NP-1在小球藻硝酸还原酶缺失突变体中的表达与活性检测

摘 要：小球藻繁殖快，培养简单，用转基因小球藻表达目的基因产物具有重要的应用前景。本研究构建了兔防御素NP-1的植物表达载体，NP-1由Ubiquitin启动子控制，含NPTII基因和硝酸还原酶基因两个筛选标记。以椭圆小球藻(Chlorella ellipsoidea)硝酸还原酶缺失突变体nrm-4为受体，采用原生质体转化的方法将目的基因转入该突变体中。用含G418的硝酸盐培养基筛选出阳性藻落，经PCR和RT-PCR检测，证明获得了转基因藻株。转基因藻株在无抗生素筛选压力的硝酸盐培养基上继代培养后进行抑菌活性检测。研究表明，转基因椭圆小球藻表达的NP-1具有正常的生物活性，并在无抗生素的培养基中稳定遗传。本研究的结果为小球藻表达外源基因提供了新的技术途径，也为NP-1的规模化生产奠定了基础。

Expression and Bioactivity Characterization of NP-1 in Transgenic Nitrate Reductase Deficient Mutant Of Chlorella ellipsoidea

Abstract：It is a potential valuable way using transgenic Cholrella as a bioreactor to produce alien proteins. In this paper, the vector containing the NP-1 gene under the control of the Ubiquitin promoter and two selective marker genes NPTII and nitrate reductase gene was constructed. The NP-1 gene was transformed into the nitrate reductase-deficient mutant nrm-4 of C. ellipsoidea via a protoplast transformation method. The transformed nrm-4 cells were screened on the medium with nitrate and G418. The transgenic lines were characterized by PCR amplification of both the NP-1 gene and NPTII genes from genomic DNA isolated from transformants. The transgenic nrm-4 was further cultured in the liquid medium with nitrate but without G418. In vitro assay results showed that the protein of the transgenic C. ellipsoidea had effective bacteria- resistance activities and it is stable without G418 selection stress. The research provided a new way to express alien genes in Chlorella, and the basis to largely produce NP-1.