摘要
用PCR技术从BacillusCalmette gu啨ｒｉｎ(BCG)基因组中扩增出抗原 85B(Ag85B)的信号肽 (SP)DNA序列 ,从pCMVMTHSP65质粒中扩增出人结核杆菌HSP65全长基因。利用DNA重组技术将以上两个片段插入质粒pBCG 2 10 0的人结核杆菌HSP70启动子下游 ,构建成分泌型原核穿梭表达质粒 (pBCG SP HSP65)。酶切鉴定、PCR和测序分析结果均表明分泌型原核穿梭表达质粒pBCG SP HSP65构建成功。利用电穿孔将该质粒转入耻垢分枝杆菌(Mycobacterialsmegmatis,MS)中 ,用卡那霉素筛选出阳性重组子。经热诱导后用SDS PAGE观察到在耻垢分枝杆菌中 65kD蛋白占总蛋白的 2 0 % ,而在重组耻垢分枝杆菌表达的 65kD蛋白占菌体总蛋白的 3 4 46% ,占裂解物上清总蛋白的 68 56% ,表明重组的HSP65基因能在耻垢分枝杆菌中高效表达 ,表达的蛋白大部分以可溶状态存在。通过Western blot证实分泌的该蛋白能与结核杆菌HSP65的抗体特异性结合 ,说明该重组蛋白具有HSP65的生物学活性。
To construct the secretive prokaryotic shuttle expression plasmid pBCG-SP-HSP6 5, the signal peptide sequence of antigen 85B amplified from Bacillus Calmette- guérin (BCG) genome by PCR and the whole HSP65 DNA sequence of human M.tuberc ulosis obtained from the plasmid pCMV-MTHSP65 by PCR were cloned into the pl asmid pBCG-2100 under the control of the promoter of Heat Shock Protein 70 (HSP 70) from human M.tuberculosis. Recombinants were electroporated into Mycob acterial smegmatis and induced by heating. Results of the induced expression w ere detected by SDS-PAGE and the biological activity of the expressed protein w as tested by Western-blot analysis. Results showed pBCG-SP-MTHSP65 was constr ucted successfully and confirmed by restriction endonuclease analysis, PCR detec tion and DNA sequencing analysis. After it was electroporated into Mycobacteri al smegmatis and induced by heating, the percentage of expressed 65kD protein in Mycobacterial smegmatis detected by SDS-PAGE was 20% in total bacterial protein. But the percentage of expressed 65kD protein in recombibinant Mycobac terial smegmatis was up to 34 46% in total bacterial protein and 68 56%in the total protein of cell lysate supernants, Which demonstrated the recombinant HSP65 gene could express in recombinant with high efficiency and the expressed p roteins were mainly soluble. Western-blot showed that the secretive proteins co uld specially combine with antibody against human M.tuberculosis HSP65 Ora lly, pBCG-SP-HSP65 was successfully constructed; HSP65 gene could express in Mycobacterial smegmatis with high efficiency via it. And the expressed prote ins possess the biological activity. So it provids experimental evidence for the application of the recombinant Mycobacterial smegmatis and the development of the vaccine against tuberculosis.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第2期170-174,共5页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目资助(No .30 370 0 75)
湖北省自然科学基金项目资助 (No .2 0 0 3ABA1 36 )~~
关键词
大肠杆菌
分枝杆菌
分泌型穿梭表达质粒
信号肽
结核轩菌HSP65
人
signal peptide, HSP65 of human Mycobacterium tuberc ulosis, a secreting prokaryotic shuttle expressing plasmid, recombinant Myco bacterial smegmatis
