Jia Xiao-Wei, Zhang Guo-Hui, Shi Hai-Yan
Department of Hepatobiliary Surgery Tianjin Fifth Central Hospital, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2012 Dec;26(6):464-6.
Express a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.
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.
The plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-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.
kod-ssb may in future be used to enhance DNA and cDNA amplification.
表达一种源自嗜热栖热菌KOD1的新型单链DNA结合蛋白（SSB），简称为kod-ssb。并评估kod-ssb对基于PCR的DNA扩增和逆转录的影响。
我们用Transrtta（DE3）表达kod-ssb，通过Ni2+琼脂糖凝胶柱亲和层析纯化kod-ssb，用SDS-PAGE进行检测。为评估kod-ssb对基于PCR的DNA扩增的影响，以人β珠蛋白基因为模板扩增5 kb、9 kb和13 kb片段。为检测kod-ssb对逆转录的影响，我们用流感细胞培养上清液提取的RNA作为模板进行qRT-PCR反应。
将质粒pET11a-kod转化到Transetta（DE3）中，获得重组菌株Transetta（pET11a-kod）。当用IPTG诱导重组菌株Transetta（pET11a-kod）时，kod-ssb得到高表达。通过SDS-PAGE检测到特异性蛋白。为证实kod-ssb能增强靶DNA合成并减少PCR副产物，以5 kb、9 kb和13 kb的人β珠蛋白基因片段作为PCR模板。当PCR反应不包含SSB蛋白时，特异性PCR产物被非特异性产物污染。当加入kod-ssb时，kod-ssb显著增强了5 kb、9 kb和13 kb靶产物的扩增，并使非特异性PCR产物最少化。为证实kod-ssb能增强靶cDNA合成，用流感细胞培养上清液提取的RNA作为qRT-PCR反应的模板。结果是当加入kod-ssb时，kod-ssb显著增强了cDNA的合成，平均Ct值为19.42，不加入kod-ssb时的平均Ct值为22.15。
kod-ssb未来可能用于增强DNA和cDNA扩增。
