大肠杆菌不耐热肠毒素亚单位的克隆、表达及A亚单位单克隆抗体的制备
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摘要
不耐热肠毒素(heat-labile enterotoxin,LT)是产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)产生的六聚体蛋白。不耐热肠毒素除了较强的毒性外,还具有很强的免疫原性以及能够辅助其他抗原通过粘膜免疫途径使机体产生特异性抗体,因而它还是良好的粘膜免疫佐剂,在粘膜免疫研究中具有重要价值。
本研究以产不耐热肠毒素44814株为材料,在产毒培养基CAYE-2培养后,收集的菌液经多粘菌素B和超声波处理,获得肠毒素粗提物。通过硫酸铵沉淀后,D-Salt Dextran Desalting Column层析脱盐获得肠毒素的初步提取物。提取物过滤除菌后,通过健康家兔回肠结扎试验和CHO细胞毒性试验,证明了所获得肠毒素的初步提取物能引起家兔回肠肠液蓄积和CHO细胞形变、死亡,符合不耐热肠毒素的生物学特性,表明提取物中含有不耐热肠毒素,从而为后续研究提供了检测抗原。
为了进一步了解不耐热肠毒素的生物学特性,根据其已发表的A、B亚单位结构基因序列,各设计一对引物,经PCR从产肠毒素大肠杆菌44814株分别扩增出LT-A、LT-B结构蛋白基因片段,并分别定向克隆入pGEX-6P-1质粒载体,分别获得重组质粒pGEX-LTA和pGEX-LTB。重组菌裂解物的SDS-PAGE及Western-blot分析证明,含有重组质粒pGEX-LTA的大肠杆菌BL21能够表达分子量约为56ku的融合蛋白GST-LTA,含有重组质粒pGEX-LTB的大肠杆菌BL21能够表达分子量约为39ku的融合蛋白GST-LTB,从而为研制LT的单克隆抗体提供了抗原。
单克隆抗体作为一种良好的检测试剂,具有较强的特异性。本研究利用初步提纯A亚单位的表达产物作为免疫原免疫6周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA检测,经三次亚克隆得到了稳定分泌抗LTA单克隆抗体的杂交瘤细胞株F8和G9,制备了腹水,并利用该单抗初步建立了间接ELISA和Dot-ELISA等特异性检测LT抗原的方法。为进一步建立特异性强、敏感性高、简便快速的LT检测方法奠定了基础。
Heat-labile enterotoixin (LT), produced by Enterotoxigenic Eshchirichia coli (ETEC), is the virulence factor of diarrhea in humans and animals. More interesting, LT also possesses strong immunogenicity and can assist other antigens delivered through mucosal pathway to produce specific antibodies, so it is a good mucosal immunization adjuvant and has significant medical and economic values in mucosal immunization research and development of vaccine for mucosal immunization.
In this research, to extract toxin, E. coli reference strain 44814 was cultured on CAYE-2 medium, and the collected bacteria were treated with polymixin B and followed by ultrasonic process. The enterotoxin extraction were obtained by precipitating the supernatant by using ammonium sulfate and removing the salts with“D-Salt Dextran Desalting Column”. The following tests on rabbit and CHO cells indicated that the enterotoxin extraction could lead the intestinal epithelium overmuch excrete and induce CHO cells distortion or death. The results confirmed that the extraction were contained heat-labile enterotoxin.
To study the biology characteristic of LT, two pairs of primers specific to LTA and LTB were designed and synthesized according to the published sequence of LT genes. Following the genes of lt-A and lt-B were amplified from strain 44814. The two fragments were subcloned into expression vector pGEX-6p-1. The resulting plasmid contained the lt-A gene was designed as pGEX-LTA and the plasmid contained the lt-B gene was designed as pGEX-LTB. By SDS-PAGE analysis and Western blotting, it confirmed that BL21 E.coli which contained pGEX-LTA was able to express large quantities of a 56-ku fusion protein (GST-LTA), and BL21 E.coli which contained pGEX-LTB was able to express large quantities of a 39-ku fusion protein (GST-LTB).
In order to identify Heat-labile enterotoixin, it is necessary to have the monoclonal antibody . The fusion protein GST-LTA was immunized the 6 weeks old BALB/c mice 4 times. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method, and the interested antibody were detected by indirect-ELISA and Dot-ELISA methods. The hybridoma cell strain F8 and G9 were obtained, which can stably secret monoclonal antibody specific for LT-A. Further more, indirect ELISA by using the monoclonal antibodies were performed to detect LT, and made a basis for further setting up a LT-testing method which has high specificity and sensibility, more over can be operated easily and quickly.
本研究以产不耐热肠毒素44814株为材料,在产毒培养基CAYE-2培养后,收集的菌液经多粘菌素B和超声波处理,获得肠毒素粗提物。通过硫酸铵沉淀后,D-Salt Dextran Desalting Column层析脱盐获得肠毒素的初步提取物。提取物过滤除菌后,通过健康家兔回肠结扎试验和CHO细胞毒性试验,证明了所获得肠毒素的初步提取物能引起家兔回肠肠液蓄积和CHO细胞形变、死亡,符合不耐热肠毒素的生物学特性,表明提取物中含有不耐热肠毒素,从而为后续研究提供了检测抗原。
为了进一步了解不耐热肠毒素的生物学特性,根据其已发表的A、B亚单位结构基因序列,各设计一对引物,经PCR从产肠毒素大肠杆菌44814株分别扩增出LT-A、LT-B结构蛋白基因片段,并分别定向克隆入pGEX-6P-1质粒载体,分别获得重组质粒pGEX-LTA和pGEX-LTB。重组菌裂解物的SDS-PAGE及Western-blot分析证明,含有重组质粒pGEX-LTA的大肠杆菌BL21能够表达分子量约为56ku的融合蛋白GST-LTA,含有重组质粒pGEX-LTB的大肠杆菌BL21能够表达分子量约为39ku的融合蛋白GST-LTB,从而为研制LT的单克隆抗体提供了抗原。
单克隆抗体作为一种良好的检测试剂,具有较强的特异性。本研究利用初步提纯A亚单位的表达产物作为免疫原免疫6周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,用间接ELISA检测,经三次亚克隆得到了稳定分泌抗LTA单克隆抗体的杂交瘤细胞株F8和G9,制备了腹水,并利用该单抗初步建立了间接ELISA和Dot-ELISA等特异性检测LT抗原的方法。为进一步建立特异性强、敏感性高、简便快速的LT检测方法奠定了基础。
Heat-labile enterotoixin (LT), produced by Enterotoxigenic Eshchirichia coli (ETEC), is the virulence factor of diarrhea in humans and animals. More interesting, LT also possesses strong immunogenicity and can assist other antigens delivered through mucosal pathway to produce specific antibodies, so it is a good mucosal immunization adjuvant and has significant medical and economic values in mucosal immunization research and development of vaccine for mucosal immunization.
In this research, to extract toxin, E. coli reference strain 44814 was cultured on CAYE-2 medium, and the collected bacteria were treated with polymixin B and followed by ultrasonic process. The enterotoxin extraction were obtained by precipitating the supernatant by using ammonium sulfate and removing the salts with“D-Salt Dextran Desalting Column”. The following tests on rabbit and CHO cells indicated that the enterotoxin extraction could lead the intestinal epithelium overmuch excrete and induce CHO cells distortion or death. The results confirmed that the extraction were contained heat-labile enterotoxin.
To study the biology characteristic of LT, two pairs of primers specific to LTA and LTB were designed and synthesized according to the published sequence of LT genes. Following the genes of lt-A and lt-B were amplified from strain 44814. The two fragments were subcloned into expression vector pGEX-6p-1. The resulting plasmid contained the lt-A gene was designed as pGEX-LTA and the plasmid contained the lt-B gene was designed as pGEX-LTB. By SDS-PAGE analysis and Western blotting, it confirmed that BL21 E.coli which contained pGEX-LTA was able to express large quantities of a 56-ku fusion protein (GST-LTA), and BL21 E.coli which contained pGEX-LTB was able to express large quantities of a 39-ku fusion protein (GST-LTB).
In order to identify Heat-labile enterotoixin, it is necessary to have the monoclonal antibody . The fusion protein GST-LTA was immunized the 6 weeks old BALB/c mice 4 times. The splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method, and the interested antibody were detected by indirect-ELISA and Dot-ELISA methods. The hybridoma cell strain F8 and G9 were obtained, which can stably secret monoclonal antibody specific for LT-A. Further more, indirect ELISA by using the monoclonal antibodies were performed to detect LT, and made a basis for further setting up a LT-testing method which has high specificity and sensibility, more over can be operated easily and quickly.
引文
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[2] O’Brien AD, RK Holmes. Shiga and Shiga–like toxins[J]. Microbiol Rev. 1987, 51: 206-220.
[3] Sack RB. Enterotoxigenic Escherichia coli: identification and characterization[J]. Infect Dis. 1980. 142(2): 279-286.
[4] Smith NW, MA Linggord. Transfer factors in Escherichis coli with particular regard to their incidence in enteropathogenic strains [J ] . J . Gen. Microbiol. 1970 ,62(2) :287 - 299.
[5] 王嘉福, 冉雪琴. 应用聚合酶链反应技术克隆猪源大肠杆菌耐热毒素基因[ J ] . 中国兽医杂志, 1997 , 23(4) :3 - 4.
[6] 王嘉福,冉雪琴,叶在荣,等. 猪源大肠杆菌热肠毒素基因的分子克隆[J ] . 畜牧兽医学报,1998 ,29 (1) :60- 65.
[7] Newsome P M, MN Buregss. Effect of Escherichia coli heat-stable enterotoxin on cyclic GMP levels in mouse intestine [J]. Infect. Immun. 1978, 22(1): 290-291.
[8] Betly MJ, VL Miller. Genetics of bacterial enterotoxin[J]. Ann. Rev Microbiol, 1986, 40: 577-605.
[9] Clements JD, RA Finkelstein. Demonstration of shared and unique immunological enterminants in enterotoxins from Vibrio cholerae and Escherichia coli[J].nfect Immun. 1978, 22(3): 709-713.
[10] Gyles CD, A. Barnum. A heat–labile enterotoxin from strains of Escherichia coli enteropathogenic for pigs[J]. J. Infect Dis. 1969, 120(3): 419-426.
[11] Clements JD, DC. Fint. Immunological and physicolchemical characterization of heat-labile enterotoxins isolated from two strains of Escherichia coli[J]. Infect .Immun. 1982, 38(2): 806-809.
[12] Sminth NW, RB Sack. Immunologic cross-reactions of entertoxin from Escherichia coli and vibrio cholerae[J] . J Infect Dis. 1973, 127(1): 164 - 170.
[13] Komase K. AB011677 Escherichia coli genes for heat-labile enterotoxin A subunit and B subunit, complete cds[gi:3062900].
[14] 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.
[15] 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.
[16] 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.
[17] 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.
[18] 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.
[19] Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers., 1983, 22(12): 2577-2637.
[20] Janin J, Miller S, Chothia C. Surface, subunit interfaces and interior of oligomeric proteins. J.Mol. Biol., 1988, 204(1): 155-164.
[21] 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.
[22] 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.
[23] van Heyningen S. in Current Topics in Membranes and Transport Vol.18: 445-471(Academic. New York, 1983).
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