蜜蜂螺原体几丁质脱乙酰酶基因的克隆、表达及酶学性质

Cloning,expression and enzymatic characterization of a chitin deacetylase gene from Spiroplasma melliferum

[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。

[Objectives]In order to explore pathogenesis of the chitin deacetylase（ CDA） in Spiroplasma melliferum CH-1,the chid gene was cloned and expressed by expression vector p ET-28a（ ＋）,followed by analyzing its enzymatic properties. [Methods]Target gene fragment was obtained through overlap extension PCR（ OE-PCR）,and site-directed mutagenesis was inserted during primer designing. Recombinant plasmid p ETchid was constructed,and positive transformats BLchid-expressed strain was sequenced. Finally,the complete heterologously expressed target protein was successfully induced by IPTG. Then the protein was purified with NTA-Ni2＋,verified by Western blotting,and its enzymatic properties were detected by UV spectrophotometry. [Results]The resulted DNA sequence showed that TGA target site was successfully mutated to TGG. In addition,clone of chid gene and complete heterologously expressed target protein was obtained. This gene contained a 672 bp coding sequence,encoding a protein with 28× 103. The protein sequence was 97% identical with S. melliferum KC3 / BC3 / IPMB4 A after blasted in NCBI. Enzyme activity of the protein was10.14 U·m L-1. The optimum temperature and optimum p H was 50 ℃ and 7. 0- 7. 5,respectively. [Conclusions]This study firstly cloned spiroplasma chitin deacetylase enzyme gene chid and obtained active CDA enzyme. All the understandings contribute to the interactions between spiroplasma and honeybee.