【作者】 叶辉；

【导师】 程备久；

【作者基本信息】 安徽农业大学 ， 生物物理学， 2005， 硕士

【摘要】 几丁质及其水解产物在病虫害防治、医药生产和饲料加工等多个领域具有广泛的用途,其专一性水解酶——几丁质酶(chitinase)已成为当前研究的热点,分离新的几丁质酶基因、构建高效表达工程菌,对实现几丁质酶产业化具有重要意义。本文采用PCR 技术,从粘质沙雷氏菌(Serratia marcescens)分离 chiB 基因,并分析了该基因的 DNA 序列,构建了原核生物表达载体, 通过 IPTG 诱导表达,分别对该基因表达的蛋白产物进行了 SDS-PAGE 分析、生物活性和功能分析。结果表明: 1. 依据己克隆的 chiB 基因序列的同源性比较,设计了三对 PCR 扩增引物,采用 PCR技术,从粘质沙雷氏菌基因组中分离出一个大小约 1500bp 的特异 DNA 片段; 2. 通过特异片段的测序和与报道的 chiB 基因的序列同源性分析,结果显示,该克隆片段是阅读框为 1500bp 的功能基因,与 Serratia marcescens X15208、AB015997 的 chiB 基因序列的同源性均达到了 99%,与 Serratia liquefaciens AF399871chiB 基因的同源性达 87 %,而与其它不同菌种的 chiB 基因同源性较低,初步表明该片段是粘质沙雷氏菌几丁质酶 chiB 基因; 3. 利用载体 pET-22b(+),构建 chiB 表达载体 pET-chiB,通过验证分析表明,所克隆的基因 chiB 已置于表达载体的正确阅读框架下; 4. 表达载体 pET-chiB 经诱导表达,结果显示其表达的蛋白为可溶性蛋白,经 SDS-PAGE 分析,结果表明该基因表达的蛋白分子量为 52kDa; 5. 通过对表达载体的 SDS-PAGE 各种参数包括时间、温度和 IPTG 浓度诱导表达分析,结果显示最佳诱导时间为 3～4 小时;最佳诱导 IPTG 浓度为 0.5mmol/L;20℃～30℃为诱导表达的适宜温度,并以 25℃为最佳; 6. 以酵母菌为指示菌做抑菌圈试验,结果显示经不同浓度的 IPTG 诱导表达的蛋白液处理后,抑菌圈大小变化十分显著,并在 IPTG 浓度为 0.5 mmol/L 时抑菌效果最明显。 7. 通过对经不同浓度的 IPTG 诱导表达的几丁质酶活力测定结果显示,当 IPTG 浓度为 0.5mmol/L 时,几丁质酶活力达到最大值 68.7U/ml,远高于原菌液几丁质酶表达的酶活力 11.63U/ml。 综上所述,本文已成功地从粘质沙雷氏菌中分离出几丁质酶 chiB 基因,并构建了高效原核表达载体 pET-chiB,同时优化了该工程菌诱导表达的参数,这些结果为几丁质酶基因的进一步研究和几丁质酶的产业化生产奠定了良好的前期工作基础。更多还原

【Abstract】 Chitin and its hydrolysate have extensive uses in a lot of domains such as prevention and cure of the plant diseases and insect pests, medicine production and fodder machining, etc. And its single hydrolase—the chitinase has already become the hotspot of current research. Separating new chitinase genes and constructing the project bacteria that can high-efficient express, which is significant to realize the industrialization of the chitinase. The paper separated chiB gene fragment from Serratia marcescens by the polymerase chain reaction (PCR) technology, and analyze the DNA sequence of chiB gene, had constructed procaryote expression vector pET-chiB and made respectively analysis on SDS-PAGE, biological activity and function with the gene expression protein production of chiB gene by IPTG inducing expression. The results were reported as follows. 1.According to analysis on sequence homology of cloned chiB genes, three couple primers were designed and a piece of special DNA fragment about 1500bp was separated from Serratia marcescens by the PCR technology; 2.Through determining the sequences of special fragment and analyzing sequence homology of reported chiB gene, the results showed, the cloned fragment is a functional gene of about 1500bp, its homology was up to 99% with the chiB gene sequence of Serratia marcescens X15208 and AB015997, 87% with Serratia liquefaciens AF399871, but it had lower homology with the chiB gene of other different bacteria. It elementarily indicated that the fragment was chitinase B gene of Serratia marcescens; 3.Utilizing vector pET-22b(+) to construct expression vector pET-chiB, through validating and analyzing, the result indicated that the cloned chiB gene had been inserted the correct reading frame of expression vector; 4.Through the expression vector induced to express, the result showed that its expressive protein was soluble. the result indicated that the protein molecular of this expressive gene was 52kDa by analysis on SDS-PAGE; 5.Through induced expressing and analyzing on various kinds of SDS-PAGE parameter including time, temperature and IPTG density, the result showed that the best induced time was 3～4 hours; the best induced IPTG density was 0.5mmol/L;20℃～30℃ was condign temperature of induced expression, and the best was 25℃; 6.Take Microzyme as demonstrative bacterium to take test on restrain bacterium circle, the result showed that the change of restrain bacterium circles were very remarkable through dealing with different densities of IPTG induced expression protein liquid, and the restrain bacterium effect was the most remarkable in 0.5 mmol/L of IPTG density; 7.Through mensurating chitinase activity under induced expression by different IPTG density, the result showed, at the 0.5mmol/L, chitinase activity reach the max 68.7U/ml, which was far higher than that of original bacterium liquid 11.63 U/ml. In sum, the paper had succeeded to separate chitinase B gene from Serratia marcescens, and construct high-efficient prokaryotic expression vector pET-chiB, optimized the induced expressive parameter of project bacterium. These results had established a favorable work foundation for the more research of chitinase gene and the industrialization production of chitinase.更多还原