卫氏肺吸虫重组抗原的制备及应用研究
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。
Objective: To express the gene of Paragonimiasis westermani (Pw) pro- cathepsin L (Pwl) which have been converted into Escherichia
coli(BL21) and purify the expressed products . Using the purified
products to test the sera of paragonimiasis patients, patients who have
other Parasitosis and normal persons with Enzyme-linked immunosorbent
assays (ELI SAs) and evaluate its specificity, sensitivity and
applicationprospect. Methods: To induce the recombinant
Pw/pRESETB/E.coli BL21 expression by IPTG and analyse the
expressed recombinant protein with SDS-PAGE. To identify its
immunological characteristics. The sera from the Pw adult antigen
immuned rabbits,nomal rabbits, Pw infected dog and normal dog,
paragonimiasis patients and schistosomiasis patients were tested with
Western blot. Then,purified the expressed products in inclusion bodies
with step by step washing and dialysing. Renatured the purified products
by Oxidation / reduction GSH to abtain the recombinant protein antigen.
Finally, the sera from the patients with paragonimiasis , schistosomiasis,
clonorchiasis sinensis, cysticerciasis or echinococcosis respectively and
normal persons were detected for corresponding antibodies with the
recombinant protein antigen and the Pw adult crude antigen , making
comparison between these two antigens. Results: A specific protein
belt was shown at about 32ku .The 32ku protein belt can only be
recognised by the sera from the rabbits immunised with PwAg, the dogs
infected with Pw and the paragonimiasis patients. The ELISA results
showed that the recombinant protein antigen has no any cross reaction
with other parasitosis and normal persons. In contrast, the crudu antigen's
cross reactions are as follows: schistosomiasis 11.43%, clonorchiasis
sinensis 4.84%, cysticerciasis 3.22%, echinococcosis5.88%, normal
person 6.67%. Conclusion: that the the specificity of the recombinant protein is very high, and can be applied to the immunodiagnosis of
paragonimisasis.
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。
Objective: To express the gene of Paragonimiasis westermani (Pw) pro- cathepsin L (Pwl) which have been converted into Escherichia
coli(BL21) and purify the expressed products . Using the purified
products to test the sera of paragonimiasis patients, patients who have
other Parasitosis and normal persons with Enzyme-linked immunosorbent
assays (ELI SAs) and evaluate its specificity, sensitivity and
applicationprospect. Methods: To induce the recombinant
Pw/pRESETB/E.coli BL21 expression by IPTG and analyse the
expressed recombinant protein with SDS-PAGE. To identify its
immunological characteristics. The sera from the Pw adult antigen
immuned rabbits,nomal rabbits, Pw infected dog and normal dog,
paragonimiasis patients and schistosomiasis patients were tested with
Western blot. Then,purified the expressed products in inclusion bodies
with step by step washing and dialysing. Renatured the purified products
by Oxidation / reduction GSH to abtain the recombinant protein antigen.
Finally, the sera from the patients with paragonimiasis , schistosomiasis,
clonorchiasis sinensis, cysticerciasis or echinococcosis respectively and
normal persons were detected for corresponding antibodies with the
recombinant protein antigen and the Pw adult crude antigen , making
comparison between these two antigens. Results: A specific protein
belt was shown at about 32ku .The 32ku protein belt can only be
recognised by the sera from the rabbits immunised with PwAg, the dogs
infected with Pw and the paragonimiasis patients. The ELISA results
showed that the recombinant protein antigen has no any cross reaction
with other parasitosis and normal persons. In contrast, the crudu antigen's
cross reactions are as follows: schistosomiasis 11.43%, clonorchiasis
sinensis 4.84%, cysticerciasis 3.22%, echinococcosis5.88%, normal
person 6.67%. Conclusion: that the the specificity of the recombinant protein is very high, and can be applied to the immunodiagnosis of
paragonimisasis.
引文
[1] 许隆祺，余森海，徐淑惠．中国人体寄生虫分布与危害[M]．北京：人民卫生出版社，2000．164-165．
[2] Blair D, Xu ZB, Agatsuma T Paragonimiasis and the genus paragonimus. Adv Parasitol, 1999(42): 113.
[3] Kagawa FT. Pulmonary paragonimiasis. Semin-Respir-Infect,1997,12(2):149-158.
[4] 陈桂光．中国并殖吸虫病研究现状．实用寄生虫病杂志，1995，3(1)：29
[5] Kim SI. Protein composition and antigenicity of trgument from Paragonimus westermani. Korean J Parasitol. 1993 ;3:269 J-Parasitol. 2000 Apr; 86(2): 333-9
[6] 凌家俭，章子豪，张耀娟．卫氏并殖吸虫成虫cDNA文库的多抗筛选．南京医科大学学报．2001，11，5；21(6)：473-475
[7] 凌家俭，章子豪，张耀娟等．卫氏并殖吸虫基因片断的亚克隆及其表达的研究．南京医科大学学报，2002
[8] Dresden MH, Deelder AM. schistosamamattsoni: hiol-proteinase properties of adult worm henoglobinase. [J] Exp Parasitol, 1979;48:190
[9] Day SR , Dalton JP, Clough KA, et al. Characterization and cloning of the cathepsin L proteinases of Schistosoma japonicum. [J]Biochem Biophys Res Commun, 1995 ;217(1): 1-9
[10] 田明礼，易新元，曾宪芳等．日本血吸虫31ku组织蛋白酶B
DNA疫苗保护性免疫效果观察．中国血吸虫病防治杂志 2001，13(4)：209-212
[11] 曾宪忠，易新元，曾宪芳．日本血吸虫组织蛋白酶B核酸疫苗血清抗体与抗生殖免疫关系的初步研究．[J]中国人兽共患病杂志 2001，17(4)：40-42
[12] 易新元，曾宪忠，曾宪芳等．日本血吸虫组织蛋白酶B DNA疫苗抗生殖免疫的研究．中国人兽共患病杂志 2000，16(1)：45-47
[13] 李雍龙，冯友仁，Conor Caffrey等．日本血吸虫组织蛋白酶的研究．中国寄生虫学与寄生虫病杂志 1998，16(2)：101-104
[14] Chung YB, Yang HJ, Kang SY, et al. Activities of different cysteine proteases of Paragonimus westermani in cleaving human IgG.Korean J Parasitol. 1997,35(2): 139-142
[15] Shin MH, Kita H, Park HY, et al. Cysteine protease secreted by Paragonimus westermani attenuates effector functions of human eosinophils stimulated with immunoglobulin G. Infect-Immun.2001,69(3): 1599-604
[16] 王英．半胱氨酸蛋白酶在寄生虫的免疫诊断和治疗中的应用．中国人兽共患病杂志 2002．18(3)：83-85
[17] Park SY, Lee KH, Hwang YB, et al. Characterization and large-scale expression of the recombinant cysteine proteinase from adult Clonorchis sinensis. J Parasitol, 2001,87(6): 1454-1458
[18] J．萨姆布鲁克，E．F．弗里奇，丁曼尼阿蒂斯．分子克隆实验指南(第二版)，1998，845
[19] 纪剑飞，张成刚．包涵体重组蛋白的纯化及复性．沈阳药科大学学报，1998，15(4)：303-307
[20] Hlodan R,Craig S,Pain R H. Protein folding and its implications for the production of recombinant proteins. Biotech Genet Eng Rev, 1991,9:47-88
[21] Kane J F, Hartley D L. Purification and analysis of recombinant protein. New York:Marcel Dekker Inc, 1991.121-146.
[22] Kim TY, Joo IJ, Kang SY, et al. Recombinant Paragonimus westermani yolk ferritin is a useful serodiagnostic antigen. J-Infect-Dis.2002 May 1; 185(9): 1373-1375
[2] Blair D, Xu ZB, Agatsuma T Paragonimiasis and the genus paragonimus. Adv Parasitol, 1999(42): 113.
[3] Kagawa FT. Pulmonary paragonimiasis. Semin-Respir-Infect,1997,12(2):149-158.
[4] 陈桂光．中国并殖吸虫病研究现状．实用寄生虫病杂志，1995，3(1)：29
[5] Kim SI. Protein composition and antigenicity of trgument from Paragonimus westermani. Korean J Parasitol. 1993 ;3:269 J-Parasitol. 2000 Apr; 86(2): 333-9
[6] 凌家俭，章子豪，张耀娟．卫氏并殖吸虫成虫cDNA文库的多抗筛选．南京医科大学学报．2001，11，5；21(6)：473-475
[7] 凌家俭，章子豪，张耀娟等．卫氏并殖吸虫基因片断的亚克隆及其表达的研究．南京医科大学学报，2002
[8] Dresden MH, Deelder AM. schistosamamattsoni: hiol-proteinase properties of adult worm henoglobinase. [J] Exp Parasitol, 1979;48:190
[9] Day SR , Dalton JP, Clough KA, et al. Characterization and cloning of the cathepsin L proteinases of Schistosoma japonicum. [J]Biochem Biophys Res Commun, 1995 ;217(1): 1-9
[10] 田明礼，易新元，曾宪芳等．日本血吸虫31ku组织蛋白酶B
DNA疫苗保护性免疫效果观察．中国血吸虫病防治杂志 2001，13(4)：209-212
[11] 曾宪忠，易新元，曾宪芳．日本血吸虫组织蛋白酶B核酸疫苗血清抗体与抗生殖免疫关系的初步研究．[J]中国人兽共患病杂志 2001，17(4)：40-42
[12] 易新元，曾宪忠，曾宪芳等．日本血吸虫组织蛋白酶B DNA疫苗抗生殖免疫的研究．中国人兽共患病杂志 2000，16(1)：45-47
[13] 李雍龙，冯友仁，Conor Caffrey等．日本血吸虫组织蛋白酶的研究．中国寄生虫学与寄生虫病杂志 1998，16(2)：101-104
[14] Chung YB, Yang HJ, Kang SY, et al. Activities of different cysteine proteases of Paragonimus westermani in cleaving human IgG.Korean J Parasitol. 1997,35(2): 139-142
[15] Shin MH, Kita H, Park HY, et al. Cysteine protease secreted by Paragonimus westermani attenuates effector functions of human eosinophils stimulated with immunoglobulin G. Infect-Immun.2001,69(3): 1599-604
[16] 王英．半胱氨酸蛋白酶在寄生虫的免疫诊断和治疗中的应用．中国人兽共患病杂志 2002．18(3)：83-85
[17] Park SY, Lee KH, Hwang YB, et al. Characterization and large-scale expression of the recombinant cysteine proteinase from adult Clonorchis sinensis. J Parasitol, 2001,87(6): 1454-1458
[18] J．萨姆布鲁克，E．F．弗里奇，丁曼尼阿蒂斯．分子克隆实验指南(第二版)，1998，845
[19] 纪剑飞，张成刚．包涵体重组蛋白的纯化及复性．沈阳药科大学学报，1998，15(4)：303-307
[20] Hlodan R,Craig S,Pain R H. Protein folding and its implications for the production of recombinant proteins. Biotech Genet Eng Rev, 1991,9:47-88
[21] Kane J F, Hartley D L. Purification and analysis of recombinant protein. New York:Marcel Dekker Inc, 1991.121-146.
[22] Kim TY, Joo IJ, Kang SY, et al. Recombinant Paragonimus westermani yolk ferritin is a useful serodiagnostic antigen. J-Infect-Dis.2002 May 1; 185(9): 1373-1375