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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 ,为进一步研究该重组抗原在莱姆病血清学诊断和基因工程亚单位疫苗研制中的应用奠定了基础
Objective To provide target antigen for the diagnosis of Lyme disease and the development of genetic engineering subunit vaccine. Methods A pair of primers was designed according to the sequence of the specific region of the 83 kD protosperm protein (p83) of Borrelia burgdorferi. The gene fragment of p83 specific fragment was amplified from the genomic DNA of Borrelia burgdorferi B31 by PCR , And the amplified product was purified and digested with BamHI and EcoRI to be cloned into the plasmid vector pBK-CMV. The recombinant clone was transformed into Escherichia coli. The recombinant plasmids were identified by double digestion with BamHI and EcoRI, PCR amplification and DNA sequencing. The recombinant plasmids were transformed into E.coli and induced by IPTG. SPS-PAGE and Western-blotting analysis were carried out. Results 1) The gene fragment of p83 specific region was amplified from the genomic DNA of Borrelia burgdorferi strain B31; 2) The amplified target gene fragment was inserted into the BamHI and EcoRI sites of pBK-CMV forward to obtain the recombinant Plasmid pBX1. 3) After induced by pBX1 in E. coli, 29kD recombinant antigen containing amino terminal fragment of β - galactosidase was obtained. Conclusion The recombinant plasmid was successfully constructed and expressed in Escherichia coli. In order to further study the diagnostic value of the recombinant antigen in serodiagnosis and genetic engineering of Lyme disease Subunit vaccine development laid the foundation for the application