鸡骨保护素（chOPG）基因的克隆、表达及生物学活性研究
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摘要
骨保护素(osteoprotegerin，OPG)最先发现于哺乳动物，是一种能抑制破骨前体细胞分化、抑制成熟破骨细胞活性并诱导其凋亡的分泌型糖蛋白。体内调节骨代谢的许多激素和细胞因子最终通过调节OPG/RANKL的表达而发挥作用。鉴于OPG在骨代谢中的重要调节作用，本文旨在克隆表达蛋鸡OPG蛋白，并对其生物学活性进行研究，有望为蛋鸡骨质疏松症的防治提供新的治疗手段。
1．建立简便有效的鸡胚破骨细胞体外培养方法，为鸡骨保护素的生物活性研究及笼养蛋鸡骨质疏松症研究提供新的试验手段。主要剔取18日龄鸡胚的长骨，纵向剖开，吹打长骨内表面，获取一定数量细胞悬液接种培养板，2 h后弃去未贴壁细胞，更换新的培养液继续培养。贴壁培养的细胞具有破骨细胞样特点：为多核大细胞，有油煎蛋形、哑铃形、椭圆形；TRAP染色阳性，能在骨片表面形成吸收陷窝。
2．在体外成功培养鸡胚破骨细胞的基础上，体外直接研究依普拉芬、地塞米松对成熟破骨细胞骨吸收功能的影响。以不同浓度的依普拉芬、地塞米松与原代分离的破骨细胞共培养6天，结果发现，10~(-7)-10~(-9)M的依普拉芬可显著抑制破骨细胞性骨吸收、使骨吸收陷窝数目和面积减少，并呈剂量依赖关系；而10~(-6)-10~(-8)M的地塞米松则促进破骨细胞性骨吸收、增加骨吸收陷窝数目面积，亦呈剂量依赖关系。
3．应用RT-PCR方法从体外培养鸡胚成骨细胞中扩增出鸡OPG基因(Genbank登录号，DQ098013)，然后将特异性片段连接到pMD18-T载体，经酶切、PCR鉴定及DNA序列测定．结果表明，扩增片段包含完整的ORF，与Genbank上登录的鸡OPG(XM_418394)同源性为99.3％；同时对鸡、人、大鼠和小鼠OPG氨基酸序列进行了多序列比对分析，结果显示，鸡OPG和人OPG的一致性为69.65％，与大鼠及小鼠OPG的一致性均为65.51％。OPG基因的成功扩增为接下来该蛋白的重组表达奠定了基础。
4．设计一对引物用于亚克隆鸡OPG成熟蛋白编码基因(不含信号肽)，然后构建鸡OPG原核表达载体pET-32a(+)-chOPGm，通过IPTG诱导表达重组鸡OPG成熟蛋白，经SDS-PAGE电泳分析，原核表达产物为63 kD的重组蛋白，以包涵体形式存在，薄层扫描显示OPG占菌体总蛋白的11.9％，western blotting分析显示，表达的OPG蛋白具有良好的免疫反应性。以经镍离子亲和层析柱纯化的OPG蛋白免疫新西兰白兔，制备抗OPG多克隆抗体，ELISA结果显示抗体效价达到1：10240。Western印迹结果表明，该抗体可以和鸡OPG酵母表达产物特异性结合。
5．设计另一对引物用于亚克隆鸡OPG成熟蛋白编码基因(不含信号肽)，然后构建鸡OPG真核表达载体pPICZa-A-chOPGm’，以电穿孔法转化酵母X-33，用Zeocin平板筛选重组子，经甲醇诱导表达后，SDS-PAGE和免疫印迹分析表达产物，由于糖基化不同，表达产物有两种，其相对分子量分别为43 kD和53 kD，表达量约为200mg/L，经Western印迹验证，有较好的抗原性。表达产物经处理后加入到体外培养的鸡胚破骨细胞上，能显著抑制成熟破骨细胞的骨吸收活性，使骨吸收陷窝个数和面积均减少，证实表达的OPG蛋白具有抑制成熟破骨细胞骨吸收活性的功能。
6．100羽56周龄ISA蛋鸡分为5组，A组为对照组，B、C、D、E组每隔3 d肌肉注射剂量分别为100μg、200μg、300μg、400μg的鸡骨保护素酵母表达冻干产物水溶物。每周采血测血钙、血磷、碱性磷酸酶、抗酒石酸酸性磷酸酶和骨钙素。每天记录产蛋量、破壳蛋数，测定蛋壳强度和厚度。最后测定骨生物力学性能和骨密度。结果表明：B、C两组总平均产蛋率显著高于对照组和其它实验组。与A组相比，用药组总平均软破壳蛋率下降，其中B、C两组差异显著(P<0.05)。各组蛋壳厚度和强度无明显差异；用药组血钙显著低于对照组，血磷无明显差异；B、C、D组碱性磷酸酶活性显著高于对照组(P<0.05)；C、D两组骨钙素水平显著高于对照组(P<0.05)；E组抗酒石酸酸性磷酸酶活性显著低于对照组(P<0.05)；C组胫骨生物力学性能和放射骨密度显著高于对照组。结论：适当剂量(约200μg)的外源OPG蛋白可以改善骨代谢、提高产蛋量。
Osteoprotegerin(OPG) was firstly found in mammalian, it was a secreted protein which could inhibit the differentiation of osteoclast precursor cells and the activity of mature osteoclast, and also induce osteoclast apoptosis. A variety of hormones and cytokines regulated the bone metabolism through the regulation of OPG/RANKL ratio in fact. Considering that OPG had a very important role in bone metabolism, there were two aims of this study, one was to find a way to obtain functional OPG protein, the other aim was to evaluate the possibilisity of gene therapy for cage layer osteoporosis.
1. The more simple and effective method of isolating the osteoclastic cells from the embryonic chicken was built in order to supply a new way to study the biological activities of osteoprotegerin as well as the cage layer osteoporosis. Tibias and humeri were isolated from 10 chicken embryos(18 d of age)and cleaned of extraneous soft tissue, split each bone lengthwise and quickly flush the inside surface of bones to abtain the cell supernatants. Cell suspension were replated at 24-well dishes, nonadherent cells were washed off after 2 h. The adherent cells had a classic characteristic of osteoclasts: be multinucleated giant cells; like a fried egg, a dumbbell and a ellipse; staining positively for TRAP in cells; forming bone absorptive lacunae on the bone slices.
2. Based on the more effective method of isolating osteoclastic cells from the long bone(tibia and humeri) of embroynic chicken, the effects of ipriflavone and dexamethasone on the bone resorption of osteoclastic cells was observed. After 6-day co-cultured, bovine bone slices were taken out for toluidine blue staining in order to idenitfy the vitality of the cultured cells. The results showed that, ipriflavone at 10-~7 to 10-~9 M reduced the number and area of bone resorption pit lacunae(P<0.05), while dexamathesone at 10-~6 to 10-~8 M could increase the the number and area of bone resorption pit lacunae. Both effects of these two drugs had a positive correlation with their dosage.
3. To abtain the encoding genes of chicken osteoprotegerin(GenBank submission number, DQ098013) from the chicken embryo osteoblasts by RT-PCR method, the chOPG DNA fragment was cloned into pMD18-T vector, DNA sequencing, restriction enzyme digestion and PCR amplification all confirmed the inserted fragment was a complete chicken OPG gene with ORF, which had the identities of 99.3% with the chicken OPG gene published in the Genbank. The ammo acid sequence phylogenetic of OPG between chicken and mammalian showed that, the chicken had 69.95% identity to human and 65.51% identity to rat and mouse. The results supplied the basis of the recombinant expression of chOPG
4. A pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the OPGm gene was inserted to the expression plasmid pET-32a(+) and expressed in Rosetta-gami(DE3) pLysS with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in Rossetta with molecular weight of 63 kD and amounted to 11.9% of the whole protein, and western blotting indicated that the expressed protein had satisfied immunobiological activity. Pure protein was obtained by Ni~(2+)-NTA chelating column for preparation of anti-OPG polyclonal antibody by immunizing New Zealand rabbit, the titer of antiserum generated was 1:10240 by ELISA. Western blotting analysis showed that it could bind with OPG protein specially expressed in Pichia Yeast.
5. Another pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the chOPGm' was inserted into vector pPICZa-A. The constructed plasmid was transformed into yeast X-33 by electroporation. The recombinant transformants were selected by Zeocin. Induced by the addition of methanol every 24 hours, the product analyzed by SDS-PAGE was sized about 43 kD and 53 kD at a yield of 200 mg per litter of culture. The result of Western blotting indicated that the recombinant protein had specific antigenicity mainly owing to heterogeneous glycosylation. The chOPG recombinant protein at certain concentration could restrain the activity of mature osteoclasts in vitro, that was to say, the number and area of the bone resorptive lacunae were decreased.
6. 100 ISA cage layers were divided into 5 groups. Group A was controll, chOPG were injected to B, C, D, E groups at the dosage about 100μg, 200μg, 300μg, 400μg per four days. Every week, hen blood was collected to check the Ca, P, AKP. Each day, the number of eggs and abnormal and broken eggs were noted. The thickness and intensity of eggshells were tested every week. At last, bone biomechanical property and bone radiographic density were measured. The total average rate of eggs of group B and C was significantly higher than that of group A and the other groups. The total average rate of abnormal and broken eggs of group B, C, D and E were all decreased, group B and C were significantly lower than group A. The thickness and intensity of eggshells between each group were not significant. The serus level of Ca of addministrated groups were significantly lower than the control while no difference of the serus level of P. The serous level of AKP of group B, C and D were significantly higher than the control; The serous level of BGP of group C and D were also significantly higher than the control; while the serous level of StrACP of group E was significantly lower than the control. The bone biomechanical property and bone radiographic density of group C was significantly higher than the control. The study demonstrated that the appropriate dosage of chOPG may improve bone metablism and the production of cage layer during the late cycle.
1．建立简便有效的鸡胚破骨细胞体外培养方法，为鸡骨保护素的生物活性研究及笼养蛋鸡骨质疏松症研究提供新的试验手段。主要剔取18日龄鸡胚的长骨，纵向剖开，吹打长骨内表面，获取一定数量细胞悬液接种培养板，2 h后弃去未贴壁细胞，更换新的培养液继续培养。贴壁培养的细胞具有破骨细胞样特点：为多核大细胞，有油煎蛋形、哑铃形、椭圆形；TRAP染色阳性，能在骨片表面形成吸收陷窝。
2．在体外成功培养鸡胚破骨细胞的基础上，体外直接研究依普拉芬、地塞米松对成熟破骨细胞骨吸收功能的影响。以不同浓度的依普拉芬、地塞米松与原代分离的破骨细胞共培养6天，结果发现，10~(-7)-10~(-9)M的依普拉芬可显著抑制破骨细胞性骨吸收、使骨吸收陷窝数目和面积减少，并呈剂量依赖关系；而10~(-6)-10~(-8)M的地塞米松则促进破骨细胞性骨吸收、增加骨吸收陷窝数目面积，亦呈剂量依赖关系。
3．应用RT-PCR方法从体外培养鸡胚成骨细胞中扩增出鸡OPG基因(Genbank登录号，DQ098013)，然后将特异性片段连接到pMD18-T载体，经酶切、PCR鉴定及DNA序列测定．结果表明，扩增片段包含完整的ORF，与Genbank上登录的鸡OPG(XM_418394)同源性为99.3％；同时对鸡、人、大鼠和小鼠OPG氨基酸序列进行了多序列比对分析，结果显示，鸡OPG和人OPG的一致性为69.65％，与大鼠及小鼠OPG的一致性均为65.51％。OPG基因的成功扩增为接下来该蛋白的重组表达奠定了基础。
4．设计一对引物用于亚克隆鸡OPG成熟蛋白编码基因(不含信号肽)，然后构建鸡OPG原核表达载体pET-32a(+)-chOPGm，通过IPTG诱导表达重组鸡OPG成熟蛋白，经SDS-PAGE电泳分析，原核表达产物为63 kD的重组蛋白，以包涵体形式存在，薄层扫描显示OPG占菌体总蛋白的11.9％，western blotting分析显示，表达的OPG蛋白具有良好的免疫反应性。以经镍离子亲和层析柱纯化的OPG蛋白免疫新西兰白兔，制备抗OPG多克隆抗体，ELISA结果显示抗体效价达到1：10240。Western印迹结果表明，该抗体可以和鸡OPG酵母表达产物特异性结合。
5．设计另一对引物用于亚克隆鸡OPG成熟蛋白编码基因(不含信号肽)，然后构建鸡OPG真核表达载体pPICZa-A-chOPGm’，以电穿孔法转化酵母X-33，用Zeocin平板筛选重组子，经甲醇诱导表达后，SDS-PAGE和免疫印迹分析表达产物，由于糖基化不同，表达产物有两种，其相对分子量分别为43 kD和53 kD，表达量约为200mg/L，经Western印迹验证，有较好的抗原性。表达产物经处理后加入到体外培养的鸡胚破骨细胞上，能显著抑制成熟破骨细胞的骨吸收活性，使骨吸收陷窝个数和面积均减少，证实表达的OPG蛋白具有抑制成熟破骨细胞骨吸收活性的功能。
6．100羽56周龄ISA蛋鸡分为5组，A组为对照组，B、C、D、E组每隔3 d肌肉注射剂量分别为100μg、200μg、300μg、400μg的鸡骨保护素酵母表达冻干产物水溶物。每周采血测血钙、血磷、碱性磷酸酶、抗酒石酸酸性磷酸酶和骨钙素。每天记录产蛋量、破壳蛋数，测定蛋壳强度和厚度。最后测定骨生物力学性能和骨密度。结果表明：B、C两组总平均产蛋率显著高于对照组和其它实验组。与A组相比，用药组总平均软破壳蛋率下降，其中B、C两组差异显著(P<0.05)。各组蛋壳厚度和强度无明显差异；用药组血钙显著低于对照组，血磷无明显差异；B、C、D组碱性磷酸酶活性显著高于对照组(P<0.05)；C、D两组骨钙素水平显著高于对照组(P<0.05)；E组抗酒石酸酸性磷酸酶活性显著低于对照组(P<0.05)；C组胫骨生物力学性能和放射骨密度显著高于对照组。结论：适当剂量(约200μg)的外源OPG蛋白可以改善骨代谢、提高产蛋量。
Osteoprotegerin(OPG) was firstly found in mammalian, it was a secreted protein which could inhibit the differentiation of osteoclast precursor cells and the activity of mature osteoclast, and also induce osteoclast apoptosis. A variety of hormones and cytokines regulated the bone metabolism through the regulation of OPG/RANKL ratio in fact. Considering that OPG had a very important role in bone metabolism, there were two aims of this study, one was to find a way to obtain functional OPG protein, the other aim was to evaluate the possibilisity of gene therapy for cage layer osteoporosis.
1. The more simple and effective method of isolating the osteoclastic cells from the embryonic chicken was built in order to supply a new way to study the biological activities of osteoprotegerin as well as the cage layer osteoporosis. Tibias and humeri were isolated from 10 chicken embryos(18 d of age)and cleaned of extraneous soft tissue, split each bone lengthwise and quickly flush the inside surface of bones to abtain the cell supernatants. Cell suspension were replated at 24-well dishes, nonadherent cells were washed off after 2 h. The adherent cells had a classic characteristic of osteoclasts: be multinucleated giant cells; like a fried egg, a dumbbell and a ellipse; staining positively for TRAP in cells; forming bone absorptive lacunae on the bone slices.
2. Based on the more effective method of isolating osteoclastic cells from the long bone(tibia and humeri) of embroynic chicken, the effects of ipriflavone and dexamethasone on the bone resorption of osteoclastic cells was observed. After 6-day co-cultured, bovine bone slices were taken out for toluidine blue staining in order to idenitfy the vitality of the cultured cells. The results showed that, ipriflavone at 10-~7 to 10-~9 M reduced the number and area of bone resorption pit lacunae(P<0.05), while dexamathesone at 10-~6 to 10-~8 M could increase the the number and area of bone resorption pit lacunae. Both effects of these two drugs had a positive correlation with their dosage.
3. To abtain the encoding genes of chicken osteoprotegerin(GenBank submission number, DQ098013) from the chicken embryo osteoblasts by RT-PCR method, the chOPG DNA fragment was cloned into pMD18-T vector, DNA sequencing, restriction enzyme digestion and PCR amplification all confirmed the inserted fragment was a complete chicken OPG gene with ORF, which had the identities of 99.3% with the chicken OPG gene published in the Genbank. The ammo acid sequence phylogenetic of OPG between chicken and mammalian showed that, the chicken had 69.95% identity to human and 65.51% identity to rat and mouse. The results supplied the basis of the recombinant expression of chOPG
4. A pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the OPGm gene was inserted to the expression plasmid pET-32a(+) and expressed in Rosetta-gami(DE3) pLysS with IPTG inducement. The SDS-PAGE result showed that the cloned recombinant protein expressed in the form of inclusion bodies in Rossetta with molecular weight of 63 kD and amounted to 11.9% of the whole protein, and western blotting indicated that the expressed protein had satisfied immunobiological activity. Pure protein was obtained by Ni~(2+)-NTA chelating column for preparation of anti-OPG polyclonal antibody by immunizing New Zealand rabbit, the titer of antiserum generated was 1:10240 by ELISA. Western blotting analysis showed that it could bind with OPG protein specially expressed in Pichia Yeast.
5. Another pair of primers were designed to sub-clone the gene encoding chicken OPG mature protein, then the chOPGm' was inserted into vector pPICZa-A. The constructed plasmid was transformed into yeast X-33 by electroporation. The recombinant transformants were selected by Zeocin. Induced by the addition of methanol every 24 hours, the product analyzed by SDS-PAGE was sized about 43 kD and 53 kD at a yield of 200 mg per litter of culture. The result of Western blotting indicated that the recombinant protein had specific antigenicity mainly owing to heterogeneous glycosylation. The chOPG recombinant protein at certain concentration could restrain the activity of mature osteoclasts in vitro, that was to say, the number and area of the bone resorptive lacunae were decreased.
6. 100 ISA cage layers were divided into 5 groups. Group A was controll, chOPG were injected to B, C, D, E groups at the dosage about 100μg, 200μg, 300μg, 400μg per four days. Every week, hen blood was collected to check the Ca, P, AKP. Each day, the number of eggs and abnormal and broken eggs were noted. The thickness and intensity of eggshells were tested every week. At last, bone biomechanical property and bone radiographic density were measured. The total average rate of eggs of group B and C was significantly higher than that of group A and the other groups. The total average rate of abnormal and broken eggs of group B, C, D and E were all decreased, group B and C were significantly lower than group A. The thickness and intensity of eggshells between each group were not significant. The serus level of Ca of addministrated groups were significantly lower than the control while no difference of the serus level of P. The serous level of AKP of group B, C and D were significantly higher than the control; The serous level of BGP of group C and D were also significantly higher than the control; while the serous level of StrACP of group E was significantly lower than the control. The bone biomechanical property and bone radiographic density of group C was significantly higher than the control. The study demonstrated that the appropriate dosage of chOPG may improve bone metablism and the production of cage layer during the late cycle.
引文
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