人抗凝血酶Ⅲ的cDNA克
发布时间：2019-05-28 07:46
【摘要】：与传统的药物相比,重组蛋白质药物具有活性高、特异性强、毒性低、生物功能明确、便于临床应用、可规模化生产的优点,因此,重组药物蛋白的生产是当今国内外药物研究的热点,其中研究的重点则是如何经济、高效的生产和应用这类蛋白。虽然有细菌、真菌、昆虫细胞、动物细胞、转基因动植物等几种外源蛋白表达系统用来生产重组药用蛋白,但它们均存在着一些难以弥补的缺陷。为探讨利用动物乳腺作为表达系统来生产药用蛋白的可行性,本研究选取人血浆中重要的抗凝蛋白-人抗凝血酶(hAT)作为研究对象,构建含有hAT cDNA基因的重组腺病毒载体Ad-hAT,将Ad-hAT注入羊乳腺内,感染羊乳腺上皮细胞,使hAT的cDNA基因在羊乳腺内高效表达,从而在羊乳汁中获得具有生物功能的重组人抗凝血酶(rhAT)。对rhAT进行分离纯化后,作用于弥散性静脉内凝血(DIC)大鼠模型,对rhAT治疗DIC大鼠的疗效进行评判。通过以上试验,为重组腺病毒载体介导人源基因在动物乳腺中高效表达rhAT以及rhAT在人类DIC疾病中的应用提供理论依据和技术支持。 本研究主要包括以下内容:(1)提取人胚胎肝脏组织中的总RNA,经RT-PCR,获得用于表达的hAT的cDNA基因序列。对获得的cDNA序列进行测序分析,初步确定所扩增的cDNA的正确性。(2)以pcDNA3.1(+)为基础质粒,构建重组真核表达质粒p3AT,将之转入体外培养的羊乳腺上皮细胞细胞中获得表达,进一步证实hAT的cDNA序列可用于表达rhAT;(3)以pShuttle-CMV为基础质粒,构建重组穿梭载体pShAT,细菌内同源重组法获得重组腺病毒载体质粒pAd-hAT;(4)将pAd-hAT转入HEK293细胞中,通过病毒包装获得重组hAT腺病毒载体Ad-hAT;Ad-hAT在HEK293细胞内大量扩增;(5)将Ad-hAT转入体外培养的羊乳腺上皮细胞中获得表达,进一步证实Ad-hAT可用于高效表达rhAT;(6)将Ad-hAT注入羊乳腺,感染乳腺上皮细胞,在乳汁中获得rhAT,对乳汁中rhAT进行检测;应用肝素琼脂糖凝胶6FF亲和层析法分离乳汁中的重组蛋白,获得rhAT冻干粉;(7)构建大鼠DIC动物模型,分别用人血浆提取的hAT(hpAT)和rhAT进行处理,检测DIC大鼠血浆中DIC指标,进行疗效判定;(8)对hpAT和rhAT对DIC大鼠的疗效进行比较,探索在DIC治疗中,利用rhAT取代hpAT的可行性。 进行上述试验研究,取得如下研究结果: (1)通过RT-PCR法从人胚胎肝脏组织中获得了hAT的cDNA基因。经DNA测序分析,所得hAT的cDNA与Genbank中的相应序列(NM_000488)一致。 (2)以pcDNA3.1+、pIRES和pShuttle-CMV为基础质粒,构建了含有hAT cDNA基因的真核表达载体p3AT以及穿梭表达载体pShAT。 载体p3AT含有新霉素抗性筛选标记基因和GFP报告基因,可对已转染的哺乳动物细胞进行抗性筛选,也可对hAT的表达进行实时观测;构建的pShAT质粒含有GFP报告基因,可对腺病毒载体进行实时蛋白表达观测和快速滴度测定,为下一步研究rhAT的表达奠定基础。 (3)将所构建并经鉴定正确的pShAT用Pme I酶切线性化,将所获得的线性化片断直接转化处于感受态且含有Adeasy-1质粒的大肠杆菌BJ5183,通过细菌内同源重组的方法获得了含有hAT cDNA基因的重组腺病毒载体质粒pAd-hAT。证实细菌内同源重组能高效获得重组腺病毒载体质粒。 (4)在培养的HEK293细胞中,进行了重组腺病毒Ad-hAT的包装与扩增,获得了重组腺病毒Ad-hAT。 (5)质粒表达载体p3AT在体外培养的羊乳腺上皮细胞中获得表达,表达含量达到了700±90 mg/L,rhAT活性为110%～120%,证实了所PT-PCR获得的hAT cDNA可用于hAT的表达;重组腺病毒载体Ad-hAT在体外培养的羊乳腺上皮细胞中获得表达,表达含量达到了900±85 mg/L,rhAT活性为120%～130%,证实了Ad-hAT在羊乳腺上皮细胞中表达的可能性,为下一步在羊乳腺内高效表达hAT奠定了基础。 (6)将Ad-hAT注入羊乳腺后,在乳汁中获得了rhAT的高效表达,21 d内共11次的检测平均表达含量达到1.58±0.21 g/L,日检测单次最高3.1 g/L,活性平均为102.5±1.44 %。应用肝素琼脂糖凝胶亲和层析法分离纯化rhAT后,分离纯化后的rhAT回收率达到54.7±1.0%,纯度达到92.5±0.5%。 (7)成功构建大鼠DIC动物模型。 (8)用rhAT和hpAT处理DIC大鼠后,rhAT和hpAT一样(P0.05),均有效地缓解了大鼠DIC病情,对治疗DIC大鼠具有良好的疗效,主要表现为:血浆纤维蛋白原浓度减少和血小板增多的趋势得到有效抑制;血浆内hAT活性和凝血酶-抗凝血酶复合物(TAT)浓度得到相应增加;谷丙转氨酶(ALT)和谷草转氨酶(AST)浓度上升的趋势得到有效抑制。结果说明,在DIC的治疗药物选择中,rhAT具有取代hpAT的可行性。 本研究利用腺病毒为载体,在羊乳腺中高效表达了具有生物活性的rhAT;应用肝素琼脂糖凝胶亲和层析法分离纯化rhAT,获得了较高纯度的rhAT冻干粉;应用rhAT作用DIC大鼠模型,取得了和hpAT一样的良好的治疗效果(P0.05)。研究结果充分说明,本试验确定的重组蛋白表达与分离纯化路线是切实可行的,该方法获得的rhAT在治疗DIC疾病方面具有和hpAT同等的疗效。这些,将促进从人血浆中提取hAT这一传统生产方式的变革,使hAT生产向高效、经济、规模化方向发展;也将进一步拓宽rhAT的应用领域,创造出更大的经济和社会效益。
[Abstract]:Compared with the traditional medicine, the recombinant protein medicine has the advantages of high activity, strong specificity, low toxicity, definite biological function, convenient clinical application and large-scale production, The focus of the study was on how to produce and apply such proteins economically and efficiently. Although several foreign protein expression systems, such as bacteria, fungi, insect cells, animal cells, transgenic animals and plants, are used to produce recombinant medicinal proteins, they all have a number of defects that are difficult to compensate. In order to study the feasibility of using animal mammary gland as the expression system to produce the medicinal protein, the important anti-coagulation protein-human anti-thrombin (hAT) in human plasma was used as the research object, and the recombinant adenovirus vector Ad-hAT containing the hAT cDNA gene was constructed, and Ad-hAT was injected into the mammary gland of the sheep. The sheep mammary epithelial cells are infected, and the cDNA gene of the hAT is efficiently expressed in the mammary gland of the sheep, so as to obtain the recombinant human anti-thrombin (rhAT) with the biological function in the milk of the sheep. After the separation and purification of rhAT, the model of disseminated intravascular coagulation (DIC) was applied to evaluate the efficacy of rhAT in the treatment of DIC rats. Through the above test, the recombinant adenovirus vector-mediated human gene is used for efficiently expressing rhAT in the mammary gland of the animal and the application of the rhAT in the human DIC disease is provided with the theoretical foundation and the technical support. The main contents of this study are as follows: (1) The total RNA in human embryonic liver tissue is extracted, and the cDNA gene of hAT for expression is obtained through RT-PCR. and sequencing and analyzing the obtained cDNA sequence to preliminarily determine the positive and negative of the amplified cDNA, and (2) constructing a recombinant eukaryotic expression plasmid p3AT by using the pcDNA3.1 (+) as a base plasmid, transferring the recombinant eukaryotic expression plasmid p3AT into an in-vitro cultured goat mammary epithelial cell cell to obtain the expression, further confirming that the cDNA sequence of the hAT can be used for expressing the rhAT, (3) constructing a recombinant shuttle vector pSh with the pShuttle-CMV as a base plasmid, in that method, the recombinant adenovirus vector plasmid pAd-hAT is obtained by the homologous recombination in the AT and the bacteria; (4) the pAd-hAT is transferred into the HEK293 cell, and the recombinant hAT adenovirus vector Ad-hAT is obtained through the virus package; and the Ad-hAT is amplified in a large amount in the HEK293 cell; and (5) the Ad-hAT is transferred into the goat mammary epithelial cell cultured in vitro the expression of the Ad-hAT can be further confirmed to be used for high-efficiency expression of rhAT; (6) the Ad-hAT is injected into the mammary gland of the sheep, the human mammary epithelial cell is infected, rhAT is obtained in the milk, the rhAT in the milk is detected, the recombinant protein in the milk is separated by the heparin agarose gel 6-FF affinity chromatography, and the rhAT is obtained The effects of hpAT and rhAT on DIC rats were compared, and the effect of hpAT and rhAT on DIC rats was compared, and in the treatment of DIC, it was found that rhAT could be used to replace hpAT. 1. Carry out the above-mentioned test and study, and obtain the The results of the following studies: (1) hA was obtained from human fetal liver tissue by RT-PCR The cDNA of T. The cDNA of hAT and the corresponding sequence in Genbank (NM _ 0) were analyzed by DNA sequencing. (2) Eukaryotic expression vector p3AT containing hAT cDNA gene was constructed with pcDNA3.1 +, pIRES and pShuttle-CMV. The shuttle expression vector pSHAT. The vector p3AT contains a neomycin resistance screening marker gene and a GFP reporter gene, and the transfected mammalian cells can be screened for resistance, and the expression of the hAT can be observed in real time; and the constructed pS The hAT plasmid contains the GFP reporter gene, and can carry out real-time protein expression observation and rapid titer determination on the adenovirus vector, And the obtained linearized fragment is directly transformed into a competent state and contains the Adeasy-1 plasmid, and the obtained linearized fragment is directly transformed into the E. coli BJ5183 which is competent and contains the Adeasy-1 plasmid, and the hAT cDNA gene is obtained through the method of homologous recombination in the bacterium. The recombinant adenovirus vector pAd-hAT. (4) The recombinant adenovirus Ad-hAT was carried out in the cultured HEK293 cells. The expression of the recombinant adenovirus Ad-hAT. (5) plasmid expression vector p3AT was obtained in the goat mammary epithelial cells cultured in vitro, the expression level of the recombinant adenovirus Ad-hAT was 700-90 mg/ L, the activity of rhAT was 110-120%, and the PT was confirmed. the hAT cDNA obtained by-PCR can be used for the expression of hAT; the recombinant adenovirus vector Ad-hAT is expressed in the goat mammary epithelial cell cultured in vitro, the expression content of the recombinant adenovirus vector Ad-hAT is 900 to 85mg/ L, the activity of the rhAT is 120 to 130 percent, and the possibility of the expression of the Ad-hAT in the goat mammary epithelial cell is confirmed, The expression of hAT in the breast of the sheep was established by the next step. (6) After the Ad-hAT was injected into the breast of the sheep, the high-efficiency expression of rhAT was obtained in the milk, and the average expression content of 11 times in 21 days was 1.58-0.21g/ L. The highest activity was 3.1 g/ L, the average activity was 102.5 to 1.44%, and the purified rhAT was isolated and purified by a heparin-agarose gel affinity chromatography, and the purified rhAT was recovered. The rate reaches 54.7% to 1.0%, and the purity is up to 92.5 to 0.5%. (7) The rat model of DIC was successfully constructed. (8) After treatment with rhAT and hpAT, rhAT and hpAT were the same (P0.05). The main performance is that the plasma fibrinogen concentration is reduced and the tendency of the platelet increase is effectively inhibited; the concentration of the hAT activity and the thrombin-antithrombin complex (TAT) in the plasma is correspondingly increased; The increase in the concentration of transaminases (ALT) and aspartate aminotransferase (AST) was effectively inhibited. In the treatment of DIC, rhAT has the feasibility of replacing hpAT. hAT, high-purity rhAT freeze-dried powder is obtained, rhAT is applied, The results of the study show that the recombinant protein expression and the separation and purification route determined by this test are practical and feasible. The rhAT obtained by the method has the same curative effect as hpAT in the treatment of DIC diseases,
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341
本文编号：2486870
[Abstract]:Compared with the traditional medicine, the recombinant protein medicine has the advantages of high activity, strong specificity, low toxicity, definite biological function, convenient clinical application and large-scale production, The focus of the study was on how to produce and apply such proteins economically and efficiently. Although several foreign protein expression systems, such as bacteria, fungi, insect cells, animal cells, transgenic animals and plants, are used to produce recombinant medicinal proteins, they all have a number of defects that are difficult to compensate. In order to study the feasibility of using animal mammary gland as the expression system to produce the medicinal protein, the important anti-coagulation protein-human anti-thrombin (hAT) in human plasma was used as the research object, and the recombinant adenovirus vector Ad-hAT containing the hAT cDNA gene was constructed, and Ad-hAT was injected into the mammary gland of the sheep. The sheep mammary epithelial cells are infected, and the cDNA gene of the hAT is efficiently expressed in the mammary gland of the sheep, so as to obtain the recombinant human anti-thrombin (rhAT) with the biological function in the milk of the sheep. After the separation and purification of rhAT, the model of disseminated intravascular coagulation (DIC) was applied to evaluate the efficacy of rhAT in the treatment of DIC rats. Through the above test, the recombinant adenovirus vector-mediated human gene is used for efficiently expressing rhAT in the mammary gland of the animal and the application of the rhAT in the human DIC disease is provided with the theoretical foundation and the technical support. The main contents of this study are as follows: (1) The total RNA in human embryonic liver tissue is extracted, and the cDNA gene of hAT for expression is obtained through RT-PCR. and sequencing and analyzing the obtained cDNA sequence to preliminarily determine the positive and negative of the amplified cDNA, and (2) constructing a recombinant eukaryotic expression plasmid p3AT by using the pcDNA3.1 (+) as a base plasmid, transferring the recombinant eukaryotic expression plasmid p3AT into an in-vitro cultured goat mammary epithelial cell cell to obtain the expression, further confirming that the cDNA sequence of the hAT can be used for expressing the rhAT, (3) constructing a recombinant shuttle vector pSh with the pShuttle-CMV as a base plasmid, in that method, the recombinant adenovirus vector plasmid pAd-hAT is obtained by the homologous recombination in the AT and the bacteria; (4) the pAd-hAT is transferred into the HEK293 cell, and the recombinant hAT adenovirus vector Ad-hAT is obtained through the virus package; and the Ad-hAT is amplified in a large amount in the HEK293 cell; and (5) the Ad-hAT is transferred into the goat mammary epithelial cell cultured in vitro the expression of the Ad-hAT can be further confirmed to be used for high-efficiency expression of rhAT; (6) the Ad-hAT is injected into the mammary gland of the sheep, the human mammary epithelial cell is infected, rhAT is obtained in the milk, the rhAT in the milk is detected, the recombinant protein in the milk is separated by the heparin agarose gel 6-FF affinity chromatography, and the rhAT is obtained The effects of hpAT and rhAT on DIC rats were compared, and the effect of hpAT and rhAT on DIC rats was compared, and in the treatment of DIC, it was found that rhAT could be used to replace hpAT. 1. Carry out the above-mentioned test and study, and obtain the The results of the following studies: (1) hA was obtained from human fetal liver tissue by RT-PCR The cDNA of T. The cDNA of hAT and the corresponding sequence in Genbank (NM _ 0) were analyzed by DNA sequencing. (2) Eukaryotic expression vector p3AT containing hAT cDNA gene was constructed with pcDNA3.1 +, pIRES and pShuttle-CMV. The shuttle expression vector pSHAT. The vector p3AT contains a neomycin resistance screening marker gene and a GFP reporter gene, and the transfected mammalian cells can be screened for resistance, and the expression of the hAT can be observed in real time; and the constructed pS The hAT plasmid contains the GFP reporter gene, and can carry out real-time protein expression observation and rapid titer determination on the adenovirus vector, And the obtained linearized fragment is directly transformed into a competent state and contains the Adeasy-1 plasmid, and the obtained linearized fragment is directly transformed into the E. coli BJ5183 which is competent and contains the Adeasy-1 plasmid, and the hAT cDNA gene is obtained through the method of homologous recombination in the bacterium. The recombinant adenovirus vector pAd-hAT. (4) The recombinant adenovirus Ad-hAT was carried out in the cultured HEK293 cells. The expression of the recombinant adenovirus Ad-hAT. (5) plasmid expression vector p3AT was obtained in the goat mammary epithelial cells cultured in vitro, the expression level of the recombinant adenovirus Ad-hAT was 700-90 mg/ L, the activity of rhAT was 110-120%, and the PT was confirmed. the hAT cDNA obtained by-PCR can be used for the expression of hAT; the recombinant adenovirus vector Ad-hAT is expressed in the goat mammary epithelial cell cultured in vitro, the expression content of the recombinant adenovirus vector Ad-hAT is 900 to 85mg/ L, the activity of the rhAT is 120 to 130 percent, and the possibility of the expression of the Ad-hAT in the goat mammary epithelial cell is confirmed, The expression of hAT in the breast of the sheep was established by the next step. (6) After the Ad-hAT was injected into the breast of the sheep, the high-efficiency expression of rhAT was obtained in the milk, and the average expression content of 11 times in 21 days was 1.58-0.21g/ L. The highest activity was 3.1 g/ L, the average activity was 102.5 to 1.44%, and the purified rhAT was isolated and purified by a heparin-agarose gel affinity chromatography, and the purified rhAT was recovered. The rate reaches 54.7% to 1.0%, and the purity is up to 92.5 to 0.5%. (7) The rat model of DIC was successfully constructed. (8) After treatment with rhAT and hpAT, rhAT and hpAT were the same (P0.05). The main performance is that the plasma fibrinogen concentration is reduced and the tendency of the platelet increase is effectively inhibited; the concentration of the hAT activity and the thrombin-antithrombin complex (TAT) in the plasma is correspondingly increased; The increase in the concentration of transaminases (ALT) and aspartate aminotransferase (AST) was effectively inhibited. In the treatment of DIC, rhAT has the feasibility of replacing hpAT. hAT, high-purity rhAT freeze-dried powder is obtained, rhAT is applied, The results of the study show that the recombinant protein expression and the separation and purification route determined by this test are practical and feasible. The rhAT obtained by the method has the same curative effect as hpAT in the treatment of DIC diseases,
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341
【引证文献】
相关期刊论文 前1条
1 李秋菊;王巍;马红霞;高云航;高见;;抗凝血酶概述[J];动物医学进展;2011年09期
，本文编号：2486870
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