CAJ | 학술논문

目的构建结核分枝杆菌rPstS1-hspX (rph)融合基因及其原核表达载体pET-23b(+)-rPstS1-hspX[pET-23b(+)-rph],表达、纯化rPstS1-HspX (rPH)融合蛋白,并分析其免疫反应性.方法采用基因拼接技术将PstS1和HspX编码基因通过多肽接头(GSGSG)的DNA序列进行连接,构建融合基因rph.将融合基因定向克隆入原核表达载体pET-23b(+),构建重组原核表达质粒pET-23b(+)-rph.将重组质粒转化大肠杆菌E.coli BL21 (DE3) pLysE感受态细胞,IPTG诱导融合蛋白表达.SDS-PAGE和Western印迹法鉴定其表达情况.用镍离子鳌合亲和层析柱纯化融合蛋白,Western印迹法初步评价融合蛋白的免疫反应性.结果融合基因rph及其原核表达载体pET-23b (+)-rph构建成功.融合蛋白rPH主要以可溶性非包涵体形式表达,相对分子质量为51 000,表达量约占菌体总蛋白的23％.经亲和层析后得到了纯度达92％的融合蛋白.Western印迹证实融合蛋白能与结核病阳性血清发生特异性免疫反应.结论成功构建了原核表达载体pET-23b(+)-rph,获得了rPH融合蛋白,为rPH融合蛋白在结核病诊断中的应用提供了依据.
목적 구건결핵분지간균rPstS1-hspX (rph)융합기인급기원핵표체재체pET-23b(+)-rPstS1-hspX[pET-23b(+)-rph],표체、순화rPstS1-HspX (rPH)융합단백,병분석기면역반응성.방법 채용기인병접기술장PstS1화HspX편마기인통과다태접두(GSGSG)적DNA서렬진행련접,구건융합기인rph.장융합기인정향극륭입원핵표체재체pET-23b(+),구건중조원핵표체질립pET-23b(+)-rph.장중조질립전화대장간균E.coli BL21 (DE3) pLysE감수태세포,IPTG유도융합단백표체.SDS-PAGE화Western인적법감정기표체정황.용얼리자오합친화층석주순화융합단백,Western인적법초보평개융합단백적면역반응성.결과 융합기인rph급기원핵표체재체pET-23b (+)-rph구건성공.융합단백rPH주요이가용성비포함체형식표체,상대분자질량위51 000,표체량약점균체총단백적23％.경친화층석후득도료순도체92％적융합단백.Western인적증실융합단백능여결핵병양성혈청발생특이성면역반응.결론 성공구건료원핵표체재체pET-23b(+)-rph,획득료rPH융합단백,위rPH융합단백재결핵병진단중적응용제공료의거.
Objective To construct the Mycobacterium tuberculosis rPstS1-hspX (rph) fusion gene and its prokaryotic expression vector pET-23b (+)-rpstS1-hspX [pET-23b (+)-rph],and to evaluate the immunoreactivity of the fusion protein PstS1-HspX [rPH ] after expression and purification.Methods The fusion gene rph was constructed using gene splicing of DNA fragment that encodes a short polypeptide liner (GSGSG),then cloned into the prokaryotic vector pET-23b(+) to generate a recombinant plasmid,pET-23b(+)-rph.The recombinant plasmid was transformed into E.coli BL21 (DE3) pLysE cells.The expression of the histidine-tagged (His-Tag) fusion protein was induced with isopropyl-β-thiogalactopyranoside (IPTG),and verified with SDS- PAGE and Western blot.The fusion protein was purified by nickel affinity chromatography and evaluated for immunoreactivity with Western blot.Results The fusion gene rph and its prokaryotic expression vector pET-23b (+)-rph were successfully constructed.The expressed 51 000 fusion protein mainly existed in soluble form,accounting for about 23％ of total proteins in E.coli.After nickel affinity chromatography,the purity of rPH reached 92％.The immunoreactivity of rph to positive serum from tuberculosis patients was confirmed by Western blot.Conclusion The successful construction of prokaryotic expression vector pET-23b (+)-rph and expression of fusion protein rPH provides experimental evidence for use of rPH fusion protein in diagnosis of tuberculosis.