梅毒螺旋体抗原在毕赤酵母中的表达纯化和单克隆抗体的制备
发布时间：2018-10-05 11:16
【摘要】：本研究首次使用毕赤酵母表达系统(Pichia pastoris)表达梅毒螺旋体(Tep onema pallidum)47 kDa、17 kDa和15 kDa三个内膜脂蛋白。毕赤酵母具有很多真核表达系统的优点,例如蛋白质的加工、折叠和转录后修饰,分泌表达,以及很强的甲醇诱导启动子等。 以梅毒螺旋体标准菌株Nichols基因组DNA为模板,扩增出47 kDa、17 kDa和15 kDa 3个成熟蛋白基因(不包含N端信号肽序列),PCR产物经酶切后分别与pPICZB和pPIC9K两个质粒相连,再转化进毕赤酵母菌株GS115。pPICZB和pPIC9K质粒不具备酵母复制起始位点,因此重组质粒通过同源序列与宿主菌株基因组重组,形成了稳定遗传的重组蛋白表达菌株BTP47-GS115、BTP17-GS115、BTP15-GS115和KTP47-GS115、KTP17-GS115、KTP15-GS115。 BTP47-GS115、BTP17-GS115和BTP15-GS115菌株经过诱导,收集菌体并用超声波破碎,通过SDS-PAGE和Western blot鉴定,确定目的蛋白在胞内形成了表达。进一步诱导表达,用Ni-NTA纯化,分别得到了His-BTP15:4.8mg/L、His-BTP17:6.6 mg/L和His-BTP47:25 mg/L的融合蛋白,ELISA鉴定均具有抗原活性。 KTP47-GS115、KTP17-GS115和KTP15-GS115菌株经过诱导,亦通过SDS-PAGE和Western blot鉴定培养上清,确定目的蛋白形成了较高量的分泌表达。其中KTP47和KTP17表达量在200mg/L培养液以上,KTP15也达到了100mg/L培养液左右,具备了大规模生产的潜力。 同时,以纯化的His-BTP47抗原免疫BALB/c小鼠,拟建立梅毒螺旋体47 kDa抗原杂交瘤细胞株。以KTP47抗原包被96孔板,建立了间接ELISA梅毒血清检测系统,用于免疫小鼠效价测定和杂交瘤细胞株的筛选。最后我们筛选出了3株能稳定分泌47 kDa抗原特异性抗体的阳性杂交瘤细胞株,命名为B2H2、C3F5
[Abstract]:In this study, Pichia pastoris expression system (Pichia pastoris) was used for the first time to express (Tep onema pallidum) 47 kDa,17 kDa and 15 kDa endometrial lipoproteins of Treponema pallidum. Pichia pastoris has many advantages of eukaryotic expression system, such as protein processing, folding and post-transcriptional modification, secretory expression, and strong methanol induced promoter. Using the genomic DNA of Treponema pallidum standard strain Nichols as a template, three mature protein genes (excluding N-terminal signal peptide) of 47 kDa,17 kDa and 15 kDa were digested with pPICZB and pPIC9K plasmids, respectively. The recombinant plasmid was transformed into Pichia pastoris GS115.pPICZB and pPIC9K plasmids without yeast replication initiation site, so the recombinant plasmid was recombined with host strain genome by homologous sequence to form stable genetic recombinant protein expression strains BTP47-GS115,BTP17-GS115,BTP15-GS115 and KTP47-GS115,KTP17-GS115,KTP15-GS115.. After induction of BTP47-GS115,BTP17-GS115 and BTP15-GS115 strains, the bacteria were collected and broken by ultrasonic, and identified by SDS-PAGE and Western blot, the expression of the target protein was confirmed in the cell. After further induction and purification by Ni-NTA, the fusion protein His-BTP15:4.8mg/L,His-BTP17:6.6 mg/L and His-BTP47:25 mg/L were identified to have antigenic activity by enzyme-linked immunosorbent assay (Elisa). After induction of KTP47-GS115,KTP17-GS115 and KTP15-GS115, the supernatants were identified by SDS-PAGE and Western blot. It was confirmed that the target protein was secreted and expressed in high quantity. The expression of KTP47 and KTP17 in the medium above 200mg/L also reached the level of 100mg/L, which has the potential of mass production. At the same time, BALB/c mice were immunized with purified His-BTP47 antigen to establish 47 kDa antigen hybridoma cell line of Treponema pallidum. The indirect ELISA syphilis serum detection system was established with KTP47 antigen coated with 96 well plate, which was used to determine the titer of immunized mice and screen hybridoma cell lines. Finally, we screened out three positive hybridoma cell lines which can stably secrete 47 kDa antigen-specific antibodies, named B2H2C3F5.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q819
本文编号：2253170
[Abstract]:In this study, Pichia pastoris expression system (Pichia pastoris) was used for the first time to express (Tep onema pallidum) 47 kDa,17 kDa and 15 kDa endometrial lipoproteins of Treponema pallidum. Pichia pastoris has many advantages of eukaryotic expression system, such as protein processing, folding and post-transcriptional modification, secretory expression, and strong methanol induced promoter. Using the genomic DNA of Treponema pallidum standard strain Nichols as a template, three mature protein genes (excluding N-terminal signal peptide) of 47 kDa,17 kDa and 15 kDa were digested with pPICZB and pPIC9K plasmids, respectively. The recombinant plasmid was transformed into Pichia pastoris GS115.pPICZB and pPIC9K plasmids without yeast replication initiation site, so the recombinant plasmid was recombined with host strain genome by homologous sequence to form stable genetic recombinant protein expression strains BTP47-GS115,BTP17-GS115,BTP15-GS115 and KTP47-GS115,KTP17-GS115,KTP15-GS115.. After induction of BTP47-GS115,BTP17-GS115 and BTP15-GS115 strains, the bacteria were collected and broken by ultrasonic, and identified by SDS-PAGE and Western blot, the expression of the target protein was confirmed in the cell. After further induction and purification by Ni-NTA, the fusion protein His-BTP15:4.8mg/L,His-BTP17:6.6 mg/L and His-BTP47:25 mg/L were identified to have antigenic activity by enzyme-linked immunosorbent assay (Elisa). After induction of KTP47-GS115,KTP17-GS115 and KTP15-GS115, the supernatants were identified by SDS-PAGE and Western blot. It was confirmed that the target protein was secreted and expressed in high quantity. The expression of KTP47 and KTP17 in the medium above 200mg/L also reached the level of 100mg/L, which has the potential of mass production. At the same time, BALB/c mice were immunized with purified His-BTP47 antigen to establish 47 kDa antigen hybridoma cell line of Treponema pallidum. The indirect ELISA syphilis serum detection system was established with KTP47 antigen coated with 96 well plate, which was used to determine the titer of immunized mice and screen hybridoma cell lines. Finally, we screened out three positive hybridoma cell lines which can stably secrete 47 kDa antigen-specific antibodies, named B2H2C3F5.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392;Q819
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