【作者】 刘剑南；

【导师】 张耀捐； 章子豪；

【作者基本信息】 南京医科大学 ， 病原生物学， 2003， 硕士

【摘要】 目的 表达并纯化含有卫氏并殖吸虫(肺吸虫)原组织蛋白酶L基因(Pw1)的重组细菌Pw-1／pRSETB／BL21，用纯化产物以ELISA检测肺吸虫病患者、其它寄生虫病患者和正常人血清，评价其敏感性，特异性和应用前景。方法 通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组菌表达，表达产物进行SDS-PAGE电泳，以卫氏肺吸虫成虫抗原免疫兔、正常兔、感染犬和正常犬、肺吸虫病患者和血吸虫病患者以及正常人血清做Western blot检测比较，以分步洗涤和透析等方法对表达产物包涵体进行纯化，纯化产物经氧化／还原型谷胱苷肽复性后即为重组蛋白抗原。用该重组蛋白抗原，以ELISA法检测肺吸虫、血吸虫、华支睾吸虫、囊虫和包虫病人以及正常人血清中相应抗体，并与卫氏肺吸虫粗抗原做比较。结果 重组菌的沉淀于约32ku处出现特异性蛋白条带，该条带只被卫氏肺吸虫成虫抗原免疫兔、感染犬和卫氏肺吸虫病患者血清所识别。经纯化复性后的重组蛋白32Ku包板做ELISA检测。结果显示，粗抗原特异性较差，其与其他寄生虫病患者和正常人血清交叉反应率分别为：血吸虫病11.43％，华支睾吸虫病4.84％，囊虫病3.22％，包虫病5.88％，正常人亦有6.67％的假阳性反应；而本研究所制备的重组蛋白抗原与上述患者及 南京医科大学颀士学位论文正常人血清均无交叉反应，表明该重组蛋白抗原应用于肺吸虫病的免疫诊断特异性较高。更多还原

【Abstract】 Objective: To express the gene of Paragonimiasis westermani (Pw) pro- cathepsin L (Pwl) which have been converted into Escherichiacoli(BL21) and purify the expressed products . Using the purifiedproducts to test the sera of paragonimiasis patients, patients who haveother Parasitosis and normal persons with Enzyme-linked immunosorbentassays (ELI SAs) and evaluate its specificity, sensitivity andapplicationprospect. Methods: To induce the recombinantPw/pRESETB/E.coli BL21 expression by IPTG and analyse theexpressed recombinant protein with SDS-PAGE. To identify itsimmunological characteristics. The sera from the Pw adult antigenimmuned rabbits,nomal rabbits, Pw infected dog and normal dog,paragonimiasis patients and schistosomiasis patients were tested withWestern blot. Then,purified the expressed products in inclusion bodieswith step by step washing and dialysing. Renatured the purified productsby Oxidation / reduction GSH to abtain the recombinant protein antigen.Finally, the sera from the patients with paragonimiasis , schistosomiasis,clonorchiasis sinensis, cysticerciasis or echinococcosis respectively andnormal persons were detected for corresponding antibodies with therecombinant protein antigen and the Pw adult crude antigen , makingcomparison between these two antigens. Results: A specific proteinbelt was shown at about 32ku .The 32ku protein belt can only berecognised by the sera from the rabbits immunised with PwAg, the dogsinfected with Pw and the paragonimiasis patients. The ELISA resultsshowed that the recombinant protein antigen has no any cross reactionwith other parasitosis and normal persons. In contrast, the crudu antigen’scross reactions are as follows: schistosomiasis 11.43%, clonorchiasissinensis 4.84%, cysticerciasis 3.22%, echinococcosis5.88%, normalperson 6.67%. Conclusion: that the the specificity of the recombinant protein is very high, and can be applied to the immunodiagnosis ofparagonimisasis.更多还原