蛇毒類凝血酶的純化與基因篩選
發(fā)布時(shí)間：2019-05-29 04:50
【摘要】：研究目的 血管損傷性止血在臨床上占有很大比例。蛇毒酶制劑作為臨床用藥與同類其它制劑相比，其效果優(yōu)良，無嚴(yán)重的副作用，使用安全，在臨床治療上有很好的發(fā)展前景。國內(nèi)關(guān)于蛇毒類凝血酶酶的研究和利用有很多報(bào)道，但是真正具有國內(nèi)知識(shí)產(chǎn)權(quán)的止血用蛋白質(zhì)還沒有，同時(shí)也存在蛇毒原料的限制。在過去8年里，我們發(fā)現(xiàn)了一個(gè)新的止血蛋白Agacutase，其含量非常低，因此，本課題對尖吻蝮蛇蛇毒的止血類凝血酶進(jìn)行分離純化和基因篩選，通過基因重組生產(chǎn)，降低生產(chǎn)成本和促進(jìn)產(chǎn)品升級打下了基礎(chǔ)。 研究方法 本研究自尖吻蝮蛇蛇毒純化一種新的具有凝血活性的類凝血酶Agacutase。通過用Sephadex G-75凝膠層析、DEAE-Sepharose Fast Flow離子交換層析和Sephadex G-25凝膠脫鹽分離純化，根據(jù)核酸蛋白檢測儀在A280的檢測值大小，收集不同組分。用SDS-PAGE、HPLC檢驗(yàn)其純度，用質(zhì)譜（MS）和凝血活性方法，對其分子量和對底物酶解活性進(jìn)行分析。 在設(shè)計(jì)上游引物時(shí)，我們根據(jù)純化得到Agacutase的蛋白測序知道的前44位氨基酸序列來設(shè)計(jì)簡并引物。運(yùn)用M13（-48）作下游引物，是為了在逆轉(zhuǎn)錄反應(yīng)時(shí)在oligodT前面加上一段便于測序的基因，以蛇毒腺總RNA為模板，用RT-PCR方法擴(kuò)增其中的類凝血酶基因序列，膠回收條帶并克隆至T載體上，通過雙向測序得到篩選類凝血酶基因目的。通過用美國NCBI數(shù)據(jù)庫對克隆的蛇毒基因進(jìn)行同源性分析研究，以獲得初步的功能信息。 研究結(jié)果 通過對蛇毒干粉進(jìn)行多步分離純化后，用HPLC檢測其純度在90%以上，SDS-PAGE電泳結(jié)果為只有單一條帶，基體輔助激光解吸/電離飛行時(shí)間質(zhì)譜(MALDI-TOF-MS)檢測結(jié)果分子量為31084Da。纖維蛋白原底物分子水解活性顯示，樣品對α亞基逐步水解，在3小時(shí)水解達(dá)到高峰，而對照組中的豬凝血酶對纖維蛋白原的α亞基和β亞基明顯水解，在1小時(shí)達(dá)到高峰。并對β亞基的水解速度明顯快于對α亞基的水解速度。 通過設(shè)計(jì)不同長度的簡并引物（I、II、III、IV和V引物），用RT-PCR方法成功擴(kuò)增出不同大小的片段：I號引物對擴(kuò)增出1200bp、800bp、600bp三個(gè)片段；II號引物對擴(kuò)增出一個(gè)500bp的片段；III號引物對擴(kuò)增出1500bp和750bp兩個(gè)片段；IV號引物對擴(kuò)增出一個(gè)750bp的片段；V號引物對擴(kuò)增出1200bp和750bp兩個(gè)片段。PCR產(chǎn)物克隆于T載體后，挑取陽性克隆，測序得到六種類凝血酶基因（基因-I、III、VI、VII、VIII和IX）。 通過NCBI的BLAST比對后，，發(fā)現(xiàn)它們分別與以下蛋白酶的氨基酸序列具有一定同源性：基因-I與蛇毒金屬蛋白酶(snake venom metalloproteinase)有最大同源性為50%，后者具有水解纖維蛋白原的α鏈功能；基因-II與活化蛋白激酶C受體(receptorfor activated protein kinase C)有最大同源性為21%；基因-III與蛇毒絲氨酸蛋白酶Da-36(snake venom serine protease Da-36)有最大同源性為78%，后應(yīng)具有裂解纖維蛋白原Aα、Bβ和γ鏈的功能；基因-IV與NADH脫氫酶亞基4(NADH dehydrogenasesubunit4)有最大同源性為95%；基因-V與rCG20216有最大同源性為77%；基因-VI與蛇毒絲氨酸蛋白酶Dav-PA(Snake venom serine protease Dav-PA)有最大同源性為80%，可能具有裂解纖維蛋白原Aα和Bβ鏈功能；基因-VII與類凝血酶Acutobin(Thrombin-like enzyme acutobin)有最大同源性為95%，應(yīng)具有水解纖維蛋白原的α鏈功能；基因-VIII與蛇種Agkistrodon Acutus類凝血酶(venom thrombin-likeenzyme)有最大同源性為60%，可能具有裂解纖維蛋白原Aα和Bβ鏈功能；基因-IX與蛇種Agkistrodon Acutus的金屬蛋白酶Aahiv的A鏈有最大同源性為55%，可能具有蛇毒金屬蛋白酶的功能。 研究結(jié)論 通過凝膠過濾層析與離子交換層析等分離純化過程從尖吻蝮蛇蛇毒干粉中得到單一的組分，該組分為一種新的具有止血活性的類凝血酶，并命名為Agacutase。 基因篩選實(shí)驗(yàn)發(fā)現(xiàn)了幾種新的蛇毒類凝血酶。根據(jù)DNA序列推導(dǎo)氨基酸序列，通過NCBI的protein BLAST比對后，發(fā)現(xiàn)其中6個(gè)與幾種已知序列的類凝血酶的同源性在50%以上，有的甚至達(dá)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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R284.2
本文編號：2487666
[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.
【學(xué)位授予單位】：廣東藥學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R284.2
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