摘要
目的:分析本地区淋病奈瑟菌临床菌株外膜蛋白pIB基因核苷酸及氨基酸序列,构建pIB基因的原核表达系统并鉴定表达产物的免疫反应性,检测标本中pIB基因的表达情况。方法:采用PCR扩增淋病奈瑟菌ATCC49226株的全长pIB基因,T-A克隆测序后与GenBank中相应序列进行相似性比较并构建pIB基因的原核表达系统。采用SDS-PAGE和BioRad凝胶图象分析系统检查目的重组蛋白rPIB表达情况,Ni-NTA亲和层析法提纯rPIB。rPIB免疫家兔以了解其抗原性并采用免疫双扩散试验检测抗血清的效价。采用Western Blot检测rPIB的免疫反应性。建立PIB-ELISA检测标本中pIB基因的表达情况。结果:与GenBank中相应序列比较,克隆的pIB基因核苷酸和氨基酸序列完全相同。rPIB表达量为细菌总蛋白的21.5%,提纯的rPIB经SDS-PAGE后显示为单一的蛋白条带。rPIB能诱导家兔产生特异性抗体,其免疫双扩散效价为1:4。pIB+淋病奈瑟菌感染的病人血清能与rPIB产生阳性Western杂交条带。PIB-ELISA检测淋病病人的脓性分泌物标本的阳性率为82.3%。结论:结果表明本研究成功地克隆并构建淋病奈瑟菌pIB基因及其原核表达系统,该表达系统能有效地表达rPIB。rPIB有良好的抗原性和免疫反应性,rPIB免疫家兔制备的抗血清特异性高。可作为研制淋病基因工程疫苗及实验室诊断方法的侯选抗原。
Objective：To analyze the nucleotide and putative amino acid sequences of pIB genes from N. gonorrhoeae isolates and to construction of the prokaryotic expression system of pIB gene To identify immunoreactivity of rPIB and detect the expression of pIB gene in specimens. Methods： Entire pIB gene of N. gonorrhoeae stain ATCC49226 was amplified using PCR, and the target amplified fragment was sequenced after T - A cloning. Sequence similarity of the cloned pIB gene was compared with the corresponding sequences in GenBank. A prokaryotic expression system of pIB gene was constructed by routine genetic engineering technique. SDS - PAGE plus BioRad Agarose Image Analysor was used to measure the expression level of target recombinant protein （rPIB）. rPIB was extracted using Ni - NTA affinity chromatography. Rabbit was immunized with rPIB to determine antigenicity of the protein, and Western Blot was applied to determine immunoreactivity of rPIB. An ELISA was established to detect the expression of pIB gene in the gonorrhea patients＇ purulent secretion specimens. Results： An entire plB gene fragment amplified from DNA of N. gonorrhoeae stain ATCC49226 was obtained. 100% similarities of nucleotide and putative amino acid sequences of the plB gene was confirmed after comparison with the corresponding sequences in GenBank. Output of rPIB was as high as 21.5% of the total bacterial proteins, and the purified rPIB showed a single protein band in gel. rPIB could induce rabbit to produce specific antibody and the immunodiffusion titer of rabbit anti - rPIB serum was 1：4. There were positive Western hybridization straps between pIB ＋ N. gonorrhoeae infected patients＇ sera and rPIB. The positive rate of the gonorrhea patients＇ purulent secretion specimens was 82. 3% by PIB- ELISA. Conelusion：plB gene of N. gonorrhoeae and its prokaryotic expression system had been successfully cloned and constructed in this study, and the expression system can efficiently express rPIB. rPIB possesses well - qualified immunoreactivity, and can be used as a candidate antigen for developing genetic engineering vaccine and laboratory diagnostic methods of gonorrhea.
出处
《中国卫生检验杂志》
CAS
2009年第5期1068-1070,共3页
Chinese Journal of Health Laboratory Technology
关键词
淋病奈瑟菌
克隆
免疫学鉴定
Neisseria gonorrhoeae
Immunological identification
