【作者】 李国才；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 预防兽医学， 2004， 博士

【摘要】 人溶菌酶(human lysozyme，hLYZ)能水解细菌细胞壁粘多糖的β-1，4糖苷键，所以对革兰阳性细菌具有直接的溶解作用，在分泌型免疫球蛋白A和补体的参与下对革兰氏阴性细菌具有间接的溶解作用，因此在医学、食品工业等领域得到十分广泛的应用。 目前使用的溶菌酶主要从鸡蛋清中提取，天然的hLYZ不仅来源有限，而且提取方法复杂、价格昂贵。目前虽有细菌和酵母表达hLYZ的报道，但普遍存在转译后加工修饰不完全、溶解宿主细胞壁和表达水平低等问题，难以进行工厂化生产。转基因动物乳腺生物反应器具有表达水平高、表达产物活性强、易于纯化和无污染等优点，是大规模生产重组蛋白的新途径。 为了研制表达hLYZ的动物乳腺生物反应器，本研究根据已发表的hLYZ序列设计引物，以人乳腺第一链cDNA为模板，用PCR方法扩增出长1.5 kb hLYZ双链cDNA。测序结果显示，其序列与已发表的从人胎盘、巨噬细胞和结肠中克隆的人hLYZ完全一致，其中包括1个447 bp的阅读框和3′端反向重复的Alu序列。将此cDNA序列推导成氨基酸序列，并与GenBank中已发表的hLYZ氨基酸序列进行比较，结果与从人胎盘、巨噬细胞和结肠中克隆的hLYZ完全相同，与从人组织细胞中克隆的hLYZ仅有1个氨基酸差异(41位的异亮氨酸→蛋氨酸替换)，但与从中国人胎盘中克隆的hLYZ具有5个氨基酸差异，分别是第10、111和128位的颉氨酸→丙氨酸替换，124位的异亮氨酸→颉氨酸替换和136位的天门冬氨酰胺→天门冬氨酸替换。因为上述5个氨基酸替换具有一定的规律性(3个为颉氨酸→丙氨酸替换)，所以这种序列差异可能是不同人种之间的遗传差异，而不是克隆过程中产生的随机错误。 将上述hLYZ cDNA克隆入真核表达载体pcDNA3，用获得的重组质粒转染COS-1细胞，经间接免疫荧光试验证明目的基因能进行正确表达。再将此cDNA扬州大学博士学位论文 克隆入自行构建的乳腺表达载体P205C3、国外引进的乳腺表达载体pBJ41和PBCI，获得的重组质粒分别命名为P205C3LYZ、PBJLYZ和PBCLYZ，经多聚乙 酞亚胺包裹后通过尾静脉注射哺乳期小鼠，经微球菌溶解试验证明三种重组载体 不仅能在乳腺细胞中表达，而且分泌在乳汁中的重组酶分别达到87、69、60m叭。将基因注射小鼠扑杀，取其12种组织提取总RNA，经Dot blotting检侧证明，三 种基因构件除在乳腺中表达外，在脾、肠或肾中也有一定的异位表达。这些试验 结果提示，自行构建的乳腺表达载体可以替代国外引进载体进行动物乳腺生物反应器研究。 用显微注射法将重组表达载体p205C3LYz注射入小鼠受精卵，获得136只F0代小鼠，用PCR和s。川出em bfotting分别检测到目的基因整合阳性鼠7只(2早5 占)和4只(1早3占);在soulthern blo伪泣g检测阳性的4只F。鼠中，1只母鼠为hLYZ表达阴性，2只雄鼠与正常母鼠交配出生的F，代母鼠为hLYZ表达阴性，1只雄鼠的4只F，代母鼠乳汁中表达的让止yZ分别为5 mg/L、75 mg几、175m叭和200m留1;从F:代开始，对表达阳性鼠进行兄妹交配，经PCR检测证明转基因的遗传符合孟德尔遗传规律;目前转基因纯合鼠已传到F7代，每代母鼠乳汁中的rhl万z表达量基本稳定，最高表达量达750m叭;将其中的3只母鼠扑杀，取其组织制备细胞裂解液和总RNA，经微球菌溶解试验和Dot blotting检测证明，目的基因除在乳腺表达外，在脾脏和小肠也有一定的异位表达。 取转基因阳性鼠的乳汁进行Westem blotting分析，结果显示乳中表达的八止yZ与人初乳中天然的LYZ具有相同的分子里;将适当稀释的转基因小鼠乳汁和人初乳在60，C、72℃、85，C、100，C水浴中孵育l、3、5、10、15、20、30 min，经微球菌溶解试验证明，比山YZ与天然址万Z具有相似的热稳定性;将乳样的pH调节为2、4、5、7.4、9、10，37℃作用30 min后进行酶活性测定，结果表明r址万Z与天然址万Z的pH活性范围相近;将不同的金属离子加入乳样中，然后进行酶活性测定，结果证明比LYZ和正常址笼Z对所试金属离子具有相似的敏感性;通过琼脂平板扩散法检测不同种类的细菌对迁止YZ和hLYZ的敏感性，检出的敏感菌在37℃培养过程中每隔3On五n取定量混合液进行琼脂平板菌落计数和绘制抑菌曲线，结果表明表达的rhl笼Z与正常址万Z具有相似的抑菌活性。 用重组载体P205C3LYZ注射山羊受精卵，第一批试验使用的供、受体羊分别李国才:人溶菌防动物乳膝生物反应器的研制为17和22只，由于胚胎移植后遇到羊痘病毒感染，导致部分羊死亡，怀孕率很低(4瓜2)，经PCR检测证明出生的3只羔羊为外源基因整合阴性;第二批试验分别使用供、受体羊巧和23只，胚胎移植后35一40天的怀孕率为14/23，尽管流产和产死胎羊相对较多，但仍获得12只存活羔羊;经Dotbl。币ng法初步检测证明，其中7只为外源基因整合阳性。更多还原

【Abstract】 Human lysozyme (hLYZ, EC3.2.1.17) hydrolyzes the glycosidic B-1, 4 linkage between N-acetyhnuramic acid and N-acetylglucosamine of the peptidoglycan polymer in the bacterial cell wall. Because of the direct exposure of polypeptidoglycan on the outside of the cell wall, hLYZ can directly lyze Gram-positive bacteria and indirectly lyze Gram-negative bacteria in the presence of slgA or complements. Furthermore, hLYZ has many other activities such as diminishing inflammation, detumescence, repairing putrescence tissues, improving blood supply in local tissues, reducing pus and anti-virus.hLYZ can be purified from human breast milk, neutrophils, placenta and urine of hemodialysis patients. Because of the limited supply and concerns over the potential risk of transmitting pathogens to the nursing infants, development of safer recombinant hLYZ is needed. Expression of rhLYZ in prokaryotic bacteria or yeast by utilizing gene-engineering techniques has some difficulties due to their lack of appropriate post-translational modifications and the potential lysis of the host cells. Animal mammary gland bioreactor is a novel alternative strategy for production of recombinant proteins with many advantages including high production capacities, easy purification, faithful translational modifications and authentic biological activities of the expressed products.To develop animal mammary gland bioreactors expressing hLYZ, 1.5 kb dscDNA for hLYZ was amplified by PCR from pooled first-stranded cDNAs from human mammary gland and subcloned into pGEM-T vector. Sequence analysis showed that the PCR-amplified cDNA was identical to that cloned from human placenta, macrophages and colon. The cDNA included an open reading frame of 447 bps and a pair of Alu elements in reverse orientation at the 3’ -noncoding region. The open reading frameencoded a polypeptide of 130 amino acids, the first 18 amino acids of which were predicted to be signal pepeptide. The mature enzyme had a predicted molecular weight of 14.7 kDa and pI of 9.02. The deduced amino acid sequence of the cDNA was also identical to that cloned from human placenta, macrophages and colon and differed by 1 or 5 amino acids from that cloned from histiocytes or Chinese placenta.The cDNA was subcloned into eukaryotic expression vector pcDNA3 and transfected into COS-1 cells following mixing with 25 kDa branched polyethylenimine(PEI). Expression of hLYZ cDNA was revealed by indirect immunofluorecence using hLYZ-specific antibody. The hLYZ cDNA was then subcloned into mammary gland-specific vectors p205C3, pBJ41 and pBCl. After mixed with 25 kDa PEI, the three recombinant expression vectors p205C3LYZ, pBJLYZ and pBCLYZ were injected into lactating mice via tail vein route. Micrococcal lysis assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands and secreted into the milk with maximal concentrations of 87, 69 and 60 mg/L, respectively, for the three recombinant vectors. The expression of the three vectors was restricted to mammary glands with some degrees of ectopic expression in spleen, intestines and/or kidney among 12 different tissues tested by dot blotting and micrococcal lysis assay. These data indicate that the self-constructed mammary gland-specific vector p205C3 can replace the other two vectors obtained from abroad for development of animal mammary gland bioreactors.The recombinant vector p205C3LYZ was used to generate transgenic mice by microinjection. A total of 136 F0 mice were obtained, of which 7 (2 5 ) and 4 (1 3 ) were showed to be transgenic by PCR and Southern blotting, respectively. Based on the micrococcal lysis assay of the milk samples, the only one female founder did not express the gene of interest. The three male founders were mated with normal mice and four female offspring of the only one male founder expressed hLYZ activities at levels of 5-200 mg/L. From the F1 generation on, the transgenic mice were mated with their sisters or brothers and a mouse colony was established with stable transmission and expre更多还原