农业科学与技术：英文版
農業科學與技術：英文版
농업과학여기술：영문판
Agricultural Science ＆ Technology
2012年
4期
723-726
,共4页
NorA基因%原核表达%多克隆抗体
NorA基因%原覈錶達%多剋隆抗體
NorA기인%원핵표체%다극륭항체
NrfA gene%Prokaryotic expression%Polyclonal antibody
[目的]为克隆大肠杆菌NrfA基因，构建pET-28a（＋）．NrfA表达载体，制备相应的多克隆抗体并鉴定。[方法]以以大肠杆菌基因组DNA为模板，PCR扩增得到NrfA基因编码区，构建pET．28a（＋）-NoCA表达载体；经IPTG诱导表达并纯化重组蛋白；再免疫新西兰雄兔，制备多克隆抗体；用ELISA方法检测抗体的效价，WesternBlotting检测抗体的特异性。[结果]适构建的表达载体pET-28a（＋）-NrfA在大肠杆菌中诱导后可以高效表达NrfA蛋白，免疫获得的多克隆抗体用ELISA检测，其效价为l：204900，经WesternBlotting分析，抗体的特异性较好。【结论】成功克隆大肠杆菌的NcfA基因，构建了表达载体，制备的NrfA多克隆抗体具有较高的效价和良好的特异性。为研究细菌有关NffA奠定了基础。
[目的]為剋隆大腸桿菌NrfA基因，構建pET-28a（＋）．NrfA錶達載體，製備相應的多剋隆抗體併鑒定。[方法]以以大腸桿菌基因組DNA為模闆，PCR擴增得到NrfA基因編碼區，構建pET．28a（＋）-NoCA錶達載體；經IPTG誘導錶達併純化重組蛋白；再免疫新西蘭雄兔，製備多剋隆抗體；用ELISA方法檢測抗體的效價，WesternBlotting檢測抗體的特異性。[結果]適構建的錶達載體pET-28a（＋）-NrfA在大腸桿菌中誘導後可以高效錶達NrfA蛋白，免疫穫得的多剋隆抗體用ELISA檢測，其效價為l：204900，經WesternBlotting分析，抗體的特異性較好。【結論】成功剋隆大腸桿菌的NcfA基因，構建瞭錶達載體，製備的NrfA多剋隆抗體具有較高的效價和良好的特異性。為研究細菌有關NffA奠定瞭基礎。
[목적]위극륭대장간균NrfA기인，구건pET-28a（＋）．NrfA표체재체，제비상응적다극륭항체병감정。[방법]이이대장간균기인조DNA위모판，PCR확증득도NrfA기인편마구，구건pET．28a（＋）-NoCA표체재체；경IPTG유도표체병순화중조단백；재면역신서란웅토，제비다극륭항체；용ELISA방법검측항체적효개，WesternBlotting검측항체적특이성。[결과]괄구건적표체재체pET-28a（＋）-NrfA재대장간균중유도후가이고효표체NrfA단백，면역획득적다극륭항체용ELISA검측，기효개위l：204900，경WesternBlotting분석，항체적특이성교호。【결론】성공극륭대장간균적NcfA기인，구건료표체재체，제비적NrfA다극륭항체구유교고적효개화량호적특이성。위연구세균유관NffA전정료기출。
[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.
