大肠杆菌不耐热肠毒素基因的克隆、改造及表达
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摘要
大肠杆菌不耐热肠毒素(heat-labile enterotoxin, LT)是肠产毒性大肠杆(Enterotoxigenic Escherichia coli,ETEC)分泌的一种热不稳定肠毒素,LT是由一个具有毒性的A亚单位(LTA)和形成环状的五个能够与神经节苷脂GM1结合而粘附于真核细胞膜上的B亚单位(LTB)组成。能引起人和其他哺乳动物的水样腹泻。LT除具有毒性外,还能够有效的启动局部及全身的体液和细胞免疫,具有很强的粘膜免疫原性和粘膜佐剂活性,在粘膜免疫研究中以及粘膜免疫疫苗开发中具有重要医学价值和经济价值。
为制备大量具有免疫活性的LT蛋白,本实验通过提取产肠毒素大肠杆菌44815菌株的基因组,采用PCR方法分别扩增不耐热肠毒素A亚基(LTA)和B亚基(LTB)的编码基因,并将其连接在pMD18-T载体上,构建成LTA和LTB基因的重组载体pMD18-T-LTA和pMD18-T-LTB。阳性克隆筛选采用蓝白斑选择和限制性内切酶XhoⅠ和NcoⅠ消化,最后进行序列测定。然后采用定点突变方法将LTA的第63位的丝氨酸改变为赖氨酸,构建成pMD18-T-LTAK63突变体。再分别将pMD18-T-LTAK63和pMD18-T-LTB通过XhoⅠ和NcoⅠ位点插入到pET-20b(+)原核表达载体,分别构建成LTA和LTB的原核表达载体pET-20b-LTAK63和pET-20b-LTB。将重组LTA和LTB表达载体分别转化宿主菌Bl21,制备工程菌进行表达。表达产物采用SDS-PAGE方法检测,目的蛋白分离纯化采用镍琼脂糖胶粒吸附法。为鉴定带有人CD5信号肽的LTAK63基因能否在真核细胞中表达,以pMD18-T-LTAK63为模板,通过PCR扩增LTAK63基因,将LTAK63基因连接在pcDNACD5sp真核表达载体上,构建成LTAK63重组质粒pcDNACD5sp-LTAK63。利用磷酸钙方法将pcDNACD5sp-LTAK63转染人胚胎肾细胞HEK 293T细胞使其瞬时表达。
本研究构建的重组质粒pMD18-T-LTA和pMD18-T-LTB的测序结果表明:所克隆的LTA基因与GenBank中大肠杆菌标准产毒株LTA基因的大小完全一致,有4个碱基发生了改变,同源性为99%。在这4个突变中有3个是无义突变,不造成氨基酸的改变,但有1个碱基的突变使谷氨酸变为赖氨酸。可见LTA基因存在着多态性;所克隆的LTB基因与GenBank中大肠杆菌标准产毒株LTB基因的完全一致,同源性为100%。利用定点突变方法构建的pMD18-T-LTAK63突变体的测序结果表明LTA的第63位的丝氨酸成功的改变为赖氨酸。构建的原核表达载体经XhoⅠ和NcoⅠ双酶切,得到了符合预期大小的特异性片段,结果证明pET-20b-LTAK63和pET-20b-LTB原核表达载体构建成功。SDS-PAGE检测宿主菌Bl21表达目的蛋白的结果显示:在细胞周质中有目的蛋白表达,分别在分子量约为28Kda(LTA)及14Kda(LTB)处有一条明显的带。而在细胞的培养基及包涵体中未见目的蛋白表达。同时通过镍吸附法纯化得到了单一目的蛋白。结果表明LTAK63和LTB在Pel信号肽的引导下进行了表达并分泌到细胞周质,而不是以包涵体形式存在,因此表达目的蛋白具有一定的活性,且易于分离纯化。在LTAK63基因进行真核表达的基础上构建的pcDNACD5sp-LTAK63真核表达载体转染HEK293T细胞,Western blot检测结果显示在分子量约为34.8KDa处有一条明显的杂交带,而空载体在相应位置未见杂交带。结果证明pcDNACD5sp-LTAK63真核表达载体构建成功,并实现了它在真核细胞中的分泌性表达。
Escherichia coli heat-labile enterotoxin (heat-labile enterotoxin, LT) was the intestinal toxicity secreted by Enterotoxigenic Eshchirichia coli (ETEC). LT was composed of subunit A (LTA) and subunit B, the former has toxicity; the latter formed five rings which can bind GM1 ganglioside on the membrane of eukaryotic cell. LT can cause diarrhea in human and other mammals. In addition to toxicity, LT was also able to trigger humoral immunity and cellular immunity, so it has the strong immunogenicity and adjuvanticity in mucous membrane and has important medical value in the study of mucosal immunity and in vaccine development.
In order to prepare LT proteins at large scale with immunocompetence, the genes encoding of heat-labile enterotoxin A subunit (LTA) and the B subunit (LTB) were amplified with PCR method from the genomic DNA of Enterotoxigenic Escherichia coli strain 44815. The amplified LTA and LTB genes were inserted into pMD18-T vector to construct pMD18-T-LTA and pMD18-T-LTB recombinant plasmids, respectively. The positive clones were identified through the blue-white screening system and double-enzyme digestion(Nco I and Xho I), vertified by sequencing. The LTA gene was modified by changing Ser63 to Lys63 with Site-directed mutagenesis on the pMD18-T-LTA recombinant plasmid. LTAK63 and LTB genes were transfered into the pET-20b (+) prokaryotic expression vectors respectively through Nco I and Xho I sites and construct the prokaryotic expression vectors of pET-20b-LTAK63 and pET-20b-LTB. Recombinant expression vectors of LTA and LTB were transformed into host bacteria Bl21 respectively for preparation of engineering bacteria. The expression products were detected by SDS-PAGE and the interest protein was separated and purified by nickel-agarose beads. In order to construct LTAK63 gene eukaryotic expression vector, the LTAK63 gene with NheI and ApaI sites was amplified by PCR using pMD18-T-LTAK63 plasmid as a template. The gene was ligased with pcDNACD5sp by NheI and ApaI sites to construct the recombinant plasmid, pcDNACD5sp-LTAK63. To identify whether LTAK63 gene with human CD5 signal peptide can express in eukaryotic cells, HEK 293T cells were applied to transiently express LTAK63 gene with the calcium phosphate transfection method.
The experimental results showed that the LTA gene amplified by PCR was consistent with the Gene Bank published sequence and the homology was 99%. Four bases of LTA gene were changed, but only one base results amino acid substitution from glutamic acid to Lysine. Therefore LTA gene has polymorphism. LTB gene amplified was in coincidence with the published in GenBank. Sequence results showed that Ser63 of LTA was changed to Lys63 successfully. The constructed prokaryotic expression vectors, pET-20b-LTAK63 and pET-20b-LTB, were correct by identification with Xho I and NcoⅠdigestion. The two proteins (MW of 28 kDa (LTA) and 14kDa (LTB)) were detected in shock–release sulution by SDS-PAGE, which indicated that the two interest proteins were expressed as secretive form. Purified protein by nickel adsorption showed the single band by SDS-PAGE. All illustrated that the LTAK63 and LTB were mediated by signal peptide Pel , can make the LTAK63 and LTB expressed and secreted to the periplasm, rather than existing in form of inclusion body, so the expressed protein has activity. and separation and purification were easy. Western blot results showed that there was a clear hybrid band at about 34.8KDa, but at the corresponding location of empty vector there was no hybrid band. Therefore the eukaryotic expression vector pcDNACD5sp-LTAK63 was constructed successfully, the secretive expression for LTA63 gene was achieved in eukaryotic cells.
为制备大量具有免疫活性的LT蛋白,本实验通过提取产肠毒素大肠杆菌44815菌株的基因组,采用PCR方法分别扩增不耐热肠毒素A亚基(LTA)和B亚基(LTB)的编码基因,并将其连接在pMD18-T载体上,构建成LTA和LTB基因的重组载体pMD18-T-LTA和pMD18-T-LTB。阳性克隆筛选采用蓝白斑选择和限制性内切酶XhoⅠ和NcoⅠ消化,最后进行序列测定。然后采用定点突变方法将LTA的第63位的丝氨酸改变为赖氨酸,构建成pMD18-T-LTAK63突变体。再分别将pMD18-T-LTAK63和pMD18-T-LTB通过XhoⅠ和NcoⅠ位点插入到pET-20b(+)原核表达载体,分别构建成LTA和LTB的原核表达载体pET-20b-LTAK63和pET-20b-LTB。将重组LTA和LTB表达载体分别转化宿主菌Bl21,制备工程菌进行表达。表达产物采用SDS-PAGE方法检测,目的蛋白分离纯化采用镍琼脂糖胶粒吸附法。为鉴定带有人CD5信号肽的LTAK63基因能否在真核细胞中表达,以pMD18-T-LTAK63为模板,通过PCR扩增LTAK63基因,将LTAK63基因连接在pcDNACD5sp真核表达载体上,构建成LTAK63重组质粒pcDNACD5sp-LTAK63。利用磷酸钙方法将pcDNACD5sp-LTAK63转染人胚胎肾细胞HEK 293T细胞使其瞬时表达。
本研究构建的重组质粒pMD18-T-LTA和pMD18-T-LTB的测序结果表明:所克隆的LTA基因与GenBank中大肠杆菌标准产毒株LTA基因的大小完全一致,有4个碱基发生了改变,同源性为99%。在这4个突变中有3个是无义突变,不造成氨基酸的改变,但有1个碱基的突变使谷氨酸变为赖氨酸。可见LTA基因存在着多态性;所克隆的LTB基因与GenBank中大肠杆菌标准产毒株LTB基因的完全一致,同源性为100%。利用定点突变方法构建的pMD18-T-LTAK63突变体的测序结果表明LTA的第63位的丝氨酸成功的改变为赖氨酸。构建的原核表达载体经XhoⅠ和NcoⅠ双酶切,得到了符合预期大小的特异性片段,结果证明pET-20b-LTAK63和pET-20b-LTB原核表达载体构建成功。SDS-PAGE检测宿主菌Bl21表达目的蛋白的结果显示:在细胞周质中有目的蛋白表达,分别在分子量约为28Kda(LTA)及14Kda(LTB)处有一条明显的带。而在细胞的培养基及包涵体中未见目的蛋白表达。同时通过镍吸附法纯化得到了单一目的蛋白。结果表明LTAK63和LTB在Pel信号肽的引导下进行了表达并分泌到细胞周质,而不是以包涵体形式存在,因此表达目的蛋白具有一定的活性,且易于分离纯化。在LTAK63基因进行真核表达的基础上构建的pcDNACD5sp-LTAK63真核表达载体转染HEK293T细胞,Western blot检测结果显示在分子量约为34.8KDa处有一条明显的杂交带,而空载体在相应位置未见杂交带。结果证明pcDNACD5sp-LTAK63真核表达载体构建成功,并实现了它在真核细胞中的分泌性表达。
Escherichia coli heat-labile enterotoxin (heat-labile enterotoxin, LT) was the intestinal toxicity secreted by Enterotoxigenic Eshchirichia coli (ETEC). LT was composed of subunit A (LTA) and subunit B, the former has toxicity; the latter formed five rings which can bind GM1 ganglioside on the membrane of eukaryotic cell. LT can cause diarrhea in human and other mammals. In addition to toxicity, LT was also able to trigger humoral immunity and cellular immunity, so it has the strong immunogenicity and adjuvanticity in mucous membrane and has important medical value in the study of mucosal immunity and in vaccine development.
In order to prepare LT proteins at large scale with immunocompetence, the genes encoding of heat-labile enterotoxin A subunit (LTA) and the B subunit (LTB) were amplified with PCR method from the genomic DNA of Enterotoxigenic Escherichia coli strain 44815. The amplified LTA and LTB genes were inserted into pMD18-T vector to construct pMD18-T-LTA and pMD18-T-LTB recombinant plasmids, respectively. The positive clones were identified through the blue-white screening system and double-enzyme digestion(Nco I and Xho I), vertified by sequencing. The LTA gene was modified by changing Ser63 to Lys63 with Site-directed mutagenesis on the pMD18-T-LTA recombinant plasmid. LTAK63 and LTB genes were transfered into the pET-20b (+) prokaryotic expression vectors respectively through Nco I and Xho I sites and construct the prokaryotic expression vectors of pET-20b-LTAK63 and pET-20b-LTB. Recombinant expression vectors of LTA and LTB were transformed into host bacteria Bl21 respectively for preparation of engineering bacteria. The expression products were detected by SDS-PAGE and the interest protein was separated and purified by nickel-agarose beads. In order to construct LTAK63 gene eukaryotic expression vector, the LTAK63 gene with NheI and ApaI sites was amplified by PCR using pMD18-T-LTAK63 plasmid as a template. The gene was ligased with pcDNACD5sp by NheI and ApaI sites to construct the recombinant plasmid, pcDNACD5sp-LTAK63. To identify whether LTAK63 gene with human CD5 signal peptide can express in eukaryotic cells, HEK 293T cells were applied to transiently express LTAK63 gene with the calcium phosphate transfection method.
The experimental results showed that the LTA gene amplified by PCR was consistent with the Gene Bank published sequence and the homology was 99%. Four bases of LTA gene were changed, but only one base results amino acid substitution from glutamic acid to Lysine. Therefore LTA gene has polymorphism. LTB gene amplified was in coincidence with the published in GenBank. Sequence results showed that Ser63 of LTA was changed to Lys63 successfully. The constructed prokaryotic expression vectors, pET-20b-LTAK63 and pET-20b-LTB, were correct by identification with Xho I and NcoⅠdigestion. The two proteins (MW of 28 kDa (LTA) and 14kDa (LTB)) were detected in shock–release sulution by SDS-PAGE, which indicated that the two interest proteins were expressed as secretive form. Purified protein by nickel adsorption showed the single band by SDS-PAGE. All illustrated that the LTAK63 and LTB were mediated by signal peptide Pel , can make the LTAK63 and LTB expressed and secreted to the periplasm, rather than existing in form of inclusion body, so the expressed protein has activity. and separation and purification were easy. Western blot results showed that there was a clear hybrid band at about 34.8KDa, but at the corresponding location of empty vector there was no hybrid band. Therefore the eukaryotic expression vector pcDNACD5sp-LTAK63 was constructed successfully, the secretive expression for LTA63 gene was achieved in eukaryotic cells.
引文
[1] Sixma T K, Pronk S E, Kalk K H, et al. Crystal structure ofa cholera toxin-related heat-labile enterotoxine from E.coli. [J] Nature.1991, 351, 371-377.
[2] Dallas W S, Falkow S. Amino acid sequence homologybetween cholera toxin and Eschrichia coli heat-labiletoxin. [J] Nature. 1980, 288, 499-501.
[3] Spicer E K, Kavanaugh W M, Dallas W S, et al. Sequence homologiesbetween A subunits of Eschrichia coli and Vibrio cholerae en-terotoxins. [J] Proc. Natl. Acad. Sci. USA 1981, 78, 50-54.
[4] Verweij WR, de Haan L , M arijke H, et al. Musosal immunoadjuvant activity of recombinant E scherich ia coli heat-labile entero toxin and its B subunit: induction of system ic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen [J ]. Vaccine, 1998, 16 (20) : 2069-2076.
[5] Kunio T , Masanori I , Yoko Y , et al . Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally coadm inistered bovine serum albumin [J ]. Vaccine,1997, 16 (2/3) : 150- 155.
[6] Komase K. AB011677 Escherichia coli genes for heat-labile enterotoxin A subunit and B subunit, complete cds. [gi:3062900]
[7] Hofstra H, Witholt B. Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin. [J]Biol Chem. 1985, 260(29):16037-16044
[8] Sixma TK, Pronk SE, Kalk KH, et al. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature, 1991, 351: 371–377
[9] Jacob CO, Leitner M, Zamir A, et al. Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli. EMBO [J ]. 1985, 4(12):3339-3343
[10] Iida T, Tsuji T, Honda T, et al. A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation. [J ]. Biol. Chem., 1989, 264(24):14065-14070
[11] Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers., 1983, 22(12):2577-2637
[12] Janin J, Miller S, Chothia C. Surface, subunit interfaces and interior of oligomeric proteins. [J ]. Mol. Biol., 1988, 204(1):155-164
[13] Goins B, Freire E. Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1. Biochemistry, 1988, 27(6):2046-2052
[14] Williams NA, Hirst TR, and Nashar TO. Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunology Today, 1999, 20(2):95-101
[15] Moss J, Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol Relat Areas Mol Biol., 1988, 61:303-379
[16] van Heyningen S. in Current Topics in Membranes and Transport Vol.18:445-471(Academic. New York, 1983)
[17] Tsuji T, Honda T, Miwatani T, et al. Analysis of receptor-binding site in Escherichia coli enterotoxin. [J ] Biol. Chem., 1985, 260(14):8552-8558
[18] Fukuta S, Magnani JL, Twiddy EM, et al. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun. 1988 Jul;56(7):1748-53
[19] Holmgren J, Fredman P, Lindblad M, et al. Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin. Infect. Immun., 1982, 38(2):424-433
[20] Burnette WN, Mar VL, Platler BW, et al. Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity. Infect. Immun., 1991, 59(11):4266-4270
[21] Lobet Y, Cluff CW, Cieplak W Jr. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. Infect. Immun., 1991, 59(9):2870-2879
[22] Tsuji T, Inoue T, Miyama A, Okamoto K, et al. A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. [J ]. Biol. Chem., 1990,265(36):22520-22525
[23] Tsuji T, Inoue T, Miyama A, Noda M. Glutamic acid-112 of the A subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity. FEBS Lett., 1991, 291(2):319-321
[24] Tsai SC, Noda M, Adamik R, et al. Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain. [J ]. Biol. Chem, 1988, 263(4):1768-1772
[25] Zhu X, Kim E, Boman AL, Hodel A, et al. ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2. Biochemistry, 2001, 40(15):4560-4568
[26]冯强,蔡绍皙,杨珺大肠杆菌不耐热肠毒素的表达及其纯化保存策略[J].生物工程学报-2003,5,532-537
[27]鲁东水,毛旭虎,吴超,邹全明大肠杆菌不耐热肠毒素B亚单位融合表达载体的构建及应用其纯化保存策略[J].第三军医大学学报-2003,14,1275-1277
[28] Bandner BC, Giuliani MM, Verhoef JC, et al The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines [J]. Vaccine 2003, 21(25-26): 3837-3844
[29] Pizza M, Giuliani MM, Fontana MR, et al. Mucosal vaccines: non toxin derivatives of LT and CT as mucosal adjuvants. Vaccines, 2001, 19:2534-2541
[30] Nashar TO, Webb HM, Eaglestone S, et al. Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets. Proc Natl Acad Sci U S A., 1996, 93(1):226-230
[31]Nashar TO, Williams NA, Hirst TR, et al. Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes. Int. Immunol., 1996, 8(5):731-736
[32] Pierce NS, Gowans JL. Cellular kinetics of the intestineal immune response to cholera toxoid in rats. [J ]. Exp. Med., 1975, 142:1550-1563
[33] Komase K. AB011677 Escherichia coli genes for heat-labile enterotoxin A subunit and B subunit, completecds[gi:3062900]. [34 ] de Haan L , Verweij W, Agsteribbe E , et al . The role of ADP-ribosylation and GM1-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibro cholerea cholera toxin. [J]. Immun Cell Biol , 1998 ,76(20) :270 - 2791
[35] Giuliani MM, Del Giudice G, Giannelli V, et al. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin withpartial knockout of ADP-ribosyltransferase activity. [J]. Exp. Med., 1998, 187(7):1123-1132
[36]赵志强,杜琳译粘膜免疫[J].生物制品快讯-2004,9,39-48
[37]李忠明主编当代新疫苗[M].高等教育出版社2000,170-181]
[38]付百年,王敏文流感疫苗的粘膜免疫中国生物制品学杂志-2003,6,383-384
[39] Conference Coverage(ICAAC). New mucosal adjuvant safe in humans. Vaccine Weekly 1997; November 3.4.
[40] Durrer P, Gluck U, Spyr C, et al. Mucosal antibody response induced with a nasal virosome-based influenza vaccine.Vaccine, 2003, 21: 4328-4334
[41] Haan L, Verweij WR, Holtrop M, et al. Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA-or IgG-mediated protective mucosal immunity. Vaccine, 2001, 19(20-22): 2898-2907
[42]张永振,徐建国,粘膜免疫系统与粘膜免疫应答诱导[J].细胞生物学杂志,2001,23(1):11-16
[43] Katsuhiro Komase et al.Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine. [J].Vaccine 1998,16: 248-231.
[44] Covone MG, Brocchi M, et al. Levels of expression and immunogenicity of attenuated Salmonella enterica serovar Typhimurium strains expressing Escherichia coli heat-labile enterotoxin. [J]. Infect and immun 1998,66:224-231.。
[45] Baudner BC, GiulianiMM ,V erhoef JC, et al. The concom itant use of the L TK63 muco sal adjuvant and of ch ito san2based delivery system enhances the immunogenicity and efficacy of intranasally adm inistered vaccines. V accine, 2003, 21: 3837-3844.
[46] Murray, Thompson WF. Rapid isolation of high-molecular-weight plant DNA[J]. Nucl Acids Res, 1980, 8: 4320~4325.
[47] Holmgren J,czerkiIlsky C,Edksson K,et a1.mucosal immunisation and adjuvsnts:a blief overview of recent advances and challenges.[J]Vaccine,2003,21(suppl2):89-95
[48] Yamamoto T, Yolota T. Host-dependent, thermo sensitive replication of an R plasmid, pJY5, isolated from enterobacter cloacae. [J]. Bacteriol., 1977, 132(3):923-930
[49]冯强,杨珺,张卫军等大肠杆菌不耐热肠毒素B亚单位的分泌表达与性质鉴定[J].免疫学杂志,2004,20(9):364-368
[50] Kunkel SL, Robertson DC. Purification and chemical characterizeation of the heat-labile enterotoxin produced by enterotoxingenic Escherichia coli. Infect. Immunol., 1979, 25(2):596-589
[51] Uesaka Y, Otsuka Y, Lin Z, et al. Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose. Microb. Pathg, 1994, 16(1):71-76
[52] Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to derect expression of cloned genes. Methods Enzymol., 1990, 185(1):60-89,
[53] Haan L, Verweij WR, Holtrop M, et al. Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA-or IgG-mediated protective mucosal immunity. Vaccine, 2001, 19(20-22): 2898-2907
[54] Yang YW, Yang JC. Calcium phosphate as a gene carrier: electron microscopy[J]. Biomaterials, 1997, 18(3): 213-217.
[2] Dallas W S, Falkow S. Amino acid sequence homologybetween cholera toxin and Eschrichia coli heat-labiletoxin. [J] Nature. 1980, 288, 499-501.
[3] Spicer E K, Kavanaugh W M, Dallas W S, et al. Sequence homologiesbetween A subunits of Eschrichia coli and Vibrio cholerae en-terotoxins. [J] Proc. Natl. Acad. Sci. USA 1981, 78, 50-54.
[4] Verweij WR, de Haan L , M arijke H, et al. Musosal immunoadjuvant activity of recombinant E scherich ia coli heat-labile entero toxin and its B subunit: induction of system ic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen [J ]. Vaccine, 1998, 16 (20) : 2069-2076.
[5] Kunio T , Masanori I , Yoko Y , et al . Recombinant cholera toxin B subunit acts as an adjuvant for the mucosal and systemic responses of mice to mucosally coadm inistered bovine serum albumin [J ]. Vaccine,1997, 16 (2/3) : 150- 155.
[6] Komase K. AB011677 Escherichia coli genes for heat-labile enterotoxin A subunit and B subunit, complete cds. [gi:3062900]
[7] Hofstra H, Witholt B. Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin. [J]Biol Chem. 1985, 260(29):16037-16044
[8] Sixma TK, Pronk SE, Kalk KH, et al. Crystal structure of a cholera toxin-related heat-labile enterotoxin from E. coli. Nature, 1991, 351: 371–377
[9] Jacob CO, Leitner M, Zamir A, et al. Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli. EMBO [J ]. 1985, 4(12):3339-3343
[10] Iida T, Tsuji T, Honda T, et al. A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation. [J ]. Biol. Chem., 1989, 264(24):14065-14070
[11] Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers., 1983, 22(12):2577-2637
[12] Janin J, Miller S, Chothia C. Surface, subunit interfaces and interior of oligomeric proteins. [J ]. Mol. Biol., 1988, 204(1):155-164
[13] Goins B, Freire E. Thermal stability and intersubunit interactions of cholera toxin in solution and in association with its cell-surface receptor ganglioside GM1. Biochemistry, 1988, 27(6):2046-2052
[14] Williams NA, Hirst TR, and Nashar TO. Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunology Today, 1999, 20(2):95-101
[15] Moss J, Vaughan M. ADP-ribosylation of guanyl nucleotide-binding regulatory proteins by bacterial toxins. Adv Enzymol Relat Areas Mol Biol., 1988, 61:303-379
[16] van Heyningen S. in Current Topics in Membranes and Transport Vol.18:445-471(Academic. New York, 1983)
[17] Tsuji T, Honda T, Miwatani T, et al. Analysis of receptor-binding site in Escherichia coli enterotoxin. [J ] Biol. Chem., 1985, 260(14):8552-8558
[18] Fukuta S, Magnani JL, Twiddy EM, et al. Comparison of the carbohydrate-binding specificities of cholera toxin and Escherichia coli heat-labile enterotoxins LTh-I, LT-IIa, and LT-IIb. Infect Immun. 1988 Jul;56(7):1748-53
[19] Holmgren J, Fredman P, Lindblad M, et al. Rabbit intestinal glycoprotein receptor for Escherichia coli heat-labile enterotoxin lacking affinity for cholera toxin. Infect. Immun., 1982, 38(2):424-433
[20] Burnette WN, Mar VL, Platler BW, et al. Site-specific mutagenesis of the catalytic subunit of cholera toxin: substituting lysine for arginine 7 causes loss of activity. Infect. Immun., 1991, 59(11):4266-4270
[21] Lobet Y, Cluff CW, Cieplak W Jr. Effect of site-directed mutagenic alterations on ADP-ribosyltransferase activity of the A subunit of Escherichia coli heat-labile enterotoxin. Infect. Immun., 1991, 59(9):2870-2879
[22] Tsuji T, Inoue T, Miyama A, Okamoto K, et al. A single amino acid substitution in the A subunit of Escherichia coli enterotoxin results in a loss of its toxic activity. [J ]. Biol. Chem., 1990,265(36):22520-22525
[23] Tsuji T, Inoue T, Miyama A, Noda M. Glutamic acid-112 of the A subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity. FEBS Lett., 1991, 291(2):319-321
[24] Tsai SC, Noda M, Adamik R, et al. Stimulation of choleragen enzymatic activities by GTP and two soluble proteins purified from bovine brain. [J ]. Biol. Chem, 1988, 263(4):1768-1772
[25] Zhu X, Kim E, Boman AL, Hodel A, et al. ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2. Biochemistry, 2001, 40(15):4560-4568
[26]冯强,蔡绍皙,杨珺大肠杆菌不耐热肠毒素的表达及其纯化保存策略[J].生物工程学报-2003,5,532-537
[27]鲁东水,毛旭虎,吴超,邹全明大肠杆菌不耐热肠毒素B亚单位融合表达载体的构建及应用其纯化保存策略[J].第三军医大学学报-2003,14,1275-1277
[28] Bandner BC, Giuliani MM, Verhoef JC, et al The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines [J]. Vaccine 2003, 21(25-26): 3837-3844
[29] Pizza M, Giuliani MM, Fontana MR, et al. Mucosal vaccines: non toxin derivatives of LT and CT as mucosal adjuvants. Vaccines, 2001, 19:2534-2541
[30] Nashar TO, Webb HM, Eaglestone S, et al. Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets. Proc Natl Acad Sci U S A., 1996, 93(1):226-230
[31]Nashar TO, Williams NA, Hirst TR, et al. Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes. Int. Immunol., 1996, 8(5):731-736
[32] Pierce NS, Gowans JL. Cellular kinetics of the intestineal immune response to cholera toxoid in rats. [J ]. Exp. Med., 1975, 142:1550-1563
[33] Komase K. AB011677 Escherichia coli genes for heat-labile enterotoxin A subunit and B subunit, completecds[gi:3062900]. [34 ] de Haan L , Verweij W, Agsteribbe E , et al . The role of ADP-ribosylation and GM1-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibro cholerea cholera toxin. [J]. Immun Cell Biol , 1998 ,76(20) :270 - 2791
[35] Giuliani MM, Del Giudice G, Giannelli V, et al. Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin withpartial knockout of ADP-ribosyltransferase activity. [J]. Exp. Med., 1998, 187(7):1123-1132
[36]赵志强,杜琳译粘膜免疫[J].生物制品快讯-2004,9,39-48
[37]李忠明主编当代新疫苗[M].高等教育出版社2000,170-181]
[38]付百年,王敏文流感疫苗的粘膜免疫中国生物制品学杂志-2003,6,383-384
[39] Conference Coverage(ICAAC). New mucosal adjuvant safe in humans. Vaccine Weekly 1997; November 3.4.
[40] Durrer P, Gluck U, Spyr C, et al. Mucosal antibody response induced with a nasal virosome-based influenza vaccine.Vaccine, 2003, 21: 4328-4334
[41] Haan L, Verweij WR, Holtrop M, et al. Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA-or IgG-mediated protective mucosal immunity. Vaccine, 2001, 19(20-22): 2898-2907
[42]张永振,徐建国,粘膜免疫系统与粘膜免疫应答诱导[J].细胞生物学杂志,2001,23(1):11-16
[43] Katsuhiro Komase et al.Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine. [J].Vaccine 1998,16: 248-231.
[44] Covone MG, Brocchi M, et al. Levels of expression and immunogenicity of attenuated Salmonella enterica serovar Typhimurium strains expressing Escherichia coli heat-labile enterotoxin. [J]. Infect and immun 1998,66:224-231.。
[45] Baudner BC, GiulianiMM ,V erhoef JC, et al. The concom itant use of the L TK63 muco sal adjuvant and of ch ito san2based delivery system enhances the immunogenicity and efficacy of intranasally adm inistered vaccines. V accine, 2003, 21: 3837-3844.
[46] Murray, Thompson WF. Rapid isolation of high-molecular-weight plant DNA[J]. Nucl Acids Res, 1980, 8: 4320~4325.
[47] Holmgren J,czerkiIlsky C,Edksson K,et a1.mucosal immunisation and adjuvsnts:a blief overview of recent advances and challenges.[J]Vaccine,2003,21(suppl2):89-95
[48] Yamamoto T, Yolota T. Host-dependent, thermo sensitive replication of an R plasmid, pJY5, isolated from enterobacter cloacae. [J]. Bacteriol., 1977, 132(3):923-930
[49]冯强,杨珺,张卫军等大肠杆菌不耐热肠毒素B亚单位的分泌表达与性质鉴定[J].免疫学杂志,2004,20(9):364-368
[50] Kunkel SL, Robertson DC. Purification and chemical characterizeation of the heat-labile enterotoxin produced by enterotoxingenic Escherichia coli. Infect. Immunol., 1979, 25(2):596-589
[51] Uesaka Y, Otsuka Y, Lin Z, et al. Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose. Microb. Pathg, 1994, 16(1):71-76
[52] Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to derect expression of cloned genes. Methods Enzymol., 1990, 185(1):60-89,
[53] Haan L, Verweij WR, Holtrop M, et al. Nasal or intramuscular immunization of mice with influenza subunit antigen and the B subunit of Escherichia coli heat-labile toxin induces IgA-or IgG-mediated protective mucosal immunity. Vaccine, 2001, 19(20-22): 2898-2907
[54] Yang YW, Yang JC. Calcium phosphate as a gene carrier: electron microscopy[J]. Biomaterials, 1997, 18(3): 213-217.