短短芽胞杆菌FJAT-0809-GLX几丁质酶基因(chiD)的克隆与原核表达

Cloning and Prokaryotic Expression of Chitinase Gene(chiD)from Brevibacillus brevis FJAT-0809-GLX

以短短芽胞杆菌FJAT-0809-GLX基因组DNA为模板,通过PCR扩增得到几丁质酶基因chiD序列,再将chiD序列连接到pMD18-T克隆载体上,形成重组载体pMD18-T-chiD,转化大肠杆菌DH5ɑ并测序,经过核苷酸和氨基酸序列分析获得几丁质酶基因chiD的序列片段为1 524 bp,编码507个氨基酸,理论蛋白质分子量约54.55 ku,其等电点约为5.77,表明该几丁质酶为酸性几丁质酶。将重组质粒pMD18-T-chiD双酶切后与表达载体pET-28a连接,构建重组表达载体pET28a-chiD,并转入大肠杆菌BL21中进行IPTG诱导表达,结果表明:重组蛋白质的分子量约55 ku,与预测的蛋白分子量结果一致。为了提高重组蛋白的表达量,对培养时间、IPTG浓度和温度3个参数进行优化,并将表达产物进行SDS-PAGE分析,最后得出重组载体pET28a-chiD的最佳诱导表达参数分别为8 h、0.5 mmol/L和28℃。这为短短芽胞杆菌来源的几丁质酶的进一步研究奠定了良好基础。

The chromosome DNA from Brevibacillus brevis FJAT-0809-GLX was treated as a template to obtain chiD chitinase gene by PCR amplification.The recombinant plasmid pMD18-T-chiD was constructed by lingking gene chiD sequence and plasmid pMD18-T.Nucleotides and amino acids sequence of chitinase gene was analyzed and the results showed that gene chiD was 1 524 bp encoding a 54.55 ku protein of 507 amino acids with pI 5.77.The chitinase gene from the recombinant plasmid was linked with the expression vector pET-28a digested with the same two restriction enzymes,and the recombination expression vector pET28a-chiD was conctructed.And the expression vector was transformed into E.coli BL21 （DE3） successfully.In order to improve protein expression index,the recombinant expression vector was induced to express and the expression product was detected by SDS-PAGE electrophoresis.The results indicated that the expressed protein molecular weight was about 55 ku,which was consistent with the prediction result.The vector pET28a-chiD was induced to express with different cultural time,isopropyl-p-D-thiogalactopyranoside（IPTG） concentrations and temperature,and the results showed that the best inducing parameters were 8 hours,0.5 mmol/L and 28 ℃.It would lay a good foundation for the further research of chiD chitinase from B.brevis FJAT-0809-GLX.