【作者】 李尧；

【导师】 茆灿泉； 李学如；

【作者基本信息】 西南交通大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 透明质酸(hyaluronic acid, HA),又名玻璃酸,普遍存在于脊椎动物和某些细菌的荚膜,是由的D-葡萄糖醛酸和N-乙酰氨基葡萄糖以p-1,3和p-1,4糖苷键交连而成的高分子链状聚合物,具有良好的生物相容性和生物降解性,在医药和化妆品领域广泛应用,近年来透明质酸的需求与日俱增,表现出良好的市场潜力和广阔的发展前景。目前,微生物发酵法已取代动物组织提取法成为透明质酸生产的主要方式。国内外发酵法生产透明质酸主要菌种为C组链球菌(Group C Streptococcus, GCS)的马链球菌马亚种和马链球菌兽疫亚种。马链球菌兽疫亚种与A组链球菌(Group A Streptococcus GAS)蛋白序列对比分析研究发现,该菌除没有链球菌致热外毒素外,几乎具有GAS业已证实的所有毒力因子。由于GCS没有宿主专嗜性,能引起多种动物,甚至人的链球菌病,因此用GCS发酵生产透明质酸,存在潜在安全风险,所以通过分子生物学的方法,构建透明质酸生产毒力基因缺失高产突变株,具有积极意义。本实验从牛鼻粘膜样品中分离出一株产透明质酸链球菌,经16S rDNA基因序列相似性分析,鉴定为Streptococcus equi subsp. Zooepidemicus (马链球菌兽疫亚种),菌株在透明质酸发酵培养基中,37℃摇瓶培养,18小时左右透明质酸产量达到最高(0.96g/L)。以该菌为出发菌株,用温度敏感基因敲除载体系统pJR700,成功构建链球菌血红素受体(Streptecoccus Heme Receptor, Shr)基因缺失突变株。研究结果为进一步敲除其他毒力基因,构建无毒透明质酸生产基因工程菌打下了基础。链球菌透明质酸合成酶是合成透明质酸的关键酶,其操纵子由hasA、hasB、hasC三个基因组成。本课题根据温度敏感载体pJR700在37℃能将同源基因整合到染色体并稳定遗传的特性,用该载体系统,构建携带hasABC基因的温度敏感性质粒载体pXL31,将该质粒载体转到血红素受体基因缺失突变株,获得含双拷贝hasABC基因工程菌。SDS-PAGE电泳证实,与血红素受体基因缺失突变株相比,工程菌分子量大约50KDa、47KDa和40KDa的三个蛋白高表达,工程菌高表达的这三个蛋白,理论上与马链球菌兽疫亚种hasA、hasB、hasC三个基因表达蛋白分子量相近；用Bitter-Muir方法测定菌株透明质酸含量结果显示,被测定的6株工程菌透明质酸产量比出发菌株提高范围在27-34%之间,其中一株菌株透明质酸产量达到1.28 g/L,比出发菌株提高34%。实验结果证实,用温度敏感基因载体系统pJR700增加染色体上基因的拷贝是可行的。这一方法研究成功,为通过基因工程的方法提高微生物代谢产物研究提供了一种新的选择。更多还原

【Abstract】 Hyaluronic Acid, also known as Hyaluron or HA, was found naturally in the tissues of all vertebrates and in the capsule of some bacteria. HA is a high molecule linear polymer which composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternatingβ-1,4 andβ-1,3 glycosidic bonds. Because of its good biocompatibility and biodegradability, HA is usded wildly in pharmaceutical and cosmetic, and showing good market needs and broad development prospects in recent years.Commercial HA is obtained by either chemical extraction from the rooster combs/umbilic-cords, or the microbial fermentation. The chemical extraction method is limited by resource availability. The microbial fermentation becomes the main processing for HA production. The main strain for HA production is the Streptococcus equi subsp. Zooepidemicus of Group C Streptococcus (GCS). Comparison vulunce factors of S. Zooepidemicus to Group A Streptococcus (GAS), the S. Zooepidemicus possess all the virulence factors which have been found in the GAS except streptococcal pyrogenic exotoxin (spe). The pathogenic GCS can infect different host and cause a variety of animals even human streptococcal disease. Therefore construction of the virulence gene deletion mutant is industrially emphasized recently for HA production.In this study we isolated a strain which produced HA from ox snout, It was identified as Streptococcus equi subsp. Zooepidemicus by 16S rDNA gene sequence analysis. The HA titer was reached 0.96g/L in flask culture at 37℃and 18 hours.We successfully constructed the deletion shr mutants by the thermosensitive vector system of pJR700 in S. Zooepidemicus. The results showed that is possible that constructing Non-virulence gene-engineering strain for HA production by knockout of virulence genes using pJR700.Streptococcus hyaluronic acid synthase (HAS) are the key enzyme for synthesis of HA in bacteria. The operon of HAS are composed of three genes which are has A, hasB and hasC. The thermosensitive vector system of pJR700 has the function, which integrate its carrying the homologue gene into chromosome of the streptococcus and the integrated homologue gene can transmit to generation. We constructed the thermosensitive delivery vector pLX31 which contain hasABC gene using pJR700, then transfered pLX31 into shr mutant of S. Zooepidemicus by electroporation, and obtain the gene engineering strain which contain two copies of hasA, hasB and hasC gene. The snalyses of SDS-PAGE indicated that the strain constructed can express exceed three protein, which molecular weight were 50KDa、47KDa and 40KDa, and in the same proteins of expression by hasA, hasB, and hasC of S. Zooepidemicus, respectively. The HA titer was increased high to 1.28g/L at 37℃and 18h in flask culture in two copies of hasABC strain. The hyaluronic acid productivity of gene-engineering strains were increased by 27 to 34% compared with the parental strain, shr mutant. Therefore, it is reasonable that we believe using pJR700 to increase the gene copy of streptococcus. Our method provides a new choice for microbial metabolic engineering research.更多还原