动物医学进展
動物醫學進展
동물의학진전
Progress in Veterinary Medicine
2015年
10期
5-9
,共5页
赵子惠%温峰琴%项海涛%曾巧英
趙子惠%溫峰琴%項海濤%曾巧英
조자혜%온봉금%항해도%증교영
牛分支杆菌%肝素结合血凝素基因%生物信息学分析%原核表达
牛分支桿菌%肝素結閤血凝素基因%生物信息學分析%原覈錶達
우분지간균%간소결합혈응소기인%생물신식학분석%원핵표체
Mycobacterium bovis%HBHA gene%biological information analysis%prokaryotic expression
应用 PCR 技术扩增牛分支杆菌肝素结合血凝素(HBHA)基因并原核表达牛分支杆菌 HBHA基因,利用在线分析软件对该基因序列及编码蛋白序列进行生物信息学分析。构建原核表达载体 pET28a (+)-HBHA,并转化至大肠埃希菌 BL21(DE3),经0.6 mmol/L IPTG 诱导目的蛋白表达,采用镍离子亲和层析纯化目的蛋白,SDS-PAGE 及 Western blot 检测表达结果。结果表明,牛分支杆菌 HBHA 基因完整的ORF 全长600 bp,编码199个氨基酸。HBHA 蛋白为碱性、亲水性蛋白质,含有12个潜在的磷酸化位点,存在抗原表位,亚细胞定位主要存在于细胞核及线粒体中,蛋白空间结构以α-螺旋、无规则卷曲、β-折叠为主。成功构建了原核表达载体 pET28a(+)-HBHA 并在大肠埃希菌中得到了高效表达,分子质量大小为28 ku,主要以可溶性形式存在。说明牛分支杆菌 HBHA 蛋白是一种抗原指数较高的亲水性蛋白,在原核系统能高效的表达,为进一步研究提供了理论基础。
應用 PCR 技術擴增牛分支桿菌肝素結閤血凝素(HBHA)基因併原覈錶達牛分支桿菌 HBHA基因,利用在線分析軟件對該基因序列及編碼蛋白序列進行生物信息學分析。構建原覈錶達載體 pET28a (+)-HBHA,併轉化至大腸埃希菌 BL21(DE3),經0.6 mmol/L IPTG 誘導目的蛋白錶達,採用鎳離子親和層析純化目的蛋白,SDS-PAGE 及 Western blot 檢測錶達結果。結果錶明,牛分支桿菌 HBHA 基因完整的ORF 全長600 bp,編碼199箇氨基痠。HBHA 蛋白為堿性、親水性蛋白質,含有12箇潛在的燐痠化位點,存在抗原錶位,亞細胞定位主要存在于細胞覈及線粒體中,蛋白空間結構以α-螺鏇、無規則捲麯、β-摺疊為主。成功構建瞭原覈錶達載體 pET28a(+)-HBHA 併在大腸埃希菌中得到瞭高效錶達,分子質量大小為28 ku,主要以可溶性形式存在。說明牛分支桿菌 HBHA 蛋白是一種抗原指數較高的親水性蛋白,在原覈繫統能高效的錶達,為進一步研究提供瞭理論基礎。
응용 PCR 기술확증우분지간균간소결합혈응소(HBHA)기인병원핵표체우분지간균 HBHA기인,이용재선분석연건대해기인서렬급편마단백서렬진행생물신식학분석。구건원핵표체재체 pET28a (+)-HBHA,병전화지대장애희균 BL21(DE3),경0.6 mmol/L IPTG 유도목적단백표체,채용얼리자친화층석순화목적단백,SDS-PAGE 급 Western blot 검측표체결과。결과표명,우분지간균 HBHA 기인완정적ORF 전장600 bp,편마199개안기산。HBHA 단백위감성、친수성단백질,함유12개잠재적린산화위점,존재항원표위,아세포정위주요존재우세포핵급선립체중,단백공간결구이α-라선、무규칙권곡、β-절첩위주。성공구건료원핵표체재체 pET28a(+)-HBHA 병재대장애희균중득도료고효표체,분자질량대소위28 ku,주요이가용성형식존재。설명우분지간균 HBHA 단백시일충항원지수교고적친수성단백,재원핵계통능고효적표체,위진일보연구제공료이론기출。
The study cloned and prokaryotic expressed the Mycobacterium bovis HBHA gene and conducted biological analysis.The Mycobacterium bovis HBHA gene was amplified by PCR and the biological infor-mation analysis was performed with an online tool and cloned into the prokaryotic expression vector pET-28a(+).The constructed recombinant plasmid was transformed into competent Escherichia coli BL21 (DE3)and the target protein was expressed by 0.6 mmol/L IPTG induction.The expressed protein was purified by Ni-sepharose affinity chromatography and analyzed by SDS-PAGE and Western-blot.The cloned ORF of Mycobacterium bovis HBHA gene consisted of 600 nucleotides.Mycobacterium bovis HBHA was a basic hydrophilic protein,which comprised 12 potential phosphorylation sites.The antigenicity was observed.HBHA mainly distributed in the nucleus and the main structures included α-helix,random coil andβ-extension.Prokaryotic expression vector pET28a(+)-HBHA was successfully constructed,which could induced the 28 ku recombinant protein of HBHA in the prokaryotic expression system.The Myco-bacterium bovis HBHA was a basic and hydrophilic protein with antigenicity.It was well expressed in pro-karyotic expression system.