运用穿梭质粒在小鼠沙眼衣原体中表达外源性基因

Expression of exogenous genes in Chlamydia muridarum by transfection with shuttle vectors

目的在能够转化小鼠沙眼衣原体的穿梭质粒pGFP：：CM中加入新的开放读码框，为研究衣原体单个蛋白功能奠定基础。方法用聚合酶链反应（PCR）扩增质粒pGFP：：CM和新的开放读码框（包括质粒蛋白pgp4的启动子、红色荧光蛋白mCherry基因和沙眼衣原体CT579的转录终止序列），将连接产物转化至大肠埃希菌感受态Stellar中，提取质粒进行PCR、酶切和测序鉴定。将重组质粒利用氯化钙法转化至无质粒的CMUT3中，荧光显微镜下观察并挑选带有荧光的转化衣原体包涵体，经过氨苄西林筛选和噬菌斑纯化后，采用间接免疫荧光染色法检测转化株CMUT3-pGFP·mCherry-CM中pgp3和glgA蛋白的表达。结果经PCR、酶切和测序鉴定后得到正确序列的重组质粒，转化CMUT3后可见含有绿色和红色荧光的衣原体包涵体。氨苄西林筛选和噬菌斑实验纯化后得到新的转化株CMUT3-pGFP-mCherry-CM。免疫荧光证实pgp3和glgA蛋白在衣原体CMUT3-pGFP-mCherry-CM和CMUT3-pGFP：：CM中表达相似。结论在质粒pGFP：：CM中成功加入开放读码框，转化衣原体后能够表达外源性基因，使衣原体转化技术能够用于衣原体蛋白质功能研究，为临床沙眼衣原体感染的防治提供新的线索。

Objective To add an open reading frame in the shuttle vector of pGFP ：： CM for transfection of exogenous genes into Chlamydia muridarum. Methods The sequence of plasmid pGFP ：： CM and new open reading frame （including promoter of pgp4, mCherry gene of red fluorescence protein and transcription termination sequence of Chlamydia trachomatis CT579 ） were amplified by polymerase chain reaction （PCR） , and the products were transfected into Stellar competent cells. The recombinant plasmids were identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transfected into plasmid-free strain CMUT3, and the GFP- and mCherry-positive inclusions were observed under the fluorescence microscope. After the ampicillin selection and plaque purification, the purified CMUT3-pGFP- mCherry-CM was identified by indirect immunofluorecesent stain using anti-pgp3 and anti-glgA antibodies. Results The correct recombinant plasmid after sequencing identification, enzyme digestion and PCR amplification was successfully transfected into CMUT3, and the GFP- and mCherry-positive inclusions were observed. The transfected strain CMUT3-pGFP-mCherry-CM was purified after ampicillin selection and plaque purification. The expression of pgp3 and glgA protein in CMUT3-pGFP-mCherry-CM was similar to that in CMUT3-pGFP ：： CM. Conclusion An open reading frame is successfully added in the plasmid pGFP ：： CM, and the new plasmid can be transfected into CMUT3 and express exogenous protein, which can be used for further study on the function of single chlamydial protein.