蜜蜂螺原体几丁质脱乙酰酶基因的克

发布时间：2018-02-03 02:05

  本文关键词： 蜜蜂螺原体 几丁质脱乙酰酶 定点突变 克隆表达 酶学性质　出处：《南京农业大学学报》2016年03期 　论文类型：期刊论文

【摘要】：[目的]通过表达载体p ET-28a(+)在大肠杆菌中克隆表达蜜蜂螺原体Spiroplasma melliferum CH-1中可能与致病相关的几丁质脱乙酰酶(chitin deacetylase,CDA)基因chid,并测定其部分酶学性质,为进一步研究该基因在蜜蜂螺原体致病过程中的作用奠定基础。[方法]利用Overlap Extension PCR(OE-PCR)定点突变技术,在引物设计时插入目的突变,通过3次PCR获得突变后的目的片段,测序验证后构建重组质粒p ETchid,挑取BLchid表达菌株。IPTG诱导表达目的蛋白,NTA-Ni2+柱纯化,Western blotting验证,紫外分光光度法测定其酶活力和酶学性质。[结果]DNA测序结果表明:chid基因中待突变位点已由TGA突变为TGG,成功克隆到chid基因全长,并得到外源表达的完整目的蛋白。该基因编码区为672 bp,编码的氨基酸构成相对分子质量约28×103的蛋白,同源性比较发现其与S.melliferum KC3/BC3/IPMB4A菌株序列相似性为97%。测得外源表达的该酶活力最高可达10.14 U·m L-1,最适温度为50℃左右,最适p H值为7.0~7.5。[结论]首次克隆了螺原体几丁质脱乙酰酶基因chid,并得到具有活性的外源目的蛋白,为后续研究螺原体与宿主蜜蜂的相互作用及其致病机制提供了重要信息。
[Abstract]:[Objective] to study the expression vector pET-28a (pET-28a). Cloning and expression of chitinase from Spiroplasma melliferum CH-1 in Escherichia coli. Chitin deacetylase. CDA) gene chidand some enzymatic properties were determined, which laid a foundation for further study on the role of CDA gene in the pathogenesis of bee spiroplasma. [Methods] the aim mutation was inserted into the primer design using Overlap Extension PCR- OE-PCR site-directed mutation technique. The target fragment was obtained by three times of PCR, and the recombinant plasmid pETchid was constructed after sequencing. The target protein was induced by BLchid expression strain. The enzyme activity and enzymatic properties were determined by UV spectrophotometry. [Results DNA sequencing showed that the mutation site in the DNA gene had been transformed from TGA to TGG, and the chid gene was cloned successfully. The encoding region of the gene is 672 BP, and the amino acids encoded constitute a protein with a molecular weight of about 28 脳 10 ~ 3. Homology analysis showed that the sequence similarity between S.melliferum KC3/BC3/IPMB4A and S.melliferum KC3/BC3/IPMB4A was 97%. The highest activity of the enzyme expressed by exogenous was 10.14%. U 路mL ~ (-1). The optimum temperature is about 50 鈩，

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