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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。
OBJECTIVE: To obtain the chlamydia trachomatis (C t) heat shock protein (cH SP) 10 gene from clinical specimens and construct eukaryotic expression recombinant plasmids for transfection of H ea cells in vitro. In order to study the biological functions of cH SP10 and C t Preparation of nucleic acid vaccine preparation. Methods The cDNA of cH SP10 gene was amplified from 40 gold standard positive clinical samples by PC R technique and cloned into pM D 18-T cloning vector. The pM D 18-T / cH SP10 foreign gene fragment was identified by restriction enzyme digestion and cloned into pcD N A3.1 (+). After sequence analysis and restriction enzyme digestion, the recombinant was transfected H ela cells, ELISA method to observe the expression of the target gene. Results The full-length cDNA of cH-SP gene was amplified by PC R, and the size was 306 bp. The sequence was identical with that of G enBan (k M 58027). The eukaryotic expression vector of cH SP10 gene was constructed. Expression of cH SP10. CONCLUSION: The eukaryotic expression vector of cH SP10 gene was constructed successfully. The recombinant plasmid can express cH SP10 in H Ela cells. It provides an experimental basis for further study on the biological function of cH SP10 and the development of C t nucleic acid vaccine.