蛇毒类凝血酶的纯化与基因筛选

发布时间：2019-05-29 04:50

【摘要】：研究目的血管损伤性止血在临床上占有很大比例。蛇毒酶制剂作为临床用药与同类其它制剂相比，其效果优良，无严重的副作用，使用安全，在临床治疗上有很好的发展前景。国内关于蛇毒类凝血酶酶的研究和利用有很多报道，但是真正具有国内知识产权的止血用蛋白质还没有，同时也存在蛇毒原料的限制。在过去8年里，我们发现了一个新的止血蛋白Agacutase，其含量非常低，因此，本课题对尖吻蝮蛇蛇毒的止血类凝血酶进行分离纯化和基因筛选，通过基因重组生产，降低生产成本和促进产品升级打下了基础。研究方法本研究自尖吻蝮蛇蛇毒纯化一种新的具有凝血活性的类凝血酶Agacutase。通过用Sephadex G-75凝胶层析、DEAE-Sepharose Fast Flow离子交换层析和Sephadex G-25凝胶脱盐分离纯化，根据核酸蛋白检测仪在A280的检测值大小，收集不同组分。用SDS-PAGE、HPLC检验其纯度，用质谱（MS）和凝血活性方法，对其分子量和对底物酶解活性进行分析。在设计上游引物时，我们根据纯化得到Agacutase的蛋白测序知道的前44位氨基酸序列来设计简并引物。运用M13（-48）作下游引物，是为了在逆转录反应时在oligodT前面加上一段便于测序的基因，以蛇毒腺总RNA为模板，用RT-PCR方法扩增其中的类凝血酶基因序列，胶回收条带并克隆至T载体上，通过双向测序得到筛选类凝血酶基因目的。通过用美国NCBI数据库对克隆的蛇毒基因进行同源性分析研究，以获得初步的功能信息。研究结果通过对蛇毒干粉进行多步分离纯化后，用HPLC检测其纯度在90%以上，SDS-PAGE电泳结果为只有单一条带，基体辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)检测结果分子量为31084Da。纤维蛋白原底物分子水解活性显示，样品对α亚基逐步水解，在3小时水解达到高峰，而对照组中的猪凝血酶对纤维蛋白原的α亚基和β亚基明显水解，在1小时达到高峰。并对β亚基的水解速度明显快于对α亚基的水解速度。通过设计不同长度的简并引物（I、II、III、IV和V引物），用RT-PCR方法成功扩增出不同大小的片段：I号引物对扩增出1200bp、800bp、600bp三个片段；II号引物对扩增出一个500bp的片段；III号引物对扩增出1500bp和750bp两个片段；IV号引物对扩增出一个750bp的片段；V号引物对扩增出1200bp和750bp两个片段。PCR产物克隆于T载体后，挑取阳性克隆，测序得到六种类凝血酶基因（基因-I、III、VI、VII、VIII和IX）。通过NCBI的BLAST比对后，，发现它们分别与以下蛋白酶的氨基酸序列具有一定同源性：基因-I与蛇毒金属蛋白酶(snake venom metalloproteinase)有最大同源性为50%，后者具有水解纤维蛋白原的α链功能；基因-II与活化蛋白激酶C受体(receptorfor activated protein kinase C)有最大同源性为21%；基因-III与蛇毒丝氨酸蛋白酶Da-36(snake venom serine protease Da-36)有最大同源性为78%，后应具有裂解纤维蛋白原Aα、Bβ和γ链的功能；基因-IV与NADH脱氢酶亚基4(NADH dehydrogenasesubunit4)有最大同源性为95%；基因-V与rCG20216有最大同源性为77%；基因-VI与蛇毒丝氨酸蛋白酶Dav-PA(Snake venom serine protease Dav-PA)有最大同源性为80%，可能具有裂解纤维蛋白原Aα和Bβ链功能；基因-VII与类凝血酶Acutobin(Thrombin-like enzyme acutobin)有最大同源性为95%，应具有水解纤维蛋白原的α链功能；基因-VIII与蛇种Agkistrodon Acutus类凝血酶(venom thrombin-likeenzyme)有最大同源性为60%，可能具有裂解纤维蛋白原Aα和Bβ链功能；基因-IX与蛇种Agkistrodon Acutus的金属蛋白酶Aahiv的A链有最大同源性为55%，可能具有蛇毒金属蛋白酶的功能。研究结论通过凝胶过滤层析与离子交换层析等分离纯化过程从尖吻蝮蛇蛇毒干粉中得到单一的组分，该组分为一种新的具有止血活性的类凝血酶，并命名为Agacutase。基因筛选实验发现了几种新的蛇毒类凝血酶。根据DNA序列推导氨基酸序列，通过NCBI的protein BLAST比对后，发现其中6个与几种已知序列的类凝血酶的同源性在50%以上，有的甚至达95%。
[Abstract]:Purpose of the study The hemostatic effect of vascular injury is much higher in the clinic. The snake venom enzyme preparation has the advantages of excellent effect, no serious side effect, safe use and good clinical treatment, There are many reports about the research and use of the snake venom thrombin enzyme in China, but there is no hemostasis protein with domestic intellectual property right, but there is also the limit of the snake venom raw material. In the past eight years, we have found a new hemostatic protein, Agacutase, which is very low in its content. Therefore, the subject is able to separate and purify the haemostatic thrombin of the venomous snake venom, and lay a foundation for the production of the gene, the production cost and the promotion of product upgrading. No, no, no. A new type of thrombin Agac with thromboplastin activity was purified by the method in this study. and utase, desalting and purifying by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-25 gel by using Sephadex G-75 gel chromatography, DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-25 gel desalting and purification, The purity was determined by SDS-PAGE and HPLC. The molecular weight and the activity of the substrate were determined by mass spectrometry (MS) and the method of coagulation. Sex analysis. At the time of designing the upstream primer, we obtained the first 44-bit amino acid sequence known from the purification of the Agacutase-based protein sequence The degeneracy primer is designed by using M13 (-48) as the downstream primer, and in order to add a section of the gene which is convenient for sequencing in the front of the oligo T in the reverse transcription reaction, the total RNA of the snake venom gland is used as a template, and the sequence of the class thrombin gene and the glue recovery strip are amplified by the RT-PCR method. cloning to a T vector, and obtaining a screening class by bi-directional sequencing Purpose of the thrombin gene. The cloned snake venom gene was analyzed by homology analysis with the NCBI database in the United States to obtain a preliminary work The results of the study were as follows: After the multi-step separation and purification of the dry powder of the snake venom, the purity was over 90% by HPLC. The results of SDS-PAGE were only single-band, matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The molecular weight of the fibrinogen substrate is 31084Da. The activity of the molecular hydrolysis of the fibrinogen substrate shows that the sample is gradually hydrolyzed to the subunit of the fibrinogen, and the peak is reached at 3 hours, while the porcine thrombin in the control group is distinct from the subunit of the fibrinogen and the subunit of the subunit. The hydrolysis is high at 1 hour, and the hydrolysis speed of the subunit is obvious. by designing degenerate primers (I, II, III, IV and V primers) of different lengths, the fragments of different sizes are successfully amplified by the RT-PCR method: the primer pair of the I is used for amplifying the three fragments of 1200bp, 800bp and 600bp; and the II primer pair A 500-bp fragment was amplified; the primer pair III was amplified with two fragments of 1500 bp and 750 bp; a 750 bp fragment was amplified by the IV primer pair; and the V-primer pair was amplified by 120. after the PCR product is cloned into the T vector, the positive clone is picked and sequenced to obtain the six-type thrombin gene (gene-I, III, VI, , VII, VIII, and IX). After the BLAST comparison of NCBI, they are found to have a certain homology with the amino acid sequence of the following protease: the gene-I and the snake venom metalloprotease have a maximum homology of 50%, The maximum homology between the gene-II and the activated protein kinase C receptor is 21%, the maximum homology of the gene-III and the snake venom serine protease Da-36 is 78%, and the gene-II has the cleavage fiber. The maximum homology of the gene-IV to the NADH dehydrogenase subunit 4 (NADH dehydro-asubunit4) is 95%, the maximum homology of the gene-V to the rCG20216 is 77%, and the gene-VI and the snake venom serine protease Dav-PA have the largest homology of 80% and may have a maximum homology of 80%. Cleavage of fibrinogen A and B-chain functions; the maximum homology of the gene-VII to the class-like thrombin-like enzyme acutobin is 95%, which should have a chain-chain function of hydrolyzing the fibrinogen; the gene-VIII has a maximum homology of 60% with the snake-like Agkistrodon Accutus-like thrombin, and may have a maximum homology of 60% Cleavage of the original A-and B-chain functions of the fibrinogen; the maximum homology of the gene-IX with the A-chain of the metal protease Ahiiv of the snake Agkistrodon Accuus is 55% and may The function of the snake venom metal protease was studied. The results of the study were as follows: the separation and purification process of gel filtration chromatography and ion exchange chromatography was used to obtain a single component in the dry powder of snake venom from the tip of the snake venom, and the group was divided into a new one with hemostatic activity. The class of thrombin, and is named Agacutase. In this paper, several new types of snake venom thrombin were identified by gene screening. The amino acid sequence was derived according to the DNA sequence.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R284.2

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