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Archive > Volume 10 Issue 3 > 2018,10(3):285-289 Prev Next

BcbZIP134的多克隆抗體制備用于柴胡皂苷生物合成的轉(zhuǎn)錄調(diào)控分析

Original articles

Polyclonal antibody preparation of BcbZIP134 for transcriptional regulation analysis on saikosaponin biosynthesis

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    摘要:
    目的：本研究的目的是使用特異性抗體來(lái)驗(yàn)證轉(zhuǎn)錄因子BcbZIP134如何調(diào)節(jié)柴胡皂苷的合成。然而，難以獲得體外表達(dá)的可溶性蛋白質(zhì)，可溶性蛋白的獲得是制備抗體的先決條件。因此我們探索了一種可溶性蛋白可以有效表達(dá)的條件，進(jìn)行特異性多克隆抗體的制備。 方法：首先在Transetta（DE3）大腸桿菌中表達(dá)柴胡BcbZIP134 cDNA（DC保留）。探索了不同濃度的IPTG（0.05和0.5mmol / L）和不同培養(yǎng)溫度（16和37℃）以有效表達(dá)靶蛋白。然后，使用Ni Sepharose 6 Fast Flow純化表達(dá)的具有His標(biāo)簽的蛋白質(zhì)。使用不同濃度的咪唑洗脫液（15,60和300mmol / L）洗脫靶蛋白。用純化的BcbZIP134蛋白免疫家兔。使用純化的多克隆抗體，通過(guò)蛋白質(zhì)印跡分析BcbZIP134在轉(zhuǎn)基因毛狀根系中的表達(dá)。 結(jié)果：在以下條件下：使用pEASY®-Blunt E1載體和Transetta（DE3）大腸桿菌，IPTG 0.5mmol / L，16℃和誘導(dǎo)過(guò)夜獲得大量的可溶性BcbZIP134重組蛋白。在兔中免疫后以高效價(jià)1：51 200獲得特異性 的BcbZIP134的抗體。多克隆抗體可以與BcbZIP134轉(zhuǎn)基因毛狀根特異性結(jié)合。 結(jié)論：獲得了BcbZIP134的可溶性重組蛋白在大腸桿菌中高效的表達(dá)系統(tǒng)，。 通過(guò)使用這種蛋白質(zhì)，制備了特異性和高性能的多克隆抗體。 此外，通過(guò)使用BcbZIP134過(guò)表達(dá)的毛狀根，通過(guò)蛋白質(zhì)印跡分析驗(yàn)證了多克隆抗體的可用性。

    Abstract:
    Objective The aim of the present study was to verify how a transcription factor BcbZIP134 regulates the synthesis of saikosaponin using specific antibody. However, it is hard to obtain this soluble protein expressed in vitro, a prerequisite for antibody preparation. So the condition in which the soluble protein can efficiently express followed by preparation of the specific polyclonal antibody were explored. Methods Firstly, the cDNA of BcbZIP134 from Bupleurum chinense was expressed in Transetta (DE3) E. coli. Different concentrations of IPTG (0.05 and 0.5 mmol/L) and different culture temperatures (16 and 37 °C) were explored for efficient expression of target protein. Then, the expressed protein with His Tag was purified using Ni Sepharose 6 Fast Flow. Different concentrations of imidazole elution (15, 60, and 300 mmol/L) were used to elute the target protein. The purified protein of BcbZIP134 was used to immunize rabbit. Using the purified polyclonal antibody, the expression of BcbZIP134 in transgenic B. chinense hairy root lines was analyzed by Western Blot. Results Under the following conditions: IPTG 0.5 mmol/L, 16 °C, and overnight, recombinant protein of BcbZIP134 was obtained in high content using pEASY?-Blunt E1 vector and Transetta (DE3) E. coli. Anti-serum against BcbZIP134 was obtained with a high titer of 1: 51 200 after immunization in rabbit. The polyclonal antibody could react with BcbZIP134 overexpressed in transgenic B. chinense hairy root lines. Conclusion An efficient protein expression system in E. coli was constructed and the soluble recombinant protein for BcbZIP134 was obtained. By using this protein, the specific and high-performance polyclonal antibody was prepared. Furthermore, by using BcbZIP134 overexpressed hairy roots, the availability of the polyclonal antibody was verified by Western Blot analysis.

    關(guān)鍵詞:
    BcbZIP134 北柴胡 多克隆抗體 原核表達(dá)

    Keywords:


    Project Supported:
    CAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-2-003); National Standardization Project of Traditional Chinese Medicine (ZYBZH-Y-JIN-34).

    Clc Number:

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