenicity Analysis of Chlamydia Trachomatis T Cell Antigen CT143 |
| 投稿时间：2011-09-28 |
| DOI： |
| 中文关键词: 沙眼衣原体 CT143 克隆表达 纯化 免疫活性 |
| 英文关键词:Chlamydia trachomatis CT143 cloning and expression purification immunoactivity |
| 基金项目:国家自然科学基金（30970165、81102230）和衡阳市科技局基金(2008KS30)资助. |
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| 中文摘要: |
| 目的克隆沙眼衣原体(Chlamydia trachomatis,Ct) T细胞抗原Ct143基因,表达并纯化该蛋白,检测其免疫活性。方法采用PCR技术从Ct 基因组中扩增Ct143基因，并构建pGEX-6p-Ct143原核表达质粒，转化E.coli XL1-Blue，IPTG诱导重组GST-CT143融合蛋白表达，经酶切后得到无GST标签的纯蛋白免疫Balb/c鼠，ELISA及Western-blot分析CT143蛋白免疫原性及免疫反应性。结果成功构建了pGEX-6p-CT143原核表达质粒，序列测定证实重组质粒插入片段与GenBank登陆的Ct D型一致，长度为843 bp；GST-CT143融合蛋白在E.coli 中获得稳定高效表达；纯蛋白与佐剂混匀后肌肉免疫小鼠，ELISA 检测免疫小鼠的血清效价为1∶12 800，Western blot 结果表明该蛋白与Ct泌尿生殖道感染病人混合血清中IgG结合,具有免疫反应性。结论成功克隆表达了Ct T细胞抗原CT143，该抗原经纯化后具有良好的免疫原性及免疫反应性。 |
| 英文摘要: |
| ObjectiveTo clone,express and purify the Chlamydia trachomatis T cell antigen CT143 and preliminarily characterize the antigenicity of purified recombinant protein.MethodsThe ORF of Ct143 was cloned and inserted into the expression vector pGEX6p-2 to construct pGEX-6p-Ct143 recombinant plasmid.The recombinant plasmid was transformed into E.coli XL1-Blue to express GST-CT143 fusion protein,then purified with Glutathione Sepharose 4B Beads and digested with PreScission Protease to remove the GST tag.The pure protein was used to immunize Balb/c mice.ELISA and Western bolt were carried out to identify the immunogenicity and immunoreactivity.ResultsThe pGEX6p-Ct143 expression plasmid was successfully constructed.DNA sequencing showed that the inserted Ct143 was about 843 bp,encoding a protein with 280 amino acids.CT143 was expressed as GST fusion proteins in E.coli and then successfully cleaved by PreScission Protease.The ELISA result showed the specific antibody titer against CT143 reached 1∶12 800 and the affinity between recombinant CT143 and IgG antibodies from pooled Ct infected patient serum was identified by Western blot.ConclusionCT143 protein was successfully expressed and purified in prokaryotic system and the specific anti-CT143 antibody was induced in BALB/c mice,which lay a foundation for the further study on CT143 protein function and vaccine development. |
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