基础医学与临床
基礎醫學與臨床
기출의학여림상
BASIC MEDICAL SCIENCES AND CLINICS
2010年
2期
117-121
,共5页
邓毛子%石春薇%王芳%付瑞玲%王春%方正明%范雄林
鄧毛子%石春薇%王芳%付瑞玲%王春%方正明%範雄林
산모자%석춘미%왕방%부서령%왕춘%방정명%범웅림
结核分枝杆菌%Ag85A%原核表达%纯化%免疫反应性
結覈分枝桿菌%Ag85A%原覈錶達%純化%免疫反應性
결핵분지간균%Ag85A%원핵표체%순화%면역반응성
Mycobacterium tuberculosis%Ag85 A%prokaryotic expression%purification%immunoreactivity
目的 通过原核表达获得结核分枝杆菌Ag85A蛋白.方法 用PCR从结核分枝杆菌1137Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达.用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性.利用PCR技术鉴定fopA基因在不同分枝杆菌的分布.结果 32 ku的Ag85A蛋白获得高效表达和纯化.表达Ag85A蛋白的fopA基因在结核分枝杆菌1137Rv、1-137Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达.结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高.结论 重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性.
目的 通過原覈錶達穫得結覈分枝桿菌Ag85A蛋白.方法 用PCR從結覈分枝桿菌1137Rv菌株中擴增齣編碼Ag85A的fbpA基因,剋隆入原覈錶達載體pProEXHTb,產生重組質粒pPro85A後,轉化至大腸桿菌感受態細胞BL21併誘導大量錶達.用鎳純化繫統純化重組Ag85A蛋白,用不同分枝桿菌感染的小鼠血清通過ELISA確定其免疫反應性.利用PCR技術鑒定fopA基因在不同分枝桿菌的分佈.結果 32 ku的Ag85A蛋白穫得高效錶達和純化.錶達Ag85A蛋白的fopA基因在結覈分枝桿菌1137Rv、1-137Ra、BCG、草分枝桿菌、土地分枝桿菌、恥垢分枝桿菌和次要分枝桿菌中均有錶達,但在牝牛分枝桿菌中未錶達.結覈病患者和結覈分枝桿菌毒株H37Rv感染小鼠血清所產生的抗Ag85A抗體滴度最高.結論 重組Ag85A蛋白已成功錶達純化,併保留瞭免疫反應性.
목적 통과원핵표체획득결핵분지간균Ag85A단백.방법 용PCR종결핵분지간균1137Rv균주중확증출편마Ag85A적fbpA기인,극륭입원핵표체재체pProEXHTb,산생중조질립pPro85A후,전화지대장간균감수태세포BL21병유도대량표체.용얼순화계통순화중조Ag85A단백,용불동분지간균감염적소서혈청통과ELISA학정기면역반응성.이용PCR기술감정fopA기인재불동분지간균적분포.결과 32 ku적Ag85A단백획득고효표체화순화.표체Ag85A단백적fopA기인재결핵분지간균1137Rv、1-137Ra、BCG、초분지간균、토지분지간균、치구분지간균화차요분지간균중균유표체,단재빈우분지간균중미표체.결핵병환자화결핵분지간균독주H37Rv감염소서혈청소산생적항Ag85A항체적도최고.결론 중조Ag85A단백이성공표체순화,병보류료면역반응성.
Objective To obtain M. Tuberculosis Ag85A protein by prokaryotic expression. Methods The fbpA gene encoding M. Tuberculosis Ag85A protein was amplified by polymerase chain reaction ( PCR) from M. Tuberculosis H37R_V strain. The PCR product was cloned into prokaryotic expression vector pProEXHTb to generate the recombi-nant plasmid pProfbpA, which was then transformed into the competence Escherichia coli BL21 cells. The recombi-nant Ag85A protein was successfully expressed by isopropyl thio-β-D-galactoside(IPTG) induction and purified by the Ni-purification system. The distribution of fbpA gene in different nonpathogenic mycobacterial strains was screened by PCR and ELISA was performed to determine the immunoreactivity of the recombinant Ag85A protein with serum from mice with mycobacterial infections. Results 32 ku Ag85A protein was successfully expressed and purified. It was confirmed by PCR and ELISA that fbpA gene presented in the genomes of M. Tuberculosis H37Rv, H37Ra, BCG, M. Smegmatis, M. Terra, M. Trivial and M. Phlei, but being absent in the genomes of M. Vaccae. The highest Ag85 A antibody titer was found in serum of TB patients and mice infected by M . Tuberculosis H37Rv .Conclusion The recombinant Ag85A protein was successfully expressed and purified.
