【作者】 孙平楠；

【导师】 张学成；

【作者基本信息】 中国海洋大学 ， 遗传学， 2007， 博士

【摘要】 十九世纪六十年代,降钙素作为一种降血钙的物质被copp发现。它在哺乳动物中起源于甲状腺C细胞,在鱼类中后腮体分泌。降钙素是一种32个氨基酸组成的多肽内激素,其蛋白结构的N端为二硫键,C端为脯胺酰胺基团。降钙素在体内的钙、磷代谢中发挥着重要的作用。它能够直接作用于破骨细胞,降低破骨细胞的活性,从而抑制骨的吸收。降钙素的这种抗骨吸收活性使降钙素能够被广泛的使用于治疗Paget综合症,骨质疏松,以及高钙血症。在所有的降钙素当中,鲑鱼降钙素的活性最高,大约是人体的30-40倍。临床上已经多年使用鲑鱼降钙素进行治疗。然而,一方面,鲑鱼降钙素的现有给药途径仅仅注射和鼻喷两种,病人的依从性差。另一方面,鲑鱼降钙素的价格昂贵也限制了它在我国的普及。如何实现鲑鱼降钙素的口服途径,降低鲑鱼降钙素的造价成为了当前研究的重点。为了达到上述目标,本文通过生物工程的方法构建了含肠激酶位点的转鲑鱼降钙素基因的酵母yAGA2-sCT,并通过细胞实验和动物实验进行了活性检测。首先,通过化学与酶法相结合,人工合成了由酿酒酵母偏爱密码子组成的鲑鱼降钙素基因。合成的产物通过电泳进行了验证。合成的鲑鱼降钙素基因被克隆到puc18载体上进行保存。蛋白结构分析显示该基因和天然鲑鱼降钙素的蛋白结构相同。然后,该基因被克隆到了穿梭载体p-sf上得到了质粒p-sct-sf。质粒p-sf由INVITROGEN公司的质粒pyd1改造而来。酵母URA3基因分别被加到了质粒pyd1表达框的两侧当作重组位点,以增加酵母转化子的稳定性。经过改造以后的质粒和原质粒一样表达配体蛋白AGA2亚基。AGA2亚基与酵母表面的AGA1亚基通过二硫键连接于酵母表面的葡聚糖上,从而实现了外源蛋白在酵母表面的表达。质粒p-sct-sf通过LiAc转化的方法转化酵母菌株EBY100,得到了转基因酵母yAGA2-sCT。同时质粒p-sf转化酵母EBY100,得到了转化子yAGA2-V5。在后继实验当中,yAGA2-V5作为对照验证重组鲑鱼降钙素的活性。利用间接荧光标记的方法,在荧光显微镜下观察到了转化子yAGA2-V5和yAGA2-sCT外源蛋白的表达。进一步的流式细胞仪分析显示,表达开始12小时之后,yAGA2-sCT中有65%的细胞表达外源蛋白,yAGA2-V5转化子中有52%的细胞表达外源蛋白。在肠激酶的作用下,转基因酵母中的外源蛋白释放到酶切液的上清当中。该上清简称为EKS。外源表达的蛋白通过细胞实验对其体外的活性进行了检测。实验中采用的是破骨细胞。破骨细胞作为一种终分化细胞,能够在骨组织上进行骨的吸收,形成骨陷窝。实验首先通过骨片切割机制作了小牛皮质骨骨片。骨片与破骨细胞进行培养的过程中加入了yAGA2-V5 EKS和yAGA2-sCT EKS。密钙息(医用鲑鱼降钙素注射液)作为培养实验的阳性对照。骨片培养一周之后,骨片在数码显微镜下拍照并计算出骨陷窝面积。结果发现yAGA2-sCT EKS能够显著减少骨陷窝的生成。即重组鲑鱼降钙素能够在体外抑制破骨细胞的活性。根据英国药典的方法进行了动物降血钙实验。动物采用了正常的Wistar雌性大鼠。实验分为阴性对照组,阳性对照组和试剂组。阴性对照组灌胃5g/kg yAGA2-V5,阳性对照组则注射200mIU/kg密钙息(医用鲑鱼降钙素注射液)。试剂组当中,灌胃了低、中、高三个剂量(0.1 g/kg, 0.5 g/kg, 5 g/kg)冷冻干燥的yAGA2-sCT。实验动物在不同的时间段(1,2,4,6,8,10小时)从眼球丛静脉毛细血管吸取全血制备血清,血清送往青岛市解放军401医院测定血钙值。实验结果表明,灌胃5 g/kg冷冻干燥的酵母yAGA2-sCT显著降低了大鼠血钙值(p<0.01)。其最大药效时间Tmax为2小时,Wistar大鼠血钙值降低到2.355±0.012mM。实验发现灌胃yAGA2-sCT能持续降低血钙值,且呈现出剂量效应。转基因酵母yAGA2-sCT的持续降血钙能力对治疗高钙血症具有特殊意义。高钙血症是最常见的伴癌内分泌综合征,晚期癌肿病人约10%有高钙血症。当此症状转化为高钙血危象时,将严重威胁患者的生命。目前治疗高钙血症的首选药物是pamidronate等双磷酸盐药物。这些双磷酸盐药物毒副作用较大,长期使用可能导致体内解毒器官病变。鲑鱼降钙素安全性强且见能迅速降低血钙值,但注射鲑鱼降钙素2小时之后药效逐渐消失,故临床上鲑鱼降钙素在治疗高钙血症时作为辅助药物。为了研究转基因酵母yAGA2-sCT对高钙血症的作用,实验首先制作了高钙血症大鼠模型。根据文献方法,连续一周灌胃正常的Wistar雌性大鼠活性维他命D3(1, 25-dihydroxy-cholecalciferol)。大鼠血钙值由2.638±0.017 mM上升到2.838±0.022 mM,显著高于普通大鼠,标志着造模的成功。高钙血大鼠实验分为阴性对照组,阳性对照组和试剂组。阴性对照组灌胃5 g/kg yAGA2-V5,阳性对照组则注射1.25 mg/kg双磷酸盐药物pamidronate。试剂组当中,灌胃了低、中、高三个剂量(0.1g/kg,0.5g/kg,5g/kg)冷冻干燥的yAGA2-sCT。处理12小时之后从眼球丛静脉毛细血管吸取全血制备血清,血清送往青岛市解放军401医院测定血钙值。实验发现,灌胃5 g/kg冷冻干燥的酵母yAGA2-sCT显著降低了模型大鼠的血钙值,血钙值从2.81±0.018 mM降低到了2.69±0.025 mM,差异显著,P < 0.01。灌胃0.5g/kg yAGA2-sCT时,血钙值降低到2.75±0.016 mM,P < 0.05。实验表明转基因酵母对高钙血症具有抑制作用。实验探讨了转基因酵母的yAGA2-sCT的生物安全性问题。在急性毒性实验中,未能检测出半致死剂量LD50。在亚急性毒性实验当中,检测了大鼠的体重变化,血液生理指标,生化指标,脏器系。各项指标均无显著变化,初步证明转基因酵母用药安全。论文探讨了鲑鱼降钙素的口服给药可能的新途径,拓宽了鲑鱼降钙素在高钙血症治疗上的应用。为鲑鱼降钙素在我国医疗事业上更广泛的使用提供了实验依据。更多还原

【Abstract】 Copp discovered calcitonin as a kind of hypocalcemic principal in 1960’s. It was secreted by parafollicular cells of the thyroid in mammals or ultimobranchial body in fish. Calcitonin is a 32-amino-acid-long peptide with an N-terminal disulfide bridge and a C-terminal polyamide residue. It plays an important role in controlling calcium and phosphorus turnover. The peptide was shown to potently inhibit bone resorption by directly acted on osteoclast. Calcitonin’s potent anti-resorptive effect has led to its use in treating Paget’s disease of bone, osteoporosis,and hypercalcemia. Among all species, salmon calcitonin is of the highest potent which is about 30-40 times to human calcitonin. Salmon calcitonin is used in medical treatment by injection or nasal administration. The high price is the other limitation of salmon calcitonin. To study the oral delivery of salmon calcitonin and to low the price of it, yeast yAGA2-sCT was made with the enterokinase site on it. The bioactivity of yAGA2-sCT was investigated by cell experiment and animal experiment.At first, the salmon calcitonin gene was composed through chemical synthesis and protease approach according to the bias codon of yeast. The production was tested by electrophoresis. The synthetic salmon calcitonin gene was clone to puc18 vector. The forecast of recombinant protein is same to the nature salmon calcitonin. Then, the gene was clone to the vector p-sf to yield p-sct-sf. Plasmid p-sf was modified from pyd1 (INVITROGEN) vector. URA3 gene was added to the two flank of the expression frame as recombinant site. Plasmid p-sf express AGA2 protein, which linked to AGA1 protein on the surface of yeast. By this way, foreign could be expressed on the surface of yeast. Transformant yAGA2-sCT was got by transform p-sct-sf into EBY100. Transformant yAGA2-V5 was also got by transform p-sf into EBY100 to be the control in followed experiment. By indirectly labelled with FITC, yAGA2-V5 and yAGA2-sCT was found to express foreign protein under UV light. Recombinant salmon calcitonin expression was detected by flow cytometer. After 12 hours’ induction, 65 % of yAGA2-sCT cell and 52 % of yAGA2-V5 cell began to express salmon calcitonin. Yeast transformants were cut by enterokinase, the solution was short for EKS.Osteoclast was used in Cell experiment. As the final differentiation cell, osteoclast is of the potent of bone resorption. At first, the bone cutter was used to make bovine bone wafers. The yAGA2-V5 EKS or yAGA2-sCT EKS was added to the cell solution with bone wafer in it. Miacalcic (salmon calcitonin injection) was used as positive control. Area of bone lacuna was calculated after a week. The yAGA2-sCT EKS inhibited the formation of bone lacuna, which suggested the recombinant salmon calcitonin inhibited the activity of osteoclast.Animal experiment was conducted according to the England pharmacopoeia. The animal used here is a normal female Wistar rat. There are three kinds of group in the experiment. The negative group: orally administered with 5g/kg yAGA2-V5; the positive group: injected 200mIU/kg Miacalcic; The dosage groups (orally administered 0.1 g/kg, 0.5 g/kg, 5 g/kg yAGA2-sCT). Rats’ serum was got from eyes in different time(1, 2, 4, 6, 8, 10 hours)and sent to Navy 401 Hospital of PLA to test. The result is, oral administration of 5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.355±0.012mM(p<0.01).The Tmax is the second hour. The dosage effect and the long-term hypocalcemic effect are found. The long-term hypocalcemic effect of yAGA2-sCT is important to hypercalcemia. Hypercalcemia is a kind of complications with cancer. About 10% advanced cancer suffers have hypercalcemia, which was a deadly factor. For treating hypercalcemia, sCT has been replaced by bisphosphonates in view of their long-term effect. In contrast to bisphosphonates, sCT remains as an alternative in treating hypercalcemia for relieving of osteogenic pain, in particular, when safety considerations have become important.To investigate the effect of yAGA2-sCT to hypercalcemia, the hypercalcemic rats model was made. The female Wistar rats were fed with 1, 25-dihydroxy-cholecalciferol for a week according to the reference. The serum calcium raised from 2.638±0.017 mM to 2.838±0.022 mM, suggested the formation of hypercalcemia. In this experiment, rats was divided to negative group (orally administered with 5g/kg yAGA2-V5), positive group(injected 1.25 mg/kg pamidronate) and dosage group(orally administered 0.1g/kg,0.5g/kg,5g/kg yAGA2-sCT). Serum calcium was got from eyes in 12 hours and sent to Navy 401 Hospital of PLA to test. It was found that oral adminstration of 5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.69±0.025 mM(p<0.01)and oral administration of 0.5g/kg yAGA2-sCT decrease the serum calcium significantly to 2.75±0.016 mM(p<0.05). It suggested the transformant yAGA2-sCT could inhibit hypercalcimia in rats.The safety of transformant was investigated. In acute toxicity experiment, the LD50 could not be finding. In subacute toxicity experiment, the change of rats’ weight, the physiologic index, the biochemical criterion and the organ coefficient was investigated. None of this changed significantly. It suggested that the transformant yAGA2-sCT is safe to rats. These results showed, recombinant S. cerevisiae could thus be considered as a new drug delivery system for salmon calcitonin in hypercalcemia therapy. The salmon calcitonin could thus use more popularly in hypercalcimia.更多还原