摘要
以干酪乳杆菌Lactobacillus casei ATCC 393(L.casei 393)作为递呈抗原口服疫苗活菌载体,将猪细小病毒保护性抗原VP2蛋白的基因片段与大肠杆菌不耐热肠毒素B亚单位(LTB)作为目的基因,构建了共表达猪细小病毒主要免疫保护性抗原VP2蛋白与LTB的重组干酪乳杆菌表达系统,经过动物免疫试验,分析比较了共表达VP2-LTB重组干酪乳杆菌与单独表达VP2重组干酪乳杆菌系统免疫效果及细胞表面表达与分泌表达两种不同表达方式的免疫效果。
根据目的基因和融合表达载体质粒的特点,应用Primer Premier 5.0软件进行设计引物。以pMD18-T-VP2质粒和pMD18-T-LTB质粒作为模板,通过PCR扩增分别得到大约1750bp的VP2基因和约375bp的LTB基因,PCR产物经酶切后,胶回收连接,并以连接产物为模板,以VP2的上游引物和LTB的下游引物扩增VP2-LTB,经PCR扩增出约2.1kb的目的基因VP2-LTB,将该基因片段克隆到pMD18-T simple载体,并进行了酶切、PCR鉴定和序列测定,重组质粒命名为pMD18-T-VP2-LTB。重组质粒经BamHI和XhoI双酶切,回收目的基因片段VP2-LTB,分别与经同样双酶切的表达载体pPG-1和pPG-2连接,电转化感受态L.casei 393,筛选阳性克隆,并进行酶切、PCR和测序鉴定,重组质粒命名为pPG-1-VP2-LTB和pPG-2-VP2-LTB,重组干酪乳杆菌命名为pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2–LTB /L.casei 393。
构建的两种重组菌pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2-LTB/L.casei 393在MRS培养液中进行培养,以2%乳糖为诱导剂进行目的蛋白的诱导表达。重组菌目的蛋白的表达和定位通过SDS-PAGE、Western blot和免疫荧光及免疫胶体金电镜鉴定。细胞表面表达型重组干酪乳杆菌经诱导后的SDS-PAGE检测结果表明,有约80kD的融合蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot结果分析表明,表达的蛋白可被鼠源PPV抗血清所识别,间接免疫荧光及免疫胶体金电镜实验结果表明,所表达的蛋白能够在干酪乳杆菌菌体表面检测到;分泌表达型重组干酪乳杆菌经诱导后,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约75kD蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot结果分析所表达的蛋白具有与天然病毒蛋白一样的抗原特异性,实验表明,重组的目的蛋白获得了分泌表达。
为检验构建的重组菌是否能诱导黏膜和系统免疫应答以及LTB作为粘膜免疫佐剂的作用,本实验以BALB/c小鼠为试验动物,进行免疫学实验。将BALB/c小鼠分为五组,每组10只,通过口服途径分别免疫接种109活菌量pPG-1/L.casei 393、pPG-1-VP2/L.casei 393、pPG-1-VP2-LTB/L.casei 393、pPG-2-VP2/L.casei 393、pPG-2-VP2-LTB/L.casei 393,每隔2w免疫一次,共免疫四次,每次连续免疫3d,一天免疫一次。于免疫前、初次免疫后第7d、21d、35d、49d采集免疫小鼠血液;免疫前、初次免疫后第1d、5d、12d、19d、26d、33d、40d、47d和54d采集免疫口服免疫组小鼠收集粪便;眼洗液和外生殖道洗液是在免疫前、初次免疫后第7d、21d、35d、49d分别以100μl的PBS冲洗眼结膜和冲洗外生殖道获得。所有收集的样品保存于–20℃备用。最后测定了小鼠粪便及眼洗液和外生殖道洗液样品中抗PPV VP2分泌型IgA及小鼠血清样本中抗PPV的特异性IgG水平。
通过ELISA方法来检测黏膜样品中特异性分泌型IgA抗体的水平,来评价黏膜免疫反应情况,在粪便、眼洗液和外生殖道洗液中均检测到了高水平的特异性IgA,与对照组相比差异显著。其中,免疫组pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2-LTB/L.casei 393的小鼠粪便、眼洗液和外生殖道样品中IgA抗体水平与免疫组pPG-1-VP2/L.casei 393和pPG-2-VP2/L.casei 393相比,差异显著(P<0.05)或极显著(P<0.01)。初步分析是共表达黏膜免疫佐剂LTB的缘故,表明LTB能加强黏膜系统反应。本研究实验结果初步认为LTB作为一种安全有效的佐剂,具有极大的应用潜力。
共表达VP2-LTB重组干酪乳杆菌免疫组与单独表达VP2重组干酪乳杆菌免疫组相比,pPG-1-VP2-LTB/L.casei 393或pPG-2-VP2-LTB/L.casei 393中小鼠血清中IgG的效价高于pPG-1-VP2/L.casei 393或pPG-2-VP2/L.casei 393组,与免疫对照组比较,差异显著,并且分泌型的重组菌免疫性更好。在本研究中,口服免疫表达VP2-LTB的重组乳酸菌不仅能诱导产生黏膜免疫,而且诱导产生了系统免疫,并且通过口服免疫产生的黏膜免疫反应不只局限于胃肠道,也引起了其他黏膜部位的免疫反应,而且共表达VP2-LTB组的重组乳酸菌免疫后的sIgA抗体水平高于单独表达VP2组的重组乳酸菌。机体通过分泌性IgA抗体在黏膜表面对病原体进行排斥和清除,这对预防细小病毒感染是至关重要的。
中和试验结果表明, pPG-1-VP2/L.casei 393组、pPG-2-VP2/L.casei 393组、pPG-1-VP2-LTB/L.casei 393组、pPG-2-VP2-LTB/L.casei 393组在免疫后49d血清的抗体中和效价分别为1:304、1:317、1:338、1:352。
本研究首次构建了重组大肠杆菌不耐热肠毒素B亚单位和猪细小病毒主要免疫保护性抗原VP2蛋白的干酪乳杆菌细胞表面表达和分泌表达系统。本研究所获得的结果表明,干酪乳杆菌可用做口服免疫传递抗原引起黏膜和系统免疫。当VP2蛋白与LTB共表达时,加强了黏膜免疫反应,LTB具有较好的免疫原性、抗原性和佐剂特性,可被用于基因工程疫苗的研制,很适合于用做黏膜疫苗的免疫佐剂。本研究为进一步研究新型、有效的猪细小病毒口服疫苗奠定了基础。
Lactobacillus casei ATCC 393 (L.casei 393) was selected as an antigen delivery vehicle for the development of live mucosal vaccine. The main protective antigen VP2 of porcine parvovirus and E.coli heat-labile toxin B subunit were selected as the target gene. We constructed recombinant L.casei 393 systems which co-expressing VP2 protein and E.coli heat-labile toxin B subunit protein. The immunogenicity responses induced by oral mucosal immunizations with recombinant strains which with LTB or without LTB were compared and two kinds of recombinant strains expressing interest protein in different cellular locations were also analyzed.
We designed primers with Primer Premier 5.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 1750bp gene fragment (VP2) and 375bp gene fragment were amplified by PCR using the plasmid pMD18-T-VP2 and the plasmid pMD18-T-LTB. The PCR products were digested by restriction enzyme, linked by T4 DNA ligase. About 2.1kbp gene fragment (VP2-LTB) was amplified by PCR using the product of linked. The interest gene fragment VP2-LTB was cloned into plasmid pMD18-T simple followed enzyme digestion, PCR identification and sequence analysis, and the recombinant plasmid was named pMD18-T-VP2-LTB. Then the pMD18-T-VP2-LTB plasmid was doubly digested by BamHI and XhoI, and the interest gene fragment VP2-LTB was obtained by agar gel purification, linked with expression vector pPG-1 and pPG-2 doubly digested by BamHI and XhoI, giving rise to pPG-1-VP2-LTB and pPG-2-VP2-LTB. The recombinant plasmids pPG-1-VP2-LTB and pPG-2-VP2-LTB were electroporated into L.casei 393 respectively, generating pPG-1-VP2-LTB/- L.casei 393 and pPG-2-VP2-LTB/L.casei 393 followed enzyme digestion, PCR identification and sequence analysis.
The recombinant strains pPG-1-VP2-LTB/L.casei 393 and pPG-2-VP2-LTB/L.casei 393 constructed in this study were induced by 2% lactose in MRS medium to express recombinant interest protein. The recombinant strain pPG-1-VP2-LTB /L.casei 393 of cell surface expression was induced and about 80kD protein was detected with SDS-PAGE according with the theoretic molecule weight. The result of Western blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescence and immunoelectron microscope test also showed that the expressed protein was displayed on the cell surface of L.casei 393. The recombinant strain pPG-2-VP2-LTB /L.casei 393 of secretion expression was induced by 2% lactose in MRS medium and about 75kD protein was detected via SDS-PAGE in the induced recombinant strain and culture supernatants according with the theoretic molecule weight. The result of Western–blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The experiment indicated that the interest protein was expressed and secreted into the culture supernatant.
To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses and the value of the LTB as mucosa adjuvant, five groups of ten female mice were immunized via oral route with pPG-1/L.casei 393, pPG-1-VP2/L.casei 393 pPG-1-VP2-LTB /L.casei 393, pPG-2-VP2/L.casei 393, pPG-2-VP2-LTB/L.casei 393 respectively. The immune pro- tocol was administered on three consecutive days at days 0, 1and 2. A booster immunization was given at days 14, 15 and 16 and a second booster was given at days 28, 29 and 30, the third boost- er was given at days 42, 43 and 44, the mice were fed with 109 recombinant strains. Serum of mice were collected before immunization and 7, 21, 35, 49 days after immunization. Fecal pellets were collected before immunization and 1, 5, 12, 19, 26, 33, 40, 47, 54 days after immunization. Ophth- almic wash and vaginal samples were obtained by washing the eyes and the vagina with 100μl phosphate-buffered saline (PBS) before immunization and 7, 21, 35, 49 days after immunization respectively. All samples were stored at–20℃until required. Finally, we determined the level of sIgA and IgG in those samples.
To assess mucosal immune responses, specific IgA levels in mucosal samples were determined by ELISA. Specific IgA reached a high level in the fecal pellets,ophthalmic and vaginal wash. In contrast,only background levels of antibodies were detected in control animals. The IgA levels of mice administered with pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 are higher than those administered with pPG-1-VP2/L.casei 393 or pPG-2-VP2/ L.casei 393 in fecal, ophthalmic and vaginal samples. This is due to co-expressing of mucosal immunoadjuvant LTB. This indicates that LTB can enhance the mucosal system response. This study has highlighted the potential of LTB as a safe and effective adjuvant.
IgG titers of serum in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 were higher than pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. It is prominence when compare with given pPG-1/L.casei 393 and the recombinant L.casei 393 of secretion expression type could elicit higher immune response level than that induced by cell surface expression type. In this study, oral administration of recombinant strains displaying VP2-LTB protein antigens induced both systemic and mucosal immune responses. Oral immunization elicited specific mucosal responses at the site of gastrointestinal tract, as well as the remote mucosal sites. And the sIgA titers in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/L.casei 393 were higher than given pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing porcine parvovirus.
The result of neutralization ability test showed that the neutralization ability of serum antibody was 1:304、1:317、1:338、1:352 which was induced by pPG-1-VP2/L.casei393、pPG-2-VP2/L.casei 393、pPG-1-VP2-LTB/L.casei 393、pPG-2-VP2-LTB/L.casei 393 respectively after 49d oral administration.
This is the first report on the cloning and expression of recombinant LTB and PPV antigen in Lactobacillus. The results obtained so far demonstrate that lactobacillus are capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. When VP2 was co-expressed with LTB, it showed much stronger mucosal immune responses. It indicated that LTB with qualified immunoreactivity, antigenicity and adjuvanticity could be used to develop genetically engineered vaccine. It should be suitably applicable as an immunoadjuvant for mucosal vaccines.All theses work established a good foundation for further study on the new and effective recombinant oral vaccine of porcine parvovirus.
引文
蔡树涛,周风兰,张锦珠.1997.免疫胶体金标记显示波形纤维与核孔复合体的结合.实验生物学报,39(2): 193-199.
曹三杰,文心田.2003.应用斑点免疫金染色法检测猪细小病毒病抗体的研究.中国兽医杂志,39(11):3-6.
常国权,杨盛华.1993.应用胶体金免疫电镜技术检测犬传染性肝炎病毒.中国兽医科技,23 (1):43-44.
陈利玉.1998.重组单核细胞增多性李斯特菌作为活疫苗载体的研究进展.国外医学(预防、诊断、治疗用生物制品分册),21(3):100-105.
程安春,汪铭书.1994.应用胶体金免疫电镜技术检测鸭肝炎病毒.中国兽医杂志,20 (6):3-4.
程道祥,徐斯良,雷瑶等.1991.福建省猪细小病毒感染血清学调查及病毒分离.中国畜禽传染病,(1):34~36.
储迅涛,吕绳凯,董永勤.1999.免疫胶体金定量测定尿微量白蛋白的研究.标记免疫分析与临床,6 (2): 93-97.
杜爱芳,王素华,胡松华.2004.鸡柔嫩艾美耳球虫5401基因真核表达载体的构建及在Vero细胞中的表达.中国畜牧兽医学会家畜寄生虫学分会第五次代表大会暨第八次学术研讨会论文集.
杜爱芳,王素华,胡松华.2004.减毒沙门氏菌为载体传递鸡球虫DNA疫苗的安全性和稳定性试验.中国畜牧兽医学会家畜寄生虫学分会第五次代表大会暨第八次学术研讨会论文集.
高熹英.2000.乳膜免疫向免疫学提出了新问题.上海免疫学杂志,20(5):257-259.
郭鹰,邹全明,朱永红等.2004. BALB/c小鼠口服幽门螺杆菌疫苗rLTB-HspA的免疫应答.免疫学杂志,20(3):201-207.
韩孝成,董齐等.2001.猪细小病毒氢氧化铝疫苗研究.中国预防兽医学报,23(5):381-385.
何家惠,侯继波,王继春.2000.江苏省4种引致猪繁殖障碍的病毒流行病学调查.畜牧与兽医,32(4):10-12.
何召庆,秦泽荣,徐摈蕊.2002.降钙素基因在乳酸杆菌中的克隆.中国兽药杂志,36(6): 20-22.
洪发.2005.弓形虫ROP2基因在大肠杆菌和卡介苗中的表达及免疫原性的研究.吉林大学博士论文.
侯强红,余兴龙,李微等.2007.猪圆环病毒2型Cap蛋白部分基因序列与大肠杆菌LTB成熟肽基因的融合表达及免疫原性研究.中国预防兽医学报,29(11):847-851.
侯世宽.1984.猪细小病毒Mu-l株的分离鉴定.中国人民解放军兽医大学学报,3(4):321-328.
黄旭雯,肖驰.1997.猪JEV和PPV疾病普查检测报告.四川畜禽,7:33.
蒋玉雯,冯军,黄安国.1990.猪细小病毒流行病学的调查.中国兽医杂志,16(3):8-10.
蒋玉雯,黄安国,冯军.1992.猪细小病毒N株的生物学和免疫学特征性研究.畜牧兽医学报,23(1):73-79.
蒋玉雯,黄安国,郑儒标.1994.猪细小病毒感染在广西的流行与防制研究.中国兽医杂志,20(8):10-11.
李刚明,姜天童,方雨铃等.1992.湖北省PPV感染的诊断及血清学调查初报.湖北畜牧兽医,(3):23-25.
李理.2004.口服免疫机制及口服疫苗的研究进展.医学分子生物学杂志,1(5):316-319.
李元刚,尹向东,王玉龙.2004.细菌活载体疫苗的研究进展.黑龙江畜牧兽医,2:63-65.
李云峰,谢明星,陈如明等.1991.鲁、豫两省猪细小病毒感染的血清学调查.中国畜禽传染病,5:39-40.
李忠明.2001.当代新疫苗.北京:高等教育出版社,2-175.
邻捷.1985.秋季初产母猪死胎病毒的分离和鉴定.农业科学学报,2:63-69.
刘云霞,张青文,周明祥.1996.电镜免疫胶体金定位水稻内生细菌的研究.农业生物技术学报,12:354-358.
卢圣栋主编.1999.现代分子生物学常用实验技术(第二版).中国协和医科大学出版.
吕建强,陈焕养,赵俊龙.2002.猪细小病毒疫苗研究进展.动物医学进展,23 (1):31-34.
孟刚.1993.碱性磷酸酶与免疫金银混合双重染色.安徽医科大学学报,28(3):235-238.
明心中,钟细苟,段载金等.1998.江西省猪细小病毒血清学调查.中国兽医杂志,24(3):25-26.
潘雪珠.1984.猪细小病毒S-1毒株的分离和鉴定.上海畜牧兽医通讯,1:5-8.
任贵强,张七斤,张和平等.2006.饲喂乳酸菌对小白鼠血清中IgG及肠道中slgA影响的研究.中国畜牧兽医,33(5):64-67.
萨姆布鲁克J,弗里奇E.F.1996.分子克隆指南(第二版).冬雁,黎孟枫等译.科学出版社,367-386; 417-428.
舒展,魏润生,韩庆彦等.2001.甘肃省集约化养猪场细小病毒病血清学检测.动物医学进展,22(4):112-113.
孙刚,郭昭林,杨本等2002.黑龙江省种猪场母猪繁殖障碍性疾病的监测.黑龙江畜牧兽医, (7):29-30.
王川庆,宋云海,范庆高等.1995.猪细小病毒的研究Ⅰ河南省部分猪场母猪繁殖障碍的流行病学调查.中国畜禽传染病,(1):45-47.
王春凤,秦泽荣,孙哲等.2001.以胸昔酸合成酶为选择压力的穿梭质粒载体的构建.微生物学通报,28(1): 42-46.
王春凤,王会岩,于守平等.2005.猪A组轮状病毒VP7基因与乳酸杆菌非抗性表达载体的重组及在大肠杆菌中的表达.畜牧兽医学报,36(9):964-968.
王东,谢琴,何存利等.1995.宁夏及临近地区猪细小病毒病的血清学调查.中国畜禽传染病,(1):43-45.
王汉中,梁莉.1996.应用套式PCR检测猪细小病毒.病毒学报,12 (2):177-181.
王红梅,李大金,袁敏敏等.2003.表达hCGp的乳酸杆菌经阴道粘膜免疫的研究.中国免疫学杂志,19(10):701-703.
王明俊.1997.兽医生物制品学.中国农业出版社,141-153;601-602.
王在时.1990.猪细小病毒及研究进展.中国兽医杂志,16(5): 44-46.
魏战勇,王学斌,崔保安等.2006.猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究.中国生物工程杂志,26(12):63-67.
吴丹. 2002.猪细小病毒NSl基因与VP2基因的克隆、表达及VP2基因免疫的研究.东北农业大学博士论文.
徐义刚.2007.共表达猪瘟病毒T细胞表位与猪细小病毒VP2蛋白重组乳酸菌系统的构建及其免疫学评价.博士论文.
杨待建,金升藻,荣俊等.1999.湖北省猪细小病毒流行的调查.中国预防兽医学报,21(5):386-390.
杨筱凤,周乐,郑瑾.2005.痢疾杆菌福氏2asf301DsbA/virG双突变减毒活候选疫苗构建和初步鉴定.微生物学报,45(5):748-752.
姚龙涛.2000.猪病毒病.上海科学技术出版社.
殷震,刘景华.1997.动物病毒学.第二版.北京:科学出版社,1150-1154.
张波,陈冠军.2002.细菌载体疫苗的研究进展.微生物学免疫学进展,30(2):86-90.
张代芬,张东,马孟根.2002.四川省部分种畜场猪细小病毒病血清抗体的检测.四川畜牧兽医,29(7):20.
张和平,张七斤,孟和毕力格等.2006.L.case Zhang对小鼠T淋巴细胞亚群及血清IgG和肠黏膜SlgA的影响.中国乳品工业,34(10):4-8.
张林波.2003.粘膜佐剂的研究进展.国外医学免疫学分朋,26(4):169-172.
赵俊龙,陈焕春,吕建强等.2003.猪细小病毒结构蛋白VP1和VP2的基因免疫研究.病毒学报,19 (1):47-51.
赵占民,甘孟侯,刘尚亮.1992.猪细小病毒研究进展.中国兽医杂志,11(3):90~96.
Aggarval P, Pandey RM, Seth P. Augmentation of HIV-1 subtype C vaccine constructs induced immune response in mice by CpG motif 1826-ODN. Viral Immunol, 2005,18(1);213-223.
Alain L S, Marie-Helene C. 2003. Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Practice&Research Clinical Gastroenterology, 17(5): 741-754.
Allan G M, Moneilly F, Meeban B, et al. 2000. Sequential study of experimental infection of pigs with porcine circoviruses and porcine parvovirus: immunostaining of cryostat sections and virus isolation. Journal of veterinary medicine series B, 4(12):81-94.
Blum S, Schiffrin E J. 2003. Intestinal microffora and homeostasis of the mucosal immune response: implications for probiotic bacterial. Curt Issues Intest Microbio1, 4(2):53-60.
Bouwkamp FT. 1985. Antibodies to porcine parvovirus in a breeding herd. Tijdschrift voor Diergeneeskunde, 110(22): 935-938.
Brown C S, Van Lent J W, Vlak J M, et al. 1991. Assembly of empty capsids by using baculorus recombinants expressing human parvovirus B19 structural protein. Virology, 65: 2702-2706.
Childers N K, Miller K L, Tong G, et al. 2000. Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antiger. Infect Immune, 68(10):5509~5516.
Choi CS, et al. 1987. Inhibition of porcine parvovirus by empty virus particles. Arch. Virol, 96:75-87.
Christopher ET. 1995. Infection and Immunity, 3241-3244.
Clements J D, Hartzog N M, Lyon FL. 1988. Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine, 6(3):269-277
Corinne G, Heide M, Denise G, et al. 2001. Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum. Infection and Immunity, 69(30):1547-1553.
De Haan L, Verweij W R, Feil I K, et al.1998. Role of GM1-binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coliheat-labile enterotoxin and its B subunit. Immunol, 94(3): 424-430.
Di Caro S, Tao H, Grillo A, et al. 2005. Effects of Lactobacillus GG on Gene’s expression pattern in small bowel mucosa. Digestive and Liver Disease, 37:320-329.
Ellis A, Atanich A, Clark E, et al. 2000. Co-infection by porcine circoviruses and porcine parvovirus in pigs with naturally acquired postwearing multisystemic wasting syndrome. Journalof veterinary diagnostic investigation, 12(1):21-27.
Enouf V, Langella P, Commissaire J, et al. 2001. Bovine rotavirus nonstructural protein 4 produced by Lactococcus Lactis is antigenic and immunogenic. Appl Environ Microbiol, 67:1423-1428.
Frech S A ,Spyr C A ,Spyr C A, et al. Improved immune responses to influenza vaccination in the elderly using an immunostimulant patch. Vaccine,2005,23(7):946-950
Friend B A, Shahani K M. 1984. Autitumor properties of lactobacilli and dairy products fermented by lactobacilli. J Food Prot, 47:717-723.
Fujisaki Y, Murakami Y, Suzuki H. 1982. Establishment of an attenuated strain of porcine parvovirus by serial passage at low temperature. Natl Inst Anim Health Q (Tokyo), 22(1):1-7.
Galdeano C M, Perdigon G. 2004. Role of viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation. J Appl Microbiol, 97(4):673-681.
Gillick JC. 1977. An outbreak of swine fetal mummification associated with parvovirus. Aust Vet J, 53(2): 105-106.
Grangette C, Muller-Alouf H, Goudercourt D, et al. 2001. Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum. Infect Immun, 69:1547-1553.
Hagiwar Y, T Tsuji, T Iwasaki, et al. 2001. Effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin (LT H44A) as an adjuvant for nasal influenza vaccine.Vaccine, 19(15-16): 2071-2079.
Hailer D, Bode C, Hammes WP. 1999. Cytokine secretion by stimulated monocytes depends on the growth phase and heat treatment of bacteria:a comparative study between lactic acid bacteria and invasive pathogens. Microbiol Immuno1, 43:925-935.
Harrod T, Martin M , Russell M W. 2001. Long-term persistence and recall of immune responses in aged mice after mucosal immunization. Oral Microbiology and Immunology, 16(3):170-177.
Harsharnjit S G. 2003. Probiotics to enhance anti-infective defences in the gastrointestinal tract. Best Practice&Research Clinical Gastroenterology, 17(5):755-773.
Hogg G G, Lenghaus C, Forman A J. 2001. Experimental porcine parvovirus infection of foetal Isabel P A, Gaspar P M. Use of lac regulatory elements for gene expression in Lactobacillus casei. Lait, 81:29-35.
Jeremy A, James V. 2003. Lactobacillus rhamnosus GG decreases TNF-αproduction in lipopolysaccharide-activated murine macrophages by a contact-independent mechanism.Cellular Microbiology, 5(4):277-285.
Joo H S, Donaldson-Wood C R, Johnson R H. et al. 1997. Pathogenesis of porcine parvovirus Infection:Pathology and immunnofluorescence in the Fetus. J. Comp. Pathol, 87:383-391.
Joshua Arrington, Ralph P. Braun, Lichun Dong, et al. 2002. Plasmid Vectors Encoding Cholera Toxin or the Heat-Labile Enterotoxin from Escherichia coli Are Strong Adjuvants for DNA Vaccines. Viro1, 76:4536-4546.
Karen M L, Patrizia B, Beatrice V, et al. 2003. Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cells. FEMS Immunol Med Microbiol, 38:165-172.
Kennedy S, Moffett D, Moneilly F. et al. 2000. Reproduction of lesions of postwearing multisystemic wasting syndrome by infection of convertional pigs with porcine circovirus type 2alone or in combination with porcine parvovirus. Journal of Comparative Pathology, 22(1):9-24.
Koenen M E, Kramer J, Hulst R, et al. 2004. Immunomodulation by probiotic Lactobacilli in layer- and meat-type chickens.Br Poult Sci, 45(3):355-366.
Krakowka S, Ellis J A, Meehan B, et al. 2000. Viral wasting syndrome of swine experimental reproduction postwearing multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Veterinary Pathology, 37(3):254-263.
Lars A, Tone K, Merete B, et al. 1998. A system for heterologous expression of bacteriocins in Lactobacillus sake. FEMS Microbiology Letters, 168(1):137-143.
Laura J P, Patricia L C. 2002. Adjuvant properties and colonization potential of adhering and non-adhering Lactobacillus spp. following oral administration to mice. FEMS Immunology and Medical Microbiology, 34:105-111.
Leenhouts K, Bolhuis A, Venema G et al. 1998. Construction of a food-grade multicopy integration systems in Lactococcus lactic. Appl Microbiol Biotech, 49:417-423.
Leman A. D. Disease of Swine. Fifth Edition. ISBN 0-8138-0440-X.pp352.
Lothar S. 2003. Genetically engineered probiotics. Best Practice&Research Clinical gastroenterology, 17(5):861-876.
Luross JA, Heaton T, Hirst TR, et al. 2002. Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+T cells. Arthritis Rheum, 46(6):1671-1682.
Lutz W, Wurm R. 1996. Serological investigation to demonstrate the presence of antibodies to the viruses causing porcine reproductive and respiratory syndrome, Aujeszky’s disease, hog cholera,and porcine parvovirus among wild boar in Northrhine Westfalia. Zeitschrift fur Jagdwissenschaft, 42(2)123-133.
Maassen C B M, Laman J D, Heijine M J, et al. 1999. Instruments for oral disease-intervention strategies: recombinant Lactobacillus Casei expressing tetanus toxin fragment C for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis. Vaccine, 17(25): 2117-2128.
Maassen C B, van Holten J C, Balk F, et al.1998. Lactobacillus GG Bacteria Ameliorate Arthritis in Lewis Rats. Vet Q, 20:422-428.
Maria L S O, Vicente M, Eliane N M, et al. 2003. Expression of streptococcus pneumoniae antigens, PsaA (pneumococcal surface antigen A) and PspA (pneumococcal surface protein A) by Lactobacillus casei. FEMS Microbiology Letters, 227:25-31.
Martinez C, Dalsgaard K, De Turiso JA, et al. 1992. Production of porcine parvivirus empty capside with high immunogenic activity. Vaccine, 10 (10): 684-690.
Mary A, Mahnel H, Zentbl. 1966. Bakt Parasitkdel.Orig, 99:399-407.
Mckay L L, Baldw in K A. Applications for biotechnology: present and future improvement in lactic acid bacteria. FCMS Microbiol Rev, 1990:87(1-2):34.
Mederlel, Le Gr and R, VaslinB, et al. 2003. Mucosal admi nistration of threerecombinant Mycobact erium bovis BCG-SIVmac251 strainst ocynomol gus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination. Vaccine, 21(27-30):4153-4166.
Medina E, Guzman C A. 2001. Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine, 19(1314):1573-1580.
Mengeling WL, Brown TT, Paul PS, et al. 1979. Efficacy ofan inactivated virus vaccine forprevention of porcine parvovious-induced reproductive failure. Am J Vet Res, 10:204-207.
Mengeling W L, Pejsak Z, Paul P S. 1984. Biological assay of artenuated strain NADL-2 and virulent strain NADL-8 of porcine parvovirus. Am J Vet Res, 45(11):2403-2407.
Mengeling W L. 1977. Diagnosing porcine parvovirus-induced reproductive failure. In Proc 20th Annu Meet Am Assoc Vet Lab Diagn, pp.237-244.
Mercenier A, Muller-Alouf H, 2000. Grangette C. Lactic acid bacteria as live vaccines. Curr Issues Mol Biol, 2(1):17-25.
Ming-Qian Wei, Catherine MR. 1995. An improved method for the transformation of Lactobacillus strains using electroporation. Journal of Microbiological Methods, 21:97-109.
Molitor T W, Joo H S, Thacker B J. 1985. Potentiating effect of adjuvants on humoral to porcine parvovirus vaccines in guinea pigs. Vet Microbiol, (3):209-218.
Muscettola M, Massai L, Tanganelli C, et al. 1994. Effects of Lactobacilli on interferon Product in young and aged mice. Annals New York Academy of science, 717:227-232.
Nashar T O, Betteridge Z E, Mitchell R N. 2001. Evidence for a role of ganglioside GM1 in antigen presentation: binding enhances presentation of Escherichia colienterotoxin B subunit (EtxB) to CD4(+) T cells. Int Immunol, 13(4): 541-551.
Nathalie R, Marie-Claude G, Camille L, et al. 2002. Comparison of the immune responses induced by local immunizations with recombinant Lactobacillus plantarum producing tetanus toxin fragment C in different cellular locations.Vaccine, 20:1769-1777.
Nicaise P, Forestier F, et al. 1993. Influence of intestinal bacterial flora on cytokine production by mouse peritoneal macrophages. Eur. Cytokine Network, 4(2):133.
Opriessnig T, Fenaux M, Yu S, Evans R B. et al.2004. Effect of porcine parvovirus vaccination on the development of PMWS in segregated early weaned pigs coinfected with type 2 porcine circovirus and porcine parvovirus. Veterinary Microbiology, 98(3-4):209-220.
Perdigon G, Alvarez S, Rachid M, Aguero G, Gobbato N. 1995. Immune system stimulation by probiotics. Journal of Dariy SCI, 78: 1597-1606.
Perdigon G, Alvarez S. 1991. Immunoadjuvant activity of oral Lactobacillus casei:influence of dose on the secretory immune response and protective capacity in intestinal infections. Jounal of Dairy Reseaeh, 58(2):485-496.
Phalipon A, Sansonetti P, et al. 1995. Live attenuated Shigella flexneri mutants as vaccine candidates against shigellosis and vectors forantigen delivery. Biologicals, 23(2):125.
Pini A. 1975. Porcine parvovirus in pig herds in southern Africa. J S Afr Vet Assoc, 46:241-244.
Pintel D, Merclinsky M J, Ward D C. 1984. Expression of minute virus of mice structural porteiros in murine cell lines tramsformed by borime papillomavirus-minuts virus of mice plasmid chmera. Virology, 52: 320-327.
Plant L J, Conway P L. 2002. Adjuvant properties and colonization potential of adhering and non-adhering Lactobacillus spp following oral administration to mice. FEMS Immunol Med Microbiol, 34(2):105-111.
Pouwels P H, Leer R J, Shaw M. 1998. Lactic acid bacteria as antigen delivery vehicles for oral immunization purposes. Int J food Microbiol, 41(2):155167.
Ranz A I, Manclus J J, Diaz-Aroca E, Cassal J I.1989. Porcine parvovirus: DNA sequence and genome organization. J Gen Virol, 70(pt10):2541-53.
Rao M, Matyas GR, Vancott TC, et al. Immunostimulatory CpG motits induce CTLresponses to HIV type 1 oligomeric gp140 envelope protein. Immunol Cell Biol, 2004,82(5);523-530.
Robinson B T, Danson D L G, 1984. Porcine parvovirus:a serological survey in the United Kingdon January 1984 to January 1985. Veterinary Record.117(23)611-612.
Robinson K, Chamberlain L M, Schofield K M, et al. 1997. Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nat Biotechnol, 15:653-657.
Rodak L, Valicek L, et al. 2005. An ELISA optimized for porcine epidemic diarrhoea virus detection in faeces. Vet Microbiol, 105(1):9-17.
Scheppler L, Vogel M, Zuercher A W, et al. Recombinant Lactobacillus johnsonii as a mucosal vaccine delivery vehicle. Vaccine, 20(23-24):2913-2920.
Schiffrin E J, Brassart D, Servin A L, et al. 1997. Immune modulation of blood leukocytes in humans by lactic acid bacteria: criteria for strain selection. Am J Clin Nutr, 66(suppl):515-520.
Seegers J F. 2002. Lactobacilli as live vaccine delivery vectors: progress and prospects. Trends Biotechno, 20(12):508-515.
Solis Pereyra B, Lemonnier D. 1993. Induction of human cytokines by bacteria used in dairy foods. Nutr Res, 13:1127-1140.
Sorensen K J. 1982. Porcine parvovirus infection in Denmark, Occurrence, significance and prophylaxis. Proceedings of the 14th Nordic Veterinary Congress, 6-9 July, Copenhagen, Denmark. 108-110.
Soriani M, Williams NA, Hirst T R. 2001. Escherichia coli enterotoxin B subunit triggers apoptosis of CD8+ T cells by activating transcription factor c-myc. Infect Inunun, 69(8):4923-4930.
Van Leengoed LA, Vos J, Gruys E. et al. 1983. Porcine Parvovirus infection: review and diagnosis in a sow herd with reproductive failure.Vet Q, 5 (3):131-141.
Vasudevacharya J, Basak S, Srinivas RV, Compans R W. 1989. Nucleotide sequence analysis of the capsid genes and the right-hand terminalpalindrome of porcine parvovirus, strain NADL-2. Virology, 173(2):368-77
Wang L, Webster D E, Wesselingh S L, et al.2004. Orally delivered malaria vaccines: not too hard tswallow. Expert Opin Biol Ther, 4(10):1585.
Yasui H, Mike A, Ohwaki M. 1989. Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer,s patches after oral administration.Journal of Dairy Scienee, 72:30.
Yasui H,Mike A,Ohwaki M. 1989. Immunogenicity of Bifidobacterium breve and change in antibody production in Peyer,s patches after oral administration.Journal of Dairy Scienee,72:30.
Zegers N D, Kluter E, van Der Stap H, et al. 1999. Expression of the protective antigen of Bacillus anthracis by Lactobacillus casei: Towards the development of an oral vaccine against anthrax. J Appl Microbiol, 87:309-314.