摘要
隐孢子虫病是一种重要的人兽共患的原虫病，该病分布广、感染率高且危害严重，目前已有200多种药物用于该病的治疗，但还没有一种具有真正的杀灭作用，以单链抗体为导向物的重组毒素已成功地用于肿瘤的治疗，为了探讨其对隐孢子虫的杀灭作用，特进行如下研究：
抗隐孢子虫子孢子表膜单克隆抗体的制备及鉴定 利用牛源微小隐孢子虫(C．parvum)超声粉碎物免疫BALB／C小鼠，取其脾细胞与SP2／O细胞融合，经3～5次有限稀释克隆化，获得4株持续分泌抗体的杂交瘤细胞株，分别命名为1D5，2E9，3D6和4G4。经检测，其分泌的抗体亚类1D5、3D6和4G4株为IgG1，2E9株为IgM。通过免疫荧光鉴定，4株单抗均作用于子孢子，其中2株针对子孢子表膜。对杂交瘤细胞株的染色体计数的结果发现染色体均数为90～98条。ELISA检测3D6、1D5、2E9和4G4的细胞培养上清效价分别为1：160，1：320，1：320和1：640，诱生小鼠腹水的抗体效价分别为1：409，600，1：819，200，1：819，200和1：1，638，400。
抗隐孢子虫子孢子单链抗体基因的克隆及序列分析 从分泌抗隐孢子虫子孢子单克隆抗体的杂交瘤细胞3D6和4G4中，分别提取其mRNA，在反转录酶的作用下，合成cDNA，经PCR扩增，分别获得抗隐孢子虫子孢子表膜单克隆抗体的轻、重链可变区基因V_L和V_H。在这两个基因中加入连接肽基因后，经进一步PCR扩增、组装合成出单链抗体(ScFv)基因，将该两个基因克隆入pMD-18T载体后，进行了序列测定。测序结果表明，获得的单克隆抗体轻、重链可变区基因V_L和V_H符合小鼠抗体可变区基因特征，为功能性重排的鼠抗体可变区基因序列，分离得到的单链抗体基因ScFv由重链可变区基因V_H-连接肽基因-轻链可变区V_L基因构成，这种构成模式较V_L-Linker-V_H基因结构具有更强的抗体亲和活性。3D6 ScFv基因全长为720bp，其中V_H基因为351bp，编码117个氨基酸，V_L基因全长为324bp，编码108个氨基酸。4G4 ScFv基因全长为717bp，其中V_H-基因为348bp，编码116个氨基酸，V_L基因全长为324bp，编码108个氨基酸。
抗隐孢子虫重组毒素ScFv-PE表达载体的构建 将获得的ScFv基因分别代替绿脓杆菌外毒素PE的受体结合区，利用原核表达载体pET28，构建了用于表达重组毒素的两个原核表达质粒3D6pET28-ScFv-PE和4G4pET28-ScFv-PE。
抗隐孢子虫重组毒素ScFv-PE在大肠杆菌中的表达 将上述质粒转化受体菌BL21(DE3)后，经IPTG诱导，成功地表达了目的蛋白。重组毒素3D6ScFv-PE40和4G4ScFv-PE40的表达量分别占菌体蛋白总量的14％和12％，表达产物以包涵体和上清两种形式存在。
抗隐抱子虫重组毒素ScFv一PE活性的检测将低温诱导获得的3D6ScFv一PE可溶
性表达产物，加入含有脱囊后的子抱子的1640培养液中，观察18h内活动的子抱子
数目和活动性的变化，结果与对照组相比，加入重组毒素组活动的子抱子数目明显
减少(P<0 .05)，活动性明显降低，提示重组毒素对隐抱子虫子抱子具有较强的杀灭
作用。
Four hybirdoma (1D5, 2E9, 3D6 and 4G4) producing McAbs against sporozoites oocyst antigens of C.parvum were developed by fusion of SP2/0 mouse myeloma cells and spleen cells from BALB/C mice immunized with sonicated oocysts.The antibody isotypes of the IDS, 3D6, 4G4 were of the IgG1 subclass,while the 2E9were of the IgM class.All McAbs were reacted with Cryptosporidium sporozoites by indirect immunofluorescent assay, two of which were reacted with surface of sporozoites.The number of chromosome of the hybirdoma cell line was 90 to 98. The ELISA liters of acites were 1:409, 600, 1:819, 200, 1:819, 200 and 1:1, 638, 400, and the ELISA liters of cell supernatant were 1:160, 1:320, 1:320 and 1:640 respectively.
Two single chain antibody ScFv directed against Cryptosporidium parvum sporozoite envelope glycoprotein was constructed by using the antibody variable region (V) genes of murine McAb against Cryptosporidium parvum, the VH and VL gene were amplified by RT-PCR from a hybridoma cell line 3D6 and 4G4, producing mouse McAb against Cryptosporidium parvum sprozoite envelope glycoprotein protein. The heavy and light chain variable regions were connected by a flexible Linker(Gly4Ser)3. The fusion gene (ScFv) were cloned into pMD-18T vector and sequenced. The nucleotide sequence results showed that the VH and VL genes were homologous with the published mouse antibody variable region gene sequences and were confirmed as functionally rearranged mouse immunoglobulin variable region genes. The 3D6 ScFv gene were 720bp, consisting of VH, Linker and VL genes , VH gene was 351bp,encoding 117 amino acids ,The VL gene was 324bp,encoding 108 amino acids .The 4G4 ScFv gene were 717bp, consisting of VH, Linker and VL genes , VH gene was 348bp,encoding 116 amino acids ,The VL gene was 324bp,encoding 108 amino acids.
To express ScFv containing chimeric immunotoxins , we conslructed a immunotoxins expressing plasmids by replacing the receptor binding domain of PE genes with ScFv gene, plasmid pET28-ScFv-PE40 encodes for a hybrid protein consisting N
terminus of ScFv and C terminus of PE, After transformed these plasmids into E.coli strain BL21(DE3) and induced by IPTG, both immunotoxins (3D6 ScFv-PE,4G4 ScFv-PE) were expressed successfully.Expressed recombinant immunotoxins 3D6 ScFv-PE take 14% of the total cell proteins , while the expression level of recombinant immunotoxins 4G4 ScFv-PE were 12%.. After cell lysis and SDS-PAGE analysis , the expression ptoducts exist in forms of inclusion body and soluble fraction.
The expression ptoducts were evaluated the cytotoxicity to Cryptosporidium parvum sprozoite, The results showed that the expression immunotoxins have specific cytotoxicity to cryptosporidium parvum sporozoite.The number of wandering sporozoites in treatment group is much less than control group, while the mobility of the sporozoite in treatment group is much less than control group.These results showed the chemeric toxin has good killing effect on C.p sporozoite.
引文
[1] Winter G.Milstein C.Man-made antibodies. Nature, 1991, 349:293-299
[2] Verma R, Boleti E, Geroge AJT. Antibody engineering. Comparision of bacterial, yeast, insect and mammalian expression systems. J Immunol Methods, 1998,216(1-2) : 165-181
[3] Kipriyanov SM, Moldenhauer G,little M. High level production of soluble single chain antibodies in small-scale Escherichia coli cultures.J Immunol Methods, 1997,200:69-77
[4] Benhar J, Rerter Y, Pai LH,et al. Administration of disulfide-stabilized Fv-immunotoxins B1(dsFv)-PE38 and B3(dsFv)-PE38 by curing large tumor xenografts in nude mice. Int J Cancer, 1995,62:351
[5] Pauza ME, Doumbia SO, Pennell CA. Construction and characterization of human CD7~specific single-chain Fv immunotoxins. J Immunol. 1997 Apr 1; 158 (7) : 3259-69．
[6] Goldberg MR, Heumbrook DC, Russo P, et al. Phase I clinical study of the recombinant oncotoxin TP40 in superficial bladder cancer. Clin Cancer Res. 1995:1(1) :57-61
[7] Kreitman R J, Chaudhary VK, Waldmann J et al. The recombinant immunotoxin anti~PE40 is cytoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia. Cancer Detect Prev, 1998,21(1) :35-38
[8] Chaudhary VK, Batra JK, Gallo MG, et al. A rapid method of cloning functional varible region antibody genes in Escherichia colias single chain toxins. PNAS USA, 1990, 87 (3) : 1066-1070
[9] Batra JK, Fitz Gerald D,Gately M, et al.Anti-Tac(Fv)-PE40, a single chain antibody Pseudomonas fusion protein directed at interlinking 2 receptor bearing cells. J Biol Chem, 1990, 265(25) : 15198-15202
[10] Brinkmann U, PaiLH, FitzGerald DJ, et al. B3(Fv)-PE38 KDEL, a single chain iommunotoxin that causes complete regression of a human carcinoma in mice. PNAS USA, 1991, 88:8616-8620
[11] Batra JK, Kasprzyk PG, Bird RE, et al. Recombinant anti~erbB2 immunotoxins cotaining Pseudomonas exotoxin. PNAS USA, 1992,89:5867-5871
[12] Brinkmann U, et al.A recombinant immunotoxin that is active on prostatc cancer cells and that is composed of the Fv region of monodonal antibody PR1 and a truncated form of Pseudomonas exotoxin. PNAS USA, 1993,90:547～551
[13] Debinski W, PuriRK, Kreitman RJ, et al.A wide range of human cancers express interleukin4(IL4) receptors that can be targeted with chimeric toxin composed of IL4 and PE. J Biol Chem, 1993,268(19):14065～14070
[14] Batra JK, JinnoY, Chaudhary VK, et al. Anti tumor activity of an immunotoxin made with anti transferring receptor and a recombinant form of Pseudomonas exotoxin A. PNAS USA, 1989,86(21):8544～8549
[15] Brinkmann U, Lee BK, Pastan I. Recombinant immunotoxins containing the V_(?)or VL domain of monoclonal antibody B3 fused to Pseudomonas exotoxin. J Immunol ,1993,150(7):2774～2782
[16] T Sreter I. Varga. Cryptosporidiosis in birds-A review. Veterinary Parasitology 87(2000):261-279
[17] Berkelman, R.L. 1994 Emerging infectious diseases in the united States 1993. Infect Dis, 70:272
[18] 徐志凯．主编．1992．实用单克隆抗体技术．西安：陕西科学技术出版社，75～77
[19] McDonald, V., R.M.A. Deer, J.M.S. Nina et al.1991Characteristics and specifity of hybirdoma antibodies against oocyst antigens of Cryptosporidium parvum from man. Parasitol Immunol, 13:251～263
[20] 张西臣，胡力生，李德昌．1991．隐孢子虫保护性单克隆抗体的制备．兽医大学学报，11(4)：309～313
[21] 宗海红，李为明，张雪等．1998．抗隐孢子虫单克隆抗体研制及其特异性鉴定，中国寄生虫学与寄生虫病杂志，16(4)：279～282
[22] 胡景辉，蒋金书．1995．抗鼠隐孢子虫单克隆抗体的研究．畜牧兽医学报，26(5)：420～425
[23] Nisson, B.O., A. Larsson. 1990; Intresplenic immunization with minute amounts of antigen. Immunol Today, 11(1):10
[24] 余传霖，叶天星，陆德源，等主编．1998，现代医学免疫学．上海：上海医科大学出版社，637
[25] 王世若，王兴龙，韩文瑜主编．现代动物免疫学，第二版，吉林人民出版社，2001
[26] Clacson T, Hoogenboom HR, Griffiths AD. Making antibody fragments using
phage display libraries, Natrue, 1991,352:624-628．
[27] Shin takeda, Nickolas A, Dorfman. Two phase approach for the expression of high affinity human anti human immunodeficiency virus immunoglobulin Fab Domains in Escherichia coli. 1995: 14(1) :9~18
[28] Pantoliano MW, Bird RE, Johoson S, et al. Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli. Biochemistry. 1991:30(42) : 10117-10125
[29] Huston JS , levinson D, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci USA, 1988:85:5879?883
[30] He M, Kang AS, Hamon M, et al. Characterization of a progesterone-binding, three-domain antibody fragment (VH/K) expressed in Escherichia coli. Immunology, 1995:84:662?68
[31] Mallender WD, Voss EW Jr. Construction, expression, and activity of a bivalent bispecific single-chain antibody. J Biol Chem, 1994; 269: 199~206
[32] 金冬雁，黎孟枫 等译.分子克隆实验指南，第二版 科学出版社 1989．
[33] Siegall CB, Chaudhary VK, FitzGerald DJ, et al. Functional analysis of domains Ⅱ, Ib, and Ⅲ of Pseudomonas exotoxin. J Biol Chem 1989; 25; 264 (24) :14256-61
[34] Pastan I, Chaudhary V.Fitgerald D J. Recombinant toxins as novel therapeutic agents. Annu Rev Biochem, 1992:61 : 331?354
[35] Nicholls PJ, Johnson VG, Andrew SM, et al. Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate. J Biol Chem 1993 Mar 5:268(7) : 5302?8 .
[36] Beattie BK, Merrill AR In vitro enzyme activation and folded stability of Pseudomonas aeruginosa exotoxin A and its C-terminal peptide. Biochemistry 1996 16;35(28) :9042?51
[37] Francisco JA, Kiener PA, Moran-Davis P, et al. Cytokine activation sensitizes human monocytic and endothelial cells to the cytotoxic effects of an anti-CD40 immunotoxin. J Immunol 1996 . 15; 157(4) : 1652~8
[38] 易学瑞.基因工程抗体表达系统研究进展。《国外医学》预防、诊断、治疗用
2000; 23(1) ;13-15
[39] Shin takeda , Nickolas A, Dorfman , Marjorie robert guroff. Two phase approach for the expression of high affinity human anti-Human immunodef iciency Virus immunoglobulin Fab domains in Escherichia coli . 1995, 14(1) ;9-18．
[40] Makrides S. Strategies for Achieving High-level Expression of Gene in Escherichia Coli Microbilological Review 1996, p:512-538．
[41] Johannes buchner , Ira pastar ulrich brinkmann. A method for increasing the yield of properly folded recombiantn fusion proteins ; single chain immunotoxins from renatruation of bacterial inclusion bodied . Analytical biochemistry . 1992． 205:263-270．
[42] Chalmers J. J, Kim E, Telford J N, et al.. Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor. Appl Environ Microbiol. 1990;56(1) : 104~11
[43] Wetzel R, Perry L. J. and Veilleux C. Mutations in human interferon gama affecting inclusion body formation identified by a general immunochemical screen. Biotechnology, 1991;9(8) : 731 ~7
[44] Arrowood MJ, Sterling CR. Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic percoll gradient. J-parastol, 1987:73 : 31
[45] Robertson L J, Campbell AT, Smith HV. In vitro excystation of Cryptosporidium parvum. Parasitologyl993;106 : 13
[46] Santana JM, et al. 1989; Effect of immunotoxins against Trypanosoma cruxi.Am J Trop Med Hyg41(2) : 177-82
[47] VillemeaCL, et al. 1984; Preparation of an immunotoxin for Acanthamoeba castellanii Biochem Biophys Res Commun. 125(1) :25~9
[48] SuroliaN, MisquithS. Cell surface receptor directed targeting of toxin to human malaria parasite, Plasmodium falciparum. FEBS Lett, 1996:396(0:57-61
[49] Tejuca M, Anderluh G, Maek P, et al. Antiparasite activity of sea-anemone cytolysins on Giardia duodenalis and specific targeting with anti-Giardia antibodies .International journal for parasitology, 1999, 29(3) : 489-498
[50] Yoshida D, Iokaa D, Matsuokaa H, et al. Bacteria expressing single-chain
immunotoxin inhibit malaria parasite development in mosquitoes. 2001, Molecular and Biochemical parasitology, 113(1) : 89~96
[51] Ramadan M, Gabr N, Bacha P, et al. Suppression of immunopathology in schisosomiasis by interleukin-2-targeted fusion toxin, DAB389IL-2． Studies of in vitro and in vivo efficacy. Cell Immunol, 1995, 166(2) :217~26