To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was ampli...To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.展开更多
目的从临床标本中获得沙眼衣原体( C t)热休克蛋白( cH SP)10 基因,构建真核表达重组质粒,利用脂质体体外转染 H ela细胞,为研究 cH SP10 生物学功能和 C t核酸疫苗的研制做准备。方法用 PC R 技术从 40 例金标法阳性的临床标本中扩增 c...目的从临床标本中获得沙眼衣原体( C t)热休克蛋白( cH SP)10 基因,构建真核表达重组质粒,利用脂质体体外转染 H ela细胞,为研究 cH SP10 生物学功能和 C t核酸疫苗的研制做准备。方法用 PC R 技术从 40 例金标法阳性的临床标本中扩增 cH SP10 基因片断,重组入 pM D 18-T 克隆载体。将 pM D 18-T/cH SP10外源基因片断经酶切鉴定后,克隆入 pcD N A3.1(+)中,经过序列分析和酶切鉴定后,运用脂质体将该重组体转染 H ela细胞,ELISA 方法观察目的基因的表达。结果 PC R 扩增得到 cH SP 基因全长,大小为 306bp,序列测定与 G enBan(k M 58027)发布的序列一致;构建得到 cH SP10 基因真核表达载体;该真核表达载体在 H ela细胞表达 cH SP10。结论成功构建 cH SP10 基因真核表达载体,该重组质粒能在 H ela细胞中表达 cH SP10,为进一步研究 cH SP10 生物学功能和 C t核酸疫苗的研制提供了实验基础。展开更多
文摘To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.
基金This topic belongs to National Family Plan Topics
文摘目的从临床标本中获得沙眼衣原体( C t)热休克蛋白( cH SP)10 基因,构建真核表达重组质粒,利用脂质体体外转染 H ela细胞,为研究 cH SP10 生物学功能和 C t核酸疫苗的研制做准备。方法用 PC R 技术从 40 例金标法阳性的临床标本中扩增 cH SP10 基因片断,重组入 pM D 18-T 克隆载体。将 pM D 18-T/cH SP10外源基因片断经酶切鉴定后,克隆入 pcD N A3.1(+)中,经过序列分析和酶切鉴定后,运用脂质体将该重组体转染 H ela细胞,ELISA 方法观察目的基因的表达。结果 PC R 扩增得到 cH SP 基因全长,大小为 306bp,序列测定与 G enBan(k M 58027)发布的序列一致;构建得到 cH SP10 基因真核表达载体;该真核表达载体在 H ela细胞表达 cH SP10。结论成功构建 cH SP10 基因真核表达载体,该重组质粒能在 H ela细胞中表达 cH SP10,为进一步研究 cH SP10 生物学功能和 C t核酸疫苗的研制提供了实验基础。
