重组人高密度脂蛋白的研究
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摘要
代谢综合征患者及高脂血症诱发的CAD患者都具有明显的脂类代谢紊乱(胆固醇升高和HDL降低),通过大量的流行病学研究已经证明血浆HDL水平与CAD呈负相关。动物试验证明,应用血浆HDL可明显预防和改善机体的脂类代谢紊乱,逆转CAD、AS的发生、发展,提示HDL有巨大的临床应用前景。
目前,HDL制剂的唯一来源是血浆,其来源受限、工艺复杂、生产成本高,直接影响其推广应用。本研究应用生物工程技术成功制备出与HDL组分相似的rhHDL制剂,并对该制品的生物学活性进行了检测,取得以下结果:
一、HDL蛋白组分的制备及中试规模的生产
1.成功构建了重组人载脂蛋白的分泌型表达载体pPICZα-rhApoX,利用巴斯德毕赤酵母稳定分泌表达了rhApoA-I、rhApoA-II、rhApoC-I、rhApoC-II和rhApoE。
2.利用80 L发酵罐进行了rhApoA-I和rhApoE中试规模发酵条件的优化,利用5 L摇瓶进行了rhApoA-II、rhApoC-I和rhApoC-II的高密度发酵试验,建立了适于大规模纯化上述五种载脂蛋白的新方法,收率分别为60%～70%,纯度达95%。
二、rhHDL的生物学活性
利用胆酸钠法,在体外将上述载脂蛋白成分与胆固醇、卵磷脂按照HDL2各成分的比例合成了rhHDL,并比较研究了rhHDL和天然HDL的生物学活性。
1.细胞水平:rhHDL能介导巨噬细胞的胆固醇流出,具有进行RCT的功能。
2.在体水平:rhHDL能调节实验性高脂血症大鼠的血脂代谢,显著降低TC、TG、AI和LDL-C,升高HDL-C;升高血清及组织中SOD活性从而抵抗自由基介导的脂质过氧化,防治动脉粥样硬化;减轻实验性高脂血症大鼠肝脏的脂肪沉积,达到对肝脏的保护作用。
体内、外分析实验表明,本研究利用胆酸钠法制备的rhHDL具有与天然HDL极为相似的生物学活性。
本研究的创新之处在于:1)建立了利用巴斯德毕赤酵母高效分泌表达rhApoA-II、rhApoC-I和rhApoE的真核表达系统;2)建立了rhApoA-I、rhApoA-II、rhApoC-I、rhApoC-II和rhApoE大规模纯化的新方法;3)利用胆酸钠法,在体外合成具有多种载脂蛋白成分的rhHDL;4)证实rhHDL具有与天然HDL极为相似的生物学活性。
High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma, which has equal content of lipid and protein. The components of protein of HDL are complicated. Identified proteins in HDL include the dominating apolipoprotein A-I (ApoA-I), ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE, recently discovered ApoM, serum amyloid A and serum amyloid A-IV. Furthermore,α-antitrypsin andα-ptyalin were identified in HDL for the first time. The complicated compositions of HDL lead to its multiple functions. HDL is a negative risk factor in coronary atherogenesis, and to raise HDL is expected to protect us against atherosclerosis. The direct anti-atherogenic activity of HDL in humans has been assumed because of due to its ability to trigger the flux of cholesterol from peripheral cells to the liver. HDL has several other potential anti-atherogenic properties, including anti-oxidation, anti-inflammation, eliminating cytotoxicity phospholipid, improving the function of vascular endodermis, anti-thrombotus, as well as facilitating fibrinolysis. At present, the way to obtain HDL is isolating it from human plasma. However, This way was restrictions on source of limited raw materials, complicated technology, potential infections and high cost restrict the development of this method. If we can obtain recombinant human HDL (rhHDL) by biotechnology, which has similar functions to that of natural HDL, the deficiency of natural HDL will be solved.
In this study, we prepared large amounts of the five kinds of recombinant human apolipoproteins (rhApoA-I, rhApoA-II, rhApoC-I, rhApoC-II and rhApoE) that consist in HDL. And recombinant human HDL having similar components to natural HDL was synthesized in vitro by the method of sodium cholate. Then the biological and pharmacodynamic functions of the rhHDL were further investigated.
The main technological processes are as follows.
1 Preparation of apolipoproteins
1.1 Acquisition of the gene of ApoX
Total RNA was isolated from human liver and used as the template for RT-PCR to obtain the DNA of ApoX (including ApoA-I, ApoA-II, ApoC-I and ApoE). Then the DNA encoding human ApoX was ligated to the clone vector pMD18-T respectively. The nucleotide sequences of the DNA were verified by sequencing with CEQ 2000 DNA analysis system and the sequences were consistent with that logged in GenBank (Accession No. NM000039, NM001643, NM001645 and NM000041).
1.2 Transformation of P. pastoris and selection of high-level expression colonies
Human ApoX DNA fragments were ligated to corresponding sites of the expression vector pPICZαC respectively. After the nucleotide sequences of the inserted DNA and flanking sequence were verified, the recombinant plasmid DNA was linearized with SacI and transformed into Pichia host cells (P. p astoris-X33) by electroporation. After induced with methanol for ApoX expression, the supernatant samples were used for SDS–PAGE analysis to identify the highly expressed rhApoX transformants.
1.3 High-density fermentation of rhApoX
After a series of experiments the optimal expression conditions for rhApoX were obtained as follows: the optimal pH of rhApoA-I was 6.5 and the optimal pH of rhApoE, rhApoA-II, rhApoC-I and rhApoC-II were 6.0, 6.0, 5.8 and 5.8 respectively. The expression of rhApoA-I and rhApoE under optimal conditions were examined in fermentor of 80 L respectively, and the expression of rhApoA-II, rhApoC-I and rhApoC-II under optimal conditions were examined in shake flask of 5 L respectively. The yield of rhApoA-I was 188 mg·L~(-1),rhApoE 120 mg·L~(-1), rhApoA-II 50 mg·L~(-1), rhApoC-I 80 mg·L~(-1) and rhApoC-II 38 mg·L~(-1).
1.4 Purification of rhApoX
The ApoX solution was purified with cation exchange chromatography (SP Sepharose XL) under pH 4.0 or pH 3.0 according to the PI of ApoX and reverse phase chromatography (SourceTM30 RPC). In regard to rhApoC-I and rhApoC-II, which molecular weigh were less than 10 kD, ultrafiltration (Vivaflow 200) was used to obtain high-purity recombinant protein. Then the purified rhApoX was concentrated by vacuum distillation to obtain high-concentration rhApoX. Then the method of purifying ApoX in pilot-scale was established. The purity was 95 % and the recovery was 60 %～70 %.
2 Preparation of rhHDL
In this study, we synthesized large-particles of mature rhHDL in vitro with rhApoX, lecithin and cholesterol, according to the components of natural HDL2 (ApoA-I 65 %, ApoA-II 10 %, ApoC-I 13 %, ApoC-II 1 % and ApoE 3%). Basing on SDS-PAGE analysis and morphological observation with electron microscope, we confirmed that the components were grouped and the diameter of particles was 50～80 nm.
3 Comparative studies on biological functions of rhHDL and natural HDL
3.1 Cholesterol efflux from macrophages triggered by rhHDL
The model of experimental hyperlipoproteinemia was established by giving lipid-riched feeds to Wistar rats. After the model was established, 3 % of thioglycollate broth (2 ml per rat) was given to the rats by peritoneal injection to induce macrophage. Four days later, the rats were executed after anaesthetized by ethoxyethane and the macrophages was collected by peritoneal lavage followed by seeded in 35-mm plates and cultured in the incubator for 3 hours. Then the non-adherent cells were washed away and 2 ml of medium (RPMI~(-1)640) was added for culturing the cells for another 24 hours before rhHDL was added. And nature HDL was also added as control. After treated with rhHDL at different dose or time, the macrophages were collected to detect the content of cholesterol. As a result, both rhHDL and natural HDL could induce cholesterol efflux from macrophages, and the ability enhanced along with the increasing of time and dose.
3.2 Effects on the blood-lipid metabolism in experimental hyperlipoproteine- mia rats
The model of experimental hyperlipoproteinemia was established by giving lipid-riched feeds to Wistar rats. After the model was established, the rats were treated with different dose of rhHDL, 1.25 mg/(kg·d) and 2.5 mg/(kg·d) respectively, contrasting with normal control group, model control group and positive medicine control group (native HDL 2.0 mg/ (kg·d)). The adjusting effects of rhHDL on TC, TG, LDL-C and HDL-C in serum were observed. At the same time, the SOD activity of serum and liver was also measured.
The results showed that both rhHDL and natural HDL could reduce the TC, TG, AI and LDL-C in serum significantly, and increase the HDL-C, regulating the blood-lipid metabolism in experimental hyperlipoproteinemia rats, and restrain fatty deposition in liver. These results suggested that rhHDL could prevent atherosclerosis by increasing the level of plasma HDL, accelerating reverse cholesterol transport to reduce cholesterol in peripheral cells. Moreover, rhHDL could significantly enhance the SOD activity both in serum and liver suggesting that rhHDL could facilitate free radical scavenging activity, resist free radical-mediated lipid peroxidation, thereby reduce the damage triggered by free radical to the body and prevent the occurrence and development of atherosclerosis.
To sum up, in this study we provided a new approach to preparing highly purified rhApoA-I, rhApoA-II, rhApoC-I, rhApoC-II and rhApoE, synthesized rhHDL by biotechnology in vitro, and demonstrated that rhHDL has the same biological functions to natural HDL.
目前,HDL制剂的唯一来源是血浆,其来源受限、工艺复杂、生产成本高,直接影响其推广应用。本研究应用生物工程技术成功制备出与HDL组分相似的rhHDL制剂,并对该制品的生物学活性进行了检测,取得以下结果:
一、HDL蛋白组分的制备及中试规模的生产
1.成功构建了重组人载脂蛋白的分泌型表达载体pPICZα-rhApoX,利用巴斯德毕赤酵母稳定分泌表达了rhApoA-I、rhApoA-II、rhApoC-I、rhApoC-II和rhApoE。
2.利用80 L发酵罐进行了rhApoA-I和rhApoE中试规模发酵条件的优化,利用5 L摇瓶进行了rhApoA-II、rhApoC-I和rhApoC-II的高密度发酵试验,建立了适于大规模纯化上述五种载脂蛋白的新方法,收率分别为60%～70%,纯度达95%。
二、rhHDL的生物学活性
利用胆酸钠法,在体外将上述载脂蛋白成分与胆固醇、卵磷脂按照HDL2各成分的比例合成了rhHDL,并比较研究了rhHDL和天然HDL的生物学活性。
1.细胞水平:rhHDL能介导巨噬细胞的胆固醇流出,具有进行RCT的功能。
2.在体水平:rhHDL能调节实验性高脂血症大鼠的血脂代谢,显著降低TC、TG、AI和LDL-C,升高HDL-C;升高血清及组织中SOD活性从而抵抗自由基介导的脂质过氧化,防治动脉粥样硬化;减轻实验性高脂血症大鼠肝脏的脂肪沉积,达到对肝脏的保护作用。
体内、外分析实验表明,本研究利用胆酸钠法制备的rhHDL具有与天然HDL极为相似的生物学活性。
本研究的创新之处在于:1)建立了利用巴斯德毕赤酵母高效分泌表达rhApoA-II、rhApoC-I和rhApoE的真核表达系统;2)建立了rhApoA-I、rhApoA-II、rhApoC-I、rhApoC-II和rhApoE大规模纯化的新方法;3)利用胆酸钠法,在体外合成具有多种载脂蛋白成分的rhHDL;4)证实rhHDL具有与天然HDL极为相似的生物学活性。
High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma, which has equal content of lipid and protein. The components of protein of HDL are complicated. Identified proteins in HDL include the dominating apolipoprotein A-I (ApoA-I), ApoA-II, ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoE, recently discovered ApoM, serum amyloid A and serum amyloid A-IV. Furthermore,α-antitrypsin andα-ptyalin were identified in HDL for the first time. The complicated compositions of HDL lead to its multiple functions. HDL is a negative risk factor in coronary atherogenesis, and to raise HDL is expected to protect us against atherosclerosis. The direct anti-atherogenic activity of HDL in humans has been assumed because of due to its ability to trigger the flux of cholesterol from peripheral cells to the liver. HDL has several other potential anti-atherogenic properties, including anti-oxidation, anti-inflammation, eliminating cytotoxicity phospholipid, improving the function of vascular endodermis, anti-thrombotus, as well as facilitating fibrinolysis. At present, the way to obtain HDL is isolating it from human plasma. However, This way was restrictions on source of limited raw materials, complicated technology, potential infections and high cost restrict the development of this method. If we can obtain recombinant human HDL (rhHDL) by biotechnology, which has similar functions to that of natural HDL, the deficiency of natural HDL will be solved.
In this study, we prepared large amounts of the five kinds of recombinant human apolipoproteins (rhApoA-I, rhApoA-II, rhApoC-I, rhApoC-II and rhApoE) that consist in HDL. And recombinant human HDL having similar components to natural HDL was synthesized in vitro by the method of sodium cholate. Then the biological and pharmacodynamic functions of the rhHDL were further investigated.
The main technological processes are as follows.
1 Preparation of apolipoproteins
1.1 Acquisition of the gene of ApoX
Total RNA was isolated from human liver and used as the template for RT-PCR to obtain the DNA of ApoX (including ApoA-I, ApoA-II, ApoC-I and ApoE). Then the DNA encoding human ApoX was ligated to the clone vector pMD18-T respectively. The nucleotide sequences of the DNA were verified by sequencing with CEQ 2000 DNA analysis system and the sequences were consistent with that logged in GenBank (Accession No. NM000039, NM001643, NM001645 and NM000041).
1.2 Transformation of P. pastoris and selection of high-level expression colonies
Human ApoX DNA fragments were ligated to corresponding sites of the expression vector pPICZαC respectively. After the nucleotide sequences of the inserted DNA and flanking sequence were verified, the recombinant plasmid DNA was linearized with SacI and transformed into Pichia host cells (P. p astoris-X33) by electroporation. After induced with methanol for ApoX expression, the supernatant samples were used for SDS–PAGE analysis to identify the highly expressed rhApoX transformants.
1.3 High-density fermentation of rhApoX
After a series of experiments the optimal expression conditions for rhApoX were obtained as follows: the optimal pH of rhApoA-I was 6.5 and the optimal pH of rhApoE, rhApoA-II, rhApoC-I and rhApoC-II were 6.0, 6.0, 5.8 and 5.8 respectively. The expression of rhApoA-I and rhApoE under optimal conditions were examined in fermentor of 80 L respectively, and the expression of rhApoA-II, rhApoC-I and rhApoC-II under optimal conditions were examined in shake flask of 5 L respectively. The yield of rhApoA-I was 188 mg·L~(-1),rhApoE 120 mg·L~(-1), rhApoA-II 50 mg·L~(-1), rhApoC-I 80 mg·L~(-1) and rhApoC-II 38 mg·L~(-1).
1.4 Purification of rhApoX
The ApoX solution was purified with cation exchange chromatography (SP Sepharose XL) under pH 4.0 or pH 3.0 according to the PI of ApoX and reverse phase chromatography (SourceTM30 RPC). In regard to rhApoC-I and rhApoC-II, which molecular weigh were less than 10 kD, ultrafiltration (Vivaflow 200) was used to obtain high-purity recombinant protein. Then the purified rhApoX was concentrated by vacuum distillation to obtain high-concentration rhApoX. Then the method of purifying ApoX in pilot-scale was established. The purity was 95 % and the recovery was 60 %～70 %.
2 Preparation of rhHDL
In this study, we synthesized large-particles of mature rhHDL in vitro with rhApoX, lecithin and cholesterol, according to the components of natural HDL2 (ApoA-I 65 %, ApoA-II 10 %, ApoC-I 13 %, ApoC-II 1 % and ApoE 3%). Basing on SDS-PAGE analysis and morphological observation with electron microscope, we confirmed that the components were grouped and the diameter of particles was 50～80 nm.
3 Comparative studies on biological functions of rhHDL and natural HDL
3.1 Cholesterol efflux from macrophages triggered by rhHDL
The model of experimental hyperlipoproteinemia was established by giving lipid-riched feeds to Wistar rats. After the model was established, 3 % of thioglycollate broth (2 ml per rat) was given to the rats by peritoneal injection to induce macrophage. Four days later, the rats were executed after anaesthetized by ethoxyethane and the macrophages was collected by peritoneal lavage followed by seeded in 35-mm plates and cultured in the incubator for 3 hours. Then the non-adherent cells were washed away and 2 ml of medium (RPMI~(-1)640) was added for culturing the cells for another 24 hours before rhHDL was added. And nature HDL was also added as control. After treated with rhHDL at different dose or time, the macrophages were collected to detect the content of cholesterol. As a result, both rhHDL and natural HDL could induce cholesterol efflux from macrophages, and the ability enhanced along with the increasing of time and dose.
3.2 Effects on the blood-lipid metabolism in experimental hyperlipoproteine- mia rats
The model of experimental hyperlipoproteinemia was established by giving lipid-riched feeds to Wistar rats. After the model was established, the rats were treated with different dose of rhHDL, 1.25 mg/(kg·d) and 2.5 mg/(kg·d) respectively, contrasting with normal control group, model control group and positive medicine control group (native HDL 2.0 mg/ (kg·d)). The adjusting effects of rhHDL on TC, TG, LDL-C and HDL-C in serum were observed. At the same time, the SOD activity of serum and liver was also measured.
The results showed that both rhHDL and natural HDL could reduce the TC, TG, AI and LDL-C in serum significantly, and increase the HDL-C, regulating the blood-lipid metabolism in experimental hyperlipoproteinemia rats, and restrain fatty deposition in liver. These results suggested that rhHDL could prevent atherosclerosis by increasing the level of plasma HDL, accelerating reverse cholesterol transport to reduce cholesterol in peripheral cells. Moreover, rhHDL could significantly enhance the SOD activity both in serum and liver suggesting that rhHDL could facilitate free radical scavenging activity, resist free radical-mediated lipid peroxidation, thereby reduce the damage triggered by free radical to the body and prevent the occurrence and development of atherosclerosis.
To sum up, in this study we provided a new approach to preparing highly purified rhApoA-I, rhApoA-II, rhApoC-I, rhApoC-II and rhApoE, synthesized rhHDL by biotechnology in vitro, and demonstrated that rhHDL has the same biological functions to natural HDL.
引文
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