霍亂毒素的純化及其單克隆抗體的制備與初步應(yīng)用
發(fā)布時間：2019-06-17 13:25
【摘要】： 目的建立從培養(yǎng)基上清分離純化霍亂毒素的有效方法,制備高純度的霍亂毒素;獲得穩(wěn)定分泌抗霍亂毒素的雜交瘤細胞株、純化抗霍亂毒素的單克隆抗體和多克隆抗體;克隆大腸不耐熱腸毒素基因、構(gòu)建其表達載體、純化并鑒定大腸不耐熱腸毒素,以對抗霍亂毒素單克隆抗體的特異性進行評價;用生物素標記抗霍亂毒素單克隆抗體、篩選最佳的夾心ELISA抗體組合、確定抗體最佳包被濃度、建立生物素—親和素—ELISA的方法檢測CT,以期為霍亂弧菌產(chǎn)毒株的診斷奠定基礎(chǔ)。 方法將霍亂弧菌O1群569B在AKI培養(yǎng)基中培養(yǎng),收集上清,用六偏聚磷酸鈉鹽析,磷酸纖維素P11柱和Immobilized D-galactose柱純化,超濾濃縮后獲得CT,SDS-PAGE、Western Blot和GM1-ELISA驗證其純度和生物學(xué)活性。以純化的CT免疫Balb/c小鼠,采用傳統(tǒng)的雜交瘤技術(shù)制備針對CT的單克隆抗體(MAb),以間接法ELISA法篩選穩(wěn)定分泌抗CT的雜交瘤細胞株,體內(nèi)法誘導(dǎo)產(chǎn)生腹水,鑒定MAb類型,采用優(yōu)球蛋白法純化IgM抗體,HiTrap rProtein A FF柱純化多克隆抗體。PCR擴增大腸桿菌E10407的LT基因,酶切后連接于pUC-19質(zhì)粒,設(shè)計引物進行點突變,然后連接于表達載體pET-30a,測序驗證序列的正確,并轉(zhuǎn)化于大腸桿菌BL21(DE3)。GM1-ELISA篩選表達LT的陽性克隆,超聲破碎重組大腸菌,用Immobilized D-galactose柱純化LT蛋白,SDS-PAGE、質(zhì)譜和Vero細胞毒性試驗驗證其純度及生物學(xué)活性。用生物素標記抗霍亂毒素單克隆抗體,篩選最佳的夾心ELISA抗體組合,確定最適的抗體包被濃度和一抗稀釋倍數(shù),建立BA-ELISA法檢測CT。確定最低檢測CT的閾值,并用BA-ELISA法檢測不同細菌的培養(yǎng)基上清。 結(jié)果獲得相對分子量約為85kDa的高純度且有生物學(xué)活性的CT蛋白和5株穩(wěn)定分泌抗CT MAb的雜交瘤細胞株,抗體類型均為IgM,經(jīng)ELISA證實其可結(jié)合CT,其中兩株結(jié)合于CT-B亞單位,其余三株的既可結(jié)合于CT-A,也可結(jié)合于CT-B。獲得了高純度的抗霍亂毒素單克隆抗體和多克隆抗體。制備了高純度且有生物學(xué)活性的LT蛋白。確定了以抗CT多抗(CTPAb)為包被抗體,生物素標記的抗CT-B IgG單抗(CTMAb-XU-22)為一抗的夾心BA-ELISA的最佳檢測組合,抗CT多抗的最佳包被濃度為20μg/ml,抗CT-B IgG單抗的最佳稀釋倍數(shù)為1:50,最低檢測CT的限值為2ng。BA-ELISA法檢測不同細菌培養(yǎng)液的上清的結(jié)果與PCR法一致,并可區(qū)分霍亂弧菌的產(chǎn)毒株和非產(chǎn)毒株。 結(jié)論本研究建立了從霍亂弧菌培養(yǎng)基上清分離純化CT蛋白的有效方法,獲得了高純度和高活性的CT和LT蛋白,制備了抗CT MAb和PAb,利用多克隆抗體的強富集能力、單克隆抗體的高度特異性以及生物素—親和素系統(tǒng)的放大作用,建立了高靈敏度的生物素—親和素—ELISA的方法,為進一步應(yīng)用此檢測方法快速檢測霍亂弧菌,對霍亂弧菌產(chǎn)毒株,特別是毒素基因轉(zhuǎn)移的新致病菌株的發(fā)現(xiàn)、監(jiān)測、預(yù)防和控制工作有很大的幫助。
[Abstract]:Objective to establish an effective method for isolation and purification of cholera toxin from culture medium, to prepare high purity cholera toxin, to obtain hybridoma cell line stably secreting anti-cholera toxin, to purify monoclonal antibody and polyclonal antibody against cholera toxin, to clone colorectal heat-resistant enterotoxin gene, to construct its expression vector, to purify and identify colorectal heat-resistant enterotoxin, and to evaluate the specificity of monoclonal antibody against cholera toxin. The monoclonal antibody against cholera toxin was labeled with biotin, the best combination of sandwich ELISA antibody was screened, the optimal coating concentration of antibody was determined, and the method of biotin-avidin-ELISA was established to detect CT, in order to lay a foundation for the diagnosis of Vibrio cholera strain. Methods Vibrio cholera O1 group 569B was cultured in AKI medium and purified by sodium hexametaphosphate salting out, cellulose phosphate P11 column and Immobilized D-galactose column. after ultrafiltration concentration, CT,SDS-PAGE,Western Blot and GM1-ELISA were obtained to verify its purity and biological activity. Balb/c mice were immunized with purified CT. Monoclonal antibody (MAb), against CT was prepared by traditional hybridoma technique. Hybridoma cell line stably secreting anti-CT was screened by indirect ELISA method. Ascitic fluid was induced by in vivo method, MAb type was identified, IgM antibody, HiTrap rProtein A FF column was purified by euglobulin method. LT gene of E. coli E10407 was amplified by PCR and ligated to pUC-19 plasmid. Primers were designed for point mutation, then ligated to the expression vector pET-30a, sequencing to verify the correct sequence, and transformed into E. coli BL21 (DE3). The positive clones expressing LT were screened by GM1-ELISA, the recombinant coliform bacteria were broken by ultrasound, and the LT protein was purified by Immobilized D-galactose column. the purity and biological activity of LT protein were verified by SDS-PAGE, mass spectrometry and Vero cytotoxicity test. The monoclonal antibody against cholera toxin was labeled with biotin, the best sandwich ELISA antibody combination was screened, the optimum antibody coating concentration and dilution multiple were determined, and the BA-ELISA method was established for the detection of CT.. The minimum threshold for detection of CT was determined, and the culture medium of different bacteria was detected by BA-ELISA. Results High purity and biologically active CT protein with relative molecular weight of 85kDa and 5 hybridoma cell lines stably secreting anti-CT MAb were obtained. The antibody types of IgM, were confirmed by ELISA to bind to CT, two of which could bind to CT-B subunit, and the other three strains could bind to both CT-A, and CT-B.. High purity monoclonal antibodies and polyclonal antibodies against cholera toxin were obtained. LT protein with high purity and biological activity was prepared. The optimum detection combination of sandwich BA-ELISA with anti-CT polyclonal antibody (CTPAb) as coating antibody and biotin-labeled anti-CT-B IgG monoclonal antibody (CTMAb-XU-22) as primary antibody was determined. the optimum coating concentration of anti-CT polyantibody was 20 渭 g / ml, anti-CT-B IgG monoclonal antibody was 1 鈮，
本文編號：2501020
[Abstract]:Objective to establish an effective method for isolation and purification of cholera toxin from culture medium, to prepare high purity cholera toxin, to obtain hybridoma cell line stably secreting anti-cholera toxin, to purify monoclonal antibody and polyclonal antibody against cholera toxin, to clone colorectal heat-resistant enterotoxin gene, to construct its expression vector, to purify and identify colorectal heat-resistant enterotoxin, and to evaluate the specificity of monoclonal antibody against cholera toxin. The monoclonal antibody against cholera toxin was labeled with biotin, the best combination of sandwich ELISA antibody was screened, the optimal coating concentration of antibody was determined, and the method of biotin-avidin-ELISA was established to detect CT, in order to lay a foundation for the diagnosis of Vibrio cholera strain. Methods Vibrio cholera O1 group 569B was cultured in AKI medium and purified by sodium hexametaphosphate salting out, cellulose phosphate P11 column and Immobilized D-galactose column. after ultrafiltration concentration, CT,SDS-PAGE,Western Blot and GM1-ELISA were obtained to verify its purity and biological activity. Balb/c mice were immunized with purified CT. Monoclonal antibody (MAb), against CT was prepared by traditional hybridoma technique. Hybridoma cell line stably secreting anti-CT was screened by indirect ELISA method. Ascitic fluid was induced by in vivo method, MAb type was identified, IgM antibody, HiTrap rProtein A FF column was purified by euglobulin method. LT gene of E. coli E10407 was amplified by PCR and ligated to pUC-19 plasmid. Primers were designed for point mutation, then ligated to the expression vector pET-30a, sequencing to verify the correct sequence, and transformed into E. coli BL21 (DE3). The positive clones expressing LT were screened by GM1-ELISA, the recombinant coliform bacteria were broken by ultrasound, and the LT protein was purified by Immobilized D-galactose column. the purity and biological activity of LT protein were verified by SDS-PAGE, mass spectrometry and Vero cytotoxicity test. The monoclonal antibody against cholera toxin was labeled with biotin, the best sandwich ELISA antibody combination was screened, the optimum antibody coating concentration and dilution multiple were determined, and the BA-ELISA method was established for the detection of CT.. The minimum threshold for detection of CT was determined, and the culture medium of different bacteria was detected by BA-ELISA. Results High purity and biologically active CT protein with relative molecular weight of 85kDa and 5 hybridoma cell lines stably secreting anti-CT MAb were obtained. The antibody types of IgM, were confirmed by ELISA to bind to CT, two of which could bind to CT-B subunit, and the other three strains could bind to both CT-A, and CT-B.. High purity monoclonal antibodies and polyclonal antibodies against cholera toxin were obtained. LT protein with high purity and biological activity was prepared. The optimum detection combination of sandwich BA-ELISA with anti-CT polyclonal antibody (CTPAb) as coating antibody and biotin-labeled anti-CT-B IgG monoclonal antibody (CTMAb-XU-22) as primary antibody was determined. the optimum coating concentration of anti-CT polyantibody was 20 渭 g / ml, anti-CT-B IgG monoclonal antibody was 1 鈮，
本文編號：2501020
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