鸡 -防御素-1,6 基因工程菌的构建及表达制备1鸡 -防御素-1,6 基因工程菌的构建及表达制备摘摘 要要-防御素是鸡体内的一种重要抗菌肽，除了具有直接的杀菌、抗病毒作用外，它还参与机体免疫反应，有重大的开发利用潜力，当前生物医学的研究热点之一。本研究以鸡 -防御素 Gal-1 和 Gal-6 为研究对象，运用分子生物学技术，从安徽三黄鸡骨髓 RNA 中克隆出 Gal-1 和 Gal-6 基因片段，并分别构建 pET-28a-Gal-6 原核表达载体和 pPIC3.5K-Gal-1 真核表达载体。研究了重组 Gal-6 在大肠杆菌系统中高水平表达、重组蛋白的纯化和抗菌活性以及在酵母系统中重组 Gal-1 的高拷贝菌株筛选。本研究分两部分：1. 重组 Gal-6 在大肠杆菌中高水平表达、纯化和抗菌活性研究从安徽三黄鸡骨髓中提取总 RNA，利用设计的引物 P1/P2 从RNA 中扩增 Gal-6 基因片段，大小约为 216 bp。将目的基因克隆到pMD-18-T 载体中，构建 pMD-18-Gal-6 克隆载体，转化大肠杆菌DH5，挑取阳性克隆进行测序。经 GenBank 的 BLAST 比对，同源性达到 100 %。利用 PCR 技术从重组质粒 pMD-18-Gal-6 中扩增目的片段，并用 Sac I 和 Hind III 进行双酶切，与同样双酶切的质粒 pET 28a (+) 进行连接，从而构建重组表达质粒 pET-28a-Gal-6，并在 BL21 中进行融合表达。Tricine SDS-PAGE 电泳表明，表达的 Gal-6 融合蛋白分子量约为 15 kD，主要以包涵体形式存在。加入诱鸡 -防御素-1,6 基因工程菌的构建及表达制备2导剂 1 h 时，开始检出有蛋白的表达，在 5 h 时其表达水平已接近最大量；诱导剂 IPTG 在 0.2-1.0 M 范围内，对重组蛋白的产量没有明显影响。融合表达蛋白经过初步分离、变性、复性、镍柱亲和层析纯化，得到纯化蛋白的浓度为 0.053 mg/mL。对纯化的融合蛋白进行生物学活性检测，结果表明对大肠杆菌、金黄色葡萄球菌都产生抗菌活性。2. Gal-1 真核表达质粒的构建和高拷贝菌株筛选利用设计的引物 P3/P4 从总 RNA 中扩增 Gal-1 成熟肽基因片段，大小约为 123 bp。将目的基因克隆到 pMD-18-T 载体中，构建 pMD-18-Gal-1 克隆载体，转化大肠杆菌 DH5，挑取阳性克隆进行测序。经 GenBank 的 BLAST 比对，同源性达到 100 %。利用 PCR 技术从重组质粒 pMD-18-Gal-1 中扩增目的片段，并用 BamH I 和 EcoR I 进行双酶切，与同样双酶切的质粒 pPIC3.5K 进行连接，构建重组表达质粒 pPIC3.5K-Gal-1。用 Sac I 将 pPIC3.5K-Gal-1 线性化后，电击转入毕赤酵母 GS115 中。通过 G418 和实时定量 PCR 筛选高拷贝转化子，并通过 PCR 证明其表型。关键词：鸡 -防御素；克隆；基因重组；蛋白表达鸡 -防御素-1,6 基因工程菌的构建及表达制备3GENE ENGINEERING BACTERIA CONSTRUCTION OF -DENFENSIN-1,6 AND EXPRESSIONABSTRACTBeta-defensin, an antimicrobial peptide in poultry, shows antimicrobial and antiviral activity and function of enhances immunity. It is one of the focus for many scholars. The study mainly applied molecular biology and molecular cloning technology. The fragments of Gal-1 and Gal-6 were cloned from the marrow of Three Yellow chicken. The prokaryotic expression vector of pET-28a-Gal-6 and eucaryotic expression vector pPIC3.5K-Gal-1 were constructed respectively. High-level expression, purification and primary biologic activity of recombinant Gal-6 in Escherichia coli were investigated. High copy screening of recombinant Gal-1 in Pichia was investigated meanwhile.This study is divided into two parts:1. High-level expression, purification and primary biologic activity of recombinant Gal-6 in Escherichia coliTotal RNA from chicken marrow was extracted. A pair of primer P1/P2 was designed to amplify Gal-6 gene. A 216 bp fragment was obtained. The fragment was cloned to pMD-18T to construct cloning vector of pMD-18-Gal-6, which was transformed into DH5. The positive clone was sequenced and compared by BLAST of www.ncbi.com. The highest degree of identity in nucleotide was 100%. The 216 bp fragment was gained from recombinant pMD-18-Gal-6 plasmid by PCR amplification, digested with Sac I and Hind III enzymes and ligated into the Sac I / Hind III-digested pET-28a plasmid. This recombinant plasmid was transformed into the E. coli BL21 cells, used for expression of the fusion protein. The molecular weight of fusion protein showed about 15 kD in Tricine SDS-PAGE analysis. The cultivation parameters of the strain harboring expression plasmid were optimized to produce the fusion protein. The optimal conditions were determined as following: cultivation at 37 in LB medium, induction with 0.2mM IPTG, and post-induction expression for 5 h. The fusion protein was purified by immobilized Ni2+-charged affinity chromatography. The analysis of spectrophotometer revealedthat concentration of purified fusion protein was 5.3mg/L. Antimicrobial assays demonstrated that 鸡 -防御素-1,6 基因工程菌的构建及表达制备4fusion protein had antimicrobial property against Escherichia coil and Staphylococcus aureus.2. Construction of eukaryotic expression vector and screening of high copy screeningA pair of primer P3/P4 was designed to amplify Gal-1 gene from total RNA. A 123 bp fragment was obtained. The fragment was cloned to pMD-18-T to construct cloning vector of pMD-18- T-Gal-1, which was transformed into DH5. The positive clone was sequenced and compared by BLAST of www.ncbi.com. The highest degree of identity in nucleotide was 100%. The 123bp fragment was gained from recombinant pMD-18-Gal-1 plasmid by PCR amplification, digested with BamH I and EcoR I enzyme and ligated into the BamH I/ EcoR I-digested pPIC-3.5K plasmid. The obtained recombinant plasmid was linearized by Sac I, and then transformed into GSI15 by electroporation. The high copy transformants were obtained by G418 and Real Time PCR screening. The phenotype of transformant was verified by PCR.These results provide scientific guidance for development and application of antibiotic of defensin in animal husbandry.Zheng Huilin (bioengeering)Supervised by Professor Zhang XingqunKEY WORDS:Gallinacin; Clone; Recombinant; Expression.鸡 -防御素-1,6 基因工程菌的构建及表达制备5目 录摘要 .1 ABSTRACT .