东方田鼠IgG3抗体相关的日本血吸虫抗原基因研究
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摘要
血吸虫病是一种分布广泛，危害严重的人畜共患寄生虫病。利用基因工程技术发展血吸虫病疫苗已成为当今寄生虫学者研究的热点。目前已鉴定出数个有免疫保护潜能的曼氏血吸虫及日本血吸虫疫苗分子。尽管血吸虫病疫苗候选抗原分子的研究有了很大的进展，但至今保护效果都不够理想，因此开辟新途径，不断寻找新的有效的候选抗原分子，以提高免疫保护作用，仍是当前血吸虫病研究的重点。
研究表明，东方田鼠作为日本血吸虫的非相容性宿主对日本血吸虫病具有先天遗传的天然抵抗力，野生东方田鼠和实验室繁殖传代的东方田鼠同样具有这一特性。已报道有39种哺乳动物可自然感染血吸虫，东方田鼠是迄今发现的唯一一种具有抗日本血吸虫病现象的啮齿类动物。机制研究表明体液免疫在其中发挥重要作用，其血清IgG3抗体与东方田鼠抗日本血吸虫病相关。据此我们用东方田鼠阴性血清免疫筛选日本血吸虫成虫cDNA文库，以期分离东方田鼠血清IgG3抗体针对的日本血吸虫特异性抗原基因并进一步研究其生物学功能，为研制和开发新的血吸虫病疫苗或治疗药物开辟新思路。
本研究利用实验室繁殖传代的东方田鼠阴性血清和辣根过氧化物酶标记的羊抗小鼠IgG3免疫筛选日本血吸虫成虫cDNA文库，经过初筛和复筛获得两个阳性克隆。应用RACE技术对两个阳性克隆的cDNA片段进行扩增，获得两个东方田鼠血清IgG3抗体相关的日本血吸虫抗原编码基因。同源序列分析表明，获得的两个基因为新的日本血吸虫抗原编码基因，国内外未见相关报道。其中N-1基因的开放阅读框长780bp，编码259个氨基酸，N-2基因的开放阅读框长336bp，编码111个氨基酸。将N-1基因克隆到pET-28a(+)原核表达载体，转化大肠杆菌BL21感受态细胞，经IPTG诱导在E.coli中表达，并以werstern-blotting试验对重组蛋白的抗原性进行分析，结果在预测位置出现了明显的识别条带，显示N-1基因重组抗原具有良好的抗原性。为了探讨该基因真核表达产物在血吸虫病防治中的作用，我们成功将此基因克隆到昆虫病毒转移载体pBacPAK-
HIS 中，为进一步探讨真核表达产物作为血吸虫候选疫苗的可行性奠
定了基础。
为了评估N－1 基因做为日本血吸虫核酸疫茵候选分子的可行性，
我们将此基因克隆到真核表达载体pCDNA3 中，构建成DNA 疫苗，并
进行动物免疫保护实验，结果实验组小鼠的减虫率达28．64％，肝脏
组织减卵率为21．73％，与对照组相比差异显著，表明此核酸疫苗应
用于日本血吸虫病的防治很有发展潜力。
本研究通过免疫筛选及RACE 技术首次克隆到两个新的东方田鼠
血清IgG3抗体相关的日本血吸虫特异性抗原编码基因，其中N－1基
因在大肠杆菌中获得表达，同时构建成核酸疫苗进行动物兔疫实验，
获得显著的免疫保护效果，为发展新的抗日本血吸虫病疫苗奠定了基
石出。
Schistosomiasis, caused by Schistosomes, is a serious parasitic zoonosis afflicting both human and animals in the world. In recent years, a great attention was paid in the study of vaccines against Schistosomiasis by genetic engineering techniques. Now several candidate antigens with potential immune protection of Schistosomiasis have been obtained.
Although a significant progress has been made in the screening of candidate antigen genes of Schistosomes, protective effects of all available antigens obtained by conventional gene engineering techniques are not so satisfied. Therefore, establishment of new techniques and strategies to screen effectively protective antigen genes would be an alternative to resolution of the above problem.
Recent studies have shown that Microtus fortis is naturally genetous resistance to Schistosomiasis and humoral immunity, especially the specific IgG3 antibody in sera of Microtus fortis could play a relatively important role for Microtus fortis against Schistosomiasis japonicum. To screen the antigen genes of Schistosotna japonicum that related to the specific IgG3 antibody and provide valuable information for searching Schistosomiasis vaccine candidates, the Schistosoma Japonicum adult worm cDNA library was immunoscreened with the sera of uninfected Microtus fortis and Goat anti-mouse IgG3-HRP.
Two positive clones were obtained and then were amplified using RACE technique (rapid amplification of cDNA ends), which resulted in acquiring two of novel genes of Schistosoma japonicum. One gene is 780 base pairs in open reading frame, encoding 259 amino acids and the other is 1169 bp.
By cloning the N-l gene into pET~28a(+) expression vector, a recombinant of pET-28a(+)/N-l was constructed and expressed in E.coli. The expression product was analyzed by SDS-PAGE and Western-blotting. The result revealed that the molecular weight of recombinant protein was approximately 35KD and can be specifically recognized by mouse serum
against Schistosoma japonicum adult worm antigen preparation (SWAP), suggesting that the recombinant protein possesses good antigenicity. Furthermore, the N-l gene was successfully cloned into plasmid pBacPAK-His, an insect virus transfer vector, which may pave the way for further study on feasibility of application on Schistosomiasis using eukaryotic expression product of N-l gene.
In order to evaluate the feasibility of N-l gene as DNA vaccine candidate in the controlling Schistosomiasis, the gene was cloned into eukaryotic expression vector pcDNAS and then animal protection experiment was carried out. 28.64% worm burden reduction and 21.73% eggs number reduction were obtained in Kunming mice immunized with recombinant plasmid, indicating the prospect for N-l gene DNA vaccine as a potential vaccine against Schistosomiasis.
In conclusion, two novel genes of Schistosoma japonicum related to the IgG3 antibody of Microtus fortis were firstly cloned by immunoscreening the Schistosoma Japonicum adult worm cDNA library and RACE technique. Furthermore, the N-l gene was expressed in E. coli successfully. The DNA vaccine contain N-l sequence was constructed and used to vaccinate the mice, significant protection was obtained when comparing with control group. Therefore, this study has provided the theoretical and methodological basis for the development of an effective vaccine against Schistosomiasis.
研究表明，东方田鼠作为日本血吸虫的非相容性宿主对日本血吸虫病具有先天遗传的天然抵抗力，野生东方田鼠和实验室繁殖传代的东方田鼠同样具有这一特性。已报道有39种哺乳动物可自然感染血吸虫，东方田鼠是迄今发现的唯一一种具有抗日本血吸虫病现象的啮齿类动物。机制研究表明体液免疫在其中发挥重要作用，其血清IgG3抗体与东方田鼠抗日本血吸虫病相关。据此我们用东方田鼠阴性血清免疫筛选日本血吸虫成虫cDNA文库，以期分离东方田鼠血清IgG3抗体针对的日本血吸虫特异性抗原基因并进一步研究其生物学功能，为研制和开发新的血吸虫病疫苗或治疗药物开辟新思路。
本研究利用实验室繁殖传代的东方田鼠阴性血清和辣根过氧化物酶标记的羊抗小鼠IgG3免疫筛选日本血吸虫成虫cDNA文库，经过初筛和复筛获得两个阳性克隆。应用RACE技术对两个阳性克隆的cDNA片段进行扩增，获得两个东方田鼠血清IgG3抗体相关的日本血吸虫抗原编码基因。同源序列分析表明，获得的两个基因为新的日本血吸虫抗原编码基因，国内外未见相关报道。其中N-1基因的开放阅读框长780bp，编码259个氨基酸，N-2基因的开放阅读框长336bp，编码111个氨基酸。将N-1基因克隆到pET-28a(+)原核表达载体，转化大肠杆菌BL21感受态细胞，经IPTG诱导在E.coli中表达，并以werstern-blotting试验对重组蛋白的抗原性进行分析，结果在预测位置出现了明显的识别条带，显示N-1基因重组抗原具有良好的抗原性。为了探讨该基因真核表达产物在血吸虫病防治中的作用，我们成功将此基因克隆到昆虫病毒转移载体pBacPAK-
HIS 中，为进一步探讨真核表达产物作为血吸虫候选疫苗的可行性奠
定了基础。
为了评估N－1 基因做为日本血吸虫核酸疫茵候选分子的可行性，
我们将此基因克隆到真核表达载体pCDNA3 中，构建成DNA 疫苗，并
进行动物免疫保护实验，结果实验组小鼠的减虫率达28．64％，肝脏
组织减卵率为21．73％，与对照组相比差异显著，表明此核酸疫苗应
用于日本血吸虫病的防治很有发展潜力。
本研究通过免疫筛选及RACE 技术首次克隆到两个新的东方田鼠
血清IgG3抗体相关的日本血吸虫特异性抗原编码基因，其中N－1基
因在大肠杆菌中获得表达，同时构建成核酸疫苗进行动物兔疫实验，
获得显著的免疫保护效果，为发展新的抗日本血吸虫病疫苗奠定了基
石出。
Schistosomiasis, caused by Schistosomes, is a serious parasitic zoonosis afflicting both human and animals in the world. In recent years, a great attention was paid in the study of vaccines against Schistosomiasis by genetic engineering techniques. Now several candidate antigens with potential immune protection of Schistosomiasis have been obtained.
Although a significant progress has been made in the screening of candidate antigen genes of Schistosomes, protective effects of all available antigens obtained by conventional gene engineering techniques are not so satisfied. Therefore, establishment of new techniques and strategies to screen effectively protective antigen genes would be an alternative to resolution of the above problem.
Recent studies have shown that Microtus fortis is naturally genetous resistance to Schistosomiasis and humoral immunity, especially the specific IgG3 antibody in sera of Microtus fortis could play a relatively important role for Microtus fortis against Schistosomiasis japonicum. To screen the antigen genes of Schistosotna japonicum that related to the specific IgG3 antibody and provide valuable information for searching Schistosomiasis vaccine candidates, the Schistosoma Japonicum adult worm cDNA library was immunoscreened with the sera of uninfected Microtus fortis and Goat anti-mouse IgG3-HRP.
Two positive clones were obtained and then were amplified using RACE technique (rapid amplification of cDNA ends), which resulted in acquiring two of novel genes of Schistosoma japonicum. One gene is 780 base pairs in open reading frame, encoding 259 amino acids and the other is 1169 bp.
By cloning the N-l gene into pET~28a(+) expression vector, a recombinant of pET-28a(+)/N-l was constructed and expressed in E.coli. The expression product was analyzed by SDS-PAGE and Western-blotting. The result revealed that the molecular weight of recombinant protein was approximately 35KD and can be specifically recognized by mouse serum
against Schistosoma japonicum adult worm antigen preparation (SWAP), suggesting that the recombinant protein possesses good antigenicity. Furthermore, the N-l gene was successfully cloned into plasmid pBacPAK-His, an insect virus transfer vector, which may pave the way for further study on feasibility of application on Schistosomiasis using eukaryotic expression product of N-l gene.
In order to evaluate the feasibility of N-l gene as DNA vaccine candidate in the controlling Schistosomiasis, the gene was cloned into eukaryotic expression vector pcDNAS and then animal protection experiment was carried out. 28.64% worm burden reduction and 21.73% eggs number reduction were obtained in Kunming mice immunized with recombinant plasmid, indicating the prospect for N-l gene DNA vaccine as a potential vaccine against Schistosomiasis.
In conclusion, two novel genes of Schistosoma japonicum related to the IgG3 antibody of Microtus fortis were firstly cloned by immunoscreening the Schistosoma Japonicum adult worm cDNA library and RACE technique. Furthermore, the N-l gene was expressed in E. coli successfully. The DNA vaccine contain N-l sequence was constructed and used to vaccinate the mice, significant protection was obtained when comparing with control group. Therefore, this study has provided the theoretical and methodological basis for the development of an effective vaccine against Schistosomiasis.
引文
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37．朱国正，汪英华，雷观愚，刘瑞三．东方田鼠的实验室饲养及其抗血吸虫感染特性．上海实验动物科学，1991，11(4)：193-198
38． 刘金明，傅志强，李浩，魏梅雄，林矫矫．东方田鼠 A D C C体外杀伤 日本血吸虫童虫效果的初步观察．寄生虫与医学昆虫学报，2001，8 (4) ：212-219
39． Fehr C,Fickov M, Hiemke C,Dahmen N. Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6ran improved method to determine 5'-and 3'-cDNA ends. Brain Res Protoc, 1999,3(3) :242-251．
40． Zhang Y, Frohman M A. Using rapid amplification of cDNA ends(RACE)to obtain full-length cDNA. Methods Mol Biol,1997,69:61-87．
41． Short RB, Liberatos JD, Teehan WH, Bruce JI. Conventional Giemsa-stained and C-banded chromosomes of seven strains of Schistosoma mansoni. J. Parasitol, 1989, 75(6) :920-926．
42． McManas EC, Tamas T,Campbell WC. Treatment of drug-resistant coccidia with acriflavine. ResVet Sci, 1972,13(5) :492-494．
43． Hota-Mitchell S, Clarke MW, Podesta RB, Dekaban GA. Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model. Vaccine, 1999,17 (11-12) : 1338-1354．
44． Waksman BH, Cook JA. A report of a conference on newer immunologic approaches to Schistosomiasis. Am J Trop Med Hyg., 1975, 24(6 Pt 1) :1037-1039．
45． Yang W, Waine GJ, McManus DP. Antibodies to Schistosoma japonicum (Asian bloodfluke) paramyosin induced by nucleic acid vaccination. Biochem Biophys Res Commun, 1995, 212(3) :1029-1039．
46． Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 1993, 259(5102) :1745．
47． Cardoso AI, Blixenkrone-Moller M, Fayolle J, Liu M, Buckland R, Wild TF. Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity. Virology, 1996, Nov 15, 225(2) :293-299
48． Zhang Y, Taylor MG, McCrossan MV, Bickle QD. Molecular cloning and characterization ofanovel Schistosoma japonicum"irradiated vaccine-specific"antigen, SJ14-3-3． Mol Biochem Parasitol, 1999; 103(1) :25-34
49． Frohman MA, Dush MK, Martin GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA, 1988, 85:8998-9002
50． Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Bio Chem, 1991,266(30) :19867-19870
51． Dune DW, Hagan P, Abath FG, et al. Prospects for immunological control of schistosomiasis. Lancet,1995,345:1488-1491
52． Wolff JA, Malone Rw, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science,1990,247(4949 pt 1) :1465．
53． Kim JJ, Ayyaroo V, Bagarazzi ML, et al. Development of a multicomponent candidate vaccine for HIV-1． Vaccine, 1997, 15(8) :879．
54． Yang W, Waine GJ, Sculley DG, Liu X, McManus DP. Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. Int J Parasitol, 1992;22(8) :1187-1191．
55． Tighe H M, Corr Mroman, et al. Gene Vaccination:Plasmid DNA is more than just a blueprint. Immunology Today, 1998, 19:89．
56． Robinson HL, Torres C A T. DNA vaccines. Immunology, 1997,9:271．
57． Kalinna BH. Becker MM, McManus DP. Engineering and expression of a full length cDNA endoding Schistosoma japonicum paranysion, Purification of the recombinant protein and its recognition protein and its recognition by infected patient sera. Acta Trop, 1997, 6(2) :111-115．
58． Becker MM, Kalinna BH,Waine GJ, et al. Gene cloning, overproduction and purification of a functimally active cytoplasimic fatty acid-binding protein(Sj-FABJP) from the human blood fluke Schistosoma japonucim. Gene, 1994, 148(2) :321-325．
59． Sun W, Liu S, Brindley PJ, et al. Bacterial expression and characterization of functional recombinant triose-phosphate isomerase form Schistosoma japonicum. Protein. Expr Purif, 1999,17(3) :410-413．
60． Waine GJ, McManus DP. Nucleic acids: vaccines of the further. Parasitol today, 1995, 10(3) : 113-116．
61． Knight M, et al. Adult Schistosome cDNA Libraries as a source of antigens for the study of experimental and human schistosomiasis. Molecular and Biochemical Parasitology,1986,18:L235．
62． Soisson LM, Masterson CP, Tom TD, McNally MT, Lowell GH, Strand M. Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen. J Immunol, 1992;149(11) :3612-3620．
63． Da' dara AA, Skelly PJ, Fatakdawala M, V isovatti S.Eriksson E, Harn DA. Comparative etticacy of the Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene fun delivery. Parasite Immunol 2001,24(4) :179-187．
64． Zhang Y, Taylor MG, Johansen Mv, Bichle QD. Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection. Vaccine, 2001,12:20(5-6) : 724-730．
65． Sahdra R , et al. Inf Immunity, 1992:60(1) :90-96
66． Shi YE, Jiang CF, Han JJ, Li YL, Ruppel A. Schistosoma japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes. Exp Parasitol, 1990, Jul;71 (1) :100-106
67． Dupre L, Kremer L, Wolowezuk I,Riveau G, CapronA, Locht C. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection. Vaccine, 2001,8:19(11-12) :1373-1380．
68． Borson N D, Sato W L, Drewes L R. A lock-docking oligo(dT)primer for 5' and 3' RACE ends (RACE). J Neurosci Methods, 1997, 73(2) :187-191．
69． Da' dara AA, Skelly PJ, Wang MM, Harn DA. Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice. Vaccine, 2001, 12;20(3-4) :359-369．
70． Herve M, Dupre L, Ban E, Schacht AM, Capron A, Riveau G. Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization. Vaccine, 2001, 14:19(31) :4549-4556．
2．王溪云，王小红．试论我国湖区血吸虫病的控制对策．中国兽医寄生虫病，2000，8(4)：40-45．
3．蔡凯平，左家铮，贺宏斌，卓尚炯，胡钢．三峡建坝后洞庭湖区泥沙淤积变化对血吸虫病流行因素的影响．实用预防医学，2000，7(1)：1-3
4．李涛，余秉圭，戴裕海．长江洪水对急性血吸虫病流行的影响及防治对策研究．中国血吸虫病防治杂志，2000，12(5)：268-272．
5．陈伟，杨先祥，黄希宝，张映皓，蔡顺祥，刘建兵，付义，李书华．1998 年洪灾对血吸虫病流行的影响．中国血吸虫病防治杂志，2000，12(4)：202-205．
6．周金春，易新元，曾宪芳，等．重组日本血吸虫副肌球蛋白诱导水牛、保护性免疫的研究．中国寄生虫病防治杂志，2000，13(4)：282-284．
7．周生华，刘述先，宋光承，徐裕信，孙文宇．日本血吸虫大陆株副肌球蛋白全基因核酸疫苗诱生免疫应答的实验研究．中华医学杂志，1999，79(11)：848-851．
8．周生华，刘述先，宋光承，徐裕信．日本血吸虫副肌球蛋白全基因克隆、测序及体内表达．中国寄生虫学与寄生虫病杂志，1999，17(4)：196-199
9．施福恢，沈纬，田锷，叶萍，吴祥甫，蔡幼民，等．日本血吸虫重组抗原 Sj GST Paramyosin和Sj 23对绵羊的保护性免疫试验．中国兽医科技，1997，27(11)：3-6．
10．刘金明，林矫矫，杨冠珍，傅志强，石耀军，蔡幼民，吴祥甫．日本血吸虫中国大陆株 26kD GST基因在大肠杆菌中的高效表达．中国生物化学与分子生物学报，2000，16(6)：759-763．
11．余传信，荫昌，殷旭仁．重组日本血吸虫中国大陆株磷酸丙糖异构酶蛋白质表达、纯化及特性分析．中国人兽共患病杂志，1999，15(4)：31-33．
12．余传信，荫昌，殷旭仁，司进．日本血吸虫(中国大陆株)磷酸丙糖异构酶(SjC-TPI)基因克隆与序列分析．中国血吸虫病防治杂志，1997，9(6)：32-325．
13．司进，荫昌，D．A．Harn，何伟，华万全，殷旭仁，梁幼生，徐明．日本血吸虫(中国大陆株)磷酸丙糖异构酶 DNA 疫苗的研制及其保护性免疫的研究．中国血吸虫病防治杂志，2000，12(6)：324-329．
14．潘漪清，邵伟娟，高诚，等．东方田鼠血液及血清生化指标的测定．上海实验动物科学，1999，19(3)：170-171
15．罗新松，刘述先，何永康，等．日本血吸虫重组GST抗原对水牛的保护性研究．实用预防医学，1996，3(4)：214-215．
16．蒋建敏，屠乐鸣，张素蛾，等．日本血吸虫 26KD基因重组重组蛋白免疫小鼠抗体内血吸虫生殖的作用．中国寄生虫学与寄生虫病杂志，1999，17(1)：9-11．
17．李传明，石佑恩．日本血吸虫26KD谷胱苷肽S-转移酶DNA疫苗的研究及其保护性免疫效果观察．中国寄生虫病防治杂志，1998，11(3)：207-211．
18．苏川，马磊，王荣芝，等．日本血吸虫(中国大陆株)22.6KD重组蛋白对小鼠的免疫保护性研究．中国寄生虫学与寄生虫病杂志，1999，17(5)：288-291．
19．王庆林，易新元，曾宪芳，周金春，等．东方田鼠感染血清免疫筛选日本血吸虫成虫 cDNA 文库．中国生物化学与分子生物学报，2001，17(5)：547-551．
20．何永康，罗新松，张新跃，喻鑫玲，等．东方田鼠天然抗血吸虫感染免疫特性的研究．中国血吸虫病防治杂志，1998，10(增刊)：73-78．
21．何永康，罗新松，喻鑫玲，林金莲，等．洞庭湖区东方田鼠天然抗日本血吸虫不同阶段抗原的抗体水平的初步观察．中国寄生虫学与寄生虫病杂志，1999，17(3)：132-134．
22．黎申恺，朱祖林，金壁如，周达仁，等．东方田鼠对日本血吸虫的不感染性．寄生虫学报，1965，2(1)：103
23．吴光，张容，杨元清，等．日本血吸虫实验感染东方田鼠的初步报告．中国医学科学院上海寄研所年报，1962，180-190．
24．傅志强，刘金明，蒋守富，李浩，等．东方田鼠抗日本血吸虫抗体水平检测与分析．中国兽医寄生虫病，2001，9(4)：6-8．
25．蒋守富，魏梅雄，林矫矫，等．东方田鼠天然抗日本血吸虫抗体及IgG亚类的初步研究．中国血吸虫病防治杂志，2001，13(1)：1-3
26．阎玉涛，刘述先，宋光承，徐裕信，等．东方田鼠天然抗体相关的日本血吸虫抗原基因筛选和克隆．中国寄生虫学与寄生虫病杂志，2001，19(3)：153-156．
27．苑纯秀，李铭，陈永军，石耀军，等．日本血吸虫中国大陆株雌、雄成虫cDNA文库的构建．生物工程学报，2000，16(6)：727-730．
28．罗新松，何永康，喻鑫玲，等．东方田鼠血清被动转移小白鼠抗血吸虫感染的保护性研究．中国人兽共患病杂志，1998，14(5)：75-76．
29．冯笑川，吴宜琴，陈淑贞，等．日本血吸虫成虫cDNA表达文库免疫筛选及抗原表达克隆的鉴定．中国血吸虫病防治杂志，1994，6(2)：68-70．
30．吴海玮，陈淑贞，王荣芝，等．日本血吸虫中国大陆株成虫 cDNA文库的免疫筛选及 PCR 初步鉴定．中国寄生虫病防治杂志，1998，10(1)：7-10．
31．刘宗传，罗新松，杨瑞青，等．东方田鼠微生物学与寄生虫学检测初报．上海实验动物科学，2000，20(1)：20-21
32．高诚，沈志敏，刘雄伟，等．不同地区东方田鼠微生物学和寄生虫学检测初步研究．上海实验动物科学，2000，20(1)：22-25
33．邵伟娟，余家璜，王晓明，沈志明．东方田鼠的血液及血清生化测定值．上海实验动物科学，1996，16(1)：46-52
34．贺宏斌，左家铮，刘柏香，等．室内繁殖和野生东方田鼠感染日本血吸虫的比较．实用寄生虫杂志，1995，3(2)：72-74
35．贺宏斌，左家铮，周利红，等．东方田鼠实验感染日本血吸虫的研究．湖南医学，1992，9(2)：65-67
36．李浩，何艳燕，林矫矫，林邦发，等．东方田鼠抗日本血吸虫病现象的观察．中国兽医寄生虫病，2000，8(2)：12-15
37．朱国正，汪英华，雷观愚，刘瑞三．东方田鼠的实验室饲养及其抗血吸虫感染特性．上海实验动物科学，1991，11(4)：193-198
38． 刘金明，傅志强，李浩，魏梅雄，林矫矫．东方田鼠 A D C C体外杀伤 日本血吸虫童虫效果的初步观察．寄生虫与医学昆虫学报，2001，8 (4) ：212-219
39． Fehr C,Fickov M, Hiemke C,Dahmen N. Rapid cloning of cDNA ends polymerase chain reaction of G-protein-coupled receptor kinase 6ran improved method to determine 5'-and 3'-cDNA ends. Brain Res Protoc, 1999,3(3) :242-251．
40． Zhang Y, Frohman M A. Using rapid amplification of cDNA ends(RACE)to obtain full-length cDNA. Methods Mol Biol,1997,69:61-87．
41． Short RB, Liberatos JD, Teehan WH, Bruce JI. Conventional Giemsa-stained and C-banded chromosomes of seven strains of Schistosoma mansoni. J. Parasitol, 1989, 75(6) :920-926．
42． McManas EC, Tamas T,Campbell WC. Treatment of drug-resistant coccidia with acriflavine. ResVet Sci, 1972,13(5) :492-494．
43． Hota-Mitchell S, Clarke MW, Podesta RB, Dekaban GA. Recombinant vaccinia viruses and gene gun vectors expressing the large subunit of Schistosoma mansoni calpain used in a murine immunization-challenge model. Vaccine, 1999,17 (11-12) : 1338-1354．
44． Waksman BH, Cook JA. A report of a conference on newer immunologic approaches to Schistosomiasis. Am J Trop Med Hyg., 1975, 24(6 Pt 1) :1037-1039．
45． Yang W, Waine GJ, McManus DP. Antibodies to Schistosoma japonicum (Asian bloodfluke) paramyosin induced by nucleic acid vaccination. Biochem Biophys Res Commun, 1995, 212(3) :1029-1039．
46． Ulmer JB, Donnelly JJ, Parker SE, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science, 1993, 259(5102) :1745．
47． Cardoso AI, Blixenkrone-Moller M, Fayolle J, Liu M, Buckland R, Wild TF. Immunization with plasmid DNA encoding for the measles virus hemagglutinin and nucleoprotein leads to humoral and cell-mediated immunity. Virology, 1996, Nov 15, 225(2) :293-299
48． Zhang Y, Taylor MG, McCrossan MV, Bickle QD. Molecular cloning and characterization ofanovel Schistosoma japonicum"irradiated vaccine-specific"antigen, SJ14-3-3． Mol Biochem Parasitol, 1999; 103(1) :25-34
49． Frohman MA, Dush MK, Martin GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA, 1988, 85:8998-9002
50． Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Bio Chem, 1991,266(30) :19867-19870
51． Dune DW, Hagan P, Abath FG, et al. Prospects for immunological control of schistosomiasis. Lancet,1995,345:1488-1491
52． Wolff JA, Malone Rw, Williams P, et al. Direct gene transfer into mouse muscle in vivo. Science,1990,247(4949 pt 1) :1465．
53． Kim JJ, Ayyaroo V, Bagarazzi ML, et al. Development of a multicomponent candidate vaccine for HIV-1． Vaccine, 1997, 15(8) :879．
54． Yang W, Waine GJ, Sculley DG, Liu X, McManus DP. Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. Int J Parasitol, 1992;22(8) :1187-1191．
55． Tighe H M, Corr Mroman, et al. Gene Vaccination:Plasmid DNA is more than just a blueprint. Immunology Today, 1998, 19:89．
56． Robinson HL, Torres C A T. DNA vaccines. Immunology, 1997,9:271．
57． Kalinna BH. Becker MM, McManus DP. Engineering and expression of a full length cDNA endoding Schistosoma japonicum paranysion, Purification of the recombinant protein and its recognition protein and its recognition by infected patient sera. Acta Trop, 1997, 6(2) :111-115．
58． Becker MM, Kalinna BH,Waine GJ, et al. Gene cloning, overproduction and purification of a functimally active cytoplasimic fatty acid-binding protein(Sj-FABJP) from the human blood fluke Schistosoma japonucim. Gene, 1994, 148(2) :321-325．
59． Sun W, Liu S, Brindley PJ, et al. Bacterial expression and characterization of functional recombinant triose-phosphate isomerase form Schistosoma japonicum. Protein. Expr Purif, 1999,17(3) :410-413．
60． Waine GJ, McManus DP. Nucleic acids: vaccines of the further. Parasitol today, 1995, 10(3) : 113-116．
61． Knight M, et al. Adult Schistosome cDNA Libraries as a source of antigens for the study of experimental and human schistosomiasis. Molecular and Biochemical Parasitology,1986,18:L235．
62． Soisson LM, Masterson CP, Tom TD, McNally MT, Lowell GH, Strand M. Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen. J Immunol, 1992;149(11) :3612-3620．
63． Da' dara AA, Skelly PJ, Fatakdawala M, V isovatti S.Eriksson E, Harn DA. Comparative etticacy of the Schistosoma mansoni nucleic acid vaccine, Sm23, following microseeding or gene fun delivery. Parasite Immunol 2001,24(4) :179-187．
64． Zhang Y, Taylor MG, Johansen Mv, Bichle QD. Vaccination of mice with a cocktail DNA vaccine induces a Th1-type immune response and partial protection against Schistosoma japonicum infection. Vaccine, 2001,12:20(5-6) : 724-730．
65． Sahdra R , et al. Inf Immunity, 1992:60(1) :90-96
66． Shi YE, Jiang CF, Han JJ, Li YL, Ruppel A. Schistosoma japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for water buffaloes. Exp Parasitol, 1990, Jul;71 (1) :100-106
67． Dupre L, Kremer L, Wolowezuk I,Riveau G, CapronA, Locht C. Immunostimulatory effect of IL-18-encoding plasmid in DNA vaccination against murine Schistosoma mansoni infection. Vaccine, 2001,8:19(11-12) :1373-1380．
68． Borson N D, Sato W L, Drewes L R. A lock-docking oligo(dT)primer for 5' and 3' RACE ends (RACE). J Neurosci Methods, 1997, 73(2) :187-191．
69． Da' dara AA, Skelly PJ, Wang MM, Harn DA. Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice. Vaccine, 2001, 12;20(3-4) :359-369．
70． Herve M, Dupre L, Ban E, Schacht AM, Capron A, Riveau G. Features of the antibody response attributable to plasmid backbone adjuvanticity after DNA immunization. Vaccine, 2001, 14:19(31) :4549-4556．