斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因表达、鉴定、组织学定位及在临床实验诊断中应用的研究
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摘要
背景：斯氏狸殖吸虫[Pagumogonimus skrjabini (Chen,1959) Chen,1963]散在流行于我国四川、重庆、甘肃、福建等15个省、市、自治区，国外尚没有该虫种流行的报道。由于人为该寄生虫的非正常宿主，幼虫侵入人体后，不能到达肺部寄生、因而不能发育至成虫，虫体滞留于童虫阶段。由于童虫在体内组织、器官间的游走、移行，常常引起游走性皮下包块或结节、脑占位性病变和内脏损伤等肺外型寄生虫表现，造成肺外多器官、组织的损害。根据其不同寄生部位临床表现多种多样，临床症状也不典型，因而误诊率非常高, 给人们的健康，尤其儿童和流行区经济建设造成极大的危害。由于该虫体在人体内寄生的特点决定了病原学诊断是比较困难的，因而免疫学检查在该型肺吸虫病的实验室诊断和鉴别诊断中尤其重要。
近年来由于多数斯氏狸殖吸虫病流行区的生态环境己经或正在发生变化，溪蟹的感染率尚较高，但体内囊蚴的感染密度较以往大大降低，这就给本病的实验研究，尤其是免疫诊断抗原的制备带来一定困难。目前，国内实验室免疫诊断用抗原仍然是使用天然抗原，主要为成虫的粗提抗原，其次为排泄分泌抗原。随着天然抗原的来源逐渐减少，直接影响免疫诊断抗原的质量以及抗原的标准化。近年来也有一些增加虫体抗原提取量的研究，如易德友报告了可以高效利用虫体成分的尿素溶解性抗原。然而这并不能从根本上解决日益突出的免疫诊断抗原的缺乏的问题。
半胱氨酸蛋白酶存在于人类、寄生生物等多种动物体内，多种寄生虫都能合成或分泌半胱氨酸蛋白酶。半胱氨酸蛋白酶作为寄生虫的主要消化酶之一，在寄生虫与宿主的相互关系上具有重要的作用。同时该酶还是许多寄生虫的免疫优势抗原，具有明显的种、属特异性。因而斯氏狸殖吸虫半胱氨酸蛋白酶的表达，对于斯氏狸殖吸虫所致疾病的致病性、免疫性、与宿主相互关系的研究，以及该病的诊断、预防，均具有重要意义。
目的：1、构建斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段－PinPointTM Xa-1 T载体重组分子。2、将重组分子转化到大肠杆菌JM109菌株中去。3、进行原核表达，获得融合蛋白，并检测其免疫反应性。4、用表达的融合蛋白的粗提物作为诊断抗原，
采用cystatin 捕获ELISA方法，对15例已经明确诊断的临床标本(5例斯氏狸殖吸虫感染的病人，5例华支睾吸虫感染的病人，5例日本裂体吸虫感染的病人)进行检测，对其在临床实验诊断的应用价值进行评估。5、进行半胱氨酸蛋白酶在成虫虫体内的组织学定位。
方法：1、将含有斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段的DH5α菌株接种于5 ml含氨苄青霉素100μg/ml的LB中37℃，250 rpm振荡过夜培养，碱裂解法提取质粒，经PCR扩增目的基因片断，胶纯化、回收后与PinPointTM Xa-1T载体相连，并导入到大肠杆菌JM109菌株中去。用特异引物扩增所有获得的氨苄抗性菌株的质粒。能够扩增出目的基因片断的阳性菌株进行测序鉴定。经测序鉴定证实为正确连接的菌株，取其过夜培养物按1:100加入到含生物素2μM的氨苄抗性的LB中37℃，振荡培养1h后，加入终浓度为100μM IPTG诱导，37℃，振荡培养4～5h使其表达编码的多肽片段。用裂解液制备抗原标本，经SDS-PAGE电泳后，考马斯亮兰染色检测其表达情况，同时将其转移到NC膜上，用生物素亲合素系统染色，观察融合蛋白表达情况。用免疫印迹初步鉴定其免疫反应性。将超声破碎法制备的重组抗原，采用重组抗原Cystatin 捕获-ELISA方法，首先以已知效价的抗血清测定抗原的效价。然后用确定效价的重组抗原分别检测斯氏狸殖吸虫、华支睾吸虫和日本裂体吸虫病人血清并对其临床应用价值作出初步评估。通过免疫组化的方法对斯氏狸殖成虫进行半胱氨酸蛋白酶体内表达的虫体组织学定位。
结果：转化实验共获得21株氨苄抗性的菌株。全部增菌后提取质粒，经过PCR扩增目的基因片段，证实有8株含有目的基因片段，分别命名为A1～A8。测序后经过与标准序列比对鉴定证实，A7正向连接，A1、A3、A4、A5、A6反向连接（A2、A8未能测出）。成功构建了PinPointTM Xa-1T-半胱氨酸蛋白酶基因片段的重组分子。分别取空菌株、正向、反向连接菌株，经过诱导表达，制备重组抗原标本，SDS-PAGE后，经考马斯亮兰染色，空菌株、正向、反向连接菌株之间无明显差异。转膜后生物素－链亲合素－碱性磷酸酶系统染色显示空菌株有一22.5kDa大小的弱带，该蛋白应为大肠杆菌本身表达的一种内源性生物素化蛋白，量较少。正向连接菌株除22.5kDa大小的弱带外，在32 kDa处有一融合蛋白条带，反向连接菌株与之相似，也出现一条表达带，但分子量远小于预期值，仅仅14kDa左右。Western-blotting显示仅正向连接菌株在32 kDa的位置有一阳性染色条带。用超声破碎法制备的重组抗原的Cystatin 捕获-ELISA检测15份已知病人血清学标本（其中斯氏狸殖吸虫感染的病人血清学标本5
份，日本裂体吸虫感染患者血清标本5份，华支睾吸虫感染病人血清标本5份，3份健康正常人血清作阴性对照），结果显示仅斯氏狸殖吸虫的病人血清出现强阳性反应，其余皆为阴性反应(p﹤0.01)。免疫组织化学染色显示在斯氏狸殖吸虫成虫虫体横切面上，肠管上皮及表皮出现阳?
Background: P.skrjabini is epidemic in 15 provinces of China including Sichuan,Chongqing,etc. As an unsuitable host, people infected with P.skrjabini often exhibit extrapulmonary type of paragonimiasis with multiple organs and tissue damage that do great harm to the people's health and economy. Cysteine protease exists in many animals including human and parasite. As a main kind of digestive enzyme of parasite, cysteine protease plays an important role in the relationship between parasite and host. Recently, more and more studies were focused on cysteine protease because the protease has extensive prospect of use, such as research of immunodiagnosis, vaccine and chemotherapy. However there was little study on cysteine protease of P.skrjabini, which is only prevalent in China.
Object: 1. To construct PinPointTM Xa-1 T- adult cysteine proteinase gene segment recombinant molecule. 2. To transform the recombinant molecule into JM109 strain. 3. To express the recombinant molecule in E.coli JM109 and primarily investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. 4. Using the crude extraction of the fusion protein, we have detected 15 serums from patients (5 infected with C.sinensis ,5 infected with P.skrjabini,5 infected with S.japonicum) by cystatin capture ELISA to evaluate the effect on immunodiagnosis. 5. Also we have located the tissue of the worm where cysteine protease is expressed in P.skrjabini.
Methods: The E coli DH5α containing target gene was inoculated in 5 ml LB medium that contained 100μg/ml ampicillin, incubated overnight at 37°C with shaking. The plasmid is extracted by Alkaline Lysis and the gene fragment is amplified with PCR. After purification with Gel-Purification Recovery, the target gene segment ligate with PinPointTM Xa-1 T vector, and is transformed into JM109 strain. All the clones, which have ampicilline resistance, are screened by amplifying specified segment with PCR. The positive clones, which can produce specified 496bp gene fragment in PCR, are sequenced to confirm having the proper orientation . Then the clone, in which target gene fragment is in the proper orientation, is inoculated with the overnight cultures in 1:100 with LB containing 2μM
biotin and 100μg/ml ampicillin. Incubate for 1 hour at 37°C with shaking and then induced by adding IPTG in final concentration of 100μM in LB medium which contains 100μg/ml ampicillin.After incubated for 4~5 hours at 37°C with shaking, the expressed fusion protein sample is prepared with Alkaline Lysis Solution ,electrophoresised by 12% SDS-PAGE , stained with Coomb's blue stain .The same sample is also transfer the proteins to nitrocellulose membrane following SDS-PAGE electrophoresis, to identify the biotinylated fusion protein by the Streptavidin Alkaline Phosphatase stain, and the fusion protein's immunoreactive property was examined with Western blotting. Using the crude extraction of the fusion protein, we have detected 15 serum from patients (5 infected with C．sinensis ;5 infected with P.skrjabini ;5 infected with S. japonicum) by cystatin capture ELISA. Also, we have located the distributing of cysteine protease expression by immunohistochemistry.
Results: Twenty one ampicillin resistance strain were harvested in the transformation test . PinPointTM Xa-1 T- cysteine protease gene fragment recombinant molecule were successfully constructed and also verified by sequencing results .A positive band was clearly fond in 32 kDa by Streptavidin-Alkaline Phosphatase stain .In the same position, the fusion protein was also detected in western blotting. Using the crude extraction of the fusion protein,we have detected 15 serum from patients (5 infected with C.sinensis ;5 infected with P.skrjabini ;5 infected with S.japonicum) by cystatin capture ELISA the results show clearly that only the samples from the patients infected with P.skrjabini have the strong positive reaction(P﹤0.01),and the others show negative reaction. Immunohistochemical study show that the po
近年来由于多数斯氏狸殖吸虫病流行区的生态环境己经或正在发生变化，溪蟹的感染率尚较高，但体内囊蚴的感染密度较以往大大降低，这就给本病的实验研究，尤其是免疫诊断抗原的制备带来一定困难。目前，国内实验室免疫诊断用抗原仍然是使用天然抗原，主要为成虫的粗提抗原，其次为排泄分泌抗原。随着天然抗原的来源逐渐减少，直接影响免疫诊断抗原的质量以及抗原的标准化。近年来也有一些增加虫体抗原提取量的研究，如易德友报告了可以高效利用虫体成分的尿素溶解性抗原。然而这并不能从根本上解决日益突出的免疫诊断抗原的缺乏的问题。
半胱氨酸蛋白酶存在于人类、寄生生物等多种动物体内，多种寄生虫都能合成或分泌半胱氨酸蛋白酶。半胱氨酸蛋白酶作为寄生虫的主要消化酶之一，在寄生虫与宿主的相互关系上具有重要的作用。同时该酶还是许多寄生虫的免疫优势抗原，具有明显的种、属特异性。因而斯氏狸殖吸虫半胱氨酸蛋白酶的表达，对于斯氏狸殖吸虫所致疾病的致病性、免疫性、与宿主相互关系的研究，以及该病的诊断、预防，均具有重要意义。
目的：1、构建斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段－PinPointTM Xa-1 T载体重组分子。2、将重组分子转化到大肠杆菌JM109菌株中去。3、进行原核表达，获得融合蛋白，并检测其免疫反应性。4、用表达的融合蛋白的粗提物作为诊断抗原，
采用cystatin 捕获ELISA方法，对15例已经明确诊断的临床标本(5例斯氏狸殖吸虫感染的病人，5例华支睾吸虫感染的病人，5例日本裂体吸虫感染的病人)进行检测，对其在临床实验诊断的应用价值进行评估。5、进行半胱氨酸蛋白酶在成虫虫体内的组织学定位。
方法：1、将含有斯氏狸殖吸虫成虫半胱氨酸蛋白酶基因片段的DH5α菌株接种于5 ml含氨苄青霉素100μg/ml的LB中37℃，250 rpm振荡过夜培养，碱裂解法提取质粒，经PCR扩增目的基因片断，胶纯化、回收后与PinPointTM Xa-1T载体相连，并导入到大肠杆菌JM109菌株中去。用特异引物扩增所有获得的氨苄抗性菌株的质粒。能够扩增出目的基因片断的阳性菌株进行测序鉴定。经测序鉴定证实为正确连接的菌株，取其过夜培养物按1:100加入到含生物素2μM的氨苄抗性的LB中37℃，振荡培养1h后，加入终浓度为100μM IPTG诱导，37℃，振荡培养4～5h使其表达编码的多肽片段。用裂解液制备抗原标本，经SDS-PAGE电泳后，考马斯亮兰染色检测其表达情况，同时将其转移到NC膜上，用生物素亲合素系统染色，观察融合蛋白表达情况。用免疫印迹初步鉴定其免疫反应性。将超声破碎法制备的重组抗原，采用重组抗原Cystatin 捕获-ELISA方法，首先以已知效价的抗血清测定抗原的效价。然后用确定效价的重组抗原分别检测斯氏狸殖吸虫、华支睾吸虫和日本裂体吸虫病人血清并对其临床应用价值作出初步评估。通过免疫组化的方法对斯氏狸殖成虫进行半胱氨酸蛋白酶体内表达的虫体组织学定位。
结果：转化实验共获得21株氨苄抗性的菌株。全部增菌后提取质粒，经过PCR扩增目的基因片段，证实有8株含有目的基因片段，分别命名为A1～A8。测序后经过与标准序列比对鉴定证实，A7正向连接，A1、A3、A4、A5、A6反向连接（A2、A8未能测出）。成功构建了PinPointTM Xa-1T-半胱氨酸蛋白酶基因片段的重组分子。分别取空菌株、正向、反向连接菌株，经过诱导表达，制备重组抗原标本，SDS-PAGE后，经考马斯亮兰染色，空菌株、正向、反向连接菌株之间无明显差异。转膜后生物素－链亲合素－碱性磷酸酶系统染色显示空菌株有一22.5kDa大小的弱带，该蛋白应为大肠杆菌本身表达的一种内源性生物素化蛋白，量较少。正向连接菌株除22.5kDa大小的弱带外，在32 kDa处有一融合蛋白条带，反向连接菌株与之相似，也出现一条表达带，但分子量远小于预期值，仅仅14kDa左右。Western-blotting显示仅正向连接菌株在32 kDa的位置有一阳性染色条带。用超声破碎法制备的重组抗原的Cystatin 捕获-ELISA检测15份已知病人血清学标本（其中斯氏狸殖吸虫感染的病人血清学标本5
份，日本裂体吸虫感染患者血清标本5份，华支睾吸虫感染病人血清标本5份，3份健康正常人血清作阴性对照），结果显示仅斯氏狸殖吸虫的病人血清出现强阳性反应，其余皆为阴性反应(p﹤0.01)。免疫组织化学染色显示在斯氏狸殖吸虫成虫虫体横切面上，肠管上皮及表皮出现阳?
Background: P.skrjabini is epidemic in 15 provinces of China including Sichuan,Chongqing,etc. As an unsuitable host, people infected with P.skrjabini often exhibit extrapulmonary type of paragonimiasis with multiple organs and tissue damage that do great harm to the people's health and economy. Cysteine protease exists in many animals including human and parasite. As a main kind of digestive enzyme of parasite, cysteine protease plays an important role in the relationship between parasite and host. Recently, more and more studies were focused on cysteine protease because the protease has extensive prospect of use, such as research of immunodiagnosis, vaccine and chemotherapy. However there was little study on cysteine protease of P.skrjabini, which is only prevalent in China.
Object: 1. To construct PinPointTM Xa-1 T- adult cysteine proteinase gene segment recombinant molecule. 2. To transform the recombinant molecule into JM109 strain. 3. To express the recombinant molecule in E.coli JM109 and primarily investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. 4. Using the crude extraction of the fusion protein, we have detected 15 serums from patients (5 infected with C.sinensis ,5 infected with P.skrjabini,5 infected with S.japonicum) by cystatin capture ELISA to evaluate the effect on immunodiagnosis. 5. Also we have located the tissue of the worm where cysteine protease is expressed in P.skrjabini.
Methods: The E coli DH5α containing target gene was inoculated in 5 ml LB medium that contained 100μg/ml ampicillin, incubated overnight at 37°C with shaking. The plasmid is extracted by Alkaline Lysis and the gene fragment is amplified with PCR. After purification with Gel-Purification Recovery, the target gene segment ligate with PinPointTM Xa-1 T vector, and is transformed into JM109 strain. All the clones, which have ampicilline resistance, are screened by amplifying specified segment with PCR. The positive clones, which can produce specified 496bp gene fragment in PCR, are sequenced to confirm having the proper orientation . Then the clone, in which target gene fragment is in the proper orientation, is inoculated with the overnight cultures in 1:100 with LB containing 2μM
biotin and 100μg/ml ampicillin. Incubate for 1 hour at 37°C with shaking and then induced by adding IPTG in final concentration of 100μM in LB medium which contains 100μg/ml ampicillin.After incubated for 4~5 hours at 37°C with shaking, the expressed fusion protein sample is prepared with Alkaline Lysis Solution ,electrophoresised by 12% SDS-PAGE , stained with Coomb's blue stain .The same sample is also transfer the proteins to nitrocellulose membrane following SDS-PAGE electrophoresis, to identify the biotinylated fusion protein by the Streptavidin Alkaline Phosphatase stain, and the fusion protein's immunoreactive property was examined with Western blotting. Using the crude extraction of the fusion protein, we have detected 15 serum from patients (5 infected with C．sinensis ;5 infected with P.skrjabini ;5 infected with S. japonicum) by cystatin capture ELISA. Also, we have located the distributing of cysteine protease expression by immunohistochemistry.
Results: Twenty one ampicillin resistance strain were harvested in the transformation test . PinPointTM Xa-1 T- cysteine protease gene fragment recombinant molecule were successfully constructed and also verified by sequencing results .A positive band was clearly fond in 32 kDa by Streptavidin-Alkaline Phosphatase stain .In the same position, the fusion protein was also detected in western blotting. Using the crude extraction of the fusion protein,we have detected 15 serum from patients (5 infected with C.sinensis ;5 infected with P.skrjabini ;5 infected with S.japonicum) by cystatin capture ELISA the results show clearly that only the samples from the patients infected with P.skrjabini have the strong positive reaction(P﹤0.01),and the others show negative reaction. Immunohistochemical study show that the po
引文
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targeted to parasitophorous vacuoles of Leishmania-infected macrophages and internalized by amastigotes of L. amazonensis and L. mexicana. J Cell Sci,1999, 112 ( Pt 15): 2559-2570
[13] Ikeda T. Cystatin capture enzyme-linked immunosorbent assay for immunodiagnosis of human paragonimiasis and fascioliasis. Am J Trop Med Hyg，1998，59(2): 286-290
[14] 龙建银,王会信 外源基因在大肠杆菌中表达的研究进展 生物化学与生物物理进展 1997,24(2):126-132
[15] Kosovsky J, Durmanova V, Kudelova M, et al. A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins. J Virol Methods, 2001, 92(2): 121-129
[16] TAE YUN KIM, SHIN-YONG KANG,SUN HYO PARK,KOM SUKONTASON, et al .Cystatin Capture Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Human Clonorchiasis and Profile of Captured Antigenic Protein of Clonorchis sinensis.Clinical and dignostic laboratary immunology, 2001, 8(6):1076-1080
[17] Ingemar BjOrk and Ewa Pol, Biphasic transition curve on denaturation of chicken Cystatin guanidinium chloride Evidence for an independently unfolding structura region. Federation of European Bioeliemieal Societies, 1992, 299(1): 66-68 .
[18] Abrahamson, M., A. Ritonja, M. A. Brown, A. Grubb, W. Machleidt, and A. J. Barrett. Identification of the probable inhibitory reactive sites of the cysteine proteinase inhibitors human Cystatin C and chicken Cystatin . J. Biol. Chem. 1987, 262:9688-9694.
[19] Anastasi, A., M. A. Brown, A. A. Kembhavi, M. J. Nicklin, C. A. Sayers, D. C. Sunter, and A. J. Barret. 1983. Cystatin , a protein inhibitor of cysteine proteinases. Improved purification from egg white, characterization, and detection in chicken serum. Biochem. J. 211:129-138.
[20] Scharfstein, J., M. Abrahamson, C. B. P. de Souza,et al. 1995. Antigenicity of Cystatin -binding proteins from parasitic protozoan de-tection by a proteinase inhibitor based capture immunoassay (PINC-ELISA). J. Immunol. Methods 182:63-72.
[21] Rhoads ML, Fetterer RH. Developmentally regulated secretion of cathepsin L-like
cysteine proteases by Haemonchus contortus. J Parasitol, 1995, 81(4): 505-512
[22] Brady CP，Dowd AJ, Brindley PJ, et al. Recombinant expression and localization of Schistosoma mansoni cathepsin L1 support its role in the degradation of host hemoglobin. Infect Immuny,1999, 67(1):368-371
[23] Michel A，Ghoneim H，Resto M，et al . Sequence, characterization and localization of a cysteine proteinase cathepsin L in Schistosoma mansoni. Mol Biochem Parasitol,1995, 73(1-2):7-18
[24] Smith AM, Dowd AJ, McGonigle S, et al. Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol Biochem Parasitol,1993, 62(1):1-8
[25] Parussini F, Duschak VG, Cazzulo JJ. Membrane-bound cysteine proteinase isoforms in different developmental stages of Trypanosoma cruzi. Cell Mol Biol Noisy le grand,1998, 44(3): 513-519