猪传染性胃肠炎病毒S蛋白在毕赤酵母中的分泌表达及免疫原性分析
|
摘要
猪传染性胃肠炎(Transmissible gastroenteritis, TGE)是一种急性、高度接触性传染病,以呕吐、严重腹泻、脱水为特征,各种年龄的猪只都可以发病,5周龄以下仔猪的病死率最高,给养猪业造成了较大的损失。TGE的病原体为猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine, TGEV),该病毒是冠状病毒科冠状病毒属成员。在众多对TGEV研究的基础上,本研究就TGEV在猪睾丸细胞(ST)上的增殖特点的认识、花青素体外抗TGEV的试验研究、TGEV纤突糖蛋白在毕赤酵母中的分泌表达及免疫原性分析三个部分对TGEV展开研究。
第一部分:猪传染性胃肠炎病毒的增殖特点的认识。
本试验复苏和传代培养了TGEV宿主细胞-ST,测定TGEV的半数细胞培养物感染剂量(TCID50),以便于病毒感染毒性的定量,显微镜下观察TGEV吸附ST细胞后引起的细胞病变现象,免疫过氧化物酶单层试验(IPMA)进一步认识TGEV的增殖规律。细胞病变观察结果显示,TGEV侵染ST细胞后可引起具有“种”的特异性的细胞病变,引起细胞核变形或碎裂,甚至导致细胞的完全破坏,IPMA证明TGEV蛋白质的合成在胞浆内。
第二部分:花青素体外抗猪传染性胃肠炎病毒的试验研究。
在已测定TGEV的TCID50的基础上,利用细胞形态学观察方法,具体研究了花青素对ST细胞的毒性及花青素对TGEV的直接灭活、治疗作用。结果表明:花青素在ST细胞上最大安全浓度为0.002g/ml(纯度5%),即1mg/ml为实际花青素的最大安全浓度;花青素在ST细胞上对抗TGEV的最佳用量为0.002g/ml 500μl;起到治疗作用的花青素用量为0.002g/ml 500μl、375μl,但是花青素只是在ST细胞感染TGEV一定时间内对该病毒起到了治疗作用,而且体外试验结果不能完全等同于体内的治疗效果,所以体内抗病毒效果有待于进一步试验加以验证。
第三部分:猪传染性胃肠炎病毒纤突糖蛋白在毕赤酵母中的分泌表达及免疫原性分析。
根据GenBank上猪传染性胃肠炎病毒纤突糖蛋白S全基因序列设计一对引物,并在5’引物和3’引物中引入EcoR I、Not I酶切位点,经PCR扩增出长2.2kb的目的基因S(其中包含A、B、C、D四个主要抗原位点),克隆于pBS-T载体,克隆载体TS经EcoR I、Not I双酶切,S基因与pPIC9k载体连接,测序验证阳性克隆,Sal I线性化重组穿梭质粒pPIC9k-S,电转化GS115感受态,G418筛选和PCR鉴定得到阳性重组子,1%甲醇诱导表达,诱导后上清经SDS-PAGE电泳、免疫印迹和薄层凝胶扫描分析,并利用离子交换技术和膜超滤方法纯化目的蛋白,将纯化后的蛋白免疫两周龄BALB/c小鼠。SDS-PAGE电泳表明,S蛋白以可溶性形式分泌表达于胞外,且表达产物的分子量约为82 000,与理论推测的分子量一致。免疫印迹结果证明,S蛋白可被TGEV猪阳性血清识别,且具有良好的免疫原性。纯化蛋白免疫小鼠后,获得了抗S蛋白的特异性血清抗体,ELISA效价最高可达1:100。
Transmissible gastroenteritis (TGE) has been reported in many parts of the world, including America, Asia and Europe. The disease is spread by the faeco-oral route and is characterised by vomiting, diarrhoea and high mortality in piglets. The causative agent is a coronavirus (TGEV), which is a member of the Coronaviridae family and belongs to the order Nidovirales. This study includes three parts.
PartⅠThe acquaintance of the proliferation of TGEV in ST cells
As the host of the TGEV, ST cell was revived and cultured. At fist, the tissue culture infective dose (TCID50) of the TGEV was determinated to measure the cytotoxicity. To confirmed the proliferation of TGEV, cytopathic effect (CPE) and Immunoperoxidase monolayer assay was observed by the Optical Microscopy. The CPE result showed that nucleus had been denatured or integrity was fully destroied. The IPMA confirmed the place of the sythesis of the protein was cytoplasm.
PartⅡExperimental study on anthocyanin resisting transmissible gastroenteritis virus in vitro
In the patrtⅠ, the TCID50 of TGEV had been determinated. By the observation of the morphological changes, the cytotoxic influence, directly inactivited action and treatment action of the procyanidins were studied. The results showed that the safe concentration was not more than 0.002 gram per milliliter, in fact 1 milligram per milliliter. The optimal dose of directly inactivited action was confirmed to be 500 microliter and the treatment dose be 500-375 microliter. This part is only about the study in vitro, since the result is not identical to the study of animals. More animal experiment on procyanidins resisting TGEV needs to be done further.
PartⅢSecretory expression of the spike glycoprotein of transmissible gastroenteritis virus in yeast Pichia pastoris and analysis on Immunogenicity of the recombinant protein
In this study, A pair of primers were disigned according to TGEV S gene sequence in Genbank. After amplified by Polymerase Chain Reaction, a 2.2 kb fragment of the amino terminal half of the S gene containing all four major antigenic sites (A, B, C and D) was sub-cloned into pBS-T vector, then S gene was ligated with vector pPIC9K after cleaved from T-vector,.the recombinant shuttle plasmids pPIC9k-S were linearized by SalI and transformed into competent Pichia pastoris GS115 by electroporation and positive clones were screened with G418 and PCR amplification and induced with 1% methanol. At last, the supernatant of medium was analyzed by SDS-PAGE and Western-blotting and purified by ion exchange chromatography and ultrafiltration. A group of 2-week old BALB/c mouse were injected with the purified protein and the serum was detected by ELISA. The results showed that the partial fragment of spike protein (S), a approximately molecular weight 82 000 soluble protein, was secreted into the culture supernatant. The western-blotting results indicated that the protein could be recognized by the positive swine’s serum of TGEV. After diluted into 1:100, the specific antibody against S protein could be detected by ELISA.
第一部分:猪传染性胃肠炎病毒的增殖特点的认识。
本试验复苏和传代培养了TGEV宿主细胞-ST,测定TGEV的半数细胞培养物感染剂量(TCID50),以便于病毒感染毒性的定量,显微镜下观察TGEV吸附ST细胞后引起的细胞病变现象,免疫过氧化物酶单层试验(IPMA)进一步认识TGEV的增殖规律。细胞病变观察结果显示,TGEV侵染ST细胞后可引起具有“种”的特异性的细胞病变,引起细胞核变形或碎裂,甚至导致细胞的完全破坏,IPMA证明TGEV蛋白质的合成在胞浆内。
第二部分:花青素体外抗猪传染性胃肠炎病毒的试验研究。
在已测定TGEV的TCID50的基础上,利用细胞形态学观察方法,具体研究了花青素对ST细胞的毒性及花青素对TGEV的直接灭活、治疗作用。结果表明:花青素在ST细胞上最大安全浓度为0.002g/ml(纯度5%),即1mg/ml为实际花青素的最大安全浓度;花青素在ST细胞上对抗TGEV的最佳用量为0.002g/ml 500μl;起到治疗作用的花青素用量为0.002g/ml 500μl、375μl,但是花青素只是在ST细胞感染TGEV一定时间内对该病毒起到了治疗作用,而且体外试验结果不能完全等同于体内的治疗效果,所以体内抗病毒效果有待于进一步试验加以验证。
第三部分:猪传染性胃肠炎病毒纤突糖蛋白在毕赤酵母中的分泌表达及免疫原性分析。
根据GenBank上猪传染性胃肠炎病毒纤突糖蛋白S全基因序列设计一对引物,并在5’引物和3’引物中引入EcoR I、Not I酶切位点,经PCR扩增出长2.2kb的目的基因S(其中包含A、B、C、D四个主要抗原位点),克隆于pBS-T载体,克隆载体TS经EcoR I、Not I双酶切,S基因与pPIC9k载体连接,测序验证阳性克隆,Sal I线性化重组穿梭质粒pPIC9k-S,电转化GS115感受态,G418筛选和PCR鉴定得到阳性重组子,1%甲醇诱导表达,诱导后上清经SDS-PAGE电泳、免疫印迹和薄层凝胶扫描分析,并利用离子交换技术和膜超滤方法纯化目的蛋白,将纯化后的蛋白免疫两周龄BALB/c小鼠。SDS-PAGE电泳表明,S蛋白以可溶性形式分泌表达于胞外,且表达产物的分子量约为82 000,与理论推测的分子量一致。免疫印迹结果证明,S蛋白可被TGEV猪阳性血清识别,且具有良好的免疫原性。纯化蛋白免疫小鼠后,获得了抗S蛋白的特异性血清抗体,ELISA效价最高可达1:100。
Transmissible gastroenteritis (TGE) has been reported in many parts of the world, including America, Asia and Europe. The disease is spread by the faeco-oral route and is characterised by vomiting, diarrhoea and high mortality in piglets. The causative agent is a coronavirus (TGEV), which is a member of the Coronaviridae family and belongs to the order Nidovirales. This study includes three parts.
PartⅠThe acquaintance of the proliferation of TGEV in ST cells
As the host of the TGEV, ST cell was revived and cultured. At fist, the tissue culture infective dose (TCID50) of the TGEV was determinated to measure the cytotoxicity. To confirmed the proliferation of TGEV, cytopathic effect (CPE) and Immunoperoxidase monolayer assay was observed by the Optical Microscopy. The CPE result showed that nucleus had been denatured or integrity was fully destroied. The IPMA confirmed the place of the sythesis of the protein was cytoplasm.
PartⅡExperimental study on anthocyanin resisting transmissible gastroenteritis virus in vitro
In the patrtⅠ, the TCID50 of TGEV had been determinated. By the observation of the morphological changes, the cytotoxic influence, directly inactivited action and treatment action of the procyanidins were studied. The results showed that the safe concentration was not more than 0.002 gram per milliliter, in fact 1 milligram per milliliter. The optimal dose of directly inactivited action was confirmed to be 500 microliter and the treatment dose be 500-375 microliter. This part is only about the study in vitro, since the result is not identical to the study of animals. More animal experiment on procyanidins resisting TGEV needs to be done further.
PartⅢSecretory expression of the spike glycoprotein of transmissible gastroenteritis virus in yeast Pichia pastoris and analysis on Immunogenicity of the recombinant protein
In this study, A pair of primers were disigned according to TGEV S gene sequence in Genbank. After amplified by Polymerase Chain Reaction, a 2.2 kb fragment of the amino terminal half of the S gene containing all four major antigenic sites (A, B, C and D) was sub-cloned into pBS-T vector, then S gene was ligated with vector pPIC9K after cleaved from T-vector,.the recombinant shuttle plasmids pPIC9k-S were linearized by SalI and transformed into competent Pichia pastoris GS115 by electroporation and positive clones were screened with G418 and PCR amplification and induced with 1% methanol. At last, the supernatant of medium was analyzed by SDS-PAGE and Western-blotting and purified by ion exchange chromatography and ultrafiltration. A group of 2-week old BALB/c mouse were injected with the purified protein and the serum was detected by ELISA. The results showed that the partial fragment of spike protein (S), a approximately molecular weight 82 000 soluble protein, was secreted into the culture supernatant. The western-blotting results indicated that the protein could be recognized by the positive swine’s serum of TGEV. After diluted into 1:100, the specific antibody against S protein could be detected by ELISA.
引文
[1]陈志瑾,丛延广,周莹冰等.毕赤酵母表达肽抗生素hPAB2β的纯化[J].免疫学杂志, 2008, 24(3): 332-336.
[2]杜晓芬,谢笔钧,杨尔宁等.莲房原花青素对二甲基苯芘蒽诱发金黄地鼠口腔癌的预防作用[J].营养学报,2005,27(3):241-244.
[3]耿珊珊,蔡东联.原花青素对血脂及氧化低密度脂蛋白的影响[J].国外医学卫生学分册,2005,32(2):76-80.
[4]侯敢,黄迪南.原花青素对肿瘤坏死因子a诱导HeIa细胞凋亡和caspase-8活化的影响[J].中草药,2005,36(9):1349-1352.
[5]洪国强,胡波,李朝霞. HIV-1膜蛋白基因gp120和gp41的合成及在毕赤酵母中的表达[J].广东医学杂志, 2008, 29(2): 198-200.
[6]贺小英,彭树英,刘艳春等.猪传染性胃肠炎病毒主要抗原基因S与M双基因共表达载体的构建[J].中国兽医杂志, 2007, 43 (11):6-9.
[7]刘忠渊,张富春,毛新芳等.利用毕赤酵母表达外源蛋白的研究[J].生物技术,2004,14(1): 56-58.
[8]李军,林继煌,陆承平等. RT-PCR检测猪传染性胃肠炎病毒[J].中国兽医学报,2001,3: 246-248.
[9]梁敏坚,洪国强,李朝霞.乙型肝炎病毒核心基因在毕赤酵母中的表达及表达产物在乙型肝炎病毒核心抗体检测中的应用评价[J].中华检验医学杂志, 2005, 28(4): 417-421.
[10]李新丽,郭二兵,于娜等.毕赤酵母表达人Neuritin蛋白的条件优化、纯化及鉴定[J].石河子大学学报, 2008, 26 (3): 316-321.
[11]李文广,张小郁.葡萄籽中原花青素的抗炎作用和机制[J].中国药理学报,2001,22(12):1117-1120.
[12]李莹,药立波,韩炯等.葡萄籽提取物原花青素诱导胃癌细胞脱落凋亡[J].中国药理学通报,2004,20(7):761-764.
[12]陆茵,孙志广,赵万洲等.葡萄籽原花青素对蛋白激酶C及鸟氨酸脱羧酶活性的影响[J].山东中医药大学学报,2003,27 (5):385-388.
[13]欧阳菁,杨林,龙綮新等.猪生长激素基因在巴斯德毕赤酵母中的高效分泌表达及产物的N2糖基化分析[J].生物工程学报, 2001, 17(5): 520-524.
[14]欧阳立明,张惠展,张嗣良等.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理学报,2000,27(2):151-154.
[15]钱刚,冯力,刘胜旺等.猪传染性胃肠炎病毒纤突蛋白基因的修饰及原核表达的研究[J].东北农业大学学报, 2005, 36 (5): 611-614.
[16]苏万国,唐永兰,邹勇等.猪传染性胃肠炎病毒在PK15和ST细胞上的增殖特性比较[J].上海畜牧畜医通讯,2000,3:4.
[17]宋淼,刘波,王越等.重组人粒细胞-巨噬细胞集落刺激因子在毕赤酵母中的表达及活性分析[J].生物技术通讯,2008, 19(6): 847-851.
[18]宋磊,刘红军,刘宁.葡激酶在毕赤酵母中的表达及纯化[J].中国实用医药, 2008, 3(24): 35-37.
[19]尚书文,侯继波,徐公豹等.禽流感病毒M2e基因与鸡IgG Fc基因在巴斯德毕赤酵母中的融合表达[J].西北农林科技大学学报, 2008, 36(1):11-22.
[20]孙怡,商学军,葛京平等.原花青素对前列腺癌PG3细胞增殖和凋亡的影响[J].山东医药,2006,46(6):12-14.
[21]温茂荣,高新,张倩等.长链胰岛素样生长因子1在毕赤酵母中的表达、纯化及活性分析[J] .军事医学科学院院刊,2008, 32(5): 439-442.
[22]王勇.毕赤酵母表达外源基因研究进展[J].微生物学免疫学进展,2004,32(1):62-66.
[23]吴春,代丽君,聂芊.原花青素对亚硝化反应的抑制作用研究[J].天然产物研究与开发,2005,17(2):213-215.
[24]吴春,陆海燕,代丽君.原花青素的抗氧化活性研究[J].哈尔滨商业大学学报,2005,21(4):457-460.
[25]吴秀香,杜莉莉,卢晓梅等.葡萄籽原花青素对小鼠脑缺血一再灌注及缺氧性损伤的影响[J].中国康复医学杂志,2006,26 (2): 145-148.
[26]徐秀云,杨湘越,兰小鹏.利用酵母重组SSB抗原建立快速斑点免疫金渗滤法检测抗SSB抗体[J].临床检验杂志, 2008, 26(6): 448-449.
[27]徐晓云,潘思轶,谢笔钧等.沙棘籽原花青素体外抗氧化活性研究[J].食品科学,2005,26(2):216-218.
[28]姚清侠,钱平,曹毅等.猪α-2干扰素在毕赤酵母中的分泌表达及其生物活性测定[J].中国兽医学报,2008, 28(12): 1456-1460.
[29]殷震,刘景华主编.动物病毒学(第2版)[M].北京科学出版社, 1997, 204-246.
[30]由倍安,高海青,张向红.葡萄籽原花青素对兔实验性动脉粥样硬化的干预研究[J].中国老年学杂志,2005,25(11):1389-1391.
[31]郑光宇.真核生物表达系统研究进展[J].喀什师范学院学报, 2004, 25(6):33-36.
[32]张小郁,李文广,郑天珍.葡萄籽中原花青素对实验动物主动脉收缩和血小板聚集的影响[J].中国应用生理学杂志,2005,21(4):383-386.
[33]郑光耀.葡萄籽原花青索提取物的生理活性、药理作用及其应用[J].林产化工通讯,2000,34(1): 28-33.
[34]张冬梅.恶性疟裂殖子表面蛋白1(MSP1)合成基因在毕赤酵母中的表达[J].生物工程学报,2000,6:723-725.
[35]张晓晖,张斌,龚培力等.莲房原花青素对大鼠心肌缺血再灌注损伤的保护作用[J].药学学报,2004,39(6):401-405.
[36]张绍松,陈永青,郑伟军等.枯草芽孢杆菌bgIS基因在酵母染色体上整合[J].生物工程学报,1995,11(3):266-271.
[37] Agarwal C, Sharma Y, Agarwal R. Studies of procyanidins on DU145 cells of human proststic cancer[J]. Mol Carcinog, 2000, 28(3): 129.
[38] Agarwal D, Stefanadis C, Toutouzas K. Statin treatment is associated threduced thermal heterogeneity in human athemselerotic plagues[J]. Carcinogen, 2002, 23(11): 1664-1669.
[39] Anchez C M, Gebauer F, Sune C, et al. Genetic evolution and tropism of transmissible gastroenteritis coronaviuses[J]. Vird, 1992, 190: 92-105.
[40] Bagchi D, Bagchi M, Stohs S J, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention[J]. Toxicology, 2000, 148 (23): 187-197.
[41] Bagchi D, Garg A, Krohn RL,et al. Oxygen free radical scavenging abilities of vitamins C and E and a grape seed proanthocyan idinextract in vitro[J]. Res Commun Mol Pathol Pharmacol, 1997, 95(2):179-189.
[42] Barbara M, Schmidt, John W. Differential effects of blueberry proanthocyanidins onandrogen sensitive and insensitive human prostate cancer cell lines[J]. Cancer Letters, 2006, 231(2):240-246.
[43] Britton P S, Kottier C M, Chen D H, et al . The use of PCR genome mapping for the characterization of TGEV strains[M]. Plenum Press, New York, 1994 :29-43.
[44] Britton P, Page K W. Sequence of t he S gene f rom a virulent British field isolate of transmissible gastroenteritis virus[J]. Virus Res, 1990, 18(1): 71-80.
[45] Bett A J, Prevec L, Graham F. Packaging capacity of human adenovirus type 5 vectors[J]. J Virol, 1993, 67:591l-5921.
[46] Castillo J, Benavente Garcia O, Lorente J, et a1. An tioxidant activity and radioprotective effects against chromoso mal damage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic andorganic compounds[J]. J Agric Food Chem, 2000, 48(5): 1738-1745.
[47] Chen H, Schifferli D M. Enhanced immune responses to vira epitopes by combining macrophage inducible expression with multimeric display on a Salmonella vector[J]. Vaccine, 2001, 19: 3009-3018.
[48] Chi-PangChuck, Chi-HangWong. Expressionof SARScoronavirus spike glycoprotein in Pichia pastoris[J]. Virus Genes, 2009, 38: 1-9.
[49] Clare J J, Rayment F B, Ballantine S P, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integration of the gene[J]. Bio Technol, 1991, 9(5): 455-460.
[50] Collins A R, Knobler R L, Powell H, et al. Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell-cell fusion[J]. Virology, 982, 119: 358-371.
[51] Correa I, Gebauer F, Bullido M J, et al. Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus[J]. J Gen Virol, 1990, 71: 271-279.
[52] Dauer A, Metzner P, Schimmer O. Proanthocyanidins from the bark of Hamamelis virginian a exhibit antlmutagenic properties against nitroaromatic compounds[J]. Planta Med, 1998 ,64(4): 324-327.
[53] Debasis B, Chandan K, Sidharthan. Molecular mechanisms of cardioprotection by a novel grape seed proanthocy anidin extract[J]. Mutation Research, 2003, 523(1): 87-97.
[54] Delmas B, Laude H. Carbohydrate induced conformational changes strongly modulated the antigenicity of coronavirus TGEV glycoproteins S and M[J]. Virus Res, 1991, 20: 107-120
[55] Delmas B, Laude H. Assembly of coronavirus spike protein into trimers and its role in epitope expression [J]. Virology, 1990, 64 (11): 5367- 5375.
[56] Delmas B, Rasschaert D, Godet M, et al. Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of the spike glycoprotein S[J]. J Gen Virol, 1990,71(1): 313-323.
[57] Dongmo A B, Kam anyi A, An chang M S, et al.Anti-inflammatory and analgesic properties of the stem bark extracts of Erythrophleum suaveolens (Caesatpiniaceae). Guillemin& Perrottet[J].J Ethnopharmacol, 2001, 77(2-3):137-141.
[58] Doyle L P, Hutchings L M. A transmissible gastroenteritis virus in pigs [J]. Am Vet Med Assoc, 1946, 108: 257–259.
[59] Eleouet J F, Rasschaert D, Lambert P, et al. Complete sequence(20 kilobases) of the polyprotein encoding gene 1 of transmissible gastroenteritis[J]. Virus virol, 1995, 206: 817-822.
[60] Godet M, L Haridon R, Vantherot J F, et al. TGEV coronavirus oRF4 encodes a membrane protein that is incorporated into virions[J]. Virology, 1992, 188: 666-675.
[61] Godet M, Stewart F, Higgins D. Processing and antigenicity of entire and anchor free S protein of TGEV expressed by recombinant Baculovirus[J]. Virology, 1991, 185: 732-740.
[62] Gomez N, Wigdorovitz A. Oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus[J]. Arch Virol, 2000, 145(8): 1725-1732.
[63] Gomez N, Carrillo J, Parra F, et al. Expression of immunogenic glycoprotein S polypeptides from TGEV in transgenic plants[J]. Virology, 1998, 249(2): 352-358.
[64] Gregg J M, Tschopp J F, Stillman G, et al. High-level expression and efficientassembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris[J]. Biotechnol Tech, 1987, 5:479-485.
[65] Halbur P, Haridon R L, Vaughn J F, et al. Experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolate AR310[J]. J Vet Diagn Invest, 1993, 5: 184-188.
[66] Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main protease[J]. J Gen Virol, 2002, 83(3): 595-599.
[67] Ho H, Kobayashi E,Takamatsu Y, et a1. Polyphenols from Eriobotrya japonica and their cytotoxicity against human oraltumor cell lines[J]. Chem Pharm Bull, 2000, 48(5): 687-693.
[68] Ho P S, Kwang J, Lee Y K. Intragastric administration of Lactbacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production[J]. Vaccine, 2005, 23(11): 1335-1342.
[69] Huangan C, Chien MS,Hu CM et al. Secreted expression of the classical Swine fever virus glycoprotein E(rns) virus in yeast and application to a sandwich blocking ELISA[J]. J Virol Methods, 2006, 132(1-2): 40-7.
[70] Huynh T H, Teel R W. Selective induction of apoptosis in human mammary cancer cells (MCF-7) by pycnogenol[J]. Anticancer Res, 2000, 20(4): 24l7-2420.
[71] Jackwood D J, Kwon H M, Saif J. Moecular dif ferent of transmissible gastroenteritis virus and porcine respiratory coronavirus strains[J]. Adv Exp Mel Biol, 1995, 380: 35-41.
[72] Jacob P, Bezalel G, Gilad G, et al. Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens[J]. Vaccine, 2003, 21: 4736-4743.
[73] Jacobs L, VanDer, Zeust B A M, et al. Characterization and translation of transmissible gastroenteritis virus mRNAs[J]. Journal of Virol, 1986, 57: 1010-1015.
[74] Jimnez G, Correa I, Melgosa M P, et al. Critical epitopes in transmissible gastroenteritis virus neutralization[J]. J Virol, 1986, 60: 131-139.
[75] Julian A, Hiscox, David C, et al. Quantification of individual subgenomic mRNA species during replication of the transmissible gastroenteritis virus[J]. VirusResearch, 1995, 36: 119-130.
[76] Kevin W, Paul Britton, Michael E G, et al. Sequence analysis of the leader RNA of transmissible gastroenteritis virus[J]. Virus gene, 1990, 4: 289-303.
[77] Kim B, Choi C. In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods[J]. J Vet Res, 2001, 65(1):33-37.
[78] Kim O, Chio C, Kim B. Detection and differentiation of porcine epidemic doarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR[J]. J Vet Res, 2000, 146(22):637-640.
[79] Krempl C, Schultze B, Laude H, et al. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus[J]. J Virol, 1997, 71:3285–3287.
[80] Kobayashi K, Kuwae S, Ohya T, et al. High level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J]. J Biosci Bioeng, 2000, 89: 55-61.
[81] Lai M M C, Patton C D, Baric R S, et al. Presence of lender sequence in the mRNA of mouse hepatitis virus[J]. Journal of virol, 1983, 46: 1027-1033.
[82] Laude H, Reeth K V, Pensaert M. Porcine respiratory coronavirus: molecular features and virus-host interactions[J]. Vet Res, 1993, 24: 125-150.
[83] Liu C, Kokuho T. DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus[J]. Virus Res, 2001, 80(122): 75-82.
[84] Mafei Facino R, Carini M, Aldini G, et a1. Sparing effect to procyanidins from Vitis vinifera on vitamin[J]. Planta Med, 1998, 61(1):343-347.
[85] Nilsson E. Field validation of a commercial block ELISA to differentiate antibody to TGEV and porcine respiratory coronavirus and to identity TGEV-infected swine herds[J]. J Vet Diagn Invest, 2002, 14(2): 97-105.
[86] Nair M P, Kandaswami C, Mahajaw S, et a1. Double-bind studyon Endotelen in Chronic venous insuficiency[J]. Biol Res, 2002, 35 (3-4): 421-431.
[87] Ogata K, Nishikawa H, Ohsugi M. A yeast capable of utilizing methanol[J]. Agric Biol Chem, 1969, 33:1519-1520.
[88] Ortego J I , Sola F , Almazan J E ,et al . Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence[J]. Virol, 2003, 308: 13-22.
[89] Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus mariti-ma) bark, pycnogenol[J]. Fre Radic Biol Med, 1999, 27(5-6): 704-724.
[90] Park J Y, Lavada M, Correa I, et al. Sequence analysis and cDNA probes hybirdization of the nucleocapsid (N) protein gene of TGEV and PCRV [J]. Korean J Vet Res, 1997, 35(3): 515-530.
[91] Paton D, Ibata G, Sands J, et al.Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus[J]. J Virol Methods, 1997, 66(2): 303-309.
[92] Paul B, Kevin W. The cloning and sequencing of the virion protein genes from a British isolate of PRCV: comparison with TGEV[J]. Journal of virus Research, 1991, 21: 181-198.
[93] Peng Shu-ying, Lv Ning, Zhang Yong, et al. Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells[J]. Virus Research, 2007, 128 (12): 52-57.
[94] Piscoya J, Rodriguez Z, Bustamante S A, et a1. Efficacy and safety of freeze-dried cat’s claw inosteoarthritis of the knee: mechanisms of action of the species Uncaria guianensis[J]. Inflamm Res, 2001, 50 (9):442-448.
[95] Pocock D. H. The Influence of pH on the Growth and Stability of Transmissible Gastroenteritis Virus in vitro[J].Achives of Virology, 1975,49: 239-247.
[96] Rein D, Lobito S, Hott, et a1. Platelet rebound effect of alcohol with drawal and wine drinking in rats. Relation to tannins and lipid peroxidation[J]. J Nutr, 2000, 130(8): 2l20-2126.
[97] Riffault S, Baric R, Levy L. In vivo induction of infernoalpha in pig by noninfectious coronavirus: tissue localization and in situ pheno typic characterization of infernoalpha producing cells[J]. J Gen Virol, 1997, 78(10): 2483-2487.
[98] Roca H, Garcia B, Rodriguez E,et al. Cloning of the Penicillium minioluteum geneencoding dextranase and its expression in Pichia pastoris[J]. Yeast, 1996, 12(12): 1187-1200.
[99] Romanos M A, Scorer C A, Clare J J. Foreign gene expression in yeast: a review[J]. Yeast, 1992, 8(6):423-488.
[100] Sanchez C M, Gebauer F, Sune C, et al. Genetic evolution and tropism of transmissible gastroenteritis coronavirus[J]. Virology, 1992, 190: 92–105.
[101] SanChez C M, Izeta A, Sanchez-Morgado J M, et al. Targeted recombination demon strates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence[J]. J Virol, 1999, 73(8): 7607-7618.
[102] Sandoval M, Charbonnet R M, Okuhama N N, et a1. Cats claw inhibits TNFa production and scavenges free radicals: role incytoprotection[J]. Free Radic Biol Med, 2000, 29(1):71-78.
[103] Sato M, Mawlik G, Loag T, et a1. Proanthocyanidin rich Extract from grape seed proanthocyanidin against ischemic reperfusion injury[J]. J Mol Cell Cardionl, 1999, 31(6): 1289.
[104] Shockley L J, Kapke P A, Lappsw. Diagnosis of porcine and bovine enteric coronaviruses infections using cloned cDNA probes[J]. J Clin Microbiol, 1987, 25: 1591-1596.
[105] Shoup D I. Active and passive immunere ponses to TGEV in swine inoculated with recombinant baculovirus expressed TGEV spike glycoprotien vaccines[J]. American Journal of Veterinary Research, 1997, 58(3): 242-250.
[106] Smerdou C, Anton I M. A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity[J]. Virus Res, 1996, 41(1): 129.
[107] Smerdou C. Characterization of TGEV S protein expression products [J]. Vet Microbiol, 1996, 48: 87-100.
[108] Sturman L S, Richard C S, Murine K V. Proteolytic cleavage of the E2 glycoprotein of murine coronavirus by trysin and separation of two different 90K cleavage fragments[J]. Journal of Virol, 1985, 56: 904-911.
[109] Subathira S, Abdul L M, Lucy P M, et a1.Variation in anti-microbial action ofDroanthocvanidins from Dorycnium rectum against rumen bacteria[J]. Phytochemistry, 2004, 65(4): 2485-2497.
[110] Sudheer K M, Mantena, Santosh K, et a1. Grape seed pro-anthocyanidins inhibit UV-radiation-induced oxidative stress andactivation of MAPK and NF-KB signaling in human epiderm alkeratinocytes[J]. Free Radical Biology& Medicine, 2006, 40(9): 1603-1614.
[111] Taguchi F, Fleming J O. Comparison of six different murine coronavius JHM variants by monoelonal antibodies against the E2 glycoprotein[J]. Virology, 1989, 169: 223-235.
[112] Takashi S, Etsuko O, Tsutomu Y, et a1. Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed[J]. Throm-bosis Research, 2005, 115(12): 115-121.
[112] Torres J M, Alonso C, Mittal S, et al. Tropism of human adenovirus type based vectors in swine and their ability to protect against TGEV[J]. Journal of Virology, 1996, 70:3770-3780.
[113] Torres J M, Sanchez C, Sune C, et al. Induction of antibodies protect in against TGEV by recombinant adenovirus expressing TGEV spike protein[J]. Virology, 1995, 213:503-516.
[114] Tuboly T, Yo W, Bailey A, et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants[J]. Vaccine, 2000, 18(19): 2023-2028.
[115] Tuboly T, Nagy E. Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus[J]. Arch Virol, 1994, 137(122): 55-67.
[116] Athmaram T N, Geetha B, GoonG K. Heterologous expression of Blue tongue VP2 viral protein fragment in Pichia pastoris[J]. Virus Genes, 2007, 35: 265-271.
[117] Vanderklei I J, Harder W, Veenhuis M. Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeast: a review[J]. Yeast, 1991, 7(3): 195-209.
[118] Vaughn E M, Halbur P G, Paul P S. Sequence comparison of porcine respiratory coronavirus isolate reveals heterogenecity in the S, 3 and 321 genes[J]. J Virol, 1995,69:3176-3184.
[119] Vaugho E M, Halbur P G, Paul P S. Use of nonradiouctive cDNA to differentiate porcine respiratory coronavirus and transmissible gastroenteritis[J]. J Vet Diagn Invest, 1996, 8(2): 241-244.
[120] Weingartl H M, Derbyshire J B. Binding of porcine transmissible gastroenteritis virus by enterocytes from weaned piglets[J]. Vet Micro Biol, 1993, 35: 23-32.
[121] Wenthworth D E, Holmer K V. Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD31)influence of N-linked glycosylation [J]. J Virol, 2001, 75(20): 9741-9752.
[122] Wesley R, Woods R, Cheung A K. Genetic basis of the pathogenesis of transmissible gastroenteritis virus[J]. Journal of virol, 1990, 64(10): 4761-4766.
[123] Wesley R, Woods R, Cheung A K. Nucleotide sequence of the E2-peplomer gene and partial nucleotide sequence of upstream polymerase gene of transmissible gastroenteritis virus (Miller strain)[J]. Adv Exp Med Biol, 1990, 276: 301-306.
[124] Yamakoshi J, Ktoka S, Loag T, et a1. Priunthocyanidin rich extract from grape seeds attenuaters the develoment of aortic atheroeclerosis in cholesterol fed rRabbits[J]. Atheroeclemsis, 1999, 142 (1): 139-142.
[125] Yamakoshi J, Kataoka S, Koga T, et a1. Proanthocyanidin-rich extract from grape seeds attenuates the developm ent of aortic atherosclerosis in cholesterol-fed rabbits[J]. Atherosclerosis, l999, l42(1): l39-149.
[126] Ye X, Krohn R L, Liu W, et a1. The cytotoxic effects of a novel lH636 grape seed proanthocyanidin extract on cultured humancancer cells[J]. Mol Cell Biochem, 1999, 196(2): 99-108.
[2]杜晓芬,谢笔钧,杨尔宁等.莲房原花青素对二甲基苯芘蒽诱发金黄地鼠口腔癌的预防作用[J].营养学报,2005,27(3):241-244.
[3]耿珊珊,蔡东联.原花青素对血脂及氧化低密度脂蛋白的影响[J].国外医学卫生学分册,2005,32(2):76-80.
[4]侯敢,黄迪南.原花青素对肿瘤坏死因子a诱导HeIa细胞凋亡和caspase-8活化的影响[J].中草药,2005,36(9):1349-1352.
[5]洪国强,胡波,李朝霞. HIV-1膜蛋白基因gp120和gp41的合成及在毕赤酵母中的表达[J].广东医学杂志, 2008, 29(2): 198-200.
[6]贺小英,彭树英,刘艳春等.猪传染性胃肠炎病毒主要抗原基因S与M双基因共表达载体的构建[J].中国兽医杂志, 2007, 43 (11):6-9.
[7]刘忠渊,张富春,毛新芳等.利用毕赤酵母表达外源蛋白的研究[J].生物技术,2004,14(1): 56-58.
[8]李军,林继煌,陆承平等. RT-PCR检测猪传染性胃肠炎病毒[J].中国兽医学报,2001,3: 246-248.
[9]梁敏坚,洪国强,李朝霞.乙型肝炎病毒核心基因在毕赤酵母中的表达及表达产物在乙型肝炎病毒核心抗体检测中的应用评价[J].中华检验医学杂志, 2005, 28(4): 417-421.
[10]李新丽,郭二兵,于娜等.毕赤酵母表达人Neuritin蛋白的条件优化、纯化及鉴定[J].石河子大学学报, 2008, 26 (3): 316-321.
[11]李文广,张小郁.葡萄籽中原花青素的抗炎作用和机制[J].中国药理学报,2001,22(12):1117-1120.
[12]李莹,药立波,韩炯等.葡萄籽提取物原花青素诱导胃癌细胞脱落凋亡[J].中国药理学通报,2004,20(7):761-764.
[12]陆茵,孙志广,赵万洲等.葡萄籽原花青素对蛋白激酶C及鸟氨酸脱羧酶活性的影响[J].山东中医药大学学报,2003,27 (5):385-388.
[13]欧阳菁,杨林,龙綮新等.猪生长激素基因在巴斯德毕赤酵母中的高效分泌表达及产物的N2糖基化分析[J].生物工程学报, 2001, 17(5): 520-524.
[14]欧阳立明,张惠展,张嗣良等.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理学报,2000,27(2):151-154.
[15]钱刚,冯力,刘胜旺等.猪传染性胃肠炎病毒纤突蛋白基因的修饰及原核表达的研究[J].东北农业大学学报, 2005, 36 (5): 611-614.
[16]苏万国,唐永兰,邹勇等.猪传染性胃肠炎病毒在PK15和ST细胞上的增殖特性比较[J].上海畜牧畜医通讯,2000,3:4.
[17]宋淼,刘波,王越等.重组人粒细胞-巨噬细胞集落刺激因子在毕赤酵母中的表达及活性分析[J].生物技术通讯,2008, 19(6): 847-851.
[18]宋磊,刘红军,刘宁.葡激酶在毕赤酵母中的表达及纯化[J].中国实用医药, 2008, 3(24): 35-37.
[19]尚书文,侯继波,徐公豹等.禽流感病毒M2e基因与鸡IgG Fc基因在巴斯德毕赤酵母中的融合表达[J].西北农林科技大学学报, 2008, 36(1):11-22.
[20]孙怡,商学军,葛京平等.原花青素对前列腺癌PG3细胞增殖和凋亡的影响[J].山东医药,2006,46(6):12-14.
[21]温茂荣,高新,张倩等.长链胰岛素样生长因子1在毕赤酵母中的表达、纯化及活性分析[J] .军事医学科学院院刊,2008, 32(5): 439-442.
[22]王勇.毕赤酵母表达外源基因研究进展[J].微生物学免疫学进展,2004,32(1):62-66.
[23]吴春,代丽君,聂芊.原花青素对亚硝化反应的抑制作用研究[J].天然产物研究与开发,2005,17(2):213-215.
[24]吴春,陆海燕,代丽君.原花青素的抗氧化活性研究[J].哈尔滨商业大学学报,2005,21(4):457-460.
[25]吴秀香,杜莉莉,卢晓梅等.葡萄籽原花青素对小鼠脑缺血一再灌注及缺氧性损伤的影响[J].中国康复医学杂志,2006,26 (2): 145-148.
[26]徐秀云,杨湘越,兰小鹏.利用酵母重组SSB抗原建立快速斑点免疫金渗滤法检测抗SSB抗体[J].临床检验杂志, 2008, 26(6): 448-449.
[27]徐晓云,潘思轶,谢笔钧等.沙棘籽原花青素体外抗氧化活性研究[J].食品科学,2005,26(2):216-218.
[28]姚清侠,钱平,曹毅等.猪α-2干扰素在毕赤酵母中的分泌表达及其生物活性测定[J].中国兽医学报,2008, 28(12): 1456-1460.
[29]殷震,刘景华主编.动物病毒学(第2版)[M].北京科学出版社, 1997, 204-246.
[30]由倍安,高海青,张向红.葡萄籽原花青素对兔实验性动脉粥样硬化的干预研究[J].中国老年学杂志,2005,25(11):1389-1391.
[31]郑光宇.真核生物表达系统研究进展[J].喀什师范学院学报, 2004, 25(6):33-36.
[32]张小郁,李文广,郑天珍.葡萄籽中原花青素对实验动物主动脉收缩和血小板聚集的影响[J].中国应用生理学杂志,2005,21(4):383-386.
[33]郑光耀.葡萄籽原花青索提取物的生理活性、药理作用及其应用[J].林产化工通讯,2000,34(1): 28-33.
[34]张冬梅.恶性疟裂殖子表面蛋白1(MSP1)合成基因在毕赤酵母中的表达[J].生物工程学报,2000,6:723-725.
[35]张晓晖,张斌,龚培力等.莲房原花青素对大鼠心肌缺血再灌注损伤的保护作用[J].药学学报,2004,39(6):401-405.
[36]张绍松,陈永青,郑伟军等.枯草芽孢杆菌bgIS基因在酵母染色体上整合[J].生物工程学报,1995,11(3):266-271.
[37] Agarwal C, Sharma Y, Agarwal R. Studies of procyanidins on DU145 cells of human proststic cancer[J]. Mol Carcinog, 2000, 28(3): 129.
[38] Agarwal D, Stefanadis C, Toutouzas K. Statin treatment is associated threduced thermal heterogeneity in human athemselerotic plagues[J]. Carcinogen, 2002, 23(11): 1664-1669.
[39] Anchez C M, Gebauer F, Sune C, et al. Genetic evolution and tropism of transmissible gastroenteritis coronaviuses[J]. Vird, 1992, 190: 92-105.
[40] Bagchi D, Bagchi M, Stohs S J, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention[J]. Toxicology, 2000, 148 (23): 187-197.
[41] Bagchi D, Garg A, Krohn RL,et al. Oxygen free radical scavenging abilities of vitamins C and E and a grape seed proanthocyan idinextract in vitro[J]. Res Commun Mol Pathol Pharmacol, 1997, 95(2):179-189.
[42] Barbara M, Schmidt, John W. Differential effects of blueberry proanthocyanidins onandrogen sensitive and insensitive human prostate cancer cell lines[J]. Cancer Letters, 2006, 231(2):240-246.
[43] Britton P S, Kottier C M, Chen D H, et al . The use of PCR genome mapping for the characterization of TGEV strains[M]. Plenum Press, New York, 1994 :29-43.
[44] Britton P, Page K W. Sequence of t he S gene f rom a virulent British field isolate of transmissible gastroenteritis virus[J]. Virus Res, 1990, 18(1): 71-80.
[45] Bett A J, Prevec L, Graham F. Packaging capacity of human adenovirus type 5 vectors[J]. J Virol, 1993, 67:591l-5921.
[46] Castillo J, Benavente Garcia O, Lorente J, et a1. An tioxidant activity and radioprotective effects against chromoso mal damage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic andorganic compounds[J]. J Agric Food Chem, 2000, 48(5): 1738-1745.
[47] Chen H, Schifferli D M. Enhanced immune responses to vira epitopes by combining macrophage inducible expression with multimeric display on a Salmonella vector[J]. Vaccine, 2001, 19: 3009-3018.
[48] Chi-PangChuck, Chi-HangWong. Expressionof SARScoronavirus spike glycoprotein in Pichia pastoris[J]. Virus Genes, 2009, 38: 1-9.
[49] Clare J J, Rayment F B, Ballantine S P, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integration of the gene[J]. Bio Technol, 1991, 9(5): 455-460.
[50] Collins A R, Knobler R L, Powell H, et al. Monoclonal antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell-cell fusion[J]. Virology, 982, 119: 358-371.
[51] Correa I, Gebauer F, Bullido M J, et al. Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus[J]. J Gen Virol, 1990, 71: 271-279.
[52] Dauer A, Metzner P, Schimmer O. Proanthocyanidins from the bark of Hamamelis virginian a exhibit antlmutagenic properties against nitroaromatic compounds[J]. Planta Med, 1998 ,64(4): 324-327.
[53] Debasis B, Chandan K, Sidharthan. Molecular mechanisms of cardioprotection by a novel grape seed proanthocy anidin extract[J]. Mutation Research, 2003, 523(1): 87-97.
[54] Delmas B, Laude H. Carbohydrate induced conformational changes strongly modulated the antigenicity of coronavirus TGEV glycoproteins S and M[J]. Virus Res, 1991, 20: 107-120
[55] Delmas B, Laude H. Assembly of coronavirus spike protein into trimers and its role in epitope expression [J]. Virology, 1990, 64 (11): 5367- 5375.
[56] Delmas B, Rasschaert D, Godet M, et al. Four major antigenic sites of the coronavirus transmissible gastroenteritis virus are located on the amino-terminal half of the spike glycoprotein S[J]. J Gen Virol, 1990,71(1): 313-323.
[57] Dongmo A B, Kam anyi A, An chang M S, et al.Anti-inflammatory and analgesic properties of the stem bark extracts of Erythrophleum suaveolens (Caesatpiniaceae). Guillemin& Perrottet[J].J Ethnopharmacol, 2001, 77(2-3):137-141.
[58] Doyle L P, Hutchings L M. A transmissible gastroenteritis virus in pigs [J]. Am Vet Med Assoc, 1946, 108: 257–259.
[59] Eleouet J F, Rasschaert D, Lambert P, et al. Complete sequence(20 kilobases) of the polyprotein encoding gene 1 of transmissible gastroenteritis[J]. Virus virol, 1995, 206: 817-822.
[60] Godet M, L Haridon R, Vantherot J F, et al. TGEV coronavirus oRF4 encodes a membrane protein that is incorporated into virions[J]. Virology, 1992, 188: 666-675.
[61] Godet M, Stewart F, Higgins D. Processing and antigenicity of entire and anchor free S protein of TGEV expressed by recombinant Baculovirus[J]. Virology, 1991, 185: 732-740.
[62] Gomez N, Wigdorovitz A. Oral immunogenicity of the plant derived spike protein from swine transmissible gastroenteritis coronavirus[J]. Arch Virol, 2000, 145(8): 1725-1732.
[63] Gomez N, Carrillo J, Parra F, et al. Expression of immunogenic glycoprotein S polypeptides from TGEV in transgenic plants[J]. Virology, 1998, 249(2): 352-358.
[64] Gregg J M, Tschopp J F, Stillman G, et al. High-level expression and efficientassembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris[J]. Biotechnol Tech, 1987, 5:479-485.
[65] Halbur P, Haridon R L, Vaughn J F, et al. Experimental reproduction of pneumonia in gnotobiotic pigs with porcine respiratory coronavirus isolate AR310[J]. J Vet Diagn Invest, 1993, 5: 184-188.
[66] Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main protease[J]. J Gen Virol, 2002, 83(3): 595-599.
[67] Ho H, Kobayashi E,Takamatsu Y, et a1. Polyphenols from Eriobotrya japonica and their cytotoxicity against human oraltumor cell lines[J]. Chem Pharm Bull, 2000, 48(5): 687-693.
[68] Ho P S, Kwang J, Lee Y K. Intragastric administration of Lactbacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production[J]. Vaccine, 2005, 23(11): 1335-1342.
[69] Huangan C, Chien MS,Hu CM et al. Secreted expression of the classical Swine fever virus glycoprotein E(rns) virus in yeast and application to a sandwich blocking ELISA[J]. J Virol Methods, 2006, 132(1-2): 40-7.
[70] Huynh T H, Teel R W. Selective induction of apoptosis in human mammary cancer cells (MCF-7) by pycnogenol[J]. Anticancer Res, 2000, 20(4): 24l7-2420.
[71] Jackwood D J, Kwon H M, Saif J. Moecular dif ferent of transmissible gastroenteritis virus and porcine respiratory coronavirus strains[J]. Adv Exp Mel Biol, 1995, 380: 35-41.
[72] Jacob P, Bezalel G, Gilad G, et al. Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens[J]. Vaccine, 2003, 21: 4736-4743.
[73] Jacobs L, VanDer, Zeust B A M, et al. Characterization and translation of transmissible gastroenteritis virus mRNAs[J]. Journal of Virol, 1986, 57: 1010-1015.
[74] Jimnez G, Correa I, Melgosa M P, et al. Critical epitopes in transmissible gastroenteritis virus neutralization[J]. J Virol, 1986, 60: 131-139.
[75] Julian A, Hiscox, David C, et al. Quantification of individual subgenomic mRNA species during replication of the transmissible gastroenteritis virus[J]. VirusResearch, 1995, 36: 119-130.
[76] Kevin W, Paul Britton, Michael E G, et al. Sequence analysis of the leader RNA of transmissible gastroenteritis virus[J]. Virus gene, 1990, 4: 289-303.
[77] Kim B, Choi C. In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods[J]. J Vet Res, 2001, 65(1):33-37.
[78] Kim O, Chio C, Kim B. Detection and differentiation of porcine epidemic doarrhoea virus and transmissible gastroenteritis virus in clinical samples by multiplex RT-PCR[J]. J Vet Res, 2000, 146(22):637-640.
[79] Krempl C, Schultze B, Laude H, et al. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus[J]. J Virol, 1997, 71:3285–3287.
[80] Kobayashi K, Kuwae S, Ohya T, et al. High level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J]. J Biosci Bioeng, 2000, 89: 55-61.
[81] Lai M M C, Patton C D, Baric R S, et al. Presence of lender sequence in the mRNA of mouse hepatitis virus[J]. Journal of virol, 1983, 46: 1027-1033.
[82] Laude H, Reeth K V, Pensaert M. Porcine respiratory coronavirus: molecular features and virus-host interactions[J]. Vet Res, 1993, 24: 125-150.
[83] Liu C, Kokuho T. DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus[J]. Virus Res, 2001, 80(122): 75-82.
[84] Mafei Facino R, Carini M, Aldini G, et a1. Sparing effect to procyanidins from Vitis vinifera on vitamin[J]. Planta Med, 1998, 61(1):343-347.
[85] Nilsson E. Field validation of a commercial block ELISA to differentiate antibody to TGEV and porcine respiratory coronavirus and to identity TGEV-infected swine herds[J]. J Vet Diagn Invest, 2002, 14(2): 97-105.
[86] Nair M P, Kandaswami C, Mahajaw S, et a1. Double-bind studyon Endotelen in Chronic venous insuficiency[J]. Biol Res, 2002, 35 (3-4): 421-431.
[87] Ogata K, Nishikawa H, Ohsugi M. A yeast capable of utilizing methanol[J]. Agric Biol Chem, 1969, 33:1519-1520.
[88] Ortego J I , Sola F , Almazan J E ,et al . Transmissible gastroenteritis coronavirus gene 7 is not essential but influences in vivo virus replication and virulence[J]. Virol, 2003, 308: 13-22.
[89] Packer L, Rimbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus mariti-ma) bark, pycnogenol[J]. Fre Radic Biol Med, 1999, 27(5-6): 704-724.
[90] Park J Y, Lavada M, Correa I, et al. Sequence analysis and cDNA probes hybirdization of the nucleocapsid (N) protein gene of TGEV and PCRV [J]. Korean J Vet Res, 1997, 35(3): 515-530.
[91] Paton D, Ibata G, Sands J, et al.Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus[J]. J Virol Methods, 1997, 66(2): 303-309.
[92] Paul B, Kevin W. The cloning and sequencing of the virion protein genes from a British isolate of PRCV: comparison with TGEV[J]. Journal of virus Research, 1991, 21: 181-198.
[93] Peng Shu-ying, Lv Ning, Zhang Yong, et al. Immune response induced by spike protein from transmissible gastroenteritis coronavirus expressed in mouse mammary cells[J]. Virus Research, 2007, 128 (12): 52-57.
[94] Piscoya J, Rodriguez Z, Bustamante S A, et a1. Efficacy and safety of freeze-dried cat’s claw inosteoarthritis of the knee: mechanisms of action of the species Uncaria guianensis[J]. Inflamm Res, 2001, 50 (9):442-448.
[95] Pocock D. H. The Influence of pH on the Growth and Stability of Transmissible Gastroenteritis Virus in vitro[J].Achives of Virology, 1975,49: 239-247.
[96] Rein D, Lobito S, Hott, et a1. Platelet rebound effect of alcohol with drawal and wine drinking in rats. Relation to tannins and lipid peroxidation[J]. J Nutr, 2000, 130(8): 2l20-2126.
[97] Riffault S, Baric R, Levy L. In vivo induction of infernoalpha in pig by noninfectious coronavirus: tissue localization and in situ pheno typic characterization of infernoalpha producing cells[J]. J Gen Virol, 1997, 78(10): 2483-2487.
[98] Roca H, Garcia B, Rodriguez E,et al. Cloning of the Penicillium minioluteum geneencoding dextranase and its expression in Pichia pastoris[J]. Yeast, 1996, 12(12): 1187-1200.
[99] Romanos M A, Scorer C A, Clare J J. Foreign gene expression in yeast: a review[J]. Yeast, 1992, 8(6):423-488.
[100] Sanchez C M, Gebauer F, Sune C, et al. Genetic evolution and tropism of transmissible gastroenteritis coronavirus[J]. Virology, 1992, 190: 92–105.
[101] SanChez C M, Izeta A, Sanchez-Morgado J M, et al. Targeted recombination demon strates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence[J]. J Virol, 1999, 73(8): 7607-7618.
[102] Sandoval M, Charbonnet R M, Okuhama N N, et a1. Cats claw inhibits TNFa production and scavenges free radicals: role incytoprotection[J]. Free Radic Biol Med, 2000, 29(1):71-78.
[103] Sato M, Mawlik G, Loag T, et a1. Proanthocyanidin rich Extract from grape seed proanthocyanidin against ischemic reperfusion injury[J]. J Mol Cell Cardionl, 1999, 31(6): 1289.
[104] Shockley L J, Kapke P A, Lappsw. Diagnosis of porcine and bovine enteric coronaviruses infections using cloned cDNA probes[J]. J Clin Microbiol, 1987, 25: 1591-1596.
[105] Shoup D I. Active and passive immunere ponses to TGEV in swine inoculated with recombinant baculovirus expressed TGEV spike glycoprotien vaccines[J]. American Journal of Veterinary Research, 1997, 58(3): 242-250.
[106] Smerdou C, Anton I M. A continuous epitope from transmissible gastroenteritis virus S protein fused to E. coli heat labile toxin B subunit expressed by attenuated Salmonella induces serum and secretory immunity[J]. Virus Res, 1996, 41(1): 129.
[107] Smerdou C. Characterization of TGEV S protein expression products [J]. Vet Microbiol, 1996, 48: 87-100.
[108] Sturman L S, Richard C S, Murine K V. Proteolytic cleavage of the E2 glycoprotein of murine coronavirus by trysin and separation of two different 90K cleavage fragments[J]. Journal of Virol, 1985, 56: 904-911.
[109] Subathira S, Abdul L M, Lucy P M, et a1.Variation in anti-microbial action ofDroanthocvanidins from Dorycnium rectum against rumen bacteria[J]. Phytochemistry, 2004, 65(4): 2485-2497.
[110] Sudheer K M, Mantena, Santosh K, et a1. Grape seed pro-anthocyanidins inhibit UV-radiation-induced oxidative stress andactivation of MAPK and NF-KB signaling in human epiderm alkeratinocytes[J]. Free Radical Biology& Medicine, 2006, 40(9): 1603-1614.
[111] Taguchi F, Fleming J O. Comparison of six different murine coronavius JHM variants by monoelonal antibodies against the E2 glycoprotein[J]. Virology, 1989, 169: 223-235.
[112] Takashi S, Etsuko O, Tsutomu Y, et a1. Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed[J]. Throm-bosis Research, 2005, 115(12): 115-121.
[112] Torres J M, Alonso C, Mittal S, et al. Tropism of human adenovirus type based vectors in swine and their ability to protect against TGEV[J]. Journal of Virology, 1996, 70:3770-3780.
[113] Torres J M, Sanchez C, Sune C, et al. Induction of antibodies protect in against TGEV by recombinant adenovirus expressing TGEV spike protein[J]. Virology, 1995, 213:503-516.
[114] Tuboly T, Yo W, Bailey A, et al. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants[J]. Vaccine, 2000, 18(19): 2023-2028.
[115] Tuboly T, Nagy E. Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus[J]. Arch Virol, 1994, 137(122): 55-67.
[116] Athmaram T N, Geetha B, GoonG K. Heterologous expression of Blue tongue VP2 viral protein fragment in Pichia pastoris[J]. Virus Genes, 2007, 35: 265-271.
[117] Vanderklei I J, Harder W, Veenhuis M. Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeast: a review[J]. Yeast, 1991, 7(3): 195-209.
[118] Vaughn E M, Halbur P G, Paul P S. Sequence comparison of porcine respiratory coronavirus isolate reveals heterogenecity in the S, 3 and 321 genes[J]. J Virol, 1995,69:3176-3184.
[119] Vaugho E M, Halbur P G, Paul P S. Use of nonradiouctive cDNA to differentiate porcine respiratory coronavirus and transmissible gastroenteritis[J]. J Vet Diagn Invest, 1996, 8(2): 241-244.
[120] Weingartl H M, Derbyshire J B. Binding of porcine transmissible gastroenteritis virus by enterocytes from weaned piglets[J]. Vet Micro Biol, 1993, 35: 23-32.
[121] Wenthworth D E, Holmer K V. Molecular determinants of species specificity in the coronavirus receptor aminopeptidase N (CD31)influence of N-linked glycosylation [J]. J Virol, 2001, 75(20): 9741-9752.
[122] Wesley R, Woods R, Cheung A K. Genetic basis of the pathogenesis of transmissible gastroenteritis virus[J]. Journal of virol, 1990, 64(10): 4761-4766.
[123] Wesley R, Woods R, Cheung A K. Nucleotide sequence of the E2-peplomer gene and partial nucleotide sequence of upstream polymerase gene of transmissible gastroenteritis virus (Miller strain)[J]. Adv Exp Med Biol, 1990, 276: 301-306.
[124] Yamakoshi J, Ktoka S, Loag T, et a1. Priunthocyanidin rich extract from grape seeds attenuaters the develoment of aortic atheroeclerosis in cholesterol fed rRabbits[J]. Atheroeclemsis, 1999, 142 (1): 139-142.
[125] Yamakoshi J, Kataoka S, Koga T, et a1. Proanthocyanidin-rich extract from grape seeds attenuates the developm ent of aortic atherosclerosis in cholesterol-fed rabbits[J]. Atherosclerosis, l999, l42(1): l39-149.
[126] Ye X, Krohn R L, Liu W, et a1. The cytotoxic effects of a novel lH636 grape seed proanthocyanidin extract on cultured humancancer cells[J]. Mol Cell Biochem, 1999, 196(2): 99-108.