發(fā)布時(shí)間：2018-10-05 11:16

【摘要】：本研究首次使用畢赤酵母表達(dá)系統(tǒng)(Pichia pastoris)表達(dá)梅毒螺旋體(Tep onema pallidum)47 kDa、17 kDa和15 kDa三個(gè)內(nèi)膜脂蛋白。畢赤酵母具有很多真核表達(dá)系統(tǒng)的優(yōu)點(diǎn),例如蛋白質(zhì)的加工、折疊和轉(zhuǎn)錄后修飾,分泌表達(dá),以及很強(qiáng)的甲醇誘導(dǎo)啟動(dòng)子等。 以梅毒螺旋體標(biāo)準(zhǔn)菌株Nichols基因組DNA為模板,擴(kuò)增出47 kDa、17 kDa和15 kDa 3個(gè)成熟蛋白基因(不包含N端信號(hào)肽序列),PCR產(chǎn)物經(jīng)酶切后分別與pPICZB和pPIC9K兩個(gè)質(zhì)粒相連,再轉(zhuǎn)化進(jìn)畢赤酵母菌株GS115。pPICZB和pPIC9K質(zhì)粒不具備酵母復(fù)制起始位點(diǎn),因此重組質(zhì)粒通過同源序列與宿主菌株基因組重組,形成了穩(wěn)定遺傳的重組蛋白表達(dá)菌株BTP47-GS115、BTP17-GS115、BTP15-GS115和KTP47-GS115、KTP17-GS115、KTP15-GS115。 BTP47-GS115、BTP17-GS115和BTP15-GS115菌株經(jīng)過誘導(dǎo),收集菌體并用超聲波破碎,通過SDS-PAGE和Western blot鑒定,確定目的蛋白在胞內(nèi)形成了表達(dá)。進(jìn)一步誘導(dǎo)表達(dá),用Ni-NTA純化,分別得到了His-BTP15:4.8mg/L、His-BTP17:6.6 mg/L和His-BTP47:25 mg/L的融合蛋白,ELISA鑒定均具有抗原活性。 KTP47-GS115、KTP17-GS115和KTP15-GS115菌株經(jīng)過誘導(dǎo),亦通過SDS-PAGE和Western blot鑒定培養(yǎng)上清,確定目的蛋白形成了較高量的分泌表達(dá)。其中KTP47和KTP17表達(dá)量在200mg/L培養(yǎng)液以上,KTP15也達(dá)到了100mg/L培養(yǎng)液左右,具備了大規(guī)模生產(chǎn)的潛力。 同時(shí),以純化的His-BTP47抗原免疫BALB/c小鼠,擬建立梅毒螺旋體47 kDa抗原雜交瘤細(xì)胞株。以KTP47抗原包被96孔板,建立了間接ELISA梅毒血清檢測(cè)系統(tǒng),用于免疫小鼠效價(jià)測(cè)定和雜交瘤細(xì)胞株的篩選。最后我們篩選出了3株能穩(wěn)定分泌47 kDa抗原特異性抗體的陽性雜交瘤細(xì)胞株,命名為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.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392;Q819

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