The Clone And Expression Analysis On Encoding Chitinase B Gene Of Serratia Marcescens

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.