【作者】 孙伟；

【导师】 朱春宝；

【作者基本信息】 上海医药工业研究院 ， 微生物与生化药学， 2004， 博士

【摘要】 β-内酰胺类抗生素(青霉素和头孢菌素)在临床上用于治疗多种细菌感染疾病,它们对人类的健康作出了巨大的贡献。但是,随着抗生素的广泛应用以及某种程度上的滥用,耐药菌在不断地产生,它们对许多β-内酰胺抗生素耐药,从而使这类药物对耐药菌感染的疾病无效。微生物来源的β-内酰胺酶的抑制剂棒酸(Clavulanic acid)及其β-内酰胺砜类化合物舒巴坦(Sulbactam)和他佐巴坦(Tazobactam),均在七十年代至八十年代被发现并证实是有效的β-内酰胺酶的抑制剂,且作为“自杀者”与β-内酰胺抗生素合并用药,可达到抑制β-内酰胺酶活力、增强β-内酰胺抗生素疗效的目的。然而,无论是砜类还是棒酸类抑制剂,对一些细菌产生的由染色体介导的Ⅰ型β-内酰胺酶的抑制作用都很小。近来人们从带棒链霉菌(Streptomyces clavuligerus)中分离到β-内酰胺酶抑制蛋白(β-lactamase inhibitory protein, BLIP),进一步研究表明其第46-51位的氨基酸残基对抑制Ⅰ型β-内酰胺酶起着关键的作用。这种新型的蛋白类β-内酰胺酶抑制剂的出现给人们带来了很多启发,关于BLIP的研究也受到人们的关注。在本研究中,我们期望通过酵母双杂交系统从随机核苷酸库中筛选到抑制β-内酰胺酶的短肽。在设计一段随机寡核苷酸时,我们将BLIP与β-内酰胺酶结合中的一些关键信息考虑进去,一方面可以扩展β-内酰胺酶抑制肽的范围,另一方面可以增加筛选成功的可能性。 在筛选新β-内酰胺酶抑制剂的研究工作中,需要纯度较高的β-内酰胺酶来验证筛选得到的短肽是否具有抑制作用。因此,首先将β-内酰胺酶基因克隆至pLY-5载体中,构建表达质粒pYG201。在pYG201质粒中β-内酰胺酶基因处于λ噬菌体P_L启动子控制下,通过42℃热激解除阻遏物(cIts857)的阻遏,高效表达β-内酰胺酶。外源蛋白高效表达易形成包含体,通过对包含体的溶解、复性和纯化过程的研究,最终获得有活性的β-内酰胺酶并达到90%以上的纯度。 通过PCR方法将随机的单链寡核苷酸大量扩增为双链,再经过一系列基因操作将扩增后的随机双链基因与pGAD424质粒载体中的激活域融合,构建随机核苷酸文库,即猎物质粒文库。通过对酵母转化条件的研究确定了转化的最佳条件,其最佳条件是:选择PEG4000为促进剂和42℃热激时间为30min,获得的转化率为1.4×10~5cfu/μg DNA。随机核苷酸文库的构建和酵母转化条件的研究为后续的酵母双杂交筛选奠定了基础。 将pYG111和pYG202(随机核苷酸文库)共同转化酵母Y153,通过初筛和复筛最终得到的三株阳性转化子,分离得到的质粒分别命名为pYG202-p1、pYG202-p2、和pYG202-p3。β-半乳糖苷酶的定量测定表明P3与β-内酰胺酶在酵母体内的相互作用最强,测得的米勒单位为10.2;P1次之,米勒单位为4.3:P2的米勒单位为2.1。用VectorNTI软件分析所测定的序列表明:通过酵母双杂交系统筛选得到的三株阳性克隆中,其随机序列之间的差异较大。但这三个肽在酵母体内均能与β-内酰胺酶发生相互作用,这1几海医药一l几业研究院博l:学位论文摘要可能与核心氨基酸序列(AAGDYY)的存在有很大关系。 通过酵母双杂交系统从一个随机DNA文库中筛选到三个编码能与p一内酞胺酶结合的短肤(Pl、PZ、和P3)的DNA序列,在本研究中将编码P1和P3的基因分别克隆到pGEX一4T-1的多克隆位点中,得到重组质粒pYG205一Pl和一p3。加入适量的IPTG诱导外源基因在宿主中表达GST-短肤融合蛋白,运用sePharose 4B亲和层析介质分离纯化GST-短肤,然后用凝血酶(thrombin)将短肤从融合蛋白中切下。体外测定Pl和P3与p一内酞胺酶的结合作用,结果表明P3可以抑制酶的活性,而Pl不能抑制酶的活性。 关键词:酵母双杂交系统p一内酞胺酶抑制蛋白随机寡核昔酸库 GST-短肤融合蛋白分更多还原

【Abstract】 β-lactam antibiotics (penicillin, cephalosporin, and their semi-synthetic derivatives) are used to treat various bacterial infections for clinical purpose. They have made great contribution to humans’ health. But as following widespread use of antibiotics and abuse to some extends, drug resistance bacteria were emerged gradually. Many bacteria are resistant to β-lactam antibiotics, thereby those drugs could not cure diseases which were brought by some drug resistance bacteia. Clavulanic acid, a β-lactamase inhibitor obtained from microbe, and compounds of β-lactam sulfone, such as sulbactam and tazobactam, were found and demonstrated to inhibit β-lactamase in 1970s and 1980s. Each was coupled with a β-lactam antibiotic to improve the potency and longevity of the partner drug. Unfortunately, those inhibitors could not inhibit TEM-1 β-lactamase mediated by chromosome to great extend. Recently, BLIP (β-lactamase inhibitory protein) was isolated from Streptomyces clavuligerus. Advanced researches indicated that the residues of 46 to51 of BLIP made critical interactions with the active site of TEM-1 β-lactamase. The new type proteinaceous β-lactamase inhibitor brought many prospect to scientists. Studies of BLIP were payed a lot of attention. In this research, we expected that small peptides which were able to inhibit β-lactamase can be screened from this random nucleotide library by yeast two hybrid system. A random oligonucleotide fragment was designed and some important information about BLIP interacting β-lactamase was designed into this random fragment. One hand it could extend spectrum of β-lactamase inhibitory peptide, the other hand it could enhance probability of screening. Researches as following:Firstly, higher homogeneous β-lactamase was needed to test peptide inhibitory activity gained by yeast two hybrid screening in this research. So β-lactamase gene was subcloned into vector pLY-5 and plasmid pYG201 was constructed. In plasmid pYG201, β-lactamase was expressed under the regulation of P_l promoter from λ phage, β-lactamase was expressed high efficiently by 42 ℃ induction which could relieve repression β-lactamase was expressed in the form of inclusion body, then the processes for wash, denaturation, refolding and purification of inclusion body were studied. Finally β-lactamase was purified up to 90% homogeneity.Random single chain oligo nucleotides were amplified to doublechain nucleotides by PCR method, then double-chain nucleotides were fused to active domain of pGAD424 by a series of molecular biology manipulation and random nucleotide library (prey plasmid library) was constructed. The optimum conditions for yeast transformation were determined as PEG4000 was used as accelerant and 30min for heat shock time. Transformation efficiencywas 1.4 X 105cfu/ugDNA under this conditions. So the following yeast two hybrid screening was based on those studies.Plasmid pYGlll and pYG202 (constructed random nucleotide library) were transformed into Saccharomyces cerevisiae Y153. three positive transformants were gained by screening and re-screening, plasmids were isolated from those positive transformants and named pYG202-pl, pYG202-p2, and pYG202-p3, respectively. Quantitative assay of P-galactosidase activity indicated that the interaction between P3 and P-lactamase in vivo was the stronges that P3 has the strongest interaction with p-lactamase in vivo, its Miller units was 10.2, whereas PI and P2 Miller units were 4.3 and 2.1, respectively. Sequence analysis suggested that there was larger difference among these random sequence of in these three positive transformants. But three peptides encoded by their gene sequence could all interact with P-lactamase in vivo. The results suggest core amino acid sequence (AAGDYY) may play a critical role provided critical function.In this work, DNA fragments coding for PI and P3 which can bind P-lactamase were obtained from a random DNA fragment bank by yeast two-hybrid system screening, and then subcloned into pGEX-4T-l, and therefore, recombinant plasmids p更多还原