HPT-ELISA方法的建立及其在转基因水稻监测中的应用

本文改进了HPT-ELISA检测法,利用一种简便并且高效的微生物表达体系,将hpt基因的全编码序列克隆到原核表达质粒pGEX-KG上,在E.Coli菌株BL21(DE3)-pLys中进行诱导表达,获得了融合蛋白GST-HPT,经Thrombin凝血酶酶切过夜,再经过柱纯化后获得不含GST且具有生物活性的HPT纯蛋白,所得蛋白纯度>90%。MALDI-TOF-MS分析表明,HPT蛋白分子量为39.4KD。用HPT纯蛋白免疫家兔,制备了高效价的多克隆抗体,进而构建了双抗夹心酶联免疫检测方法,其灵敏度为0.31ng/ml。应用该HPT-ELISA方法,测定了不同生育期转Bt基因水稻植株中HPT蛋白的表达水平以及成熟期稻米中的HPT蛋白含量。结果表明,不同生育期转Bt基因水稻植株中HPT蛋白的表达量约为15.67~60.12ng/g.FW,转Bt基因稻米中HPT蛋白的含量为5.28ng HPT/g.FW,而在非转基因亲本的植株和稻米中均未检测到HPT蛋白。

ESTABLISHMENT OF HPT-ELISA METHOD AND ITS APPLICATION IN HPT-MONITORING OF TRANSGENIC PLANTS

A simple HPT-ELISA method was developed for quantitative and qualitative determination of HPT protein.Hpt gene fragment was inserted into the prokaryotic vector pGEX-KG,and transferred into E.coli BL21 for expression of HPT fusion protein.After overnight cleavage with Thrombin and purification by liquid chromatography,the final HPT proteins were obtained with purity of 90%.The analysis of MALDI-TOF-MS demonstrated that molecular weight is 39.4kD.The HPT protein was used to immunize rabbit for the preparation of polyclonal antibodies with high titer.The specificity of HPT-antibody(HPT-Ab) was detected by Western blot,which showed specific binding reaction between the antibodies and the purified HPT protein.Then the double-Ab sandwich ELISA method for HPT protein was established with the sensitivity of 0.31 ng/ml.The method was applied to determine HPT content in Bt transgenic rice.The results showed that the HPT expression level in the plants of Bt transgenic rice ranged from 15.67 to 60.12 ng/gFW during the whole growth,and its content in the flour of the Bt-rice was 5.28 ng/gFW.Relatively,no HPT protein was detected in non-Bt rice.