论文标题:重组人A20的分子设计及酵母表达研究
Molecular Design of Recombinant Human A20 and Its Expression in Pichia Pastoris
论文作者 吴丽娟
论文导师 康格非,论文学位 博士,论文专业 临床检验诊断学
论文单位 重庆医科大学,点击次数 518,论文页数 136页File Size1398k
2004-05-01论文网 http://www.lw23.com/lunwen_9039217/ 锌指蛋白A20;生物信息学;基因表达;巴斯德毕赤酵母菌;系统性全身炎症反应
zinc finger protein A20;bioinformatics;gene expression;Pichia pastoris;Systemic inflammatory response syndrom
脓毒症失控性炎症反应导致体内免疫机能紊乱，诱发脓毒性休克，甚至MODS，是临床病人死亡的重要原因。近年来在炎症的信号转导途径研究中，人们发现了一种被称为A20的内源性炎症调控蛋白，该蛋白可以下调炎症信号转导途径重要膜受体TLR4的表达和核转录因子NF-κB、AP-1的活性，因此，在维护体内促炎细胞因子与抑炎细胞因子的平衡和炎症的内源性细胞保护中发挥重要作用。鉴于临床对失控性炎症反应干预药物的渴求和病人血样中A20表达水平的及时快速检测的需要，本课题运用分子设计的理论和方法，自行设计了rhA20，并从穿膜肽融合蛋白毕赤酵母表达载体yevCFP的构建和hA20基因的克隆着手，开展了毕赤酵母表达rhA20的一系列相关研究，为深入研究rhA20的抗炎作用效果与机制,以及制备hA20单克隆抗体，开展临床样品A20表达的ELISA定量分析等奠定必备的基础。本课题采用的主要研究方法和取得的阶段性结果有：1.首先将蛋白质分子设计的理论和方法引入rhA20的分子设计, 通过生物信息学软件的反复分析比对，我们选择了二级结构、理化性质等与天然hA20相对接近的设计方案。结果rhA20设计全长809个氨基酸，预测分子量93kD。2.利用自行设计的多对PCR引物，采用巢式梯度RT-PCR技术，从LPS诱导的人外周血白细胞总RNA中成功克隆得到2361bp长的hA20 cDNA并插入pCRR4-TOPOR载体，测序结果表明克隆基因与GenBank公布的hA20基因一致。3. 采用自行设计的单链PCR基因扩增技术，通过多轮单引物PCR体系的交替扩增以及相应扩增产物的混合变性和退火，成功制备了大量80bp长的CPP-Linker双链寡核苷酸。4.通过限制性核酸内切酶消化反应、目的DNA片段的分离纯化、T4 DNA连接酶催化的基因拼接反应、热休克基因转化技术、菌落PCR技术等基因工程技术操作，我们成功构建收获了穿膜肽融合蛋白毕赤酵母表达载体yevCFP，并将克隆的hA20 2361bp基因片段插入该载体得到yevCFP-rhA20表达质粒。5.用线性化的yevCFP-rhA20质粒电转化毕赤酵母GS115感受态细胞，通过HIS-营养缺陷平板、YPD-G418梯度平板的筛选，以及PCR技术对rhA20基因在GS115基因组中的整合的鉴定，得到了GS115/ yevCFP-rhA20阳性高拷贝菌株。采用甲醇诱导法，成功实现了rhA20在巴斯德毕赤酵母GS115中的分泌表达。表达蛋白分子量约93kD,全长表达产物占整个诱导培养基总蛋白的1.1%，全长蛋白的表达量达到22.84±0.17μg/ml水平。6.通过对BMMY培养基的改造和培养条件的优化，发现表达产物被毕赤酵母合成的蛋白水解酶降解可以通过降低诱导培养基pH值、添加低剂量EDTA、添加蛋白水解酶作用底物、降低诱导培养温度等措施在一定程度上得到缓解，以保护产物的获取。7.建立了rhA20初步分离纯化的超滤、(NH4)2SO4沉淀方法。8.利用免疫荧光染色技术和激光共聚焦扫描成像证实酵母表达rhA20能够在40min分钟内被THP1细胞摄取，进入细胞后主要分布在细胞质中。9. 体外实验显示，rhA20能显著抑制LPS对THP1内NF-κB的激活作用。综上所述，rhA20是一种基于CPP和hA20基因序列人工设计的抗炎基因工程重组蛋白，本课题设计和实现了rhA20在毕赤酵母GS115中的分泌表达，收获的产物经初步鉴定具有主动穿越THP1细胞膜的能力和一定的抗炎效果。此外，在上述研究中，双链CPP-Linker制备的单链PCR方法是对PCR扩增技术的补充和发展；课题构建的yevCFP载体可以作为一种基因表达工具，用于动物细胞胞内蛋白的表达研究。
Uncontrolled inflammatory response leading to immunity dysfunction and septic shock, even multiple organ dysfunction syndrome, is one of the most critical event in clinical patient’ mortality. In recent years, among the research of the inflammatory signal transduction, a new regulatory protein named A20 was recognized. It down-regulate the expression of TLR4, an important membrane receptor in inflammatory signal transduction, and can also down-regulate the activities of nuclear transcription factor NF-κB and AP-1. So, it plays a crucial role in keeping the balance between pro-inflammatory and anti-inflammatory cytokines and was taken as one of the intrinsic protecting proteins for tissues and cells. Regarding on the urgent drug requirement in clinical therapy of sepsis and the clinical diagnosis requirement for detecting the expression levels of A20 in samples at the first moment by ELISA, we designed a rhA20 molecule under the theory and methods of molecular design. We put great efforts to carry out a series of researches around expression A20 in Pichia pastoris from constructing the expression vector (yeast expression vector for cell-penetrating peptide fusion protein, yevCFP) as well as cloning human A20 cDNA. These are essential foundations for studying the anti-inflammatory effects and molecular mechanism, and preparing monoclonal antibody of rhA20. The latter is ready to develop a diagnostic ELISA kit for detecting the levels of A20 in clinical samples. The main research methods, results and conclusions were summarized as follow:A. Based on the molecular designing methods, we selected a regime of rhA20. Bioinformatics analysis suggested that the predict secondary structure、important physical and chemical characters of designed rhA20 relatively approach to natural hA20. The full-length of designed rhA20 is 809 amino acids. The predict molecular weight is 93kD.B. Total RNA of peripheral blood leucocytes stimulated by LPS was isolated and purified. Multi-pair PCR primers were designed. The 2361bp cDNA fragment of hA2O ORF was cloned by nested gradient RT-PCR and inserted into pCRR4-TOPOR vector by genetic engineering method. The results of sequencing analysis confirmed that the sequence of 2362bp cDNA fragment is the same as that in GenBank.C. Using our designed single-primer PCR, after many alternation amplification with different single primers and subsequently degenerating and annealing of their corresponding products in a PCR tubule, we successfully prepared a considerable quantity of CPP-Linker with 80bp length. D. Using genetic engirneering methods such as endonuclease digestion, DNA ligation catalysed by T4 DNA ligase、transformation and colony PCR etc, we successfully constructed yevCFP. The expression plasmid of rhA20, called yevCFP-rhA20, was obtained after insert the 2363bp DNA fragment into the yevCFP vector.E. Electroporation of Pichia pastoris was carried out with linearized yevCFP-rhA20. High copy clones were obtained through screening with MD/-His and YPD/G418. Pichia integrnants with full hA20 ORF gene were confirmed by PCR. The rhA20 was expressed by Pichia pastoris strain GS115 with methanol induction. The molecular weight of expressed rhA20 is about 93kD. The full expressing products hold 1.1% in total proteins of expressing supernatant and the expressing amount achieves to 22.84±0.17μg/ml.F. The recipe of BMMY medium was reformed and the culture condition was optimized. It was found that the degradation phenomena of expressing products can be controlled by a certain extent of decreasing the pH of medium, adding a low dosage of EDTA, adding the substrate of protease, as well as decreasing the induction temperature. G. The purified methods of expressing rhA20, including ultrafiltration and (NH4)2SO4 precipitation, were established. H. Using immunofluorescence staining, the laser confocal microphotographs showed that rhA20 could come into THP1 cells in 40 minutes and the distribution of rhA20 was mostly in cytop
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