莱姆病螺旋体83kD原浆柱蛋白特异性区段的基因克隆与表达研究  

MOLECULAR CLONING AND EXPRESSION OF THE SPECIFIC REGION OF THE GENE ENCODING THE 83kD PROTOPLASMIC CYLINDER PROTEIN FROM BORRELIA BURGDORFERI B31 STRAIN

作　　者：谢勇恩[1] 鲍朗[1] 晏菊芳[1] 邱洪宇 方之茂[1] 张会东[1] 

机构地区：[1]华西医科大学基础医学院钩端螺旋体病研究室,成都610041

出　　处：《中国人兽共患病杂志》2000年第5期20-24,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助项目!(批准号:39770663)

摘　　要：目的　为莱姆病血清学诊断和基因工程亚单位疫苗研制提供靶抗原。方法　根据莱姆病螺旋体 83kD原浆柱蛋白 (p83)特异性区段的基因序列 ,设计一对引物 ,采用PCR技术从莱姆病螺旋体B31株基因组DNA中扩增p83特异性区段的基因片段 ,纯化扩增产物 ,用BamHI和EcoRI双酶切后定向克隆入质粒载体pBK -CMV ;转化大肠杆菌筛选重组克隆。用BamHI和EcoRI双酶切、PCR扩增和DNA序列测定对重组质粒进行鉴定 ,将重组质粒转化大肠杆菌 ,IPTG诱导表达后进行SPS -PAGE和Western -blotting分析。结果　 1)从莱姆病螺旋体B31株基因组DNA中扩增出p83特异性区段的基因片段 ;2 )将扩增所得的目的基因片段正向插入pBK -CMV的BamHI和EcoRI位点 ,获得重组质粒pBX1。 3)pBX1在大肠杆菌中诱导表达后获得 2 9kD含 β -半乳糖苷酶氨基末端片段的重组抗原。 结论　成功构建了莱姆病螺旋体 83kD原浆柱蛋白特异性区段的基因重组表达载体 ,并在大肠杆菌中获得了稳定的表达 。Aim\ To provide the target antigen for the development of a Lyme disease vaccine and serodiagnosis reagent.Methods\ According to the published gene seqence encoding the 83kd protoplasmic cylinder protein (p83) from Borrelia burgdorferi B31 strain, we selected the region from 1420bp to 1896 bp of the p83 gene as the target sequence to design a pair of oligonucleotide primers, obtained the gene fragment by using polymerase chain reaction, and constructed the recombinant plasmid by cloning the gene fragment into plasmid vector pBK-CMV. The recombinant plasmid was identified by restriction enzyme analysis and PCR amplification and DNA sequencing. Then, the recombinant plasmid was tranformed into E.coli XL-1 Blue MRF' and induced with IPTG. The expression product was analyzed by using SDS-PAGE and western-blotting. Results\ 1) The gene fragment encoding the specific region of the 83kD protoplasmic cylinder protein from Borrelia burgdorferi B31 strain was successfully amplified. 2) The purified PCR product was cloned into the BamHI and EcoRI site of the plasmid vector pBK-CMV, and a recombinant plasmid pBX1 was obtained. 3) Plasmid pBX1 can stably express a 29kD recombinant fusion protein in E.coli XL-1Bue MRF'. Conclusion\ A recombinant plasmid which contains the gene fragment encoding the specific region of the 83kD protoplasmic cylinder protein from Borrelia burgdorferi was successfully constructed. The recombinant plasmid can stably express a 29kD recombinant fusion protein in E.coli XL-1 Blue MRF'. These results provided the basis for further study the usefullness of the fusion protein in serodiagnosis and vaccine for Lyme disease.

关 键 词：莱姆病螺旋体 83kD原浆柱蛋白 基因克隆 

分 类 号：R514.02[医药卫生—内科学]