EXPRESSION OF RECOMBINANT-DNA INTRODUCED INTO CHLAMYDIA-TRACHOMATIS BY ELECTROPORATION
EXPRESSION OF RECOMBINANT-DNA INTRODUCED INTO CHLAMYDIA-TRACHOMATIS BY ELECTROPORATION
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DOI：
10.1139/m94-093
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发表时间：
1994-07-01
影响因子：
2.8
通讯作者：
WYRICK, PB
中科院分区：
文献类型：
--
作者：
TAM, JE;DAVIS, CH;WYRICK, PB
采用电穿孔法将DNA导入专性寄生细菌沙眼衣原体的初级体中。实验的DNA来源是由沙眼衣原体全长7.5 kb的质粒与大肠杆菌质粒pBGS9构建而成的嵌合质粒pPBW100。为了直接筛选携带pPBW100的沙眼衣原体，将衣原体启动子P-7248与无启动子氯霉素乙酰转移酶（chloramphenicol acetyltransferase, cat）盒式基因融合到质粒中。用含有pPBW100的电穿孔初级体感染McCoy鳗鱼后，通过原位杂交和Southern杂交分析在耐氯苯衣原体包涵体中检测到质粒DNA；（ii）物理和生化证据表明，电穿孔沙眼衣原体合成了氯霉素乙酰转移酶；（iii） P-7248::Cat的表达受发育调控，发生在衣原体网状体发育的早期阶段；（iv）虽然P-7248::Cat的表达主要是短暂的，但在四次传代后观察到氯霉素耐药沙眼衣原体的罕见情况。
Electroporation was used to introduce DNA into the elementary bodies of the obligate parasitic bacterium Chlamydia trachomatis. The source of DNA for these experiments was the chimeric plasmid pPBW100, which was constructed from the well-characterized 7.5-kb plasmid of C. trachomatis and the Escherichia coli plasmid pBGS9. To select directly for C. trachomatis carrying pPBW100, an in-frame gene fusion between the chlamydial promoter P-7248 and a promoterless chloramphenicol acetyltransferase (cat) cassette was incorporated into the plasmid. After infection of McCoy eels with electroporated elementary bodies containing pPBW100, the following were observed: (i) the plasmid DNA was detected inside the chloromphenicol-resistant chlamydial inclusions by in situ and Southern hybridization analyses; (ii) both physical and biochemical evidence showed that chloramphenicol acetyltransferase was synthesized by the electroporated C. trachomatis; (iii) expression of P-7248::Cat was developmentally regulated and occurred during the early stages of chlamydial reticulate body development; and (iv) although the expression from P-7248::Cat was mainly transient, there were rare instances where chloramphenicol-resistant C. trachomatis were observed after four passages.