新型超耐热菌的单链DNA结合蛋白表达和纯化及其增强DNA、cDNA的合成

Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding proteinand enhancement the synthesis of DNA and cDNA

目的表达和纯化一种新的来自于超耐热菌（thermococcuskodakarensis）KODl的单链DNA结合蛋白（缩写为kod—ssb），并探讨其对DNA、cDNA合成的影响。方法采用Transrtta（DE3）表达kod-ssb，用镍柱亲和层析纯化，经SDS—PAGE分析检测表达纯化后的kod—ssb。使用PCR及qRT／qPCR检测kod-ssb对DNA、cDNA合成的影响。结果将质粒pETlla—kod转化Transetta（DE3）中，得到重组菌株Transetta（pETlla—kod），经IPTG诱导。由SDS—PAGE分析可见表达的约40×103的特异条带；使用人类B球蛋白基因作为模板分别扩增5kbp，9kbp和13kbp目的片段，结果加了kod—ssb的PCR目的片段比没加的产量高很多，而且kod—ssb显著降低了PCR反应的非特异性扩增。以流感细胞培养上清抽提的RNA为模板，实施RT-／qPCR反应。结果加kod的平均Ct值是19．42，而没加的为22．15。结论kod-ssb在未来将被用于增强DNA和cDNA的合成。

Objective Express a novel species of single-stranded DNA-binding protein （SSB） derived from Thermococcus kodakarensis KODl,abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription. Methods We express kod-ssb with the Transrtta （DE3）, and kod-ssb was purified by affinity chromatography on a Ni2＋ Sepharose column, detected by SDS- PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction. Results The plasmid pETlla - kod was transformed into Transetta （DE3） and the recombinant strain Transetta（pETll a-kod）was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta（pETlla-kod）was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15. Conclusions kod-ssb may in future be used to enhance DNA and cDNA amplification.