Expression and purification of a novel thermophilic bacterial single-stranded DNA-binding protein and enhancement the synthesis of DNA and cDNA
10.3760/cma.j.issn.1003-9279.2012.06.019
- VernacularTitle: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
- Author:
Xiao-Wei JIA
1
;
Guo-Hui ZHANG
;
Hai-Yan SHI
Author Information
1. 300450,天津市第五中心医院肝胆外科
- Keywords:
Thermococcus;
DNA;
Polymerase chain reaction
- From:
Chinese Journal of Experimental and Clinical Virology
2012;26(6):464-466
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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.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 pET11 a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta(pET11a-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.Conclusions kod-ssb may in future be used to enhance DNA and cDNA amplification.
