幽门螺杆菌重组Bb-vacA-hpaA候选疫苗的构建
摘要
目的
构建幽门螺杆菌vacA-hpaA融合基因；构建大肠杆菌-双歧杆菌穿梭表达质粒pGEX-vacA-hpaA;分析重组质粒pGEX-vacA-hpaA在大肠杆菌BL21中的表达;电穿孔转化两歧双歧杆菌(Bb),构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。
方法
以质粒pQE-vacA、pET-hpaA为模板,PCR扩增获得vacA和hpaA编码基因序列；依次构建重组质粒pQE-hpaA, pQE-vacA-hpaA,以重组质粒pQE-vacA-hpaA为模板,PCR扩增构建融合基因vacA-hpaA。将融合基因vacA-hpaA定向连接至大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-vacA-hpaA。电穿孔将pGEX-vacA-hpaA导入BL21, SDS-PAGE分析pGEX-vacA-hpaA在大肠杆菌BL21中的表达；Western blot鉴定表达蛋白的抗原性。最后将pGEX-vacA-hpaA电转化导入Bb,构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。
结果
琼脂糖凝胶电泳证实vacA-hpaA融合基因全长1500bp左右,测序结果显示其与预期结果一致。双酶切证实vacA-hpaA融合基因成功插入pGEX-1λT质粒,成功构建重组质粒pGEX-vacA-hpaA并成功转入大肠杆菌BL21。SDS-PAGE结果显示重组质粒在大肠杆菌BL21中经IPTG诱导4h表达了约85KDa的目的蛋白,Western blot分析显示该蛋白能分别与兔抗VacA和兔抗HpaA免疫血清发生特异性结合。最后PCR及双酶切鉴定证实重组质粒pGEX-vacA-hpaA成功转入两歧双歧杆菌Bb,成功获得rBb-vacA-hpaA候选疫苗。
结论
1.成功构建vacA-hpaA融合基因。
2.成功构建幽门螺杆菌穿梭表达载体pGEX-vacA-hpaA。
3.幽门螺杆菌穿梭表达质粒pGEX-vacA-hpaA能在大肠杆菌BL21中经IPTG诱导表达,而且所表达的重组蛋白同时具有VacA和HpaA蛋白单独的抗原性。
4.成功构建幽门螺杆菌rBb-vacA-hpaA候选疫苗。
Objective
To compose the vacA-hpaA fusion gene of Helicobacter pylori; To construct the Escherichia coil-Bifidobacterium shuttle plasmid pGEX-vacA-hpaA; To analyse the plasmid pGEX-vacA-hpaA induced by IPTG in BL21; To transform the recombinant plasmid into Bifidobacterium bifidum by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.
Methods
Amplified the vacA and hpaA genes by PCR from plasmids pQE-vacA and pET-hpaA; Constructed recombinant plasmids pQE-hpaA and pQE-vacA-hpaA in order, and amplified the vacA-hpaA fusion gene by PCR from recombinant plasmid pQE-vacA-hpaA. Linked the vacA-hpaA fusion gene to Escherichia coil-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA. Introduced the recombinant plasmid pGEX-vacA-hpaA into BL21 by electroporation, analysed the target protein induced by IPTG in BL21 for 4h by SDS-PAGE and Western blot.Finally introduced the recombinant plasmid pGEX-vacA-hpaA into Bb by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.
Results
Agarose gel electrophoresis identified the vacA-hpaA fusion gene was about 1500bp, sequence analysis confirmed the sequence of the vacA-hpaA fusion gene was in accordance with the expected result. Double-digestion demonstrated the vacA-hpaA fusion gene was correctly cloned into the multiple cloning site of Escherichia coil-Bifidobacterium expression shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA, and the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into BL21. SDS-PAGE showed the recombinant plasmid pGEX-vacA-hpaA was able to express a 85Kda target protein in E.coil BL21 induced by IPTG for 4h, Western blot showed the target protein could combine specifically with the rabbit VacA immune surum and the rabbit HpaA immune surum. Amplifying the plasmid from the rBb selected in MRS medium with 100μg/ml AMP by PCR and double-digestion demonstrated that the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into Bb and the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori was constructed successfully.
Conclusion
1. Amplified the vacA-hpaA fusion gene successfully.
2. Constructed the recombinant plasmid pGEX-vacA-hpaA successfully.
3. The recombinant plasmid pGEX-vacA-hpaA was able to express the target protein in E.coil BL21 induced by IPTG, and the target protein had specific antigenicity of both VacA and HpaA.
4. Constructed the Helicobacter pylori recombinant candidate vaccine Bb-vacA-hpaA successfully.
构建幽门螺杆菌vacA-hpaA融合基因；构建大肠杆菌-双歧杆菌穿梭表达质粒pGEX-vacA-hpaA;分析重组质粒pGEX-vacA-hpaA在大肠杆菌BL21中的表达;电穿孔转化两歧双歧杆菌(Bb),构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。
方法
以质粒pQE-vacA、pET-hpaA为模板,PCR扩增获得vacA和hpaA编码基因序列；依次构建重组质粒pQE-hpaA, pQE-vacA-hpaA,以重组质粒pQE-vacA-hpaA为模板,PCR扩增构建融合基因vacA-hpaA。将融合基因vacA-hpaA定向连接至大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-vacA-hpaA。电穿孔将pGEX-vacA-hpaA导入BL21, SDS-PAGE分析pGEX-vacA-hpaA在大肠杆菌BL21中的表达；Western blot鉴定表达蛋白的抗原性。最后将pGEX-vacA-hpaA电转化导入Bb,构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。
结果
琼脂糖凝胶电泳证实vacA-hpaA融合基因全长1500bp左右,测序结果显示其与预期结果一致。双酶切证实vacA-hpaA融合基因成功插入pGEX-1λT质粒,成功构建重组质粒pGEX-vacA-hpaA并成功转入大肠杆菌BL21。SDS-PAGE结果显示重组质粒在大肠杆菌BL21中经IPTG诱导4h表达了约85KDa的目的蛋白,Western blot分析显示该蛋白能分别与兔抗VacA和兔抗HpaA免疫血清发生特异性结合。最后PCR及双酶切鉴定证实重组质粒pGEX-vacA-hpaA成功转入两歧双歧杆菌Bb,成功获得rBb-vacA-hpaA候选疫苗。
结论
1.成功构建vacA-hpaA融合基因。
2.成功构建幽门螺杆菌穿梭表达载体pGEX-vacA-hpaA。
3.幽门螺杆菌穿梭表达质粒pGEX-vacA-hpaA能在大肠杆菌BL21中经IPTG诱导表达,而且所表达的重组蛋白同时具有VacA和HpaA蛋白单独的抗原性。
4.成功构建幽门螺杆菌rBb-vacA-hpaA候选疫苗。
Objective
To compose the vacA-hpaA fusion gene of Helicobacter pylori; To construct the Escherichia coil-Bifidobacterium shuttle plasmid pGEX-vacA-hpaA; To analyse the plasmid pGEX-vacA-hpaA induced by IPTG in BL21; To transform the recombinant plasmid into Bifidobacterium bifidum by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.
Methods
Amplified the vacA and hpaA genes by PCR from plasmids pQE-vacA and pET-hpaA; Constructed recombinant plasmids pQE-hpaA and pQE-vacA-hpaA in order, and amplified the vacA-hpaA fusion gene by PCR from recombinant plasmid pQE-vacA-hpaA. Linked the vacA-hpaA fusion gene to Escherichia coil-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA. Introduced the recombinant plasmid pGEX-vacA-hpaA into BL21 by electroporation, analysed the target protein induced by IPTG in BL21 for 4h by SDS-PAGE and Western blot.Finally introduced the recombinant plasmid pGEX-vacA-hpaA into Bb by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.
Results
Agarose gel electrophoresis identified the vacA-hpaA fusion gene was about 1500bp, sequence analysis confirmed the sequence of the vacA-hpaA fusion gene was in accordance with the expected result. Double-digestion demonstrated the vacA-hpaA fusion gene was correctly cloned into the multiple cloning site of Escherichia coil-Bifidobacterium expression shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA, and the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into BL21. SDS-PAGE showed the recombinant plasmid pGEX-vacA-hpaA was able to express a 85Kda target protein in E.coil BL21 induced by IPTG for 4h, Western blot showed the target protein could combine specifically with the rabbit VacA immune surum and the rabbit HpaA immune surum. Amplifying the plasmid from the rBb selected in MRS medium with 100μg/ml AMP by PCR and double-digestion demonstrated that the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into Bb and the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori was constructed successfully.
Conclusion
1. Amplified the vacA-hpaA fusion gene successfully.
2. Constructed the recombinant plasmid pGEX-vacA-hpaA successfully.
3. The recombinant plasmid pGEX-vacA-hpaA was able to express the target protein in E.coil BL21 induced by IPTG, and the target protein had specific antigenicity of both VacA and HpaA.
4. Constructed the Helicobacter pylori recombinant candidate vaccine Bb-vacA-hpaA successfully.
引文
1. International agency for reseach on cancer. Schistosomes, liver fluker and Helicobacter Pylori. I ARC monographs on the evalition on carcinogenic risks to human. Vol61, Lyon:IASC,1994.
2. Dirk Kuck, Bernhard Kolmerer, Chrisitf lking-konert, etal. Vacuolating Cytotoxin of Helicobacter pylori Induced Apoptosis in the Human Gastric Epithelial cell line AGS. Infect Immun.2001;69(8):5080-5087.
3. Alm RA, Ling LS, Moir DT, etal.Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature.1999; 397:176-180.
4. Evans DG, Evans DJ Jr, Lampert HC, etal. Restriction fragment length polymorphism in the adhesin gene HpaA of Helicobacter pylori. Am J Gastroenterol.1995; 90(8):1282-1288.
5. Elisabet Carlsohn, Johanna Nystroom, Ingrid Bolin, etal. HpaA Is Essential for Helicobacter pylori Colonization in Mice.Infect Immun.2006;74 (2):920-926.
6. Lundstrom AM, Bolin I, Bystrom M, etal. Recombinant HpaA purified from Escherichia coli has biological properties similar to those of native Helicobacter pylori HpaA. APMIS.2003; 111(3):389-397.
7. Voland P, N Hafsi, M Zeitner, etal. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infect Immun.2003; 71:3837-3843.
8.吴利先,杨致邦,林珊珊,等.幽门螺杆菌临床株粘附素HpaA基因的克隆表达及在诊断中的价值.中国人兽共患病杂志.2004；20(4)：275-278.
9. 吴利先,杨致邦,林珊珊,等.幽门螺杆菌融合蛋白HpaA-CtxB的研制及免疫原性研究.中国免疫学杂志.2004；20(5)：306-310.
10.刘淼,杨致邦,林珊珊,等.幽门螺杆菌细胞空泡毒素基因毒性片段的克隆与表达.重庆医科大学学报.2003；28(6)：720-723
11. Nystrom J, Svennerholm AM. Oral immunization with HpaA affords therapeutic protective immunity against H. pylori that is reflected by specific mucosal immune responses. Vaccine.2006; Dec 26.
12.徐灿,李兆申,屠振兴,等.幽门螺杆菌HpaA减毒鼠伤寒沙门菌核酸疫苗的构建及免疫原性检测.解放军医学杂志.2004;29(8)：667-669,673.
13.毛小琴,杨致邦,张绍兰,等.幽门螺杆菌重组VacA蛋黄抗体的体外活性.世界华人消化杂志.2006；14(8)：789-794.
14.庸建国,茹炳椹,德长洁等.蛋白质工程的研究.北京大学学报(自然科学版).1998;34(2)：342-348.
15.崔立斌,马清钧.新生肽的折叠与重组蛋白可溶性表达.生物工程进展.1998;18(1)：36-40.
16.王慧珍.蛋白质肽链的翻译后加工.佛山科学技术学院学报.2000；18(21)：70-75.
17.杨运桂,童芹,郑卫东等.分子伴侣过分表达对蛋白质分泌及可溶性的影响.中国生物化学与分子生物学报.2000；16(3)：382-387.
18. Lacham-Kaplan O, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.Stem Cells.2006;24(2):266-273.
19.蓝景刚,胡宏.双歧杆菌的免疫调节作用研究进展.中国微生态学杂志.2002;14(3)：181-185.
20.张金洁,王立生,潘令嘉等.双歧杆菌的完整肽聚糖对实验性大肠癌诱导型一氧化氮合酶表达的影响.中国微生态学杂志.2001；13(1)：11-12.
21. Witz IP, Levy-Nissenbaum O. The tumor microenvironment in the post-PAGET era. Cancer Lett.2006; 242(1):1-10.
22. Wei MQ, Ellem KAO, Dunn P, etal. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur J Cancer.2007; 43(3):490-496.
23. Yazawa K, Fujimor IM, Amand J, etal. Bifidobacterium longum as a delivery system for cancer gene therapy:Selective localization and growth in hypoxic tumor. Cancer gene Therapy.2000; 7(2):269-274.
24.侯鑫,刘俊娥.大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中 的表达.微生物学报.2006；46(3)：347-352.
25.韩庆旺,徐叶芬,傅更锋,等.大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达.生物技术通讯.2006;17(1)：18-21.
26. XU YF, Zhu LP, Hu B, etal. A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy. Cancer Gene Ther.2007; 14(2):151-157.
27. Yi C, Huang Y, Guo ZY, etal. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta pharmacologica sinica.2005; 26(5): 629-634.
28. Yi C, Huang Y, Guo ZY, etal. Construction of Bifidobacterium infantis/CD targeting gene therapy system. Chin-Germ J Clin Oncol.2005; 4(4):244-247.
29.罗耀玲,王勇,黄雪萍,等.大肠埃希菌热不稳定肠毒素B亚单位在B. infantis中的表达.中国微生态学杂志.2008;20(2)：113-115
30.周必英,陈雅棠,李文桂,等.细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定.中国人兽共患病学报.2009；25(6)：502-506.
31.谢婷,李文桂,曾丽蓉.结核分枝杆菌重组Bb-ESAT-6疫苗的构建、鉴定和表达及其保护力.中国人兽共患病学报.2008;24(6)：555-561.
32. Yazawa K, Fujimori M, Nakamura T, etal. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Can Res Treatment.2001; 66(2):165-170.
33. Rhim SL, Park MS, Ji GE. Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum. Biotech Lett.2006; 28(3): 163-168.
34.云雪霞,胡静,陈清.霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达.中国人兽共患病学报.2007;23(2)：168-171.
35.杨梅,李文桂,朱佑明.多房棘球绦虫重组Bb-EmII/3-Eml4-3-3疫苗的构建及鉴定.中国人兽共患病学报.2007；23(10)：1026-1029.
36. Parche S, Beleut M, Rezzonico E, et al. Lactose-over-glucose preference in Bifidobacterium longum NCC2705:glcP, encoding a glucose transporter, is subject to lactose repression. J Bacteriol.2006;188(4):1260-1265.
37. Vitali B, Wasinger V, Brigidi P, etal. A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry.2005;5(7):1859-1867.
38. Sgorbati B, Scardovi V, Lebblanc DJ, etal.Plasmids in the genus Bifidobacterium. J Gen Microbiol.1982; 128(9):2121-2131.
39. Bourget N, Simonet JM, Decaris B.Analysis of the genome of the five Bifidobacterium breve strain:plasmid content pulsed-field gel electrophoresis genome size estimation and loci number.FEMS Microbiol Lett.1993; 110(1):11-20.
40. Matteuzzi D, Brigidi P, Rossi M, etal.Characterization and molecular clonging of Bifidobacterium longum cryptic plasmid pMB1. Lett Appl Microbiol.1990; 11(4):220-225.
41. Rossi M, Brigidi P, Gonzalez A, etal.Characterization of the plasmid pMB1 from Bifidobacterium longum and its use of shuttle vector construction.Pes microbiol.1996; 147(3):133-143.
42. Tanaka K, Samura K, Kano Y. Structural and functional analysis of pTB6 from Bifidobacterium longum. Biosci Biotechnol Biochem.2005;69(2):422-425.
43. Pablo AM, Ana BF, Baltasar M. Screening for Plasmids among Human Bifidobactefia Species:Sequencing and Analysis of pBC1 from Bifidobacterimn catenulatum L48. Plasmid.2007;57(2):165-174.
44. Guglielmetti S, KARP M, Mora D, et al. Molecular Characterization of Bifidobacterium longum Biovar longum NAL8 Plasmids and Construction of a Novel Replicon Screening System. Appl Microbiol Biotechnol.2007;74(5): 1053-1061.
45. Lee JH, O'sullivan DJ. Sequence Analysis of two Cryptic Plasmid from Bifidobacterium longum DJOIOA and Construction of a Shuttle Cloning Vector. Appl Environ Microbiol.2006;72(1):527-535.
46. Rossi M, Brigidi P, Matteuzzi D. An efficient transformation system for bifidobacterium species. Lett Appli Microbiol.1997; 24:33-36.
47. Argnani A, Leer RJ, Luijk N, etal.A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiol.1996; 142: 109-114.
1 Witz IP, Levy-Nissenbaum O. The tumor microenvironment in the post-PAGET era. Cancer Lett,2006,242(1):1-10.
2 Wei MQ, Ellem KAO, Dunn P, et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur J Cancer, 2007,43(3):490-496.
3 Yazawa K, Fujimor IM, Amand J, et al. Bifidobacterium longum as a delivery system for cancer gene therapy:Selective localization and growth in hypoxic tumor. Cancer gene Therapy,2000,7(2):269-274.
4 吴瑜,易成,王树人,等.B. infantis对小鼠黑色素瘤模型肿瘤组织的靶向性.四川大学学报(医学版),2003,34(3)：435-438.
5 Tanaka K, Samura K, Kano Y. Structural and functional analysis of pTB6 from Bifidobacterium longum. Biosci Biotechnol Biochem,2005,69(2):422-425.
6 Pablo AM, Ana BF, Baltasar M. Screening for Plasmids among Human Bifidobactefia Species:Sequencing and Analysis of pBC1 from Bifidobacterimn catenulatum L48. Plasmid,2007,57(2):165-174.
7 Guglielmetti S, KARP M, Mora D, et al. Molecular Characterization of Bifidobacterium longum Biovar longum NAL8 Plasmids and Construction of a Novel Replicon Screening System. Appl Microbiol Biotechnol,2007,74(5): 1053-1061.
8 Lee JH, O'sullivan DJ. Sequence Analysis of two Cryptic Plasmid from Bifidobacterium longum DJOIOA and Construction of a Shuttle Cloning Vector. Appl Environ Microbiol,2006,72(1):527-535.
9 侯鑫,刘俊娥.大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达.微生物学报,2006,46(3)：347-352.
10韩庆旺,徐叶芬,傅更锋,等.大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达.生物技术通讯,2006,17(1)：18-21.
11 Xu YF, Zhu LP, Hu B, et al. A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy. Cancer Gene Ther,2007,14(2):151-157.
12 Yi C,Huang Y,Guo ZY, et al. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta pharmacologica sinica,2005,26(5): 629-634.
13 Yi C, Huang Y, Guo ZY,et al. Construction of Bifidobacterium infantis/CD targeting gene therapy system. Chin-Germ J Clin Oncol,2005,4(4):244-247.
14 叶鹏,李著华,杨彦彪,等.肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT的构建.肿瘤防治研究,2009,36(2)：86-90
15 Chen JC, Chang YS,Wu SL, et al. Inhibition of Escherichia coli heat-labile enterotoxin-induced diarrhea by Chaenomeles speciosa. J Ethnopharmacol,2007, 113(2):233-239.
16 Rezaee MA, Rezaee A, Moazzen ISM, et al. Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae. J Microbiol,2005,43(4):354-360.
17 罗耀玲,王勇,黄雪萍,等.大肠埃希菌热不稳定肠毒素B亚单位在B. infantis中的表达.中国微生态学杂志,2008,20(2)：113-115
18 Lightowlers MW, Jensen O, Fernandez E, et al. Vaccination trials in Australia and Argentina confirm the effectiveness of the Eg95 hydatid vaccine in sheep. Int J Parasitol,1999,29(4):531-534.
19 Fu YC, Martinez C, Chalar C, et al. A new potent antigen from Echinococcus granulosus associated with muscles and tegument. Mol Biochem Parasitol,1999, 102(1):43-52.
20 Fu YC, Marchal S, Marchal T, et al. Cellular immune response of lymphnodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66kDa protein of Echinococcus granulosus. Vet Immunol Immunopathol,2000,74(34):195-208.
21 周必英,陈雅棠,李文桂,等.细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定.中国人兽共患病学报,2009,25(6)：502-506.
22 谢婷,李文桂,曾丽蓉.结核分枝杆菌重组Bb-ESAT-6疫苗的构建、鉴定和表达及其保护力.中国人兽共患病学报,2008,24(6)：555-561.
23 Yazawa K, Fujimori M, Nakamura T, et al. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Can Res Treatment,2001,66(2):165-170.
24 Rhim SL, Park MS, Ji GE. Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum. Biotech Lett,2006,28(3): 163-168.
25 云雪霞,胡静,陈清.霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达.中国人兽共患病学报,2007,23(2)：168-171.
26杨梅,李文桂,朱佑明.多房棘球绦虫重组Bb-EmⅡ/3-Em14-3-3疫苗的 构建及鉴定.中国人兽共患病学报,2007,23(10)：1026-1029.
27 Parche S, Beleut M, Rezzonico E, et al. Lactose-over-glucose preference in Bifidobacterium longum NCC2705:glcP, encoding a glucose transporter, is subject to lactose repression. J Bacteriol,2006,188(4):1260-1265.
28 VitaliB, Wasinger V, Brigidi P, et al. A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry.2005,5(7):1859-1867.
2. Dirk Kuck, Bernhard Kolmerer, Chrisitf lking-konert, etal. Vacuolating Cytotoxin of Helicobacter pylori Induced Apoptosis in the Human Gastric Epithelial cell line AGS. Infect Immun.2001;69(8):5080-5087.
3. Alm RA, Ling LS, Moir DT, etal.Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature.1999; 397:176-180.
4. Evans DG, Evans DJ Jr, Lampert HC, etal. Restriction fragment length polymorphism in the adhesin gene HpaA of Helicobacter pylori. Am J Gastroenterol.1995; 90(8):1282-1288.
5. Elisabet Carlsohn, Johanna Nystroom, Ingrid Bolin, etal. HpaA Is Essential for Helicobacter pylori Colonization in Mice.Infect Immun.2006;74 (2):920-926.
6. Lundstrom AM, Bolin I, Bystrom M, etal. Recombinant HpaA purified from Escherichia coli has biological properties similar to those of native Helicobacter pylori HpaA. APMIS.2003; 111(3):389-397.
7. Voland P, N Hafsi, M Zeitner, etal. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infect Immun.2003; 71:3837-3843.
8.吴利先,杨致邦,林珊珊,等.幽门螺杆菌临床株粘附素HpaA基因的克隆表达及在诊断中的价值.中国人兽共患病杂志.2004；20(4)：275-278.
9. 吴利先,杨致邦,林珊珊,等.幽门螺杆菌融合蛋白HpaA-CtxB的研制及免疫原性研究.中国免疫学杂志.2004；20(5)：306-310.
10.刘淼,杨致邦,林珊珊,等.幽门螺杆菌细胞空泡毒素基因毒性片段的克隆与表达.重庆医科大学学报.2003；28(6)：720-723
11. Nystrom J, Svennerholm AM. Oral immunization with HpaA affords therapeutic protective immunity against H. pylori that is reflected by specific mucosal immune responses. Vaccine.2006; Dec 26.
12.徐灿,李兆申,屠振兴,等.幽门螺杆菌HpaA减毒鼠伤寒沙门菌核酸疫苗的构建及免疫原性检测.解放军医学杂志.2004;29(8)：667-669,673.
13.毛小琴,杨致邦,张绍兰,等.幽门螺杆菌重组VacA蛋黄抗体的体外活性.世界华人消化杂志.2006；14(8)：789-794.
14.庸建国,茹炳椹,德长洁等.蛋白质工程的研究.北京大学学报(自然科学版).1998;34(2)：342-348.
15.崔立斌,马清钧.新生肽的折叠与重组蛋白可溶性表达.生物工程进展.1998;18(1)：36-40.
16.王慧珍.蛋白质肽链的翻译后加工.佛山科学技术学院学报.2000；18(21)：70-75.
17.杨运桂,童芹,郑卫东等.分子伴侣过分表达对蛋白质分泌及可溶性的影响.中国生物化学与分子生物学报.2000；16(3)：382-387.
18. Lacham-Kaplan O, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.Stem Cells.2006;24(2):266-273.
19.蓝景刚,胡宏.双歧杆菌的免疫调节作用研究进展.中国微生态学杂志.2002;14(3)：181-185.
20.张金洁,王立生,潘令嘉等.双歧杆菌的完整肽聚糖对实验性大肠癌诱导型一氧化氮合酶表达的影响.中国微生态学杂志.2001；13(1)：11-12.
21. Witz IP, Levy-Nissenbaum O. The tumor microenvironment in the post-PAGET era. Cancer Lett.2006; 242(1):1-10.
22. Wei MQ, Ellem KAO, Dunn P, etal. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur J Cancer.2007; 43(3):490-496.
23. Yazawa K, Fujimor IM, Amand J, etal. Bifidobacterium longum as a delivery system for cancer gene therapy:Selective localization and growth in hypoxic tumor. Cancer gene Therapy.2000; 7(2):269-274.
24.侯鑫,刘俊娥.大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中 的表达.微生物学报.2006；46(3)：347-352.
25.韩庆旺,徐叶芬,傅更锋,等.大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达.生物技术通讯.2006;17(1)：18-21.
26. XU YF, Zhu LP, Hu B, etal. A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy. Cancer Gene Ther.2007; 14(2):151-157.
27. Yi C, Huang Y, Guo ZY, etal. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta pharmacologica sinica.2005; 26(5): 629-634.
28. Yi C, Huang Y, Guo ZY, etal. Construction of Bifidobacterium infantis/CD targeting gene therapy system. Chin-Germ J Clin Oncol.2005; 4(4):244-247.
29.罗耀玲,王勇,黄雪萍,等.大肠埃希菌热不稳定肠毒素B亚单位在B. infantis中的表达.中国微生态学杂志.2008;20(2)：113-115
30.周必英,陈雅棠,李文桂,等.细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定.中国人兽共患病学报.2009；25(6)：502-506.
31.谢婷,李文桂,曾丽蓉.结核分枝杆菌重组Bb-ESAT-6疫苗的构建、鉴定和表达及其保护力.中国人兽共患病学报.2008;24(6)：555-561.
32. Yazawa K, Fujimori M, Nakamura T, etal. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Can Res Treatment.2001; 66(2):165-170.
33. Rhim SL, Park MS, Ji GE. Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum. Biotech Lett.2006; 28(3): 163-168.
34.云雪霞,胡静,陈清.霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达.中国人兽共患病学报.2007;23(2)：168-171.
35.杨梅,李文桂,朱佑明.多房棘球绦虫重组Bb-EmII/3-Eml4-3-3疫苗的构建及鉴定.中国人兽共患病学报.2007；23(10)：1026-1029.
36. Parche S, Beleut M, Rezzonico E, et al. Lactose-over-glucose preference in Bifidobacterium longum NCC2705:glcP, encoding a glucose transporter, is subject to lactose repression. J Bacteriol.2006;188(4):1260-1265.
37. Vitali B, Wasinger V, Brigidi P, etal. A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry.2005;5(7):1859-1867.
38. Sgorbati B, Scardovi V, Lebblanc DJ, etal.Plasmids in the genus Bifidobacterium. J Gen Microbiol.1982; 128(9):2121-2131.
39. Bourget N, Simonet JM, Decaris B.Analysis of the genome of the five Bifidobacterium breve strain:plasmid content pulsed-field gel electrophoresis genome size estimation and loci number.FEMS Microbiol Lett.1993; 110(1):11-20.
40. Matteuzzi D, Brigidi P, Rossi M, etal.Characterization and molecular clonging of Bifidobacterium longum cryptic plasmid pMB1. Lett Appl Microbiol.1990; 11(4):220-225.
41. Rossi M, Brigidi P, Gonzalez A, etal.Characterization of the plasmid pMB1 from Bifidobacterium longum and its use of shuttle vector construction.Pes microbiol.1996; 147(3):133-143.
42. Tanaka K, Samura K, Kano Y. Structural and functional analysis of pTB6 from Bifidobacterium longum. Biosci Biotechnol Biochem.2005;69(2):422-425.
43. Pablo AM, Ana BF, Baltasar M. Screening for Plasmids among Human Bifidobactefia Species:Sequencing and Analysis of pBC1 from Bifidobacterimn catenulatum L48. Plasmid.2007;57(2):165-174.
44. Guglielmetti S, KARP M, Mora D, et al. Molecular Characterization of Bifidobacterium longum Biovar longum NAL8 Plasmids and Construction of a Novel Replicon Screening System. Appl Microbiol Biotechnol.2007;74(5): 1053-1061.
45. Lee JH, O'sullivan DJ. Sequence Analysis of two Cryptic Plasmid from Bifidobacterium longum DJOIOA and Construction of a Shuttle Cloning Vector. Appl Environ Microbiol.2006;72(1):527-535.
46. Rossi M, Brigidi P, Matteuzzi D. An efficient transformation system for bifidobacterium species. Lett Appli Microbiol.1997; 24:33-36.
47. Argnani A, Leer RJ, Luijk N, etal.A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiol.1996; 142: 109-114.
1 Witz IP, Levy-Nissenbaum O. The tumor microenvironment in the post-PAGET era. Cancer Lett,2006,242(1):1-10.
2 Wei MQ, Ellem KAO, Dunn P, et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumours. Eur J Cancer, 2007,43(3):490-496.
3 Yazawa K, Fujimor IM, Amand J, et al. Bifidobacterium longum as a delivery system for cancer gene therapy:Selective localization and growth in hypoxic tumor. Cancer gene Therapy,2000,7(2):269-274.
4 吴瑜,易成,王树人,等.B. infantis对小鼠黑色素瘤模型肿瘤组织的靶向性.四川大学学报(医学版),2003,34(3)：435-438.
5 Tanaka K, Samura K, Kano Y. Structural and functional analysis of pTB6 from Bifidobacterium longum. Biosci Biotechnol Biochem,2005,69(2):422-425.
6 Pablo AM, Ana BF, Baltasar M. Screening for Plasmids among Human Bifidobactefia Species:Sequencing and Analysis of pBC1 from Bifidobacterimn catenulatum L48. Plasmid,2007,57(2):165-174.
7 Guglielmetti S, KARP M, Mora D, et al. Molecular Characterization of Bifidobacterium longum Biovar longum NAL8 Plasmids and Construction of a Novel Replicon Screening System. Appl Microbiol Biotechnol,2007,74(5): 1053-1061.
8 Lee JH, O'sullivan DJ. Sequence Analysis of two Cryptic Plasmid from Bifidobacterium longum DJOIOA and Construction of a Shuttle Cloning Vector. Appl Environ Microbiol,2006,72(1):527-535.
9 侯鑫,刘俊娥.大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达.微生物学报,2006,46(3)：347-352.
10韩庆旺,徐叶芬,傅更锋,等.大肠杆菌-双歧杆菌穿梭表达载体的构建及内皮抑素基因的表达.生物技术通讯,2006,17(1)：18-21.
11 Xu YF, Zhu LP, Hu B, et al. A new expression plasmid in Bifidobacterium longum as a delivery system of endostatin for cancer gene therapy. Cancer Gene Ther,2007,14(2):151-157.
12 Yi C,Huang Y,Guo ZY, et al. Antitumor effect of cytosine deaminase/5-fluorocytosine suicide gene therapy system mediated by Bifidobacterium infantis on melanoma. Acta pharmacologica sinica,2005,26(5): 629-634.
13 Yi C, Huang Y, Guo ZY,et al. Construction of Bifidobacterium infantis/CD targeting gene therapy system. Chin-Germ J Clin Oncol,2005,4(4):244-247.
14 叶鹏,李著华,杨彦彪,等.肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT的构建.肿瘤防治研究,2009,36(2)：86-90
15 Chen JC, Chang YS,Wu SL, et al. Inhibition of Escherichia coli heat-labile enterotoxin-induced diarrhea by Chaenomeles speciosa. J Ethnopharmacol,2007, 113(2):233-239.
16 Rezaee MA, Rezaee A, Moazzen ISM, et al. Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae. J Microbiol,2005,43(4):354-360.
17 罗耀玲,王勇,黄雪萍,等.大肠埃希菌热不稳定肠毒素B亚单位在B. infantis中的表达.中国微生态学杂志,2008,20(2)：113-115
18 Lightowlers MW, Jensen O, Fernandez E, et al. Vaccination trials in Australia and Argentina confirm the effectiveness of the Eg95 hydatid vaccine in sheep. Int J Parasitol,1999,29(4):531-534.
19 Fu YC, Martinez C, Chalar C, et al. A new potent antigen from Echinococcus granulosus associated with muscles and tegument. Mol Biochem Parasitol,1999, 102(1):43-52.
20 Fu YC, Marchal S, Marchal T, et al. Cellular immune response of lymphnodes from dogs following the intradermal injection of a recombinant antigen corresponding to a 66kDa protein of Echinococcus granulosus. Vet Immunol Immunopathol,2000,74(34):195-208.
21 周必英,陈雅棠,李文桂,等.细粒棘球绦虫重组Bb-Eg95-EgA31融合基因疫苗构建及鉴定.中国人兽共患病学报,2009,25(6)：502-506.
22 谢婷,李文桂,曾丽蓉.结核分枝杆菌重组Bb-ESAT-6疫苗的构建、鉴定和表达及其保护力.中国人兽共患病学报,2008,24(6)：555-561.
23 Yazawa K, Fujimori M, Nakamura T, et al. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Can Res Treatment,2001,66(2):165-170.
24 Rhim SL, Park MS, Ji GE. Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum. Biotech Lett,2006,28(3): 163-168.
25 云雪霞,胡静,陈清.霍乱弧菌肠毒素B亚单位基因在大肠杆菌和双歧杆菌的表达.中国人兽共患病学报,2007,23(2)：168-171.
26杨梅,李文桂,朱佑明.多房棘球绦虫重组Bb-EmⅡ/3-Em14-3-3疫苗的 构建及鉴定.中国人兽共患病学报,2007,23(10)：1026-1029.
27 Parche S, Beleut M, Rezzonico E, et al. Lactose-over-glucose preference in Bifidobacterium longum NCC2705:glcP, encoding a glucose transporter, is subject to lactose repression. J Bacteriol,2006,188(4):1260-1265.
28 VitaliB, Wasinger V, Brigidi P, et al. A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry.2005,5(7):1859-1867.