摘要
目的 构建GFP -MOMP融合蛋白真核表达重组质粒pEGFP/MOMP ,利用脂质体体外转染HeLa细胞 ,观察MOMP在真核细胞中的表达。 方法 PCR法从D型Ct基因组中扩增MOMP全长基因 ,克隆至 pEGFP真核表达载体的相应位点 ,进行序列分析和酶切鉴定后 ,脂质体介导重组体pEGFP/MOMP转染HeLa细胞 ,荧光显微镜、Westernblot鉴定MOMP基因的表达。 结果 PCR扩增得到约 1.2kb的特异性MOMP基因片段 ;成功的构建真核表达重组体pEGFP/MOMP ;重组质粒pEGFP/MOMP在HeLa表达出约 71kDa大小的融合蛋白。 结论 GFP -MOMP能够在体外真核细胞中表达 ,为进一步研究该蛋白的生物学功能及核酸疫苗的制备提供实验依据。
Objective To construct the recombinant plasmid pEGFP/MOMP, and transfect it into HeLa cells to express the major outer membrane protein(MOMP). Methods MOMP gene was amplified from the genomic DNA of Chlamydia trachomatis PCR, the gene was cloned into appropriate site of pEFFP-C1 vector. After identifying by sequencing and restrictive enzymes digestion, The recombinant plasmid was transfected into HeLa cells using Liposome. The expressed protein was identified by fluorescent microscope and Western Blot. Results The specific MOMP gene about 1.2kb was obtained,pEGFP/MOMP was successfully constructed .About 71kDa recombinant protein was expressed in HeLa cells. Conclusion GFP-MOMP was successfully expressed in eukaryotic cells which lay the foundation for studying the biological activities and the development of the Chlamydia trachomatis vaccine against this pathogen.
出处
《实用预防医学》
CAS
2004年第6期1089-1091,共3页
Practical Preventive Medicine
基金
湖南省自然科学基金 (0 4JJ30 63)
湖南省教育厅课题 (0 3C379)
湖南省卫生厅课题 (B2 0 0 3 - 0 79)
关键词
融合蛋白
转染
真核表达
Fusion protein
Transfection
Eukaryotic expression