钩端螺旋体外膜脂蛋白LipL32的克

发布时间：2018-01-05 20:11

本文关键词：钩端螺旋体外膜脂蛋白LipL32的克隆、表达及其在钩端螺旋体抗体检测中的应用　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 钩端螺旋体病(简称钩体病)是由致病性钩端螺旋体(简称钩体)引起的一种人兽共患的自然疫源性疾病。钩体血清型别众多,临床表现多种多样。目前钩体病血清学的主要检测方法是国家标准即显微镜凝集试验(MAT),需要培养各种血清型的钩体活菌,操作繁琐,安全性差且需要暗视野显微镜,无法在基层开展该项目的检测工作。钩体外膜脂蛋白LipL32是各种致病性钩体中高度保守的抗原成份,可刺激机体产生中和抗体。因此该抗原有可能应用于抗体检测。本课题用PCR法扩增致病性钩端螺旋体黄疸出血群(56601株)LipL32蛋白的全长基因片段,与表达载体pET-3Oa(+)连接并在大肠杆菌BL21(DE3)中表达重组蛋白,Western blot鉴定免疫活性后用电洗脱法进行纯化。纯化后的LipL32重组蛋白用间接ELISA和夹心ELISA法对不同的人和动物血清进行检测,以MAT为参照标准进行比较。主要结果如下: 1.纯化的重组蛋白用间接ELISA和夹心ELISA法对致病性钩体15株标准菌株的兔免疫血清全部检出,对7株腐生性钩体菌株的兔免疫血清全部未检出,与MAT检测结果相符。表明该重组蛋白能区别腐生性和致病性钩体感染。进一步确证黄疸出血群(56601株)外膜脂蛋白LipL32基因在致病性钩体中高度保守。 2.重组蛋白检测确诊钩体病人的9例急性期和恢复期双份血清标本,急性期9份血清,MAT有2份出现阳性,间接ELISA和夹心ELISA法分别可检出3份和2份阳性,进口ELISA检测试剂盒则是2份阳性,4份可疑。对于恢复期9份血清,MAT检出9份阳性,间接ELISA和夹心ELISA法均检出8份阳性,进口ELISA则是1份阳性,7份可疑。提示该重组蛋白包被的间接ELISA和夹心ELISA在钩体病检测方面与MAT相似,恢复期优于进口试剂盒。 3.重组蛋白检测不同血清标本,45份MAT阳性标本,间接ELISA法敏感性为71.11%(32/45),夹心ELISA敏感性为80.00%(36/45),而进口ELISA试剂盒检测则是阳性13份,占28.89%(13/45),25份可疑占55.56%(25/45)。在特异性检测方面,检测MAT阴性血清59份(其中健康体检血清43份,非钩体发热病人16份,包括恙虫病阳性和出血热阳性各8份)间接ELISA和夹心ELISA法特异度均为96.61%(57/59),进口ELISA检测健康体检血清14份,均为阴性,检测非钩体发热病人16份,则出现1份阳性,12份可疑。对梅毒螺旋体质控阳性血清10份,四种方法均为阴性。证明该重组抗原在敏感性方面略高于进口试剂盒,特异性与MAT相同,大大好于进口试剂盒,在钩体流行病学大样本人群调查中可用于初筛。 4.重组蛋白应用于夹心ELISA法检测鼠血清标本,以MAT为标准。检测274份标本,夹心ELISA的敏感性为86.75%(131/151),特异性为99.19%(122/123),符合率为92.34%(253/274)。结果表明夹心ELISA对鼠血清钩体抗体的检测显示较好的敏感性和特异性。夹心ELISA操作简单,特别适用于大样本钩体病现场血清流行病学宿主动物的调查。
[Abstract]:Leptospirosis (Leptospirosis) is a natural zoonotic disease caused by pathogenic leptospirosis (Leptospira). There are many serotypes of leptospirosis. There are many clinical manifestations. At present, the main detection method of leptospirosis serology is the national standard, I. E. microscope agglutination test, which requires the cultivation of various serotypes of live Leptospira bacteria, and the operation is cumbersome. The safety of Leptospira outer membrane lipoprotein LipL32 is a highly conserved antigen component of various pathogenic leptospirosis due to its poor security and the need for dark field microscope. The detection of Leptospira outer membrane lipoprotein (LipL32) can not be carried out at the grass-roots level. Can stimulate the body to produce neutralizing antibodies. Therefore, the antigen may be used in antibody detection. In this study, the full-length gene fragment of LipL32 protein of pathogenic leptospira jaundice haemorrhage strain 56601 was amplified by PCR. The recombinant protein was ligated with pET-3Oa() and expressed in E. coli BL21DE3. Western. The purified LipL32 recombinant protein was detected by indirect ELISA and sandwich ELISA methods. The main results are as follows: 1. The purified recombinant protein was detected by indirect ELISA and sandwich ELISA for rabbit immune serum of 15 standard strains of pathogenic leptospira. The rabbit immune serum of 7 strains of Leptospira rot was not detected. The results of MAT showed that the recombinant protein could distinguish between rancid and pathogenic leptospirosis, and further confirmed that 56601 strains of jaundice haemorrhage. The outer membrane lipoprotein (LipL32) gene is highly conserved in pathogenic leptospirosis. 2. Two serum samples of 9 patients with Leptospira confirmed by recombinant protein were detected in acute phase and convalescence stage, and 2 of 9 serum samples were positive in acute phase. Indirect ELISA and sandwich ELISA methods could detect 3 and 2 positive samples respectively, while imported ELISA kits were 2 positive and 4 suspicious. 9 sera were found in convalescent stage. 9 cases were positive in MAT, 8 cases were positive in indirect ELISA and sandwich ELISA method, and 1 case was positive in imported ELISA. It was suggested that the indirect ELISA and sandwich ELISA coated with the recombinant protein were similar to those of MAT in the detection of leptospirosis, and the recovery period was better than that of the imported kit. 3.The sensitivity of indirect ELISA assay for detecting 45 MAT positive samples from different serum samples was 71.11 and 32 / 45). The sensitivity of sandwich ELISA was 80.000.36 / 45, while the imported ELISA kit was positive in 13 samples, accounting for 28.8913 / 45). In terms of specificity, 59 MAT negative sera were detected (43 in health examination and 16 in patients with non-leptospirosis). The heterogeneity of indirect ELISA and sandwich ELISA were 96.61% and 57 / 59, including 8 cases of tsutsugamushi disease positive and 8 cases of haemorrhagic fever respectively. 14 healthy serum samples were detected by imported ELISA, all of them were negative, 16 cases of non-leptospirosis fever patients were detected, 12 cases were suspicious and 10 cases were positive for Treponema pallidum (Treponema pallidum) quality control. All the four methods were negative. It was proved that the recombinant antigen was slightly more sensitive than the imported kit, and the specificity was the same as that of MAT, which was much better than that of the imported kit. It can be used for primary screening in large sample population survey of leptospirosis. 4. The recombinant protein was used to detect mouse serum samples by sandwich ELISA, and 274 samples were detected by MAT. The sensitivity of sandwich ELISA was 86.75 / 151, and the specificity was 99.19 / 122 / 123). The coincidence rate was 92.34 / 253 / 2740.The results showed that sandwich ELISA showed better sensitivity and specificity for detection of rat serum leptospira antibody. Sandwich ELISA was simple to operate. It is especially suitable for seroepidemiological host animals in large sample of leptospirosis.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R446.6

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