假海源菌ZJCN121基因组文库的构建及岩藻多糖酶基因的筛选-微生物学专业论文.docxVIP

I II Abstract Fucoidan can be hydrolyzed into low molecular weight fucoidan(LMWF) by fucoidanase. LMWF were well in solubility, good absorption, anti-tumor, and could enhance immune. They had a variety of functions, resulting broad prospects for development and application in the medical and health care. The process of preparation of LMWF with fucoidanase was safety, mild for reaction conditions, and had low requirement on equipment. Enzymic method had high specificity, their products were pure and easy to separate and purify. Also enzymic method did not have high requirements on raw materials. However, obtained natural fucoidanase from the environment was difficult, and its process was complex. So the study of fucoidanase gene, its expression and regulation has far-reaching significance. In this study, the marine bacteria ZJCN121 strain was used to construct genomic expression library by molecular cloning methods. Preliminary estimated the molecular weight of fucoidanase from ZJCN121 by the SDSelectrophoresis. Genomic DNA of ZJCN121 strain was extracted by CTAB/NaCl. 7μL total genomic DNA was partially digested with 1.5μL HindⅢ at 37 ℃. SanPrep column DNA gel extraction kit wad used to purify these DNA fragments between 3kb and 8kb. Connected the genomic DNA fragments with the dephosphorylated expression vectors pET-32a, then transformed them into Escherichia coli BL21(DE3). Transformants were selected randomly to identify their stability and to analysis the insert fragments’ size. The results showed that the average weight of the exogenous fragments was about 6kb, the transformations were genetic and stable. The library of the marine bacteria ZJCN121 had about two thousand transformants. In the foundation of the ZJCN121 strain genomic library, used Zobell 2216E medium contained 3% of fucoidin to screen positive clones. Stained with 1% Congo red solution and eluted twice with 1mmol/L NaCl. Observed and screened again by measuring their enzyme activity, a positive c